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CARDIOVASCULAR

ARD-353 [4-((2R,5S)-4-(R)-(4-Diethylcarbamoylphenyl)(3-hydroxyphenyl)methyl)-2,5-dimethylpiperazin-1-ylmethyl)benzoic Acid], A Novel Nonpeptide Receptor Agonist, Reduces Myocardial Infarct Size without Central Effects

Enhance Biotech Inc., Durham, North Carolina (M.J.W., J.D.S.H., S.J.O., K.W., W.P., K.-J.C.); Medical College of Wisconsin, Department of Pharmacology and Toxicology, Milwaukee, Wisconsin (G.J.G.); and Alza Corporation, Mountain View, California (P.J.G.)

Received July 26, 2005; accepted September 26, 2005.

	Abstract

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A novel -receptor selective compound, ARD-353 [4-((2R,5S)-4-(R)-(4-diethylcarbamoylphenyl)(3-hydroxyphenyl)methyl)-2, 5-dimethylpiperazin-1-ylmethyl)benzoic acid], was evaluated for activity on infarct size in a rat model of acute myocardial infarction. ARD-353 was characterized as having receptor selectivity using radioligand binding and had no apparent selectivity between receptor subtypes as determined by [³H] cyclic [D-Pen²,D-Pen⁵]enkephalin (₁) and [³H]Deltorphin II (₂) competition binding. ARD-353 also showed selective receptor agonist activity in mouse-isolated vas deferens. There was no evidence of any seizure-like convulsions when ARD-353 was administered to mice either i.v. or p.o., implying minimal penetration of the blood-brain barrier. ARD-353 decreased infarct size in a left anterior descending coronary artery (LAD) occlusion model of myocardial infarction. In animals pretreated with ARD-353 (i.v.) and then subjected to 30 min of LAD occlusion followed by 90 min of reperfusion, infarct size was reduced in a dose-dependent manner compared with vehicle-treated controls. The effects of ARD-353 on infarct size were blocked by the ₁-opioid selective antagonist 7-benzylidenenaltrexone, indicating a significant role for the ₁-opioid receptor in the cardioprotective mechanism of ARD-353. ARD-353 (0.3 mg/kg i.v.) produced significant protection when administered 5 min and 12 and 48 h before ischemic insult or when given immediately after the ischemic insult (at the start of reperfusion). Given the lack of central nervous system effects and beneficial efficacy in the rat model of myocardial ischemia, it is felt that ARD-353 is the first nonpeptide -receptor agonist with true potential for clinical use before surgically induced ischemia or in an emergency setting.

Patients undergoing repeated balloon angioplasty have been shown to experience significant protection through adaptation of the myocardium to mild ischemic periods, which was sensitive to blockade by the nonspecific opioid receptor antagonist naloxone (Tomai et al., 1999). An opioid role in human preconditioning was further demonstrated by Xenopoulos et al. (1998) with the application of intracoronary morphine (15 µg/kg) as a mimic for preconditioning, as evidenced by its effectiveness in reducing ST segment changes during percutaneous transluminal coronary angioplasty. Despite these promising results with morphine, no exclusive receptor agonists are available for current clinical use, and most synthetic nonpeptide receptor agonists have been shown to produce seizure-like convulsions in animals (Comer et al., 1993; Broom et al., 2002a,b).

The effects of receptor activation on broader cardiovascular function remain unclear. Historically agonists have been considered pressor in nature, although these findings seem to be specific to the site of administration (Holaday, 1983). Further complexity in the historical data stems from the assessment of cardiovascular effects of endogenous opioids in anesthetized and conscious animals. Interactions with anesthetics seem to reduce the pressor effects of opioids, while increasing the depressor effects of µ opioids (Holaday, 1983). Several studies have focused on the central effects of opioid agonists and antagonists administered directly into specific brain regions such as the paraventricular nucleus (Bachelard and Pitre, 1995) or the ventrolateral periaquaductal gray region (Keay et al., 1997; Cavun et al., 2004), indicating a possible role for central receptors in the modulation of cardiac rhythm and blood pressure in response to various stimuli including visceral nociception (Cavun et al., 2004). However, only opioid receptor-selective agonists (and not µ or receptor-selective agonists) produced changes in baseline cardiac rhythm of isolated rat hearts (Wong et al., 1990). More recently, Farias et al. (2003) have demonstrated both a vagolytic and vagotonic activity of the agonist, methionine-enkephalin-arginine-phenylalanine, applied locally to the sinoatrial node of dogs.

