Introduction

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Overactive bladder is a highly prevalent disorder in humans characterized by the symptoms of urinary urgency and frequency with or without urge incontinence. About 75% of the patients are women with incidence escalating with age. Although clinical data provide evidence that muscarinic receptor antagonists are effective in the treatment of overactive bladder, their optimal efficacy is compromised by mechanism-based adverse events such as dry mouth, constipation, and blurred vision. These agents are also contraindicated for treating the symptoms in men with outflow obstruction due to the potential to exacerbate urinary retention. Accordingly, a need exists to identify drugs with different mechanisms of action to improve efficacy and increase treatment options for patients with symptoms of urgency and frequency arising from different underlying causes.

In some patients, symptoms of overactive bladder correspond with the urodynamic finding of bladder instability, i.e., the presence of involuntary bladder contractions during filling cystometry. Bladder instability has been hypothesized to arise primarily from functional changes in detrusor smooth muscle structure and function (myogenic etiology). It has been suggested that supersensitivity to agonists, increase in gap junctions, and enhanced electrical coupling between smooth muscle cells could enable widespread dissemination of depolarization signals, leading to spontaneous nonvoiding contractions (Brading, 1997; Mills et al., 2000a). Bladder instability in animal models has been reliably produced by partial urethral obstruction. Although this technique has been described using various species (Mattiasson and Uvelius, 1982; Malmgren et al., 1987; Kato et al., 1988; Radzinski et al., 1991; Azadzoi et al., 1996), the pig is thought to represent a more appropriate model (Sibley, 1985) than either rat or dog to emulate bladder instability in humans. Pigs are similar to humans with respect to overall size, voiding behavior, and baseline values of cystometric flows and pressures (Speakman et al., 1987; Guan et al., 1994). Detrusor strips from obstructed pigs and humans also exhibit similar physiological properties in vitro. These include 1) increases in spontaneous contractile activity, including the presence of fused tetanic contractions; 2) evidence of cholinergic denervation; and 3) altered agonist sensitivity (Brading, 1997). Recently, similar findings were noted in strips obtained from patients with idiopathic instability, suggesting that such properties might be common features of detrusor instability regardless of etiology (Mills et al., 2000a).

It has been hypothesized that K_ATP channel openers (KCOs), by "stabilizing" the smooth muscle, can effectively inhibit unstable bladder contractions without affecting normal reflex voiding, thereby offering an attractive rationale for treating bladder overactivity. In isolated detrusor muscle preparations, KCOs have been shown to suppress action potential firing in smooth muscle cells and inhibit phasic myogenic contractions (Petkov et al., 2001; Shieh et al., 2001). In vivo, cromakalim has been shown to inhibit spontaneous nonvoiding contractions in obstructed pigs, whereas the ability to void was maintained (Foster et al., 1989). However, limited bladder selectivity (versus vascular effects) has thus far limited the clinical utility for at least the first generation KCOs. More recently, preclinical evidence for improvements in in vivo selectivity has emerged from the second generation KCOs exemplified by ZD6169 (Howe et al., 1995) and WAY-133537 (Wojdan et al., 1999). In light of the fundamental role of K_ATP channels in the modulation of myogenic bladder contractions, compounds with superior efficacy, and with improved bladder selectivity, could further opportunities in exploiting their potential for the treatment of bladder overactivity.

In this study, we describe the in vivo efficacy and selectivity of a novel K_ATP channel opener, (9S)-9-(3-bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-8(4H)-one 1,1-dioxide (A-278637), that potently and selectively activates K_ATP channels in bladder smooth muscle and suppresses contractions of pig and human bladder smooth muscle strips (Gopalakrishnan et al., 2002). Our studies demonstrate that A-278637 exhibited enhanced efficacy in suppressing unstable bladder contractions and demonstrated improved bladder versus cardiovascular selectivity in a myogenic model of obstructive bladder instability in pigs. Portions of this work were presented previously as an abstract (Brune et al., 2001).

