Cell experiment:

CHO cells (105) are seeded in each well of a 24-well culture plate and subcultured. Three days later, the medium is exchanged with 250 μL/well Hanks' balanced salt solution containing 0.1 mM 3-isobutyl-1-methylxanthine, pH 7.4. The cells are incubated with each compound (isoproterenol, Mirabegron, BRL37344, and CL316,243 at final concentrations of 10-10 to 10-4 M) for 10 min at 37℃, after which incubation is stopped by the addition of 250 μL of 0.2 M HCl. cAMP concentration in the reaction mixture is measured by radioimmunoassay using an 125I-cAMP assay system using a gamma counter. Fifty microliters of reaction mixture is incubated with 50 μL of succinyl agent for 10 min at room temperature, after which the reaction is stopped by the addition of 400 μL of buffer solution. Fifty microliters of succinylated sample is incubated with 50 μL of 125I-cAMP and 50 μL of anti-cAMP antibody for 24 h at 4℃. At the end of the incubation period, 250 μL of charcoal suspension is added and centrifuged for 10 min at 2800g at 4℃. Two hundred and fifty microliters of supernatant is transferred into a tube and counted for 1 min using a gamma counter. The intrinsic activity (I.A.) relative to isoproterenol for each β-adrenoceptor agonist is calculated using the maximal response of each compound[1].

Animal experiment:

Rats[1] Male (350 to 400 g) and female (225 to 290 g) Wistar rats are used. The free-form doses of 0.03, 0.1, 0.3, 1 and 3 mg/kg for Mirabegron and 0.0272, 0.0907, 0.272, 0.907, and 2.72 mg/kg for oxybutynin are used in this study.

Mirabegron is a selective β3-adrenoceptor agonist with EC50 of 22.4 nM.

Mirabegron (YM178) increases cyclic AMP accumulation in Chinese hamster ovary (CHO) cells expressing human β3-adrenoceptor (AR). EC50 value is 22.4 nM. EC50 values of Mirabegron for human β1- and β2-ARs are 10,000 nM or more, respectively. EC50 of Mirabegron in rat bladder strips precontracted with 10-6 M Carbachol (CCh) is 5.1 μM, whereas that in human bladder strips precontracted with 10-7 M CCh is 0.78 μM. Mirabegron concentration-dependently increases the accumulation of cAMP in CHO cells expressing human β3-ARs, with an EC50 value and I.A. of 22.4 nM and 0.8, respectively. Mirabegron has little agonistic effect on β1- and β2-ARs. Compared by EC50 value, Mirabegron is approximately one third as potent as isoproterenol. The maximal relaxant effects of Mirabegron are 94±1%, that of CCh, indicating that Mirabegron acts a full agonist in the rat bladder. The maximal relaxant effects of Mirabegron is 89.4±2.3%[1].

Mirabegron (YM178) produces a dose-dependent decrease in the frequency of rhythmic bladder contraction in anesthetized rats. In contrast, Mirabegron does not decrease the amplitude of rhythmic bladder contraction at up to 3 mg/kg i.v.. On the contrary, Oxybutynin significantly increases the frequency of rhythmic bladder contraction and decreased its amplitude at doses of 0.272 mg/kg i.v. or more[1].

References:
[1]. Takasu T, et al. Effect of (R)-2-(2-aminothiazol-4-yl)-4'-{2-[(2-hydroxy-2-phenylethyl)amino]ethyl} acetanilide (YM178), a novel selective beta3-adrenoceptor agonist, on bladder function. J Pharmacol Exp Ther. 2007 May;321(2):642-7.