The benzhydrylpiperazine compound presented here, ARD-353 (Fig. 1), was selected for the assessment of its potential cardioprotective efficacy in a rodent model of cardiac ischemia and reperfusion injury after drug administration at various preischemic and postischemic time points. The studies presented herein were designed to characterize the radioligand binding affinity and intrinsic activity of ARD-353 at the , µ, and opioid receptors and the effect of ARD-353 on CNS and cardiovascular function in rodents and to describe the cardioprotective efficacy of ARD-353 in rodents and its induction of acute and delayed phases of protection.

View larger version (13K): [in this window] [in a new window]   Fig. 1. The structure of ARD-353.

	Materials and Methods

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This study was performed under approved Institutional Animal Care and Use Committee protocols and in accordance with U.S. Department of Agriculture regulations.

Radioligand Binding
Membrane Preparation for Radioligand Binding. The brains from male albino Sprague-Dawley rats were obtained from Dynal Biotech (Lake Success, NY) and cerebellum from male albino guinea pigs from Accurate Chemical & Scientific (Westbury, NY). After tissues were rinsed with ice-cold 50 mM Tris-HCl buffer, pH 7.4 (25°C) containing the following protease inhibitors: 50 µg/ml soybean trypsin inhibitor, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 10 µg/ml leupeptin, 200 µg/ml Bacitracin, and 0.5 µg/ml Aprotinin, the brains were homogenized in 5 to 10 volumes/g wet weight of ice-cold 50 mM Tris buffer containing protease inhibitors. A motor-driven glass-Teflon homogenizer (nominal clearance, 0.13-0.18 mm) was used to prepare the homogenate that was then centrifuged at 6000g for 15 min at 4°C. The resulting supernatant was centrifuged at 41,000g for 30 min. The membrane pellet was resuspended in 10 volumes/g wet weight of 10 mM Tris-sucrose (0.32 M) buffer and sonicated with a tissue grinder (10 s, low speed; Polytron, Kinematica, Basel, Switzerland). The homogenate was centrifuged at 41,000g for 30 min at 4°C. The resulting membrane pellet was resuspended in 50 mM Tris buffer with protease inhibitors at a final protein concentration that ranged from 40 to 50 µg/ml. The membrane fraction was frozen under liquid N₂ and stored at -80°C before use in radioligand binding studies. Protein content was determined by the method of Bradford (1976).

Competition Binding. Membrane fractions were incubated with either 0.1 nM [³H]DPDPE (a -opioid receptor agonist; ₁) or [³H]Deltorphin II (₂) (specific activity, 50.6 Ci/mmol; n = 5), 0.1 nM [³H]DAMGO (a µ-opioid receptor agonist; specific activity, 50.0 Ci/mmol; n = 8), or 0.1 nM [³H]U69593 (a -opioid receptor agonist; specific activity, 41.4 Ci/mmol; n = 4) in 2 ml of 10 mM Tris-HCl buffer containing 5 mM MgCl₂ and protease inhibitors. Incubation was carried out for 90 min at 25°C to permit the complete equilibration of the radioligand with its receptor. The reaction termination was achieved by rapid filtration through Whatman GF/C glass fiber filters using a cell harvester (model M-48R; Brandel Inc., Gaithersburg, MD) followed by two 5-ml rinses with ice-cold 50 mM Tris buffer. Nonspecific binding was defined as that radioligand bound in the presence of 1 x 10^-6 M naloxone. Filters were counted by liquid scintillation spectrometry (LS 6500; Beckman Coulter, Fullerton, CA) at an efficiency, determined by external standards, of 40 to 45%.

