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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
3-Adrenoceptor Agonist, on Bladder FunctionInstitute for Drug Discovery Research, Astellas Pharma Inc., Ibaraki, Japan (T.T., M.U., S.S., Te.M., I.N., Ta.M., M.S., K.M.); and Department of Urology, Fukushima Medical University, Fukushima, Japan (H.U., O.M.)
Received October 18, 2006; accepted February 8, 2007.
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Abstract |
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3-adrenoceptor (AR). The half-maximal effective concentration (EC50) value was 22.4 nM. EC50 values of YM178 for human 1- and 2-ARs were 10,000 nM or more, respectively. The ratio of intrinsic activities of YM178 versus maximal response induced by isoproterenol (nonselective -AR agonist) was 0.8 for human 3-ARs, 0.1 for human 1-ARs, and 0.1 for human 2-ARs. The relaxant effects of YM178 were evaluated in rats and humans bladder strips precontracted with carbachol (CCh) and compared with those of isoproterenol and 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (CGP-12177A) (3-AR agonist). EC50 values of YM178 and isoproterenol in rat bladder strips precontracted with 10–6 M CCh were 5.1 and 1.4 µM, respectively, whereas those in human bladder strips precontracted with 10–7 M CCh were 0.78 and 0.28 µM, respectively. In in vivo study, YM178 at a dose of 3 mg/kg i.v. decreased the frequency of rhythmic bladder contraction induced by intravesical filling with saline without suppressing its amplitude in anesthetized rats. These findings suggest the suitability of YM178 as a therapeutic drug for the treatment of symptoms of overactive bladder such as urinary frequency, urgency, and urge incontinence.
3-adrenoceptor (AR) is one of three -AR subtypes, termed 1-to 3-AR. 3-AR is a G protein-coupled receptor identified by genomic cloning of human cells in the late 1980s (Emorine et al., 1989
) that is sparsely distributed in humans. Functional 3-AR-mediated responses have been observed in human brown and white fat cells, where they mediate lipolysis; and in gall bladder, stomach, small intestine, prostate, colon (Berkowitz et al., 1995), and bladder, where they evoke relaxation (Fujimura et al., 1999). Although more than 15 years has passed since 3-AR was identified, the therapeutic potential of 3-AR agonists in humans remains unclear. BRL37344, CL316,243, and CGP-12177A are representative 3-AR agonists that were optimized using rodent -ARs (Arch et al., 1984; Langin et al., 1991; Dolan et al., 1994). These compounds have lower potency for human 3-ARs than for rodent receptors, however, and they act as only partial agonists in humans (Igawa et al., 1999, 2001). Subsequent recognition of important pharmacological differences between rodent and human 3-ARs has led to the development of novel 3-AR agonists that are potent and highly selective toward human 3-ARs (Hu and Jennings, 2003).
Although -ARs play an important role in bladder relaxation in mammals, considerable functional interspecies differences among -AR subtypes have been identified. In human bladder smooth muscle, 3-AR mRNA expression is predominant, with this subtype accounting for 97% total -AR mRNA (Yamaguchi, 2002; Nomiya and Yamaguchi, 2003). In accordance with expression levels, human bladder relaxation is mainly induced through 3-AR and not 1-or 2-ARs (Igawa et al., 1998, 1999; Yamazaki et al., 1998; Takeda et al., 1999). Recently, it was shown that 3-AR agonists can improve bladder overactivity in rat experimental models (Woods et al., 2001; Kaidoh et al., 2002), suggesting the usefulness of 3-AR agonists in the treatment of overactive bladder (OAB). YM178 (Fig. 1) was synthesized by Astellas Pharma Inc. (Ibaraki, Japan). Here, we report for the first time the pharmacological profile of YM178 and its effects on bladder smooth muscle, as investigated in vitro and in vivo.
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Materials and Methods |
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1- and 2-ARs were purchased from Dr. Lefkowitz (Duke University Medical Center, Durham NC). CHO cells were from the American Type Culture Collection (Manassas, VA). Lipofectin, G-418 sulfate, Ham's F-12 medium, penicillin/streptomycin (100 units/100 µg/ml), and Hanks' balanced salt solution were from Invitrogen (Carlsbad, CA). Fetal bovine serum was from Bioserum (Parkville, VIC, Australia). Trypsin-EDTA was from the Research Institute for Microbial Diseases (Osaka University, Osaka, Japan). HEPES sodium salt was from Wako Pure Chemicals (Osaka, Japan). 125I-cAMP assay system was from Yamasa Shouyu Co., Ltd. (Chiba, Japan).
