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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

DPI-221 [4-((-S)--((2S,5R)-2,5-Dimethyl-4-(3-fluorobenzyl)-1-piperazinyl)benzyl)-N,N-diethylbenzamide]: A Novel Nonpeptide Receptor Agonist Producing Increased Micturition Interval in Normal Rats

Enhance Biotech, Inc., Durham, North Carolina

Received for publication June 17, 2005
Accepted July 12, 2005.

	Abstract

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There is a wealth of information from animal models and clinical opioid-analgesic use that indicates a significant role for opioid receptors in the modulation of bladder activity. The novel benzhydrylpiperazine compound DPI-221 [4-((-S)--((2S,5R)-2,5-dimethyl-4-(3-fluorobenzyl)-1-piperazinyl)benzyl)-N,N-diethylbenzamide] was characterized as having receptor selectivity using radioligand binding (K_i = 2.0 ± 0.7 nM, receptor; 1800 ± 360 nM, µ receptor; and 2300 ± 680 nM, receptor), and agonist activity was demonstrated in the mouse isolated vas deferens where DPI-221 inhibited electrically induced contractions with an IC₅₀ value of 88 ± 7.5 nM. In the guinea pig isolated ileum, DPI-221 had no effect on electrically induced contractions at concentrations as high as 1 µM. Sterile saline was infused (7 ml/h) into the bladder of Sprague-Dawley rats, via a transmural catheter; DPI-221 (1.0 to 20 mg/kg p.o.) significantly increased the interval between micturition events, whereas peak void pressure was not significantly decreased by any dose of DPI-221. The micturition effects of 10 mg/kg p.o. DPI-221 were blocked by naltrindole, indicating a receptor mechanism of action. In isolated rat bladder strips, DPI-221 was ineffective at relaxing detrusor muscle precontracted with carbachol. The most crucial safety aspect of agonist administration is the incidence of seizure-like convulsions in rodents. DPI-221 produced no convulsions at doses up to 100 mg/kg p.o. in mice, although rapid bolus i.v. injection of 5 mg/kg produced convulsions in 3% of mice tested. These findings indicate a good safety profile for DPI-221 administered orally, with potent efficacy in modifying bladder activity.

The ability of morphine and other opioids to inhibit reflex activity of the urinary bladder was demonstrated by several groups in the early 1980s (Brent et al., 1983; Dray and Metsch, 1984a,b,c; Hisamitsu and de Groat, 1984; Jubelin et al., 1984). Unlike peripherally limited opioid antagonists, centrally administered naloxone blocked morphine-induced inhibition of reflex bladder activity (Dray and Metsch, 1984a,b,c), indicating a central nervous system-based mechanism of morphine's action. In the cat urinary bladder, activation of preganglionic neurons, positive for leucineenkephalin-like immunoreactivity, inhibited postganglionic, cholinergic innervation of the urinary bladder via a receptor mechanism (de Groat and Kawatani, 1989). These data indicate a -receptor-mediated inhibitory role of the preganglionic neurons in control of cholinergic regulation of bladder activity. More recently, investigations into the naloxone-sensitive inhibition of micturition events with the widely used analgesic tramadol, an opioid receptor agonist and inhibitor of noradrenaline and serotonin reuptake, continue to highlight the role of the opioid systems in modulation of bladder activity (Pandita et al., 2003; Pehrson and Andersson, 2003).

The opioid receptor system constitutes three receptor subtypes, , µ, and (Chang et al., 1979), and all three receptors play a role in bladder control and micturition events (Dray and Nunan, 1985, 1987; Sheldon et al., 1987, 1989). There is a growing volume of information supporting a role for the receptor in these processes. Dray et al. (1985) demonstrated that the receptor agonists [2-D-penicillamine, 5-L-penicillamine]-enkephalin, [2-D-penicillamine, 5-L-cysteine]-enkephalin, and cyclic [D-Pen²,D-Pen⁵] enkephalin (DPDPE) produced dose-related inhibition of reflex bladder contractions when administered either i.c.v. or i.t.