Intrinsic Activity
Tension development in isolated vas deferens was measured as described previously (Chang et al., 1993). After cervical dislocation, vasa deferentia were isolated from male CD-1 mice (Charles River Laboratories, Inc., Wilmington, MA) weighing 20 to 25 g. Muscles were suspended in individual organ baths containing Mg-free Krebs-Henseleit solution (37°C, aerated with O₂-CO₂, 95:5) of the following composition: 117.5 mM NaCl, 4.75 mM KCl, 2.6 mM CaCl₂, 1.2 mM KH₂PO₄, 24.5 mM NaHCO₃, and 11 mM glucose.

The vas deferens segments were positioned between platinum electrodes (Radnoti Glassware Technology Inc., Monrovia, CA) and connected to a Grass (Grass-Telefactor, West Warwick, RI) FTO3 isometric force transducer using suture silk at a resting tension of 0.5 g. Muscles were stimulated to contract by administering 400-ms pulse trains (1-ms duration, supramaximal voltage, 10 Hz) with a Grass S88 stimulator. IC₅₀ values for , µ, and receptor activity were derived by the blockade of all but one receptor subtype through the application of combinations of specific inhibitors for each respective receptor subtype ( antagonist, 3 µM TIPP; µ antagonist, 1 µM CTOP; antagonist, 15 µM nor-binaltorphimine) before exposure to cumulative concentrations of ARD-353 (Chang et al., 1993).

Evaluation of Central Effects in Mice
Male CD-1 mice (Charles River Laboratories, Inc.) weighing 20 to 25 g were used to assess the central effects of ARD-353. Mice received a single dose of ARD-353, either s.c. via the tail vein (10 or 50 mg/kg; 1 ml/kg bolus; n = 10/dose) (100 mg/kg) or orally (100 mg/kg). BW373U86 (10 mg/kg s.c.) was used as a positive control. They were then assessed for the following.

Seizure-Like Convulsions. Mice were observed for 1 (i.v.) or 2 (p.o. and s.c.) h after dosing. A seizure-like event was recorded if a mouse had uncontrollable clonic (or tonic/clonic) muscle movements that encompassed its entire body, usually followed by a brief cataleptic period.

Catalepsy. Catalepsy was determined by placing the animals' front paws on a horizontal bar held 2 to 3 inches from the cage floor. Cataleptic animals made no attempt to remove their paws.

Loss of Righting Reflex. Loss of righting reflex was determined by laying the animal on its side in the cage. Loss of righting reflex was determined as making little or no effective attempts to return to a normal, upright posture.

Strong Antinociception. Response latency to short-term pain was evaluated with the tail-pinch test. An arterial clamp was placed on the base of the tail until the mouse demonstrated audible vocalizations, bit the clamp, demonstrated a tail-flick escape response, or showed no response for 20 s. The time, in seconds, to any response (defined as the tail-pinch response latency) was recorded by means of a stopwatch. Immediate response to the application of the arterial clamp was recorded as a latency of 1.0 s and scored as no antinociception. After application of the arterial clamp, if the maximum 20-s time period elapsed with the animal demonstrating no escape response, the animal received a full (or 100%) antinociceptive score.

Baseline response latency was determined for each animal before drug administration and is recorded as the latency at 0 min. Thereafter, mice were injected with the test compound or control (dextrose), and their response latency to tail-pinch was measured.

Antinociceptive effects of ARD-353 were evaluated at the following postdosing time points: s.c. and p.o., tail-pinch response latency was evaluated at 0, 10, 30, 60, 90, and 120 min after oral or s.c. administration of DPI-221 or dextrose; and i.v., tail-pinch response latency was evaluated at 0, 2, 5, 10, 20, and 30 min after i.v. administration of DPI-221 or dextrose.

Hemodynamic Measurements
Blood pressure and heart rate were measured in five male Sprague-Dawley rats (Harlan, Indianapolis, IN) (225-300g) that received 3, 20, and 40 mg/kg i.v. ARD-353 (1 ml/kg, bolus). Animals were anesthetized under N₂O/O₂/isoflurane. Both the jugular vein and carotid artery were catheterized to allow for i.v. compound dosing and arterial blood pressure measurements, respectively. Animals were allowed to recover from anesthesia before any measurements were taken, and all hemodynamic measurements were obtained from conscious, restrained animals. After exposure to ARD-353, the positive inotrope, isoproterenol (2 mg/kg i.v.; 1 ml/kg bolus) was administered to confirm hemodynamic responsiveness and catheter patency.

