J. Smooth Muscle Res. (2009) 45 (4): 187–195 187

Original

Effects of four different α1-adrenoceptor antagonists on α-adrenoceptor agonist-induced contractions in isolated mouse and hamster ureters

Shinya KOBAYASHI1, Yoshitaka TOMIYAMA1, Kazuyasu MARUYAMA1, Yuji HOYANO1, Yoshinobu YAMAZAKI1 and Hiroshi KUSAMA1

1Pharmacology, Research and Development, Kissei Pharmaceutical Co. Ltd., Nagano 399-8304, Japan

Received April 16, 2009; Accepted June 18, 2009

Abstract

Purpose: To compare the efficacy of the selective α1A-adrenoceptor antagonist silodosin with those of doxazosin, terazosin, and alfuzosin against α-adrenoceptor

agonist-induced contractions in mouse and hamster ureters. Methods: The four α1- adrenoceptor antagonists were evaluated against norepinephrine-induced phasic contractions in mouse isolated ureteral preparations and against phenylephrine-induced sustained contractions in hamster isolated ureteral preparations using a functional

experimental technique. Results: In mouse ureters, silodosin (a selective α1A-

adrenoceptor antagonist), doxazosin (a nonselective α1-adrenoceptor antagonist),

terazosin (a nonselective α1-adrenoceptor antagonist), and alfuzosin (a nonselective α1- adrenoceptor antagonist) all shifted the norepinephrine concentration-response curve to

the right. The rank order of potencies (pKB value) was silodosin (9.47 ± 0.16) > doxazosin (8.62 ± 0.15) > terazosin (8.39 ± 0.16) > alfuzosin (8.03 ± 0.12). In hamster ureters, all four antagonists shifted the phenylephrine concentration-response curve to the right, the rank order of potencies being silodosin (10.09 ± 0.13) > doxazosin (8.22 ± 0.16) > terazosin (7.75 ± 0.15) > alfuzosin (7.70 ± 0.10). In each case, silodosin was much more potent than the other three drugs. Conclusion: In this study, silodosin suppressed both mouse and hamster ureteral contractions more potently than doxazosin, terazosin,

or alfuzosin. Hence, this α1A-adrenoceptor antagonist warrants further study as a potentially very useful medication for stone passage in urolithiasis patients.

Key words: α1-adrenoceptor antagonists, contraction, ureter

Introduction

Management of urinary stones still occupies an important place in everyday urological practice as 8–15% of the population in western countries are afflicted by this condition (Pak, 1998). Such stones are mostly lodged in the distal part of the ureter (Menon et al., 1998). The

Correspondence to: Dr. Shinya Kobayashi, Pharmacology R&D, Kissei Pharmaceutical Co. Ltd., 4365-1 Kashiwabara, Hotaka, Azumino, Nagano 399-8304, Japan Phone: +81-263-82-8820 Fax: +81-263-81-1045 e-mail: [email protected] 188 S. KOBAYASHI et al. expulsion of ureteral stones is probably facilitated by several factors, such as relaxation of ureteral smooth muscle, peristalsis, and diuresis (Micali et al., 2006).

α1-Adrenoceptors have been demonstrated in the ureter of the pig, dog, and horse and are more prevalent than other adrenoceptors in ureteral smooth muscle (Weiss et al., 1978; Morita et al., 1994). Moreover, α1-adrenoceptor antagonists have been shown to inhibit ureteral contraction in humans (Küpeli et al., 2004). Since several α1-adrenoceptor mRNAs (α1a-, α1b-,

α1d-) and proteins (α1A-, α1B-, α1D-) are reportedly expressed in the human ureter, α1- adrenoceptor antagonists would be expected to act as facilitators of ureteral stone passage (Itoh et al., 2007; Park et al., 2007). Indeed, Ukhal et al. (1999) demonstrated a decade ago that the

α1-adrenoceptor antagonist doxazosin accelerated ureteral stone passage. More recent studies have shown that other α1-adrenoceptor antagonists (terazosin and alfuzosin) could be used to augment spontaneous stone expulsion and to reduce the time to expulsion of ureteral stones (Pricop et al., 2004; Mohseni et al., 2006).

Recently, Sasaki et al. (2008) demonstrated that α1A-adrenoceptors mediate ureteral contractions in humans, and we showed that ureteral contraction was mediated mainly via the

α1A-adrenoceptor in mice, hamsters, and dogs (Tomiyama et al., 2007; Kobayashi et al., 2009a;

Kobayashi et al., 2009b). However, it is unclear which of the currently available α1-adrenoceptor antagonists is the most suitable for stone discharge. The aim of this study was to compare the efficacy of the new α1A-adrenoceptor antagonist silodosin with that of three nonselective α1- adrenoceptor antagonists (doxazosin, terazosin, and alfuzosin) against α-adrenoceptor agonist- induced contractions in both mouse and hamster ureters.