Clinical experience with µ-receptor-based analgesic compounds such as morphine indicates a side effect profile that includes urinary retention, respiratory depression, muscle rigidity, nausea and vomiting, and a high addiction potential, a profile not suitable for the treatment of OAB. In stark contrast, activation of the receptor system presents with no addiction or reinforcing potential in rhesus monkeys (Negus et al., 1994, 1995, 1998) and no respiratory depression, muscle rigidity, or nausea and vomiting (O'Neill et al., 1997).

Chang et al. (1993) described one of the first nonpeptide receptor agonists [(±)BW373U86] with chemistry suitable for production as a clinical drug. However, (±)BW373U86 produces the most common rodent side effect of high levels of central receptor activation, convulsions (Comer et al., 1993; Broom et al., 2002a,b). These tonic-clonic convulsions are observed with other nonpeptide agonists that are now readily available as research tools and are a direct function of the rate of drug delivery (Comer et al., 1993; Broom et al., 2002a,b). The right shift in convulsant threshold of receptor agonists produced by increasing the i.v. bolus infusion time strongly suggests that the rate of brain penetration is a critical factor in the determination of this convulsant threshold. The inherently slower absorption rate following oral administration might be expected to improve the convulsant threshold of agonists. However, the currently available nonpeptide agonists are not orally bioavailable and as such are poor drug candidates for many urologic indications.

Building on the original chemistry of (±)BW373U86 and the work of Chang et al. (1993), we have developed a highly selective, nonpeptide agonist that is orally available and produces no convulsions in rodents except following rapid bolus i.v. doses. Herein, we report the radioligand binding affinity (K_i), intrinsic activity, and convulsant profile of DPI-221 (Fig. 1). The effects of DPI-221 on rat micturition events are investigated, along with the associated receptor mechanism of action.

View larger version (13K): [in this window] [in a new window]   Fig. 1. The chemical structure of DPI-221.

	Materials and Methods

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These studies were conducted under approved Institutional Animal Care and Use Committee protocols at Enhance Biotech, Inc. (formerly Ardent Pharmaceuticals, Inc.) and in accordance with U.S. Department of Agriculture regulations.

Receptor Binding Affinity
Membrane Preparation for Radioligand Binding. The brains from male albino Sprague-Dawley rats were obtained from Pel-Freez Biologicals (Rogers, AR) and cerebellum from male albino guinea pigs from Accurate Chemical & Scientific (Westbury, NY). The tissue was 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 and 1 mM EDTA, 10 µg/ml leupeptin, 200 µg/ml bacitracin, and 0.5 µg/ml aprotinin. Brains were minced and homogenized in 5 to 10 volumes/g wet weight in ice-cold 50 mM Tris buffer containing protease inhibitors. The homogenate was prepared using a glass-Teflon homogenizer (nominal clearance, 0.13-0.18 mm). The homogenate was centrifuged at 6000g for 15 min at 4°C, and the resulting supernatant was centrifuged at 41,000g for 30 min at 4°C. The membrane pellet was resuspended in 10 volumes/g wet weight of 10 mM Tris-sucrose (0.32 M) buffer and rehomogenized with a Polytron tissue grinder (10 s, low speed). 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 prior to use in radioligand binding studies. Protein concentration was determined using the method of Bradford (1976).

Radioligand Binding. Membrane fractions were incubated with DPI-221 (3 x 10^-11 to 6 x 10^-5 M) or naloxone (1 x 10^-6 M) plus 0.1 nM of the -opioid receptor agonist [³H]DPDPE (specific activity 50.6 Ci/mmol; n = 7), 0.1 nM of the µ-opioid receptor agonist [³H]DAMGO (specific activity 50.0 Ci/mmol; n = 7), or 0.1 nM of the -opioid receptor agonist [³H]U69593 (specific activity 41.4 Ci/mmol; n = 5) 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 was terminated 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
Vas Deferens Studies. Tension development in isolated mouse vas deferens was measured as described previously (Chang et al., 1993). Following 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 under 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. An IC₅₀ value for DPI-221 inhibition of electrically induced contractions was produced through the addition of cumulative concentrations (1 x 10^-10 to 1 x 10^-7 M; n = 8) of DPI-221 to the bathing solution (Chang et al., 1993).