Modeling of Cardioprotective Efficacy
Surgery and Left Anterior Descending Coronary Artery (LAD) Occlusion. The methods used were adapted from those described previously by Fryer et al. (1999). Male Sprague-Dawley rats (Harlan) (225-300g) were anesthetized by i.p. administration of urethane (1-1.5 g/kg). A tracheotomy was performed, and the animal was cannulated with PE-240 tubing, which was then connected to a respirator (model 683; Harvard Apparatus Inc., Holliston, MA). The animal was ventilated at 40 breaths/min with O₂. Atelectasis was prevented by maintenance of a positive end respiratory pressure of 5 to 10 mm H₂O. This respiratory rate and ventilation have been shown to maintain proper blood pH, P_O2, and P_CO2 in these animals. Body temperature was maintained at 37°C by use of a heating pad and monitored using rectally placed temperature probes connected to a digital thermometer. The left carotid was cannulated to measure blood pressure and heart rate via a Grass PT300 pressure transducer connected to a PowerLab/16sp data module and a personal computer. The right jugular vein was cannulated to allow administration of test compound or vehicle and patent blue dye.

A left thoracotomy was performed to expose the heart at the fifth intercostal space. The pericardium was removed, and the atrial appendage was moved to allow access to the LAD. A snare was made by passing a 6-0 ligature beneath the artery, and the ligature was passed through a flared section of PE-205 tubing. With the PE-205 tubing placed on the myocardial surface, the ligature could be tightened around the LAD and clamped to provide a readily reversible constriction or occlusion of blood flow.

Treatments. A dose-response curve for the cardioprotective effects of ARD-353, compared with pH-matched 5% dextrose vehicle (n = 9), was produced with i.v. ARD-353 doses of 0.01 (n = 5), 0.03 (n = 6), 0.3 (n = 6),1(n = 5), and 3 (n = 5) mg/kg administered 5 min before LAD occlusion in a 1 ml/kg dosing volume as a bolus.

Antagonism of ARD-353 by 7-benzylidenenaltrexone (BNTX) was investigated using a single dose of BNTX (1 mg/kg i.v.; 1 ml/kg bolus) or DMSO vehicle (n = 9) administered 10 min before dosing with either ARD-353 (0.3 mg/kg i.v.; 1 ml/kg bolus; n = 9) or saline vehicle (n = 7). LAD occlusion was performed immediately after ARD-353 (or saline) dosing. Additional controls were run with ARD-353 alone (0.3 mg/kg i.v.; 1 ml/kg bolus; n = 7), saline vehicle (n = 5) or no drug or vehicle (sham group; n = 16) immediately before LAD occlusion.

The time course of effects of ARD-353 administered before occlusion was investigated using 0.3 mg/kg i.v. ARD-353 (1 ml/kg bolus) administered 5 min and 12, 24, 48, and 72 h before occlusion (n = 6 for all groups except 72 h where n = 5 and vehicle where n = 9).

The effect of ARD-353 on reperfusion injury occurring postischemia was assessed through the administration of 0.3 mg/kg i.v. ARD-353 (1 ml/kg bolus) immediately after (0 min; n = 5) and 30 min after (n = 8) removal of the LAD occlusion.

Determination of Cardioprotective Effects. After administration of vehicle or test compound, the LAD was occluded for 30 min, followed by a 90-min reperfusion period where the ligature was unclamped and loosened from the LAD. After 90 min of reperfusion, the ligature was again tightened to occlude the LAD, and patent blue dye (0.4 ml of 10% w/v in saline) was injected. Once the dye had stained the heart, except in the area at risk, clearly demarcating the total area at risk (AAR), the heart was then quickly removed from the animal, and the atria, valvular material and right ventricular wall were removed leaving just the left ventricle. The left ventricle was then sectioned transversely into five to six slices. The area defined as normal (dyed blue) was separated from the AAR (not dyed blue), and the tissue was placed in separate 20-ml vials containing 100 mM KH₂PO₄ and 0.187% 2,3,5-triphenyltetrazolium chloride and incubated at 37°C for 5 to 10 min. Infarcted areas appeared whitish after 2,3,5-triphenyltetrazolium chloride staining, whereas noninfarcted, or viable, areas appeared reddish in color. Tissues were then placed in separate vials containing a 10% buffered formaldehyde solution overnight for fixing.