Cell Culture. CHO cells expressing human 3-AR were constructed by transfecting cDNA of human 3-AR into CHO cells by the Lipofectin method. Stable transfectants were selected with 600 µg/ml G-418 sulfate. CHO cells expressing each type of human -AR were cultured at 37°C in a humidified atmosphere with 5% CO2 in Ham's F-12 medium, supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, 0.5 mg/ml G-418 sulfate, and 10% (v/v) fetal bovine serum. The cells were subcultured weekly, with the supernatant aspirated and trypsin-EDTA added for 10 min to detach the cells from the culture dish, followed by the addition of fresh medium and transfer of the cells to new Petri dishes.
cAMP Accumulation. Cells (105) were seeded in each well of a 24-well culture plate and subcultured. Three days later, the medium was exchanged with 250 µl/well Hanks' balanced salt solution containing 0.1 mM 3-isobutyl-1-methylxanthine, pH 7.4. The cells were incubated with each compound (isoproterenol, YM178, BRL37344, and CL316,243 at final concentrations of 10–10 to 10–4 M) for 10 min at 37°C, after which incubation was stopped by the addition of 250 µl of 0.2 M HCl. cAMP concentration in the reaction mixture was measured by radioimmunoassay using an 125I-cAMP assay system using a gamma counter (ALOKA, Tokyo, Japan). Fifty microliters of reaction mixture was incubated with 50 µl of succinyl agent for 10 min at room temperature, after which the reaction was stopped by the addition of 400 µl of buffer solution. Fifty microliters of succinylated sample was incubated with 50 µl of 125I-cAMP and 50 µl of anti-cAMP antibody for 24 h at 4°C. At the end of the incubation period, 250 µl of charcoal suspension was added and centrifuged for 10 min at 2800g at 4°C. Two hundred and fifty microliters of supernatant was transferred into a tube and counted for 1 min using a gamma counter. The intrinsic activity (I.A.) relative to isoproterenol for each -adrenoceptor agonist was calculated using the maximal response of each compound.
Animals. Male (350 to 400 g) and female (225 to 290 g) Wistar rats were purchased from Charles River Japan, Inc. (Kanagawa, Japan) and for in vitro and in vivo study, respectively.
Relaxant Activity in Isolated Rat Bladder Smooth Muscle. After anesthetizing rats with diethyl ether, they were sacrificed and the whole bladder was removed. Bladder strips (approximately 3 x 10 mm) were prepared and suspended under a loading tension of 1 g in Krebs-Henseleit solution (118.4 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 25.0 mM NaHCO3, and 11.1 mM glucose) and oxygenated with a gas mixture of 95% oxygen and 5% carbon dioxide at 37°C. Contractile response was measured with an isotonic transducer (TB-611T; Nihon Kohden, Tokyo, Japan) and registered on an ink-writing recorder (SR6211, SR6221; Graphtec, Tokyo, Japan). After stabilization for 30 to 60 min, 10–6 M carbachol (CCh) was added to induce repeated contractile responses at 30- to 60-min intervals. After the response to CCh had almost equalized, the strips were washed, and the contractile response to 10–6 M CCh was recorded again. After the contractile response had stabilized, a test compound or vehicle was added cumulatively at approximately 10-min intervals in the concentration range of 10–9 to 10–4 M, and the relaxant effect was recorded. At the end of each experiment, 10–4 M papaverine was added to obtain the maximal relaxant response, which was considered a 100% response and used to calculate the percentage of relaxation for each compound (n = 5).
Relaxant Activity in Isolated Human Bladder Smooth Muscle. This study was conducted at Fukushima Medical University in six male patients (mean age of 69.5 ± 1.8 years; range 64 to 75 years) with normal bladders undergoing radical cystectomy for malignancy. Bladder muscle strips from the anterior portion of the bladder dome obtained from these patients at surgery were placed immediately in preoxygenated Krebs-Henseleit solution at 4°C, and the strips were transported to the laboratory. Each strip (approximately 3 x 10 mm) was prepared and suspended under a loading tension of 1 g in Krebs-Henseleit solution oxygenated with a gas mixture of 95% oxygen and 5% carbon dioxide at 37°C. Contractile response was measured with an isotonic transducer (TB-621T; Nihon Kohden) and registered on an ink-writing recorder. After stabilization for 60 min, 10–7 M CCh was added at 60-min intervals to induce a repeated contractile response. After the responsive to CCh had almost equalized, each strip was washed, and the contractile response to 10–7 M CCh was recorded again. After the contractile response had stabilized, test compound or vehicle was added cumulatively at approximately 10-min intervals in the concentration range of 10–9 to 10–4 M, and the relaxant effect was recorded. At the end of each experiment, 10–4 M papaverine was added to obtain the maximal relaxant response, which was considered a 100% response and used to calculate the percentage relaxation for each compound (n = 4–6).