Guinea Pig Ileum Studies. Tension developed in response to electrical stimulation of guinea pig isolated ileum was recorded as described previously by Gengo et al. (2003b). Male albino guinea pigs (Charles River Laboratories) weighing 300 to 500 g were euthanized by decapitation and an 8-cm section of ileum removed and divided into 2- to 3-cm segments. Individual segments were suspended in standard organ baths (Radnoti) and continuously bathed in Krebs-Henseleit solution (37°C, aerated with O₂/CO₂, 95:5) of the following composition: 117.5 mM NaCl, 4.75 mM KCl, 2.4 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 24.5 mM NaHCO₃, and 11 mM glucose. Tissues were suspended from Grass FTO3 isometric force transducers under a resting tension of 1 g, and contractions were elicited by a field stimulation of 0.1-Hz pulses of 0.5-ms duration at supramaximal voltage using platinum electrodes and a Grass S88 stimulator. DPI-221 effects on electrically induced contractions of the ileum were examined through the addition of cumulative concentrations (1 x 10^-7 to 1 x 10^-6 M; n = 7) of DPI-221 to the bathing solution. The maximum DPI-221 concentration tested was limited by the solubility of the compound in these assay conditions.

Direct Effects on Detrusor Tension
Bladder Isolated Rings. Male Sprague-Dawley rats (Harlan, Indianapolis, IN) were anesthetized with isoflurane in an N₂O/O₂ vehicle (65% N₂O; 35% O₂) and then killed by exsanguination. The bladder was removed and placed into warm (37°C) Krebs' solution that was made up fresh daily and consisted of the following (in distilled water, pH 7.2): 119.0 mM NaCl, 4.4 mM KCl, 20.0 mM NaHCO₃, 1.2 mM NaH₂PO₃, 1.2 mM MgCl₂, 2.5 nM CaCl₂, and 11.0 mM glucose.

Bladders were dissected free of any adhering fat or serosa and then cut into longitudinal strips of muscle 4 x 1 x 0.5 mm. These were suspended in a Perspex organ bath of 10.0-ml volume using a fine silk suture. The bladder strips were constantly perfused at a rate of 1 ml/min with Krebs' solution aerated with 95% oxygen and 5% carbon dioxide, and the temperature was maintained at 37°C throughout the experiments. The bladder muscle strips were attached to an isometric force transducer (Kent Scientific Corp., Torrington, CT) connected to a personal computer and held at a resting tension of 2 g for a 3- to 4-h equilibration period. Data were acquired using Workbench software (Strawberry Tree, Sunnyvale, CA).

DPI-221 on Carbachol-Precontracted Muscles. After equilibration to the resting tension, an EC₅₀ concentration of carbachol (360 nM) was used to precontract the muscle strips. The carbachol-induced increase in tone was allowed to plateau to a stable precontracted state before DPI-221 solutions were added directly to the tissue bath to produce bath concentrations of 1, 3, and 10 µM. Each concentration was allowed to establish a stable tension, and then a sufficient additional volume of DPI-221 was added to produce the next highest bath concentration of interest. Tissue viability was confirmed with 10 µM oxybutynin as a positive control added to the tissue bath after completion of DPI-221 concentrations.

Micturition Events in Conscious Restrained Rats
Cystometry. The cystometry methods used were similar to those of Pandita et al. (2003). Male Sprague-Dawley (Harlan) rats weighing 200 to 250 g were anesthetized with pentobarbital, 65 mg/kg i.p., and placed in a supine position. A 10-mm-long midline incision was made in the abdominal wall, and the urinary bladder was cannulated, via an incision at the dome, with a polyethylene cannula (PE50) that was permanently sutured in place. The cannula was s.c. positioned and exteriorized in the retroscapular region. Compounds were administered i.v. or orally (p.o.) via catheters inserted into the jugular vein or stomach, respectively, and exteriorized in the retroscapular area. Rats were treated with penicillin G i.m., 1 mg/kg (1658 units), to prevent infection and allowed to rest 3 days after implantation before testing commenced.

Three days after cannula implantation animals were placed, conscious, into Plexiglas tube-shaped rat restrainers (Plas-Labs Inc., Lansing, Michigan) where they remained for the duration of the study. Maximal restraint times were 4 h for dose-response studies and 6 h for the duration of action study. Once animals were restrained, the bladder cannula was attached to a syringe pump, and sterile saline was pumped into the bladder at a rate of 7 ml/h. A pressure probe (Grass PT200) was attached to the bladder infusion line with a three-way hub to record micturition pressure changes. Data were acquired using PowerLab hardware (ADInstruments Pty Ltd., Castle Hill, Australia) and software (Chart 4) on a personal computer.