Infarcted areas were dissected from noninfarcted areas, and the amount of normal; area at risk, noninfarcted; and infarcted tissues were measured gravimetrically. Data were presented as infarct size (IS) as a percentage of the AAR (i.e., %IS/AAR) for statistical analysis of cardioprotective effects (decreased %IS/AAR).

Sources of Drugs
[³H]DPDPE, [³H]DAMGO, [³H]Deltorphin II, and [³H]U69593 were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Purities were greater than 98%. CTOP and TIPP were purchased from Peninsula Laboratories (Belmont, CA). Naltrindole and all other chemicals were reagent grade and purchased from Sigma-Aldrich (St. Louis, MO). Novel compounds described in this study were synthesized at Ardent Research Laboratories (Durham, NC) using standard protocols.

Calculations and Statistics
Pharmacological data were analyzed by nonlinear regression of the concentration-response curves to determine EC₅₀, IC₅₀, or K_i values using the computer program Prism (GraphPad Software Inc., San Diego, CA). Statistical comparisons for in vivo infarct reduction were carried out using analysis of variance followed by a Tukey's post hoc test or a Dunnett's post hoc test where comparisons were made only with vehicle controls.

	Results

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Competition Binding. ARD-353 bound with high affinity to receptors with no apparent selectivity for either of the putative receptor subtypes (₁ K_i, 1.9 ± 0.1 nM; ₂ K_i, 1.9 ± 0.4 nM; mean ± S.E.M.), demonstrating a greater than 1000-fold selectivity for the over the µ receptor (K_i, 2700 ± 900; Fig. 2). ARD-353 bound to the receptor with such low affinity that 50% displacement of ligand was not achieved at the highest concentration tested. As such, the extrapolation of the curve gave a mean K_i value of 19,300 ± 1900 nM. The binding of ARD-353 to the and µ receptors was best fit by a one-site, nonlinear regression curve (and the same was used for the extrapolation).

View larger version (14K): [in this window] [in a new window]   Fig. 2. Competition binding of ARD-353 versus 1- (), 2- ( ), µ- (), and - () specific ligands ([3H]DPDPE, [3H]Deltorphin II, [3H]DAMGO, and [3H]U69593, respectively) provided evidence that ARD-353 is highly -opioid receptor selective. Ki values (mean ± S.E.M.; n = 5-8) were calculated using nonlinear curve analysis and the Cheng-Prusoff equation. Data were best fit using a one-site model as determined by F test of the respective curves.

Intrinsic Activity. ARD-353 produced receptor-mediated, concentration-dependent inhibition of electrically evoked developed tension in the vas deferens with a corresponding mean IC₅₀ (±S.E.M.) of 18.0 ± 3.6 nM (Fig. 3). Under conditions selective for µ receptor activation, ARD-353 inhibited contractions with an IC₅₀ value of 2600 ± 98 nM. Low levels of inhibition of vas deferens contractions were produced by ARD-353 under conditions selective for receptors such that 50% inhibition was not elicited via this receptor. Computer extrapolation of the low levels of effect seen under selective conditions predicted an apparent IC₅₀ value of 13,500 ± 2780 nM at the receptor.

View larger version (13K): [in this window] [in a new window]   Fig. 3. The effect of ARD-353 on electrically induced developed tension in mouse isolated vas deferens was investigated in the presence of selective antagonists for the µ (1 µM CTOP; ), (12 nM nor-binaltorphimine; ), or (3 µM TIPP; ) opioid receptors. IC50 values were determined from nonlinear curve fitting (mean ± S.E.M.; n = 4-8).