Rhythmic Isovolumetric Reflex Bladder Contraction. Rats were anesthetized with urethane (1 g/kg i.p.), and a flank incision was made. Both ureters were tied and cut at the side of the kidney. A midline abdominal incision was made and a polyethylene-50 cannula was inserted into the bladder through the urethra and ligated around the urethra. Urine in the bladder was removed through the cannula by gently pressing on the abdomen. The bladder cannula was connected to a pressure transducer (TP-400T; Nihon Kohden). At least 10 min after the operation, physiological saline at room temperature was infused into the bladder through the cannula at 2.4 ml/h, and the saline infusion was terminated after initiation of spontaneous rhythmic bladder contractions. At least 30 min after the rhythmic bladder contraction stabilized, drug was intravenously administered at escalating doses in a volume of 1 ml/kg through a polyethylene-50 cannula inserted into the left femoral artery. Rats were excluded if the rhythmic bladder contraction did not stabilize or was repeatedly stopped by saline administration. The frequency and amplitude of the rhythmic bladder contractions were evaluated for 10 min (minutes 5 to 15 after dosing). For each parameter, the saline administration value was taken as the pretreatment value.
YM178 was dissolved in saline containing 10% dimethyl acetamide and 5% Cremophor-EL (Nacalai Tesque, Kyoto, Japan), and oxybutynin chloride was dissolved in saline. A saline solution containing 10% dimethyl acetamide and 5% Cremophor-EL was used as a vehicle control. Subsequent dilutions of all drugs and vehicle were prepared in saline. The free-form doses of 0.03, 0.1, 0.3, 1 and 3 mg/kg for YM178 and 0.0272, 0.0907, 0.272, 0.907, and 2.72 mg/kg for oxybutynin were used in this study.
Statistical Analysis. Results are expressed as the mean ± S.E.M. or mean with 95% confidence intervals. EC50 values were calculated by nonlinear regression analysis. Statistical analysis was performed using Student's t test. Statistical significance was defined as a P value less than 0.05. All data analyses were performed using SAS statistical software (SAS Institute, Cary, NC).
Ethical Considerations. The animal experiments were performed in compliance with the International Guiding Principles for Biomedical Research Involving Animals. The protocol for this study was approved by the Animal Ethics Committee of Astellas Pharma Inc. The human bladder muscle study was approved by the Ethics Committee of Fukushima Medical University.
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Results |
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-ARs. YM178 concentration-dependently increased the accumulation of cAMP in CHO cells expressing human 3-ARs (Fig. 2C), with an EC50 value and I.A. of 22.4 nM and 0.8, respectively (Table 1). BRL37344 and CL316,243 also concentration-dependently increased the accumulation of cAMP in these cells (Fig. 2C), with EC50 values of 457 and 4,430 nM and I.A. of 0.6 and 0.5, respectively (Table 1). YM178 and CL316,243 had little agonistic effect on 1- and 2-ARs (Table 1; Fig. 2, A and B). In contrast, BRL37344 activated 1- and 2-ARs, with EC50 values of 12,900 and 360 nM and I.A. of 0.5 and 0.7, respectively (Table 1). YM178 did not induce cAMP elevation in untransfected CHO cells (data not shown).
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Relaxant Effects of YM178, Isoproterenol, and CGP-12177A in Rat Bladder Strips Precontracted with CCh. Both YM178 and isoproterenol concentration-dependently relaxed rat bladder smooth muscle strips precontracted with 10–6 M CCh with EC50 values of 5.1 and 1.4 µM, respectively (Table 2; Fig. 3). Compared by EC50 value, YM178 was approximately one third as potent as isoproterenol. The maximal relaxant effects of YM178 and isoproterenol were 94.0 ± 1.0 and 78.0 ± 1.5%, respectively, that of CCh, indicating that YM178 acts a full agonist in the rat bladder (Table 2). In contrast, CGP-12177A relaxed this contraction by only 19.4 ± 1.2% at the highest concentration of 10–4 M, so the EC50 value for this compound could not be determined (Table 2).