Dose-Response Curve. A stable baseline of all micturition values (interval, baseline pressure, and peak developed pressure) was maintained for 1 h prior to administration of any test compound. Data were produced only from animals with micturition intervals greater than 2 min and less than 15 min. A single dose of DPI-221 [0.5 (n = 11), 1 (n = 10), 2 (n = 8), 10 (n = 15), or 20 mg/kg (n = 5)] or pH-matched 5% dextrose vehicle (n = 13) was administered via the stomach cannula (p.o.), and cannula patency was confirmed at the end of the study for all animals with a positive control dose of the muscarinic antagonist oxybutynin. DPI-221 was slowly administered over the course of 1 min, and data were acquired for 3 h (or until any effects had obviously reached a plateau). Data were collected over a 1-h predrug baseline and over a 1-h period starting 1 h after drug administration. The mean peak developed voiding pressure and the mean interval between pressure peaks over the 1-h data collection window were measured and expressed as the percentage of the predrug baseline values for each animal.

Duration of Action of DPI-221. To assess the duration of action of DPI-221, rats received a single p.o. (stomach cannula) dose of 10 mg/kg of DPI-221, and bladder pressures were recorded for up to 4 h after dosing. Data were analyzed in 40-min bins.

Naltrindole Blockade of DPI-221 Micturition Effects. To investigate whether the effects of DPI-221 on micturition events were sensitive to blockade of the receptor, the receptor-selective antagonist naltrindole was used in an i.v. dosing regimen designed and reliably used in our laboratories to provide stable levels of receptor blockade over an extended (>2-h) experimental period. Naltrindole (NTI) was administered i.v. (0.75 mg/kg) 35 min prior to oral dosing of DPI-221 at the lower of the maximally effective doses previously tested (10 mg/kg p.o.), 5 min after DPI-221 dosing (n = 8) or pH-matched 5% dextrose vehicle (n = 8), and a third time 40 min later. Control DPI-221 or dextrose vehicle groups (n = 15 and 13, respectively) received three injections of saline as the vehicle for NTI doses. The sequential dosing was used to maintain an effective inhibitory concentration of NTI in the blood at all times during the course of the experiment. Data were analyzed from a 1-h window sampled 1 h after DPI-221 dosing.

Convulsant Effects in Mice
The convulsant effects of DPI-221 were investigated using male CD-1 mice (Harlan). Using individual dosing groups of 10 animals, DPI-221 was administered as a rapid bolus i.v. (tail vein) at doses of 3, 5, and 10 mg/kg (n = 30, 29, and 70) or via oral gavage/feeding tube over 1 min at doses of 60 and 100 mg/kg p.o. (n = 10 and 10) and at 30 and 100 mg/kg s.c. (n = 10 and 10). Animals were observed for 1 h following i.v. dosing and 2 h following p.o. dosing. A convulsion was recorded if a mouse had uncontrollable clonic (or tonic-clonic) movements that encompassed its entire body, usually followed by a brief cataleptic period. Convulsion incidence was expressed as a percentage of the number of animals receiving the dose.

Arterial Blood Gas and Antinociception in Rats
Male albino Wistar Hannover rats (Harlan) weighing 200 to 300 g were anesthetized with 2% isoflurane in a 30% O₂ and 70% N₂O vehicle. Catheters were inserted in the femoral artery and external jugular vein. Sixty minutes after surgery, DPI-221 was administered at 10 mg/kg i.v. or 60 mg/kg p.o. (by gavage), and antinociception was measured using the tail-pinch method, and arterial blood gas was analyzed at 0, 4, 8, 16, and 32 min following DPI-221 dosing as per the methods of Gengo et al. (2003a).

Sources of Drugs
[³H]DPDPE, [³H]DAMGO, and [³H]U69593 were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Purities were greater than 98%. NTI 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) and Burroughs Wellcome (Research Triangle Park, NC) using standard protocols. DPI-221 was manufactured at Ardent Pharmaceuticals, Inc. as a free-base, and all solutions were made based on dry weight of free-base (molecular weight 487.66) that was then dissolved in 1 ml of ethanol and either converted to an HCl salt and dried under nitrogen for aqueous dosing solution preparation or diluted directly from the ethanol stock for in vitro studies.