Methods

Animals This study was conducted according to guidelines approved by the Laboratory Animal Committee of Kissei Pharmaceutical Co. Ltd. and conforms to current Japanese Law. Male ICR mice (35.0–47.2 g; Nihon SLC, Hamamatsu, Japan) and male Syrian hamsters (67.0–97.2 g; Nihon SLC) were maintained under a 12-hour light/12-hour dark cycle with free access to water and standard laboratory food until the day of the experiment.

Isolation of ureters Mice and hamsters were anaesthetized with sodium pentobarbital (30 mg/kg i.p.), and then euthanized with an overdose of the same agent. Ureters were carefully dissected free from the surrounding fatty and fibrous tissues.

Selection of α1- adrenoceptor agonists In our preliminary experiments on the mouse isolated ureter, the concentration-response curve for norepinephrine (nonselective α-adrenoceptor agonist) was more reproducible than that for phenylephrine (selective α1-adrenoceptor agonist). However, the reverse was true in the hamster isolated ureter. Accordingly, we selected norepinephrine for the mouse study, but phenylephrine for the hamster study. α1-Adrenoceptor antagonists on the ureter 189

Functional experiments on mouse ureters Ureters were cut so as to make tubular preparations (each 15 mm long). Each preparation was suspended longitudinally in a 10 mL organ bath containing Krebs solution (composition in mM: NaCl 118.1, KCl 4.7, CaCl2 2.5, MgSO4 7H2O 1.2, NaHCO3 25.0, KH2PO4 1.2, glucose 11.1), which was maintained at 37°C and continuously gassed with a mixture of 95% oxygen and 5% carbon dioxide. The responses to various agents were assessed by means of an isometric force- transducer and measuring system (TB-611T, AP-601G, and RPM-6004; Nihon Kohden, Tokyo, Japan) connected to a polygraph (Recti-Horiz-8K; GE Marquette Medical System Japan, Tokyo, Japan). An initial resting tension of 1,000 µN was exerted on each segment prior to an equilibration period of 60 min before the start of the experiment. The isolated ureter of the mouse did not develop spontaneous contractions. Desipramine (1 × 10–7 mol/L), corticosterone (1 × 10–5 mol/L), yohimbine (1 × 10–7 mol/L), and propranolol (1 × 10–6 mol/L) were present throughout the experiments to block neural uptake of norepinephrine, extraneural uptake of norepinephrine, α2-adrenoceptor-induced responses, and β-adrenoceptor-induced responses, respectively. Norepinephrine was cumulatively added in 0.5-log increments every 5 min. The effect of norepinephrine on ureteral motility was evaluated by summing all the contractions occurring during the 5-min period after the application of the drug. The response to a given concentration of norepinephrine was expressed as a percentage of the maximal response achieved in each preparation. Each tissue was equilibrated with either vehicle or with one of the

α1-adrenoceptor antagonists for 60 min before the addition of norepinephrine. Each preparation was exposed to only the antagonist and/or one agonist.

Functional experiment on hamster ureters Ureters were cut to make tubular preparations (each 20 mm long). An initial resting tension of 1,000 µN was exerted on each segment before being allowed to equilibrate for at least 60 min before the start of the experiment. Spontaneous contractions were hardly observed at all in the isolated hamster ureter. The other experimental conditions and the equipment used were the same as those described for mouse ureters. Phenylephrine was added cumulatively in 0.5-log increments every 2.5 min. Each preparation was equilibrated with vehicle or with one of the α1- adrenoceptor antagonists for 60 min before the addition of phenylephrine, with the phenylephrine-induced sustained contractions being expressed as a percentage of the maximal response achieved in each preparation. Each preparation was exposed to only the antagonist and/or one agonist.

Drugs Silodosin (KMD-3213: (–)-1-(3-hydroxypropyl)-5-[(2R)-2-({2-[2-(2,2,2-trifluoro-ethoxy)- phenoxy]ethyl}amino)propyl]-2,3-dihydro-1H-indole-7-carboxamide) was synthesized by Kissei Pharmaceutical Co. Ltd. (Nagano, Japan). Doxazosin mesylate, terazosin hydrochloride, desipramine hydrochloride, corticosterone, yohimbine hydrochloride, (±)-propranolol hydrochloride, (–)-norepinephrine bitartrate, and (R)-(–)-phenylephrine hydrochloride were purchased from Sigma (St. Louis, MO). Alfuzosin hydrochloride was from SynFine Research (Ontario, Canada), dimethyl sulphoxide (DMSO) from Nacalai tesque (Kyoto, Japan), and 190 S. KOBAYASHI et al.