Evaluation of Central Effects in Mice. There were no observations of seizure-like convulsions, catalepsy, or loss of righting reflex in mice that received ARD-353 (Table 1). BW373U86 produced seizure-like convulsions and catalepsy in several animal on every day tested. In total, convulsions and catalepsy were observed in 86% of animals tested with BW373U86. No antinociceptive effects were observed in the tail-pinch test (Table 2).

Hemodynamics. ARD-353 had no effect on heart rate or mean arterial pressure at 3, 20, or 40 mg/kg i.v. in conscious rats, whereas all animals tested responded to the positive control, isoproterenol, with increased heart rate and decreased mean arterial pressure (Table 3). The decreased mean arterial pressure with isoproterenol was accompanied by increased chronotropic and inotropic responses of the heart (data not shown).

Modeling of Cardioprotective Effects: Dose-Response Curve. Control animals that underwent 30 min of LAD occlusion followed by 90 min of reperfusion demonstrated a mean infarct size (as a percentage of the area at risk, IS/AAR; mean ± S.E.M.) of 54.8 ± 2.7%. ARD-353 significantly reduced IS/AAR (p < 0.05; Fig. 4) when dosed 5 min before ischemia at a dose as low as 0.03 mg/kg (43.7 ± 1.9%) and was maximal at 0.3 mg/kg (32.4 ± 3.9%). Intravenous administration of ARD-353 at 0.01 mg/kg had no effect on infarct size (55.2 ± 1.8%). The area at risk, as a percent of total left ventricle, was not significantly different between treatment groups (Table 4).

View larger version (10K): [in this window] [in a new window]   Fig. 4. ARD-353 decreased infarct size (as a percentage of the area at risk, mean ± S.E.M.; n = 5-10) in a dose-dependent manner when administered i.v. to male Sprague-Dawley rats 5 min before a 30-min occlusion of the LAD and 90-min reperfusion period. *, p < 0.05 versus control; **, p < 0.01 versus control.

Antagonism of ARD-353 with BNTX. The putative ₁ receptor selective antagonist, BNTX, completely blocked the protective effects of i.v. 0.3 mg/kg ARD-353 (Fig. 5). There was no significant difference in IS/AAR (mean ± S.E.M.) among saline 54.3 ± 2.0%), DMSO (52.9 ± 2.9%), or sham (53.7 ± 2.5%), whereas ARD-353 produced significant protection from ischemic damage (IS/AAR, 38.0 ± 3.0%; p < 0.05). BNTX alone had no effect on infarct size produced by a 30-min LAD occlusion (54.0 ± 4.0%) but completely blocked the beneficial effects of ARD-353 when administered 10 min before the ARD-353 dose (IS/AAR, 55.8 ± 1.7%).

View larger version (24K): [in this window] [in a new window]   Fig. 5. The cardioprotective effects of 0.3 mg/kg i.v. ARD-353 administered before 30 min occlusion of the LAD and 90 min of reperfusion in male Sprague-Dawley rats was completely blocked by the 1 receptor antagonist BNTX administered at 1 mg/kg i.v. 10 min before ARD-353 and LAD occlusion (mean ± S.E.M.; n = 5-16). Sham, full surgery and occlusion but no vehicle or drug administration. DMSO was the vehicle control for BNTX dose, and saline was the vehicle control for the ARD-353 dose. *, p < 0.05 versus all controls.

View larger version (11K): [in this window] [in a new window]   Fig. 6. ARD-353 (0.3 mg/kg i.v.) significantly attenuated infarct size when administered 5 min or 12 or 48 h before the 30-min LAD occlusion and 90-min reperfusion period in male Sprague-Dawley rats (mean ± S.E.M.; n = 5-9). Mild, nonsignificant, effects were observed at the 24-h pretreatment time point. *, p < 0.05 versus vehicle control.

View larger version (10K): [in this window] [in a new window]   Fig. 7. ARD-353 (0.3 mg/kg i.v.) significantly attenuated the effect of 30-min LAD occlusion and 90-min reperfusion when administered immediately after removal of the LAD occlusion (0 min) but had little effect 30 min into the reperfusion period (mean ± S.E.M.; n = 5-8). *, p < 0.05.