Calculations and Statistics
Receptor binding and intrinsic activity data were analyzed by nonlinear regression of the concentration-response curves to determine the K_i or IC₅₀ values using the computer program Prism (GraphPad Software Inc., San Diego, CA).

Micturition event recordings provided significant differences in group sizes and variances because of cannula twisting or chewing by the rats. Once the pressure trace was compromised, no further analysis was performed on that trace. Micturition parameters for DPI-221 doses were expressed as the percent change from baseline and compared with vehicle control using a Kruskal-Wallis analysis of variance followed by Dunn's post hoc test. Data from the duration of action study were compared with baseline and between measurement time points using the same tests.

	Results

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Receptor Binding Affinity
In competition radioligand binding assays, DPI-221 demonstrated receptor selectivity (Fig. 2) with binding affinities of (K_i ± S.E.M.) 2.0 ± 0.7 nM at the receptor (n = 7); 1800 ± 360 nM at the µ receptor (n = 7), and 2300 ± 680 nM at the receptor (n = 5). The data were best fit using a one-site sigmoidal model with a pseudo-Hill slope of close to 1.0.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Receptor binding competition curves. Membrane fractions were incubated with 0.1 nM of the receptor agonist [3H]DPDPE (; n = 7), 0.1 nM of the µ receptor agonist [3H]DAMGO (; n = 7), or 0.1 nM of the receptor agonist [3H]U69593 (; n = 5). Displacement of bound ligand by DPI-221 is expressed in terms of the remaining bound ligand as a percentage of total specific binding in control conditions (percentage of control).

View larger version (10K): [in this window] [in a new window]   Fig. 3. The concentration-response effects of DPI-221 on electrically stimulated contractions of the mouse vas deferens ( receptor dominated; ; n = 8) or guinea pig ileum (µ and receptor dominated; ; n = 7). DPI-221 was added to the tissue bath in increasing concentrations to produce a cumulative concentration-response curve for opioid receptor-induced relaxation.

Direct Effects on Detrusor Tension
At concentrations up to and including the maximum achievable concentration in physiological buffering solutions (10 µM), DPI-221 produced no significant relaxation of carbachol-induced contraction of bladder muscle strips. The muscarinic antagonist oxybutynin (10 µM) completely relaxed the tissues, indicating the viability of each preparation. A representative trace for a single bladder strip is presented in Fig. 4.

View larger version (14K): [in this window] [in a new window]   Fig. 4. A sample trace of the effect of DPI-221 on the carbachol-induced tension of a bladder strip isolated from a male Sprague-Dawley rat. DPI-221 (1 and 10 µM) had no significant effect on carbachol-induced tension (500 nM). Oxybutynin (10 µM) completely antagonized carbachol-induced tension.

View larger version (28K): [in this window] [in a new window]   Fig. 5. DPI-221 p.o. micturition interval and pressure dose-response curve. In catheterized rats undergoing constant infusion (7 ml/h) of sterile saline into the bladder, DPI-221 (p.o.) dose-dependently increased the time interval between micturition events. DPI-221 (p.o.) produced a nonsignificant trend toward decreasing micturition-developed bladder pressure. *, p < 0.05 versus vehicle control; **, p < 0.01 versus vehicle control.

Duration of Action of DPI-221. Oral administration of DPI-221 (10 mg/kg) followed by data sampling in 40-min windows demonstrated a slow increase in micturition interval that reached a peak at around 2 h after dosing (Fig. 6). Micturition interval remained stable at 25% above baseline for the rest of the recording period (up to 120 min). The variability of the later time points was larger than that seen at initial time points owing to diminishing pressure trace integrity over the 6-h study period. Bladder pressure traces were rendered unreadable by animals that twisted or chewed cannulae. The effect of DPI-221 on peak void pressure described above was supported in this study. There was also a statistical trend in the effect of DPI-221 (p < 0.05 vs. baseline) that paralleled the time course of the significant effects on micturition interval (Fig. 6).