	Materials and Methods

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Materials. A-278637, WAY-133537, and ZD6169 were synthesized at Abbott Laboratories. Nifedipine was purchased from Sigma-Aldrich (St. Louis, MO). Compounds were dissolved in a solution containing equal parts of a hydroxypropyl--cyclodextrin solution (100 g/200 ml; Sigma-Aldrich) and sterile water and dosed at a volume of 0.1 mg/kg. All protocols were approved by the Institutional Animal Care and Use Committee of Abbott Laboratories.

Cystometry in Obstructed Pigs. The method for producing partial outlet obstruction in pigs was modified from protocols published previously (Sibley, 1985; Speakman et al., 1987; Mills et al., 2000b) described elsewhere in more detail (Fey et al., 2002). Briefly, female Landrace/Yorkshire pigs (~12 weeks old; 14-20 kg) were obstructed with a 7.5-mm silver omega ring placed around the proximal urethra using an inguinal approach. Ring placement was confirmed at necropsy in all animals. Seventeen to 20 weeks after placement of the ring, the pigs were instrumented with telemetry transducer/transmitters (Data Sciences, St. Paul, MN) for the measurement of carotid arterial pressure (unit 1: TA11PA-C40) and intravesical/abdominal pressures (unit 2: TL11M3-D70-PCP). A port catheter (TI-9; Access Technologies; Skokie, IL) was placed subcutaneously in the side of the abdomen and its distal catheter secured in the bladder lumen. Animals were treated with amoxicillin and buprenorphine for 3 to 5 days postsurgery and allowed to recover for 10 to 14 days before testing.

Compound Evaluation. For urodynamic testing, pigs were anesthetized with a mixture of telazol (4.4 mg/kg i.m.) and xylazine (2.2 mg/kg i.m.), intubated, and maintained on isoflurane/oxygen in the supine position. Anesthesia level and bladder volume (via the port catheter) were adjusted to establish a regular unstable contraction pattern and stable mean arterial pressure (MAP). After a 30-min baseline period, up to two increasing doses of test compounds were administered i.v. at 30-min intervals. Each dose was administered over a 5-min period. Blood samples were obtained from the contralateral ear at 15 and 28 min after each dose for subsequent determination of plasma concentrations by liquid chromatography/mass spectroscopy. A total of 24 pigs was used to complete the present study. In the interest of keeping the total number of animals to a minimum, pigs were routinely tested on more than one occasion to profile more than one compound. However, each animal was used only once to profile any given dose of any given compound and was allowed to recover for at least 7 days between experiments.

Data Acquisition and Analysis. Radiotelemetry signals were acquired using the Data Sciences ART system interfaced to a Ponemah physiology platform (Gould, Valley View, OH). Bladder contraction amplitude, frequency, duration, and area under the bladder pressure curve (AUC) were determined using the Ponemah CYS analysis module (Gould). Data were averaged over the entire 30-min postdosing period and expressed as mean ± S.E.M. percent change from baseline values. Estimated intravenous doses of each compound required to reduce unstable contraction AUC by 35% and MAP by 10% were estimated from the dose-response graphs using GraphPad Prism (GraphPad Software, San Diego CA) for comparison of compound profiles.

	Results

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General Characteristics. Under isoflurane anesthesia, voiding contractions were inhibited and highly rhythmic, spontaneous nonvoiding contractions were observed (Fig. 1; Table 1). Mean contraction amplitude and duration were 13 ± 1 cm of H₂O and 36 ± 2 s, respectively. Typically, these contractions occurred about once per minute (Table 1). The baseline values for mean arterial pressure, 91 ± 3 mm Hg, were similar to those routinely observed in conscious pigs (data not shown). Baseline predose control values for cystometric parameters, mean arterial pressure, and heart rate were not statistically different between dose groups (analysis of variance; p > 0.05). The onset of compound-induced effects on both bladder and arterial pressure was immediate, and the time course was comparable for both parameters. In all cases, the duration of effects exceeded the 30-min postdosing observation period; however, the duration was not specifically determined due to the desire to limit the length of time that animals were maintained under anesthesia.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Representative trace of spontaneous nonvoiding (unstable) bladder contractions in an isoflurane-anesthetized unstable pig, illustrating the dose-dependent inhibitory effect of A-278637. Shown are baseline contractions and after i.v. administration of A-278637 (10 and 30 nmol/kg).