	Discussion

Top Abstract Materials and Methods Results Discussion References

In line with the wealth of information highlighting a cardioprotective role of the opioid system in IPC (Schultz et al., 1997a-c) and the IPC-mimicking effects of -receptor agonists such as BW373U86 and TAN-67 (Schultz et al., 1998; Patel et al., 2001), ARD-353 demonstrated significant cardioprotective effects in a rodent model of short-term ischemic insult. The potency, however, of ARD-353 at protecting the myocardium from ischemic damage and reperfusion injury was much greater (0.03-3.0 mg/kg i.v. bolus) than that reported with TAN-67 (10 mg/kg by 15-min i.v. infusion) but of a similar order of magnitude as that reported with BW373U86 (0.02-0.1 mg/kg i.v. bolus). The cardioprotective potency of ARD-353 may reflect the pharmacokinetic and physicochemical properties of the benzhydrylpiperazine class of compounds (including BW373U86) compared with the isoquinoline-based structure of TAN-67 (Knapp et al., 1995) because all three compounds have high affinity for the receptor [K_i, 1.9 nM for ARD-353, 1.8 nM for BW373U86 (rat brain; Chang et al., 1993), and 0.7 nM for TAN-67 (human clone; Knapp et al., 1995)] and good, full agonist activity in the vas deferens (ARD-353 EC₅₀, 18 nM; BW373U86 EC₅₀, 0.2 nM; Chang et al., 1993) or adenylyl cyclase assays (TAN-67 EC₅₀, 1.7 nM; Knapp et al., 1995).

Building on the protocol of Yellon and Baxter (1995), Fryer et al. (1999) examined the short-term and delayed effects of -receptor activation on ischemic damage through the administration of TAN-67 at multiple time points before LAD occlusion. In a similar paradigm, ARD-353 produced acute and delayed phases of protection with a null period separating them: ARD-353 was effective when given at 5 min and 12 and 48 h before ischemia. No statistically significant effect was reached when ARD-353 was administered at either 24 or 72 h before ischemia. The difference in the timing of the null period for ARD-353 compared with TAN-67 may again be due to the pharmacokinetics of the compounds tested since Patel et al. (2001) produced clear cardioprotective effects 24 h before ischemia with another benzhydrylpiperazine, BW373U86.

Although the cardioprotective potency of ARD-353 in rats fits well with the other benzyhydrylpiperazine tested in this model, BW373U86, the mechanistic study using the putative ₁ receptor antagonist BNTX indicates a similar mechanism of action for TAN-67 and ARD-353. BNTX completely blocked the cardioprotection produced by both TAN-67 and ARD-353 (suggesting an exclusive ₁ mechanism). When BW373U86 was administered 24 h before ischemia, however, BNTX only partially blocked the cardioprotective effects of BW373U86 (Patel et al., 2001), suggesting a mixed mechanism of action of BW373U86 in eliciting the delayed phase of protection. Patel et al. (2001) have suggested that the delayed phase of protection for BW373U86 may be partially the result of an initial burst of oxygen-derived free radicals; indeed, the free-radical scavenger N-(2-mercaptopropionyl) glycine completely blocked the delayed effects of BW373U86. The cardioprotective effects of ARD-353 have only been challenged in a short-term dosing setting to date, and the complexities of the opioid receptor versus a nonreceptor-mediated effect in the delayed protection seen with ARD-353 will be examined in future studies.

As shown by Gross et al. (2005) with the -selective agonist, fentanyl isothiocyanate, ARD-353 was still cardioprotective when administered immediately postocclusion. This effect, combined with the lack of any measurable central effects, may indicate a critical component of the clinical utility of ARD-353 in an emergency setting after a myocardial infarction event. Although in the rodent paradigm, the post-ischemic protection was not evident when the drug was administered 30 min postreperfusion, it is helpful to consider the differences between the rodent model and the clinical condition. In the instance of the laboratory model of LAD occlusion, a large coronary vessel is closed for 30 min, producing ischemia in a very large proportion of the ventricular wall, and then this blockage is removed completely and full circulation allowed to return, giving an accelerated and immediate model of reperfusion injury. Although this would seem to be an excellent model of the acute ischemic events observed in angioplastic interventions, there is little evidence to suggest that this rapid return of perfusion occurs in the recovery phase of a spontaneous myocardial infarct; as such, the clinical time window for dosing with ARD-353 postinfarct may be considerably larger than that seen in the rodent model.