View larger version (12K): [in this window] [in a new window]   Fig. 6. The duration of action of DPI-221. In male catheterized rats undergoing constant infusion (7 ml/h) of sterile saline into the bladder, DPI-221 (10 mg/kg p.o.) increased micturition interval () and decreased micturition pressure (). Maximum effect was reached approximately 120 min after gastric infusion. *, p < 0.05 versus baseline.

View larger version (49K): [in this window] [in a new window]   Fig. 7. Naltrindole blockade of DPI-221 effects on micturition parameters. In male catheterized rats undergoing constant infusion (7 ml/h) of sterile saline into the bladder, the increase in micturition interval in response to DPI-221 (10 mg/kg p.o.) was blocked by the opioid antagonist NTI (0.75 mg/kg x3). The small decrease in pressure induced by DPI-221 was also antagonized by NTI. *, p < 0.05 versus vehicle control; **, p < 0.01 versus vehicle control.

Arterial Blood Gas and Antinociception in Rats
There were no antinociceptive effects of DPI-221 administered either i.v. or orally. Similarly, there was no deviation in arterial blood gases from normal, control levels with DPI-221 dosed either i.v. or p.o.

	Discussion

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DPI-221 is a highly -receptor-selective compound with good agonist potency in the standard mouse vas deferens assay for receptor activity and no µ (or ) receptor activity in vitro (guinea pig ileum assay) or in vivo, as evidenced by the lack of antinociceptive (tail-pinch test) or respiratory depressant effects in rats. When administered orally to conscious, restrained rats, DPI-221 significantly prolonged micturition interval without altering basal or peak void pressure at doses as low as 1.0 mg/kg. This effect is thought to be more indicative of changes in micturition urge and neuronal control than of inhibition of muscle activity. The maximal effect produced by DPI-221 was an average extension of micturition interval of 32% (10 and 20 mg/kg p.o.). The size of this effect is thought to be somewhat constrained by the physiological limitations of rat bladder size (up to 1.5 ml for 200-230-g rats; Jeong and Lee, 2000) and the 7 ml/h rate of bladder filling. At the higher doses tested (10 and 20 mg/kg p.o.), a small decrease in void pressure was observed with DPI-221. In isolated bladder strips, DPI-221 had no effect on carbachol-induced tension, indicating that the decreased void pressure was probably not the result of a direct muscarinic effect on detrusor tone or contractility. Throughout the course of the study, basal bladder pressure remained stable and was not affected by DPI-221 treatment, suggesting that the decreased void pressure does not indicate retention of urine due to ineffective voiding contractions. DPI-221 effects on bladder compliance, voiding resistance, absolute void volume, or its indirect effects on bladder contractility cannot be determined from the data presented here. The physiological relevance of the decreased peak void pressure at higher doses of DPI-221 is not clear from this data.

The effects of DPI-221 as a -selective agonist support the growing volume of evidence that the receptor system plays an important role in the regulation of reflex and voluntary bladder activity. Activation of the opioid system with morphine and antagonists such as naloxone centrally (Dray and Metsch, 1984a,b,c) and at spinal and supraspinal levels (Dray and Nunan, 1985, 1987) alters reflex activity of the urinary bladder in intact animals. Building on this earlier evidence that had used somewhat generalized (if µ-favoring) opioid receptor tools, Sheldon et al. (1987, 1989) and Dray et al. (1985) utilized peptide-derived compounds with greater selectivity for µ, , or receptors to further elucidate the differential roles of these receptor subtypes. These studies, in combination with leucine-enkephalin immunolocalization in preganglionic neurons of the bladder-associated ganglia in cats (de Groat and Kawatani, 1989) and the i.c.v. and i.t. efficacy of DPDPE (and [2-D-penicillamine, 5-L-penicillamine]-enkephalin and [2-D-penicillamine, 5-L-cysteine]-enkephalin) in inhibiting reflex bladder contractions (Dray et al., 1985) and potentiating the effects of morphine (Sheldon et al., 1989), clearly indicate a role for the receptor in control of the urinary bladder. As mentioned previously, however, there have been no nonpeptide compounds available for clinical use or testing prior to this time because of their poor oral availability and rapid central nervous system penetration (i.e., low convulsion threshold). The compound introduced herein, DPI-221, is a novel receptor agonist with characteristics far more suited to development as a clinical therapeutic agent.