Effect of A-278637 on Unstable Bladder Contractions. As shown in Fig. 1, A-278637 caused dose-dependent inhibition of unstable contractions. Statistically significant mean reductions in total contraction AUC of 26 ± 6, 51 ± 4, and 75 ± 11% were observed after doses of 3, 10, and 30 nmol/kg i.v., respectively (Table 2). At the lowest dose of 3 nmol/kg i.v., there was no change in contraction amplitude, duration, or AUC per contraction. This indicates that the observed decrease in total area was derived entirely by a decrease in the contraction frequency. At higher doses (10 and 30 nmol/kg), additional decreases in frequency were observed along with dose-dependent decreases in amplitude. Contraction duration did not change after any dose tested, despite marked decreases in other contraction parameters (Table 2). For comparative purposes, the dose of compound that suppressed contraction AUC by 35% (AUC pED_35%) was chosen because this efficacy was generally achieved by all KCOs within the dose range tested in this model. The estimated log moles per kilogram dose of A-278637 to inhibit contraction AUC by 35% (AUC pED_35%) was 8.38 (corresponding to 4.2 nmol/kg or 1.7 µg/kg; Table 3). The plasma concentration of A-278637 corresponding to the ED_35% was 1.7 ng/ml (4.1 nM).

Comparison of Profile of A-278637 with Other KCOs. WAY-133537 caused dose-dependent inhibition of unstable contractions with a profile that was qualitatively similar to that of A-278637. However, 10- to 30-fold higher doses of WAY-133537 were required to produce similar efficacy (Table 2; Fig. 2). Like A-278637, the effects of WAY-133537 on AUC at lower doses were also derived from decreases in frequency with dose-dependent reductions in amplitude occurring at higher doses and with little or no effect on contraction duration at any dose tested. The estimated AUC pED_35% was 6.96 (corresponding to 109 nmol/kg or 35 µg/kg; Table 3).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Dose-response curves for the inhibition of unstable bladder contraction AUC by KATP channel openers A-278637 (), WAY-133537 (), and ZD6169 (), and the L-type calcium channel antagonist nifedipine (). Data expressed as mean ± S.E.M. percent change from predose control period. Number of animals tested was three to five per dose as indicated in Table 2.

Effect of Nifedipine on Unstable Bladder Contractions. In vitro studies have shown that smooth muscle relaxation by KCOs is primarily due to membrane hyperpolarization leading to attenuated calcium influx through dihydropyridine-sensitive L-type calcium channels (Quayle et al., 1997). Accordingly, the profile of an agent that directly inhibits L-type calcium channels on unstable contractions was evaluated. Nifedipine inhibited unstable contractions at 100 nmol/kg i.v. but not at 30 nmol/kg i.v. The decrease in total AUC at 100 nmol/kg i.v. (24%) was similar to efficacy observed after the same dose of WAY-133537 (28%) or after 3 nmol/kg i.v. A-278637 (26%; Table 2). However, in contrast to the profile of the KCOs (A-278637 and WAY-133537), the inhibition caused by nifedipine seems to be primarily due to a decrease in contraction amplitude and not frequency (Table 2). Administration of higher doses of nifedipine to achieve greater bladder efficacy was limited by the degree of hypotension observed immediately after dosing (data not shown). The estimated potency of nifedipine in this model (AUC pED_35% = 6.87) was similar to WAY-133537 (6.96) (Table 3).

