Underactive bladder (UAB) is the clinical manifestation of detrusor underactivity (DU), which is defined as a contraction of reduced strength and/or duration resulting in prolonged bladder emptying and/or a failure to achieve complete bladder emptying within a normal time span. Available treatments for UAB include, Bethanechol, a non-selective muscarinic agonist, which mimics the action of acetylcholine (ACh) in bladder,  but the  effectiveness of the cholinomimetic, Bethanechol in UAB is compromised by its off-target side effects.  Approved gastroprokinetic agent, Acotiamide was recently shown to reduce the post-void residual urine volume (PVR) of detrusor underactivity (DU) patients. However, a direct excitatory effect of Acotiamide on rat and human bladder is not known.

Longitudinal, urothelium intact bladder strips were removed from euthanized male Sprague-Dawley rats (10-12 weeks old) and mounted in 37°C organ bath constantly gassed with 95% oxygen-5% carbon dioxide for isometric tension studies. Strips were stretched to 1 g of tension for eliciting spontaneous phasic contractions. Human bladder strips were procured from 2 female deceased organ donors in the age range 30-49 years through local tissue bank in full ethical compliance. Electrical field stimulation  (EFS: 5 ms pulses, 1-64Hz, 5s train at 10V)  evoked contractions sensitive to 1 μM tetrodotoxin (TTX), a neuronal Na+ channel blocker were considered nerve evoked (neurogenic) in rat and human bladder strips.   Frequency-response curves with the above stated EFS parameters with one stimulation at each frequency at 1-min intervals were constructed on equilibrated strips at baseline and after addition of Acotiamide 1-8µM in the bath. Considering that atropine sensitive (ACh mediated) EFS contractile response are dominant at EFS frequencies ≥ 10Hz, we also assessed the contractile responses to repeat 10Hz stimulation  every 60s with 5ms pulse trains for 5s at 10V until a stable plateau of 4-5 contractile responses was obtained before and after addition of Acotiamide.  EFS evoked contraction amplitude was normalized by the response to 120mM KCl.  Effect of Acotiamide on spontaneous  contractions was assessed on strips not exposed to EFS.

Addition of Acotiamide 2µM significantly enhanced the contraction amplitude evoked at 8-32Hz during frequency response curve by 20% compared to only 10% with Acotiamide 1µM (Two -way ANOVA followed Sidak’s multiple comparison; *p<0.01, panel A of attached figure) and with repeat stimulation at 10Hz (Paired Student’s t test. *p<0.05).   Acotiamide 2µM did not affect the carbachol response (panel C of attached figure), but higher Acotiamide concentrations enhanced the spontaneous contractility (panel B of attached figure) and suppressed carbachol response. Contractions of human bladder strips evoked at 16-32Hz during frequency response curve and with repeat stimulation at 10Hz were also significantly enhanced by Acotiamide 2µM.   α,βme-ATP (30 μM), an ATP analogue reduced Acotiamide mediated enhancement at 1-8Hz by 15-30% but only by 10% at 32Hz. Lack of any excitatory effect by Acotiamide in absence of EFS indicates prejunctional action for Acotiamide 2µM, which is abolished by 1 μM tetrodotoxin.

TTX sensitive contractions elicited by EFS is known to evoke a frequency dependent release of neurotransmitters from post-ganglionic nerves of mammalian bladder with dominant release of ATP at lower frequencies ≤ 10Hz and the ACh release being dominant at higher frequencies ≥ 10Hz.  Significant effect of Acotiamide 2µM in rat and human bladder was noted at EFS frequencies >8Hz, which suggests enhancement of the cholinergic neurotransmission in bladder because the enhancement was sensitive to both exposure  to TTX (1μM) and atropine (5μM).  TTX sensitivity for the excitatory effect of Acotiamide 2µM and absence of any effect on the rat and human bladder contractility in absence of EFS and on carbachol evoked contractile response, suggest modulation of prejunctional receptors and rules out any direct stimulation of post-junctional receptors.  The peak response measured at 32 Hz was only reduced by 10% by α,βme-ATP (30 μM), an ATP analogue, used to desensitize purinergic receptors.  Acotiamide possess binding affinity for all the muscarinic receptors except for M3 receptors with the reported IC50 of ~ 1.8µM and since dose regimen of Acotiamide used for DU in clinic typically generates plasma concentration in the range of 2µM, the findings described here are easily translatable to the bedside.

This is a first report demonstrating a direct excitatory effect of Acotiamide in neural modulation of rat and human bladder contractility. These findings from an ex vivo experimental setup are consistent with the reported decrease in PVR of DU patients and the resolution of urinary retention in female patients following thrice daily oral treatment with Acotiamide 100 mg for 2 weeks. Findings highlight the therapeutic potential of drugs lacking antimuscarinic action on M3 receptors  for UAB treatment.

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