It is well established in clinical settings, particularly in the analgesic use of morphine, that µ agonist agents induce urinary retention. This was illustrated in our rat model as a large increase in basal pressure to above that of peak voiding pressure and a complete ablation of all voiding pressure changes (Holt et al., 2005). The effects of DPI-221 on micturition interval without altering pressure also contrasts starkly with the effects of antimuscarinic agents, such as oxybutynin, in the same conscious rat model. Oxybutynin produced variable changes in micturition interval, tending toward decreased interval, and dose-dependently decreased peak void pressure in this rat model (Holt et al., 2005). These findings would suggest that the receptor mechanism may actually offer a cleaner therapeutic tool for limiting micturition urge and frequency without significantly altering bladder mechanics or producing urinary retention.

One of the critical advantages of DPI-221 over other currently available receptor agonists is its oral efficacy in models of micturition events. This is a crucial factor in the development of this compound as a drug in clinical use and is a major improvement over the original compounds from this chemical class, such as BW373U86. In addition to the obvious clinical suitability of an oral medication for overactive bladder, the infusion rate dependence of receptor-mediated convulsions (Comer et al., 1993) would theoretically be avoided by the relatively slow rate of gastric absorption as compared with i.v. bolus administration. This theoretical premise appears to hold true in the case of DPI-221. Intravenous doses of DPI-221 produce convulsions when administered in a rapid bolus of 5 or 10 mg/kg, whereas an oral dose of 60 or 100 mg/kg or s.c. dose of 100 mg/kg produced no convulsions. The obvious interpretation of these findings is that oral administration does not carry the convulsion-based safety concerns of current receptor agonists. However, these findings are also pertinent to further considerations of the mechanism of action of DPI-221.

DPI-221 increased micturition interval in intact animals but had no effect on carbachol-developed tension in isolated rat bladder detrusor muscle strips. This would seem to discount any direct action of DPI-221 on bladder muscle or at the cholinergic neuromuscular junction as the mechanism of interval prolongation. However, although oral dosing of DPI-221 does not produce convulsions, the presence of convulsions following i.v. dosing indicates that DPI-221 can cross the blood-brain barrier. As such, no inference can be made as to the localization of effect to more peripheral sites, such as at the preganglionic level described by de Groat and Kawatani (1989) or central sites such as the pontine micturition center or periaquaductal gray area (Fowler, 2002).

What remains clear from the studies presented here, however, is that DPI-221 modulates micturition interval through a naltrindole-sensitive, receptor-mediated mechanism and that the effects on micturition interval last beyond 4 h after oral dosing. Taken together with the suitability of DPI-221 for formulation and development, the lack of convulsant effects after oral dosing, and the high potency in extending micturition interval, these findings indicate that DPI-221 would be an appropriate candidate for development as a clinically useful therapy for overactive bladder and urinary incontinence.

In light of the selectivity of DPI-221 and the naltrindole sensitivity of its micturition effects, demonstration of the therapeutic efficacy and clinical utility of DPI-221 would provide a unique opportunity to validate the animal models of a receptor mechanism as chemically meaningful and therapeutically useful. Alongside the clinical development of DPI-221, follow-up animal studies will further define the mechanism and localization of action of the -mediated effects of DPI-221 on urinary bladder activity.

	Footnotes

 
Some of these data were presented at Experimental Biology 2004, Washington, DC, April 17-21, 2004 [Watson MJ, Gengo PJ, Pendergast WJ, Chang JP, Wei V, Sanford IJ, Millard DJ, and Chang K-J (2004) The effects of a novel -opioid receptor agonist on micturition in rats. FASEB J 18:3191].

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

doi:10.1124/jpet.105.090498.

ABBREVIATIONS: OAB, overactive bladder; DPDPE, cyclic [D-Pen²,D-Pen⁵] enkephalin; (±)BW373U86, (±)-4-((-R^*)--((2S^*,5R^*)-4-allyl-2,5-dimethyl-1-piperazinyl-3-hydroxybenzyl)-N,N-diethylbenzamide; DPI-221, 4-((-S)--((2S,5R)-2,5-dimethyl-4-(3-fluorobenzyl)-1-piperazinyl)benzyl)-N,N-diethylbenzamide; 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; NTI, naltrindole.

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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