Hypothesis / aims of study
Overactive bladder (OAB) is a diagnosis which encompasses bladder hyperactivity and hypersensitivity and affects a large number of patients worldwide. The symptoms experienced by patients vary, especially when comparing OAB patients with an otherwise healthy bladder to those with concomitant inflammation. Further, it is well-known that the most common treatment against OAB, namely antimuscarinics, is less effective in patients with concomitant cystitis. Not long ago, mirabegron, a selective β3-adrenoceptor agonist, was approved for the treatment of OAB and studies have indicated that one benefit of mirabegron is its efficacy also during cystitis. While the most common treatment against OAB worldwide is still monotherapy, a combination therapy including an antimuscarinic and mirabegron has been suggested as the new golden standard treatment. While a direct effect on the detrusor is assumed, some reports on mirabegron have suggested that part of its relaxatory effect is exerted via release of nitric oxide (NO).
The aim of the current study was to compare how micturition patterns are affected by combination drug therapy against OAB in health and cystitis and if the efficacy of the drugs can be linked to altered release of NO. To the best of our knowledge this is the first study to perform such a comparison in wake animals.
Study design, materials and methods
A total of 32 adult male Sprague-Dawley rats were used in the current study, which followed national guidelines for the care and use of laboratory animals and was approved by the local ethics committee (permit #196/2013). Each rat was pre-treated twice daily with either saline (1 mL/kg s.c.) or a combination of the antimuscarinic drug tolterodine (0.05 mg/kg/day s.c.) and the β3-adrenoceptor agonist mirabegron (0.6 mg/kg/day s.c.) for a period of 10 days. Sixty hours prior to spending 16 h in a metabolism cage, the rats were treated with either cyclophosphamide (CYP; 100 mg/kg i.p.) to induce experimental cystitis or saline (4 mL/kg i.p.), serving as controls. This yielded four groups (saline pre-treated controls, drug pre-treated controls, saline pre-treated inflamed and drug pre-treated inflamed) with n = 8 in each group. During the time in the metabolism cage, for each rat, the micturition pattern was recorded and the total volume of urine was collected. The collected urine was rapidly frozen at -60 C and used for voltammetric measurement of nitric oxide content. After the metabolic cage experiment the animals were euthanized and their bladders were excised and fixed in 4% paraformaldehyde for future immunohistochemical analysis. All data analysis and statistical comparisons were performed using GraphPad Prism 8.01 (GraphPad Software Inc., San Diego, USA). Two-way ANOVA followed by Tukey’s correction for multiple comparisons was used to compare micturition parameters and data collected from the urine samples. Immunohistochemical analysis was performed qualitatively. All data are expressed as mean±SEM.
In control rats, the volume/micturition was not affected by the combination drug therapy (1.03±0.14 mL and 0.95±0.11 mL in saline pre-treated and drug pre-treated control rats, respectively). Likewise, no difference could be seen when comparing number of micturitions per hour (1.03±0.23 and 0.94±0.15 mict/h in saline pre-treated and drug pre-treated control rats, respectively; Fig 1). However, induction of cystitis with CYP caused a non-significant decrease in volume/micturition (from 1.03±0.23 to 0.74±0.06 mL) and a significant increase in the number of micturitions per hour (from 1.03±0.23 to 1.86±0.18 mict/h; p = 0.0097; Fig 1). When pre-treating the CYP-treated (inflamed) rats with the combination of tolterodine and mirabegron, the micturition pattern was normalized and no significant differences as compared to control rats were seen (1.21±0.12 mL vol/mict and 1.07±0.09 mict/h, respectively). Similarly, the micturition parameters in the inflamed rats which were pre-treated with the combination of drugs was significantly different as compared to saline pre-treated inflamed rats (p = 0.032 for vol/mict and p = 0.014 for mict/h, respectively).
There were large interindividual variations in water consumption within each group and CYP pre-treated rats tended to drink more than saline pre-treated rats. However no significant differences could be detected when comparing the groups (17±4, 14±4, 25±6 & 23±5 mL in saline pre-treated controls, drug pre-treated controls, saline pre-treated inflamed and drug pre-treated inflamed, respectively).
Release of NO, measured by voltammetry as amount of nitrite in urine, increased significantly upon treatment with the drug combination, both in controls and rats with CYP-induced cystitis (from 25.86±1.96 in saline pre-treated controls to 48.54±2.88 and 53.42±4.28 µM in drug pre-treated controls and drug pre-treated inflamed, respectively; p<0.01 in both comparisons; Fig 2). Further, induction of cystitis with CYP per se increased the release of NO (from 25.86±1.96 in saline pre-treated controls to 42.99±5.51 µM in saline pre-treated inflamed; p < 0.05; Fig 2).
Immunohistochemical analysis showed an up-regulation of β3-receptors in the saline pre-treated inflamed group. Drug treatment seemed to, if anything, decrease the expression but only in the detrusor, not in the urothelium. As a final point, the immunohistochemical analysis showed clear signs of inflammation in the CYP-treated groups, namely a thickening of the urothelium and less intact smooth muscle tissue.
Interpretation of results
Induction of cystitis with CYP alters the micturition pattern, causing a state of overactivity. This is in line with previous studies. The current data show that the altered micturition parameters were normalized by a combination therapy which is suggested as the golden standard drug therapy against OAB. The drug combination does not, however, affect healthy bladder parameters. This study does not take side effects into account, but clearly shows the beneficial effects of the combination therapy during cystitis.
It is evident that treatment with a combination of tolterodine and mirabegron increases the release of nitric oxide. This needs to be argued to be mainly caused by mirabegron, since muscarinic antagonism most likely inhibits release of nitric oxide, if anything. This is highly interesting in the context of understanding the mechanism of effect of mirabegron. However, it should be noted that inflammation per se increases the levels of NO and the level of NO is not significantly higher when comparing levels of NO during cystitis with and without drug pre-treatment. Tentatively, this is an indication of maximum NO synthase activity.
The immunohistochemical analysis demonstrates the methodological robustness since signs of inflammation are evident after CYP-treatment. Interestingly, there was an up-regulation of β3-adrenoceptors during cystitis. This could indicate the possibility of an increased, or at least maintained, efficacy of mirabegron during inflammation. However, the drug treatment per se caused a slight decrease in the expression of β3-adrenoceptors.