Fig. 1. Typical tracings of effects of vehicle and silodosin on norepinephrine-induced phasic contractions in isolated ureteral preparations of the mouse, with concentrations shown as log [norepinephrine] (mol/L).

sodium pentobarbital from Dainippon Pharmaceutical (Osaka, Japan). Silodosin, doxazosin mesylate, terazosin hydrochloride, alfuzosin hydrochloride, and corticosterone were dissolved in 100% DMSO. Silodosin was diluted with Hartmann’s solution of the following composition

(w/v %): NaCl 0.6, KCl 0.03, CaCl2 0.02, and lactic acid 0.31. The other agents were diluted with distilled water. At the maximal concentration reached in the bath (0.001%), neither DMSO nor Hartmann’s solution had any effect on our preparations.

Data analysis

Data are expressed as the mean ± standard error of the mean (SEM). The pKB value was calculated using the following formula: pKB = log (CR-1)-log [antagonist], where CR is the ratio of norepinephrine or phenylephrine concentrations that induced a similar response (i.e., half- maximal response) between the presence and absence of the test drug. Tukey’s test was performed to assess the differences in pKB value between all possible pairs of groups, with P<0.05 considered to be statistically significant.

Results

Effects of α1-adrenoceptor antagonists in isolated ureters of the mouse Figure 1 shows typical tracings of the effect of silodosin on the phasic contractions induced by norepinephrine in the isolated ureter of the mouse. All four α1-adrenoceptor antagonists tested concentration-dependently inhibited norepinephrine-induced phasic contractions, and each drug produced a rightward shift in the concentration-response curve for norepinephrine

(Fig. 2). The rank order of antagonist potencies (pKB value) was silodosin (α1A-adrenoceptor selective; 9.47 ± 0.16) > doxazosin (nonselective α1-adrenoceptor antagonist; 8.62 ± 0.15) > terazosin (nonselective α1-adrenoceptor antagonist; 8.39 ± 0.16) > alfuzosin (nonselective α1- adrenoceptor antagonist; 8.03 ± 0.12). The pKB value for silodosin was significantly greater (P<0.01) than those obtained for the other three antagonists. α1-Adrenoceptor antagonists on the ureter 191

Fig. 2. Antagonism by silodosin (A), doxazosin (B), terazosin (C), and alfuzosin (D) of norepinephrine-induced phasic contractions of the isolated ureter of the mouse. Means ± SEM from 7 experiments.

Effects of α1-adrenoceptor antagonists in isolated ureters of the hamster Figure 3 shows typical tracings of the effect of silodosin on the sustained contractions induced by phenylephrine in the isolated ureter of the hamster. Each of the four α1- adrenoceptor antagonists tested inhibited phenylephrine-induced sustained contractions in a concentration-dependent manner, and the concentration-response curve was shifted to the right by each drug tested (Fig. 4). The rank order of antagonist potencies was silodosin (10.09 ± 0.13)

> doxazosin (8.22 ± 0.16) > terazosin (7.75 ± 0.15) > alfuzosin (7.70 ± 0.10). The pKB value for silodosin was significantly greater (P<0.0001) than those obtained for the other three antagonists.

Discussion

α1-Adrenoceptor antagonists, such as doxazosin, terazosin, and alfuzosin, have been reported to increase the stone-expulsion rate in patients with ureteral stone (Pricop et al., 2004; Mohseni et al., 2006), effects that may be related to their inhibitory actions against the ureteral contraction induced by norepinephrine. We recently reported that α1A-adrenoceptors mediate ureteral contractions in mice, hamsters, and dogs (Tomiyama et al., 2007; Kobayashi et al., 2009a; Kobayashi et al., 2009b). In the present study, we used pharmacological techniques to compare the efficacy of the selective α1A-adrenoceptor antagonist silodosin with those of three 192 S. KOBAYASHI et al.

Fig. 3. Typical tracings of effects of vehicle and silodosin on phenylephrine-induced sustained contractions in isolated ureteral preparations of the hamster, with concentrations shown as log [phenylephrine] (mol/L).