Comparison of Potencies in Vivo and in Vitro. The potency of KCOs and nifedipine to suppress spontaneous nonvoiding contractions in obstructed pigs in vivo was compared with potency to suppress myogenic phasic activity of normal pig detrusor strips in vitro. Figure 3 shows a correlation between the in vivo AUC pED_35% values and the corresponding log EC₅₀ values of KCOs to inhibit spontaneous phasic activity in vitro. The rank order of potency, YM934 > A-278637 > ()-cromakalim > WAY-133537 > ZD6169, was similar both in vitro and in vivo. The potencies were positively linearly correlated (r² = 0.97), and the equation of the best-fit line was y = 0.54x + 3.2. The 95% confidence limits for the slope were from 0.42 to 0.69, indicating that the slope was significantly less than 1. As previously noted, the reduction in the area of contraction AUC by lower doses of A-278637 was solely derived from reductions in contraction frequency. This is similar to the profile in vitro where the contraction frequency was suppressed at lower concentrations than other parameters (Gopalakrishnan et al., 2002). At higher concentrations, significant dose-dependent suppression of contraction amplitude was noted in both in vivo and in vitro studies.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Comparison of the potencies of compounds to inhibit spontaneous nonvoiding bladder contractions in obstructed pigs in vivo to their potency to inhibit spontaneous contractions of detrusor strips in vitro. The x-axis represents log moles per kilogram (i.v.) to inhibit contraction AUC by 35% (pED35), and the y-axis represents log IC50 values for inhibition of spontaneous contractions by 50% (pIC50). The in vitro data for suppression of myogenic contractions for WAY-133537, ZD6169, ()-cromakalim, YM-934, and nifedipine were taken from Buckner et al. (2002). Data for A-278637 were taken from Gopalakrishnan et al. (2002). The dashed line represents 1:1 correlation (slope of unity). Equation of best-fit regression line for KCOs (squares) was y = 0.54 x + 3.2; r2 = 0.97. Although depicted, the data for nifedipine was not included in the regression analysis.

Selectivity for Bladder Versus Hypotensive Effects. Although KCOs caused dose-dependent reductions in MAP, there are apparent differences between compounds as to the extent of hypotension at a given level of bladder contraction inhibition, suggesting differences in bladder selectivity. For example, as shown in Table 3, a 5-fold higher dose of nifedipine was required to decrease AUC ED_35% than to decrease MAP by 10% (MAP ED_10%). In contrast, the AUC ED_35% of A-278637 was 3-fold lower than its MAP ED_10%. By this comparison, A-278637 was approximately 15-fold more bladder-selective than nifedipine (Table 3). Table 3 also indicates that the absolute bladder selectivity of A-278637 (3-fold) was approximately 5-fold better than either WAY-133537 or ZD6169, both of which reduced MAP with selectivity ratios of 0.5 and 0.6, respectively.

	Discussion

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Results of the present study demonstrate that A-278637, a novel K_ATP channel opener (Gopalakrishnan et al., 2002), potently inhibits unstable myogenic contractions in a pig model of bladder instability secondary to partial outlet obstruction. The bladder versus vascular selectivity of A-278637 was found to be superior to other KCOs, WAY-133537 and ZD6169, agents previously reported to be bladder-selective in vivo. Furthermore, the bladder efficacy of KCOs in general and A-278637 in particular was superior to nifedipine, a calcium channel blocker that was also effective in suppressing unstable contractions. These data support the notion that bladder-selective KCOs could be useful in the treatment of irritative symptoms secondary to benign prostatic obstruction in men as well as idiopathic overactive bladder in women.

Although the present study did not include nonobstructed control pigs, as reported from our laboratory previously (Fey et al., 2002), spontaneous bladder contractions were observed only after several weeks of obstruction. This is consistent with the observations of Sibley (1985) who did not detect spontaneous contractions in four of four sham-operated nonobstructed control pigs during filling cystometry and that of Mills et al. (2000b) in 14 conscious, unobstructed telemetry-implanted pigs. These data, taken together, suggest that spontaneous nonvoiding contractions are secondary to obstruction, not related to telemetry implantation per se, and are not a normal finding during routine filling cystometry in unobstructed control pigs, as reported in humans (Salvatore et al., 2001).