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Relaxant Effects of YM178, Isoproterenol, and CGP-12177A in Human Bladder Strips Precontracted with CCh. Both YM178 and isoproterenol concentration-dependently relaxed human bladder smooth muscle strips precontracted with 10–7 M CCh with EC50 values of 0.78 and 0.28 µM, respectively (Table 3; Fig. 4). The maximal relaxant effects of YM178 and isoproterenol were 89.4 ± 2.3 and 85.6 ± 2.7%, respectively (Table 3). In contrast, CGP-12177A relaxed this contraction by only 48.2 ± 7.2% at the highest concentration of 10–4 M, indicating an EC50 value for this agonist of 10–4 M or more (Table 3).
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Rhythmic Isovolumetric Reflex Bladder Contraction. YM178 produced a dose-dependent decrease in the frequency of rhythmic bladder contraction in anesthetized rats (Fig. 5A). In contrast, it did not decrease the amplitude of rhythmic bladder contraction at up to 3 mg/kg i.v. (Fig. 5B). On the contrary, oxybutynin significantly increased the frequency of rhythmic bladder contraction and decreased its amplitude at doses of 0.272 mg/kg i.v. or more (Fig. 5, A and B).
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Discussion |
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3-AR over 1-or 2-AR. The agonistic potency of YM178 for human 3-ARs was 20 and 200 times greater than that of BRL37344 and CL316,243, respectively. In addition, the intrinsic activity of YM178 for human 3-ARs was higher than that of BRL37344 and CL316,243. To date, 3-AR has been cloned in many species, including humans (Emorine et al., 1989), rats (Granneman et al., 1991), and mice (Nahmias et al., 1991), and subtle species-dependent differences in pharmacological response have been identified. In particular, BRL37344 and CL316,243 are less potent and efficacious in stimulating human receptors than rodent receptors (Liggett, 1992; Dolan et al., 1994). The EC50 values of BRL37344 and CL316,243 in stimulating human 3-ARs were larger, and less selectivity of BRL37344 for 3-ARs versus 2-ARs was demonstrated in the present study compared with previous studies (Dolan et al., 1994; Wilson et al., 1996). Given that EC50 value and I.A. can vary according to experimental conditions, such as receptor expression level (Wilson et al., 1996), this may have been due to the difference in receptor density in each cell type. Although we unfortunately did not measure the receptor expression levels of human 3-AR-expressing cells, we nevertheless considered it possible to use these cells to rank the order of potency of 3-AR agonists, and we obtained a ranking (EC50) of YM178 > isoproterenol > BRL37344 > CL 316,243. These data suggest that YM178 is different from earlier 3-AR agonists, namely, in having full and selective agonistic activity for human 3-ARs. In the present study using rat and human bladder muscle, YM178 showed a similar high potency and I.A. to isoproterenol. Although a difference in bladder muscle -AR subtype expression between humans and rats has been identified (Fujimura et al., 1999; Yamaguchi, 2002), YM178 showed full agonistic activity in bladder strips of both species. In contrast, CGP-12177A showed only a slight relaxing effect, even at the highest concentration, in both species. CGP-12177A is known to be a partial agonist for 3-ARs (Langin et al., 1991), and our present results are consistent with previous reports (Yamazaki et al., 1998; Igawa et al., 1999). In addition, the earlier 3-AR agonists BRL37344A and CL316,243 are reported to show strong relaxing effects (like isoproterenol) in rat bladder strips (Yamazaki et al., 1998; Longhurst and Levendusky, 1999), but not in human bladder strips (Igawa et al., 2001). We used a different concentration of CCh in rats and humans for precontraction of bladder strips, which may have contributed to the difference in EC50 values between them. Furthermore, there was a difference in EC50 values for YM178 between CHO cells expressing 3-ARs and in human bladder, with values for human bladder being higher. We consider that this is attributable to a difference in experimental conditions, as follows. First, receptor expression level in bladder tissue may differ from that in CHO cells expressing 3-ARs. Wilson et al. (1996) demonstrated differences in EC50 values and I.A. for isoproterenol, BRL37344, and CGP12177 in three different 3-ARs expression levels in CHO cells. Second, this discrepancy was caused by restricted drug diffusion into structured tissues, which hinders equilibrium conditions. Third, the EC50 value for CHO cells expressing human 3-ARs was calculated as cAMP accumulation, whereas that for human bladder was calculated as relaxant activity under carbachol-precontracted conditions. Longhurst and Levendusky (1999) reported that isoproterenol is approximately 100-fold less potent against carbachol- versus KCl-induced contraction of the rat bladder. Furthermore, Frazier et al. (2005) demonstrated that isoproterenol is about 10-fold less potent against KCl precontraction versus passive tension of the rat bladder. The difference in EC50 values for YM178 in CHO cells expressing 3-ARs and in human bladder is therefore considered to be mainly attributable to the difference in experimental conditions. Although we did not examine the effect of 3-AR antagonists in the present study, Igawa et al. (1999) has reported that the relaxant effect of isoproterenol in human bladder is mediated via 3-AR using a 3-AR antagonist. Furthermore, Nomiya and Yamaguchi (2003) demonstrated that 3-AR mRNA is expressed predominantly in human bladder. Moreover, we confirmed here that YM178 showed full agonistic activity for human 3-ARs but not for human 1- and 2-ARs, and that it had little affinity for any other receptors or channels at a concentration of 10–6 M. We therefore consider that the relaxant effect of YM178 in human bladder was mediated via 3-ARs.
