No efficient pharmacological therapy is currently available to relieve the symptoms of men suffering from chronic pelvic pain syndrome (CPPS) [1]. Some studies have indicated that anti-inflammatory drugs can lead to symptom relief in men with CPPS.  Among the tested drugs, it was shown that the COX-2 inhibitor celecoxib had the highest penetration and retention in the prostate [2]. In recent years, drugs that affect soluble guanylate cyclase (sGC) have been suggested as possible treatment alternatives for various urological diseases. Most interest has been given to sGC activators, for instance BAY 60-2770, which can stimulate formation of cyclic guanosine monophosphate (cGMP) in the absence of nitric oxide (NO) [3]. The aim of the present study was to investigate the effects of BAY 60-2770, celecoxib and a combination of both on bladder function in a rat model of CPPS.

In the present study, 48 adult male Sprague-Dawley rats (300-450 g; Charles River Laboratories, Calco, Italy) were used. The animals were age matched and randomly divided into eight groups (n = 6 per group). In group 1-4, saline (10 µl) was injected into the dorsal lobe of the prostate, serving as control.  In group 5-8, rats were instead injected with zymosan (0.1 mg in 10 µl saline), to create a functional model for CPPS. Between day 8-20 post intraprostatic injection, the rats were given daily subcutaneous injections with either dimethylsulfoxide (DMSO; 0.05 ml/day; group 1 & 5), celecoxib (20 mg/kg/day; group 2 & 6), BAY 60-2770 (0.5 mg/kg/day; group 3 & 7) or a combination of celecoxib and BAY 60-2770 (group 4 & 8). On day 20, metabolic cage experiments were performed. The obtained parameters during the 16 h period were total number of micturitions, total urine volume and total water intake. From these parameters, micturition frequency and volume per micturition were calculated. 
Immediately following each metabolic cage experiment, on the 21st day post intraprostatic injection, cystometry was performed. Briefly, the femoral artery and vein were catheterized to allow for blood pressure monitoring and drug administration, respectively. Via a careful incision in the bladder dome, a pressure sensing catheter and cannula were placed in the urinary bladder and subsequently fixed with a ligature. Bladder pressure, residual urine volume, bladder capacity, compliance, voiding time and non-voiding contractions (NVCs) were measured during cystometry. Bladder compliance was calculated by dividing the volume change (ΔV) by the change in intravesical pressure (ΔP) that occurred during bladder filling (ΔV/ΔP). Saline was infused via the cannula to induce simulated micturition cycles. This was repeated three times before measuring cholinergic and purinergic contractile responses with methacholine (1, 2 and 5 µg/kg, i.v.) and ATP (5, 10 and 100 µg/kg, i.v.), respectively. 
Statistical calculations were performed using GraphPad Prism version 9.0.2 (GraphPad Software Inc., San Diego, USA). One-way or two-way ANOVA followed by Tukey`s correction for multiple comparisons was used for statistical comparisons and the data are presented as mean±SEM. Statistical significance was regarded for p-values < 0.05.

Induction of CPPS led to increased urinary frequency and, correspondingly, lower micturition volume. The symptoms of bladder overactivity were improved by treatment with celecoxib and normalized by treatment with BAY 60-2770 or a combination of celecoxib and BAY 60-2770 (Table 1). Cystometry revealed an increased number of non-voiding contractions (NVCs), lower bladder compliance and longer voiding time following induction of CPPS. A significant improvement in NVCs and compliance was observed after treatment with BAY 60-2770 and the combination therapy. However, voiding times were only improved by combination treatment. Induction of CPPS led to significantly reduced cholinergic and purinergic contractile responses. Treatment with BAY 60-2770 improved the cholinergic and purinergic responses, and they were completely normalized by combination therapy (Fig 1).

In the present in vivo study, the sGC activator BAY 60-2770, the COX-2 inhibitor celecoxib and a combination of both drugs were investigated as potential treatment alternatives for bladder dysfunction in a rat model of CPPS. Both afferent (NVCs, compliance) and efferent (voiding time, cholinergic and purinergic contractile responses) parameters were examined. Metabolic cage experiments confirmed experimentally induced bladder overactivity that could be interpreted as CPPS having a sensitizing effect on bladder afferent signalling. Cystometry revealed significant negative effects on both afferent and efferent signalling in the bladder after the induction of CPPS. In addition, induction of CPPS led to significantly reduced cholinergic and purinergic contractile responses. Taken together, these findings support the idea of prostate-to-bladder cross-organ sensitization leading to symptoms of bladder overactivity. 
It was hypothesized that changes in bladder function caused by CPPS as well as local changes in the prostate could be ameliorated by treatment with BAY 60-2770. In addition, celecoxib was tested since it (a) is the only current anti-inflammatory drug recommended for treatment of prostatitis and (b) has been shown to increase the bioavailability of NO. Significant improvement in micturition frequency, NVCs and bladder compliance showed that the induced changes in afferent bladder signalling could be normalized by BAY 60-2770 alone or in combination with celecoxib. These findings are in line with previous studies that concluded BAY 60-2770 to be an effective treatment alternative for bladder overactivity [3]. The suggested mechanism of action is direct activation of sGC, thereby increasing cGMP levels, which in turn leads to relaxation of bladder smooth muscle. However, the drug clearly has positive effects on afferent signalling as well.
Prostatitis is commonly associated with increased voiding time. Similarly, in the current study, induction of CPPS lead to increased voiding times. The observed increase was significantly improved by treatment with BAY 60-2770 alone or in combination with celecoxib. Tentatively, BAY 60-2770 has an inhibitory/relaxatory effect on prostate smooth muscle, leading to decreased voiding times. However, additional sites of action cannot be excluded. 
The reduced muscarinic and purinergic receptor-induced bladder contractility following induction of CPPS is interpreted as a demonstration of the effect of CPPS on efferent signalling and the contractile properties of the detrusor. Both the muscarinic and purinergic responses were improved by treatment with BAY 60-2770 alone and completely normalized by combination therapy. This further supports the beneficial effects of BAY 60-2770 alone or in combination with celecoxib on bladder dysfunction caused by CPPS. 
The current findings shed a light on the underlying mechanisms of cross-organ sensitization between the prostate and urinary bladder, suggesting that down-regulation or dysfunction of the NO-cGMP pathway plays a significant role in the negative effects on bladder function that are seen during CPPS.

The current animal model for induction of CPPS led to distinct signs of bladder overactivity. The sGC activator BAY 60-2770, alone or in combination with celecoxib, was shown to effectively normalize micturition frequency and restore bladder function. Clinical studies are required to investigate the efficacy and safety of BAY 60-2770, both alone and in combination with celecoxib, for treatment of male patients with CPPS.

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