Fig. 4. Antagonism by silodosin (A), doxazosin (B), terazosin (C), and alfuzosin (D) of phenylephrine-induced sustained contractions of isolated ureters of the hamster. Means ± SEM from 5 experiments.

nonselective α1-adrenoceptor antagonists (namely, doxazosin, terazosin, and alfuzosin) against α-adrenoceptor agonist-induced contractions in both mouse and hamster ureters. Our results showed that all four drugs antagonized the norepinephrine-induced contraction in the mouse isolated ureter. The antagonist potency of silodosin in this experiment (pKB value

= 9.47) was much greater than those of doxazosin (pKB value = 8.62), terazosin (pKB value =

8.39), and alfuzosin (pKB value = 8.03). The pKB values we obtained for doxazosin, terazosin, α1-Adrenoceptor antagonists on the ureter 193

and alfuzosin in the mouse ureter were in good agreement with the pA2 values reported for α1A- adrenoceptors in isolated preparations of human prostate, rat vas deferens, and the trigone of the rabbit urinary bladder (Lefèvre-Borg et al., 1993; Kenny et al., 1996; Hancock et al., 2002). The above data indicate that silodosin is the most effective of the four drugs on the mouse ureter. In our experiments on the isolated ureter of the hamster, silodosin had a strong inhibitory effect on the phenylephrine-induced contraction. The antagonist potency of silodosin (pKB value

= 10.09) was about two orders of magnitude greater than those of doxazosin (pKB value = 8.22), terazosin (pKB value = 7.75), and alfuzosin (pKB value = 7.70). The pKB values we obtained for doxazosin, terazosin, and alfuzosin in the hamster ureter corresponded well to the pA2 values reported for α1A-adrenoceptors in the human prostate gland, rat vas deferens, and the trigone of the rabbit urinary bladder (Lefèvre-Borg et al., 1993; Kenny et al., 1996; Hancock et al., 2002). Our data therefore demonstrate that silodosin is more potent than the other three drugs in the hamster ureter.

Recently, three types of α1-adrenoceptor mRNAs and proteins were reported to be expressed in human ureteral smooth muscle (Itoh et al., 2007; Park et al., 2007). It might therefore be expected that nonselective α1-adrenoceptor antagonists would be effective for the expulsion of ureteral stones. Actually, Yilmaz et al. (2005) reported a few years ago that in humans, doxazosin and terazosin reduced the time to expulsion of distal ureteral stones with equal efficacy. In our study, the antagonist potency of doxazosin was almost the same as that of terazosin in each of the two species, in good agreement with the above clinical finding. The potencies of the various α1-adrenoceptor antagonists in our study would presumably be reflected by their clinical efficacies towards stone passage. The efficacies displayed by silodosin in the present experiments on the mouse ureter (pKB value = 9.47) and hamster ureter (10.09) were much greater than those reported for the mouse aorta (α1D-adrenoceptor-mediated response; pA2 value = 8.25) (Hosoda et al., 2005), rat aorta (α1D-adrenoceptor-mediated response; pA2 value

= 7.88) (Tatemichi et al., 2006a), dog carotid artery (α1B-adrenoceptor-mediated response; pKB value = 7.54) (Tatemichi et al., 2006b), or human mesenteric artery (α1B-adrenoceptor-mediated response; pA2 value = 7.47) (Murata et al., 2000). Moreover, Akiyama et al. (1999) demonstrated in anesthetized rats that silodosin suppressed an α1A-adrenoceptor-mediated response (the increase in intraurethral pressure induced by phenylephrine) without seriously altering mean blood pressure, with selectivity (α1A-adrenoceptor-mediated response against hypotensive effect) being markedly greater for silodosin (29.7) than for terazosin (0.52). In the present study, the effect of terazosin in the two ureters was quite similar to those of doxazosin and alfuzosin, and reportedly there is close similarity among these three drugs in that each of them displays almost completely nonselective antagonistic activities against the three subtypes of α1-adrenoceptor

(α1A, α1B, and α1D) (Martin, 1999). Accordingly, doxazosin and alfuzosin, like terazosin, would not be expected to be selective for the ureters versus the cardiovascular system. Since human ureteral contraction is mediated mainly via the α1A-adrenoceptor (Sasaki et al., 2008), a selective

α1A-adrenoceptor antagonist such as silodosin would probably be more effective for the treatment of ureteral stones than a nonselective α1-adrenoceptor antagonist, and would be less likely to have cardiovascular side effects. 194 S. KOBAYASHI et al.

In summary, in this study silodosin inhibited α-adrenoceptor agonist-induced contractions in both mouse and hamster ureters more potently than doxazosin, terazosin, or alfuzosin. This result argues in favor of this α1A-adrenoceptor antagonist potentially being a very useful medication for stone passage in patients with ureteral stone.

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