The effect of KCOs on various urodynamic parameters characterizing the morphology and frequency of unstable contractions demonstrates a differential inhibition profile that is distinct from that of a prototypical calcium channel blocker. For example, as shown in Table 2, A-278637 at low doses (3 nmol/kg) primarily reduced the frequency component of contractions without significant changes in the amplitude or duration. Similar effects were noted with WAY-133537 and, to a lesser extent, with ZD6169. The predominant effect of KCOs on the frequency of spontaneous contractions is quite analogous to that reported in vitro for the suppression of myogenic contractions in pigs (Buckner et al., 2002) and is consistent with the idea that small increases in K⁺ conductance could move the resting membrane potential away from the threshold for action potential firing, thereby limiting the initiation of phasic contractions (Petkov et al., 2001; Shieh et al., 2001). At higher doses, A-278637 (10 and 30 nmol/kg) also significantly decreased the amplitude of contractions, which presumably reflects attenuation of intracellular calcium levels resultant to K_ATP channel activation. On the other hand, the suppression of unstable contractions evoked by nifedipine (100 nmol/kg) was predominantly driven by a decrease in the amplitude of contractions with no change in the frequency (Table 2). Our in vivo results with nifedipine are consistent with the notion that the amplitude of contractions depends predominantly on calcium entry through L-type calcium channels during membrane depolarization.

As shown in Table 1, baseline unstable bladder contractions in obstructed pigs were approximately 10 to 15 cm of H₂O in amplitude with duration of about 40 s. In contrast, the baseline unstable contractions in obstructed rats were smaller in amplitude (~6 cm of H₂O) and of shorter duration (19 s) but were about 2-fold more frequent (Fabiyi et al., 2002). Despite these distinctions in baseline contraction morphology and differences in experimental protocols, including species, anesthetic agent, and differences in duration and method of obstruction, the effective doses of ZD6169 and WAY-133537 to inhibit unstable contraction AUC by 35% (0.66 and 0.11 nmol/kg i.v., respectively) were comparable with those previously reported in obstructed rats (1.4 and 0.07 µmol/kg i.v.; Fabiyi et al., 2002). These data demonstrate that the rank order of potencies of KCOs to inhibit unstable contraction AUC is comparable in both rats and pigs, suggesting no apparent differences in K_ATP channel function between these species. However, there are apparent differences between rats and pigs regarding the effect of KCOs on contraction parameters from which the AUC measurement is derived. For example, the primary effect of the WAY-133537 in obstructed pigs was on contraction frequency with no change in duration (Table 2). On the other hand, decreases in contraction AUC were driven by simultaneous and parallel decreases in frequency, amplitude, and duration in obstructed rats (Fabiyi et al., 2002). The reasons for these differences remain to be elucidated and could be protocol-related. Alternatively, it is conceivable perhaps that differences in resting membrane potential, distinctions in ion channels governing membrane potential, or local humoral factors such as nucleotides may regulate bladder smooth muscle K_ATP channel activity differently in rats and pigs.

In this study, the estimated doses required to cause 35% inhibition of contraction AUC were used to compare compound potencies and selectivity. This level of efficacy was chosen because it allows simultaneous comparison of all compounds without extrapolation because 50% inhibition was not attained with nifedipine and ZD6169 due to hypotension and solubility limitations, respectively. However, as indicated in Fig. 2, the AUC dose-response curves are sufficiently parallel such that similar overall conclusions as to the relative potency of these compounds are obtained regardless of the efficacy level chosen for comparison. For example, estimated AUC ED_50% values for A-278637, WAY-133537, and ZD6169 (8.7, 161.1, and 1111 nmol/kg i.v.) were all greater to a similar extent (1.5-, 1.7-, and 2.1-fold) than their corresponding ED_35% values (4.2, 109, and 661 nmol/kg i.v.). The therapeutic relevance of reductions in unstable contraction AUC and to what degree of efficacy in this preclinical model translates to clinical efficacy and symptom improvement in overactive bladder remains to be elucidated.