We also compared the effects of YM178 and oxybutynin on rhythmic bladder contraction induced by saline bladder filling in anesthetized rats. The results showed that YM178 did not affect the amplitude of rhythmic bladder contractions at doses at which it reduced contraction frequency. Likewise, CL316,243 suppressed mechanically or chemically induced bladder overactivity and improved urine storage function without affecting voiding function (Woods et al., 2001; Kaidoh et al., 2002; Takeda et al., 2002). Together, these results suggest that the activation of 3-ARs increases bladder capacity without influencing bladder contraction or residual urine volume during the voiding phase in commonly used animal models of bladder overactivity. This characteristic distinguishes it from oxybutynin, an antimuscarinic agent that significantly decreases the amplitude of rhythmic bladder contraction caused by blockade of muscarinic M3 receptors in bladder smooth muscle.
It is well known that the mammalian bladder is under dual autonomic nervous system control. Specifically, sympathetic nerves play an important role in the urine storage phase. Norepinephrine induces bladder relaxation and improves compliance via -ARs. Given that these receptors play an important role in relaxation and improvement of compliance of the mammalian bladder, it has been suggested that the activation of bladder -ARs might be of therapeutic relevance to the treatment of OAB conditions (Yamaguchi, 2002). 3-AR is the main subtype in human bladder muscle (Fujimura et al., 1999; Yamaguchi, 2002), whereas in rats not only 3-ARs but also 2-ARs contribute to bladder relaxation (Yamazaki et al., 1998). To date, clinical treatment of OAB has involved the use of antimuscarinic agents, but disadvantages such as insufficient efficacy, antimuscarinic agent resistance in patients, and adverse events, including dysuria and dry mouth (Yarker et al., 1995), have lead to calls for the development of more potent and better tolerated drugs.
With regard to why 3-ARs do not affect voiding function, the following mechanism may be considered. Acetylcholine released from parasympathetic nerves during the voiding phase activates postjunctional muscarinic M2 receptors and inhibits adenylate cyclase activity mediated by -ARs. At the same time, acetylcholine also stimulates muscarinic M3 receptors and activates the phosphatidylinositol-Ca2+ recruitment system (Igawa, 2000). In rats, Hegde et al. (1997) demonstrated that muscarinic M2 receptors oppose -adrenoceptor mediated bladder relaxation both in vitro and in vivo. A recent report (Furuno et al., 2006) supports this premise, showing an enhanced effect of isoproterenol on bladder relaxation in M2 receptor knockout mice. In addition, such muscarinic M2 receptor-mediated inhibition of adenylate cyclase has also been demonstrated in cultured human bladder cells (Daniels et al., 1999). Thus, under the administration of YM178, the relaxation of bladder smooth muscle in the voiding phase may be canceled by muscarinic M2-receptor activation, and YM178 may therefore not affect muscarinic M3 receptor-mediated bladder contraction. This may in turn suggest that YM178 has little risk of causing the urinary retention noted with antimuscarinic agents.
In conclusion, our study shows that YM178 has good selectivity and agonist potency for human 3-ARs. YM178 does not directly inhibit voiding bladder contractions, and it may therefore represent a promising choice for the treatment of overactive bladder with or without lower urinary tract symptoms such as those seen with benign prostatic hypertrophy.
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Footnotes |
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ABBREVIATIONS: -AR, -adrenoceptor; OAB, overactive bladder; YM178, (R)-2-(2-aminothiazol-4-yl)-4'-{2-[(2-hydroxy-2-phenylethyl)amino]ethyl} acetanilide; CHO, Chinese hamster ovary; I.A., intrinsic activity; CCh, carbachol; CL, confidence limit; CGP-12177A, 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride; BRL37344, (±)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid sodium; CL316,243, disodium 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate.
Address correspondence to: Dr. Toshiyuki Takasu, Pharmacology Research Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585 Japan. E-mail: toshiyuki.takasu{at}jp.astellas.com
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