From the preliminary investigations into the potential safety concerns with ARD-353, it seems to be limited to the periphery and is not likely to enter the CNS in that i.v. doses as high as 50 mg/kg produced no convulsant events or any other CNS-related effects. In addition, there were no signs of hemodynamic complications resulting from an interaction of ARD-353 with the cardiovascular system. This potential for a silent central and hemodynamic profile, supported with data from higher species and clinical studies, would be a vital component in the potential application of ARD-353 to use in a clinical setting.

Although the effectiveness of ARD-353 as a cardioprotective agent has been demonstrated in the rat model of myocardial infarct in two laboratories and the scale of effect in this model agrees well with that reported in other laboratories (Schultz et al., 1998; Fryer et al., 1999; Gross et al., 2005), there is little support for, or against, a direct correlation between efficacy in the rodent models of myocardial ischemia and efficacy in clinical studies across compound classes. As specific test populations are described more explicitly, and so-called surrogate measures for myocardial damage are validated as early measures in place of long, large-scale mortality studies, so more data on the efficacy of compounds and the correlation with preclinical data are anticipated. Despite the lack of support for predictive validity of the rodent model of cardioprotection, the opioid receptor mechanisms are supported by clinical studies (Xenopoulos et al., 1998; Tomai et al., 1999) and anecdotal reports of potential benefit received from postinfarct treatment with morphine in the clinical setting. The true potential for ARD-353 as a clinically useful therapeutic providing protection against damage incurred because of myocardial ischemia remains to be proven through clinical trials, and the full safety profile to be defined across multiple animal species and through clinical studies will be a critical part in the progression of ARD-353 as a novel cardioprotective agent.

	Footnotes

 
Some of these data were presented at Experimental Biology, 2005 [Watson MJ, Holt JDS, O'Neill SJ, Wei K, Pendergast W, Gengo PJ, Gross GJ, and Chang K-J (2005) Acute and chronic cardioprotective effects of a novel, -opioid receptor agonist ARD-353 in rats (Abstract). FASEB J 19:A1562] and the International Society for Heart Research, 2005 [Holt JDS, Watson MJ, and Chang K-J (2005) ARD-353: a cardioprotective receptor agonist active before ischemia and immediately post-ischemic insult (Abstract). J Mol Cell Cardiol 38:834].

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.092742.

ABBREVIATIONS: IPC, ischemic preconditioning; ARD-353, 4-((2R,5S)-4-(R)-(4-diethylcarbamoylphenyl)(3-hydroxyphenyl)methyl)-2,5-dimethylpiperazin-1-ylmethyl)benzoic acid; CNS, central nervous system; DPDPE, cyclic [D-Pen²,D-Pen⁵] enkephalin; DAMGO, [D-Ala,N-Me-Phe,Gly-ol]-enkephalin; U69593 [GenBank] , (+)-(5,7,8)-N-methyl-N-(7-(1-pyrrolidinyl)-1 oxaspiro[4.5] dec-8-yl)benzeneacetamide; TIPP, Tyr-Tic-Phe-Phe; CTOP, cyclic [D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂]; BW373U86, (±)-[1(S^*),2,5]-4-{[2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-hydroxypheny)methyl}-N,N-diethylbenzamide; LAD, left anterior descending coronary artery; BNTX, 7-benzylidenenaltrexone; DMSO, dimethyl sulfoxide; AAR, area at risk; IS, infarct size; TAN-67, 2-methyl-4a -(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a -octahydro-quinolino[2,3,3-g]isoquinoline.

Address correspondence to: Jonathon Holt, Enhance Biotech Inc., 631 United Drive, Suite 200, Durham, NC 27713. E-mail: jholt{at}enhancebiotech.com

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