Comparison of KCO Profiles in Vivo versus in Vitro. Figure 3 illustrates the relationship between the potencies of structurally distinct KCO chemotypes and nifedipine to inhibit spontaneous contractions of detrusor strips in vitro to their potency to inhibit unstable bladder contractions in vivo. The results of the correlation analysis (r² = 0.97) suggest a strong positive linear relationship between results obtained in both assays. For example, the rank order of potency to inhibit myogenic contractions of detrusor tissue strips from young normal pigs in vitro was similar to that noted for inhibition of spontaneous nonvoiding bladder contractions of intact anesthetized older obstructed pigs in vivo. These results are consistent with previous observations from our laboratory, suggesting a strong 1:1 relationship between KCO potencies to inhibit in vitro spontaneous myogenic contractions of detrusor strips from either normal or obstructed pigs (Milicic et al., 2001). In addition, unstable contractions in vivo were not inhibited by the ganglionic blocker hexamethonium or by the muscarinic antagonist tolterodine (Fey et al., 2002). The excellent in vitro versus in vivo correlation of rank order potencies of KCOs, together with the lack of effects of ganglionic blockers and muscarinic receptor antagonists support the hypothesis that the unstable contractions are of myogenic origin. However, it should be noted that clinical symptoms of overactive bladder could have multiple potential etiologies that may not be necessarily modeled in this system, including increased afferent nerve activity. Therefore, the degree of efficacy and selectivity required in this preclinical model that translates to substantial improvements in the symptoms of overactive bladder in humans remains to be determined.

In Vivo Selectivity of A-278637: Comparison with ZD6169 and WAY-133537. ZD6169 and WAY-133537 were chosen for comparison because previous reports have suggested enhanced in vivo bladder selectivity versus cardiovascular effects for these agents. For example, Howe et al. (1995) reported that, in conscious rats after oral dosing, ZD6169 caused dose-dependent increases in bladder capacity with potency approximately 6-fold greater than cromakalim and a bladder selectivity ratio of 187. Wojdan et al. (1999) reported that WAY-133537 was approximately 18-fold more potent than ZD6169 to inhibit number of unstable contractions in conscious obstructed rats (ED₅₀ values of 0.13 and 2.4 mg/kg p.o., respectively). Selectivity ratios versus hypotensive effects for ZD6169 and WAY-133537 were 3 and 18, respectively (Wojdan et al., 1999). In the present study, although modest improvements in selectivity of these compounds relative to nifedipine were evident, no absolute bladder selectivity was observed (Fig. 4; Table 3). Selectivity differences between the present study and previous reports may be attributed to differences in methodology such as routes of administration and resultant differences in pharmacokinetics and/or the formation of putative metabolite(s). For example, the efficacy and selectivity reported by Howe et al. (1995) at relatively low doses for several hours does not seem to parallel the time course of plasma concentrations of the parent compound based on pharmacokinetic studies (L. King, unpublished observations). Species differences are not a likely explanation because no bladder selectivity was observed with ZD6169 and WAY-133537 after intravenous dosing in an analogous obstructed rat model (Fabiyi et al., 2002). Preferential distribution of the compound to the detrusor tissue may also not be a factor because bladder and arterial pressure changes occurred simultaneously shortly after dosing.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Dose-response curves for suppression of unstable contraction AUC and for reductions in MAP by KCOs A-278637, WAY-133537, and ZD6169 (A-C), and the L-type calcium channel antagonist nifedipine (D) in anesthetized obstructed pigs. Number of animals tested was three to five per dose as indicated in Table 2.