Characterization of the muscarinic cholinoceptors in the human detrusor

Contractions of the human detrusor are thought to be mediated mainly via cholinergic muscarinic receptors. In the present study, we used a receptor-binding technique with 1-quinuclidinyl[phenyl 4-3H]benzilate ((-)3H-QNB) as radioligand to directly demonstrate the presence of muscarinic receptors in homogenates of the human detrusor. The binding of (-)3H-QNB was of high affinity (KD = (1.2 +/- 0.1) X 10(-10) M), saturable (Ro = 160 +/- 15 fmol./mg. protein) and possessed the pharmacological specificity expected of an interaction with muscarinic receptors. Muscarinic receptor antagonists were bound to a virtually uniform population of sites, whereas muscarinic receptor agonists recognized more than one population of muscarinic binding sites. The affinities of a series of antimuscarinic drugs, determined in competition experiments with (-)3H-QNB, were found to correlate with the capacity to inhibit carbachol-induced contractions in isolated human bladder muscle. Binding data together with the functional data indicated that the human detrusor does not contain any significant number of muscarinic spare receptors. The results suggest that a selective effect on the muscarinic receptors of human bladder is not possible to obtain with presently available antimuscarinic agents.     

Pharmacological characterization of a novel investigational antimuscarinic drug, fesoterodine, in vitro and in vivo

OBJECTIVE

To investigate the primary pharmacology of fesoterodine (a novel antimuscarinic drug developed for treating overactive bladder) and SPM 7605 (its active metabolite, considered to be the main pharmacologically active principle of fesoterodine in man) against human muscarinic receptor subtypes, and to investigate in vitro and in vivo functional activity of these agents on the rat bladder compared with existing standard agents.

MATERIALS AND METHODS

The displacement of radioligand binding by fesoterodine, SPM 7605 and standard agents in membrane preparations of Chinese hamster ovary (CHO) cells expressing the different human muscarinic receptors (M1–M5) was characterized. Agonistic and antagonistic activities were studied using different CHO cell lines stably expressing the human recombinant muscarinic receptor subtypes. The effects of fesoterodine and SPM 7605 on isolated bladder strips contracted by carbachol or electrical field stimulation (EFS) were investigated. In vivo the effects of fesoterodine and SPM 7605 on micturition variables were assessed using continuous cystometry in conscious female Sprague‐Dawley rats, and compared to those of oxybutynin and atropine.

RESULTS

In vitro SPM 7605 potently inhibited radioligand binding at all five human muscarinic receptor subtypes with equal affinity across all five. Fesoterodine had a similar balanced selectivity profile but was less potent than SPM 7605. Both substances were competitive antagonists of cholinergic agonist‐stimulated responses in human M1‐M5 cell lines and had a similar potency and selectivity profile to the radioligand‐binding studies. In rat bladder strips, fesoterodine and SPM 7605 caused a rightward shift of the concentration‐response curve for carbachol with no depression of the maximum, and concentration‐dependently reduced contractions induced by EFS. The potency of both drugs was similar to that of atropine and oxybutynin. In the presence of the esterase inhibitor neostigmine, the concentration‐response curve of fesoterodine was shifted to the right, suggesting that part of the activity was caused by metabolism to SPM 7605 by tissue enzymes. In vivo, low doses (0.01 mg/kg) of fesoterodine and SPM 7605 reduced micturition pressure and increased intercontraction intervals and bladder capacity, but did not affect residual volume.

CONCLUSIONS

Fesoterodine and its active metabolite, SPM 7605, are nonsubtype selective, competitive antagonists of human muscarinic receptors, but SPM 7605 has greater potency than the parent compound. Pharmacodynamic studies in the rat bladder in vitro confirm the competitive muscarinic antagonist profile of these agents in a native tissue preparation, and in vivo studies in the rat showed effects on bladder function consistent with a muscarinic antagonist profile.     

The effect of aging on cholinergic receptor binding in the rat urinary bladder

Binding sites with high affinity and specificity for [³H]quinuclidinyl benzilate (QNB), a cholinergic muscarinic receptor antagonist, were found in homogenates of rat urinary bladder. Atropine and scopolamine inhibited 50% of the specifically bound [³H]QNB (IC₅₀) at a concentration of approximately 2 nM, whereas the agonist oxotremorine had an IC₅₀ of 580 nM. By contrast, the IC₅₀ for the ganglionic nicotinic antagonist mecamylamine was greater than 100,000 nM. These values are similar to those reported for [³H]QNB binding sites in rat brain. [³H]QNB binding was characterized in rat bladder obtained from animals aged 6, 16, and 26 months. No differences in either the number of binding sites or the affinity constant for the radioligand were noted between these groups, suggesting that there are no agerelated alterations in muscarinic receptor recognition sites in this organ.     

Muscarinic receptor antagonists for overactive bladder

Overactive bladder (OAB) is a syndrome characterized by urinary urgency, with or without urgency urinary incontinence, usually with frequency and nocturia. OAB symptoms are often associated with detrusor overactivity (DO). Like OAB symptoms, the prevalence of DO increases with age and can have a neurogenic and/or myogenic aetiology. Bladder outlet obstruction can be a contributing factor in DO, possibly through cholinergic denervation of the detrusor and supersensitivity of muscarinic receptors to acetylcholine, although the prevalence of OAB is similar in men and women across age groups. Acetylcholine is the primary contractile neurotransmitter in the human detrusor, and antimuscarinics exert their effects on OAB/DO by inhibiting the binding of acetylcholine at muscarinic receptors M(2) and M(3) on detrusor smooth muscle cells and other structures within the bladder wall. Worldwide, there are six antimuscarinic drugs currently marketed for the treatment of OAB: oxybutynin, tolterodine, propiverine, trospium, darifenacin, and solifenacin. Each has demonstrated efficacy for the treatment of OAB symptoms, but their pharmacokinetic and adverse event profiles differ somewhat due to structural differences (tertiary vs quaternary amines), muscarinic receptor subtype selectivities, and organ selectivities. Antimuscarinics are generally well tolerated, even in special populations (e.g. men with bladder outlet obstruction, elderly patients, children). The most frequently reported adverse events in clinical studies of antimuscarinics are dry mouth, constipation, headache, and blurred vision; few patients withdraw from clinical trials because of adverse events. Development of an antimuscarinic with functional selectivity for the bladder would reduce the occurrence of antimuscarinic adverse events. The therapeutic potential of several other agents, such as alpha(3)-adrenoceptor agonists, purinergic receptor antagonists, phosphodiesterase inhibitors, neurokinin-1 receptor antagonists, opioids, and Rho-kinase inhibitors, is also under investigation for the treatment of OAB.     

Human muscarinic receptor binding characteristics of antimuscarinic agents to treat overactive bladder

PURPOSE: We characterized the binding affinities of several antimuscarinic agents in human muscarinic receptors. MATERIALS AND METHODS: Competitive inhibitory effects of antimuscarinic agents on specific NMS [H] (PerkinElmer Life Sciences, Boston, Massachusetts) binding were examined in human tissue homogenates and in CHO-K1 cell membranes expressing human muscarinic receptor subtypes. RESULTS: Oxybutynin, propiverine, tolterodine, the respective metabolites DEOB, DPr-P-4(N-->O) and 5-HM, and darifenacin inhibited in concentration dependent fashion specific [(3)H]NMS binding in homogenates of the human bladder and parotid gland as well as in membranes of CHO-K1 cell lines expressing human muscarinic M(1) to M(5) receptor subtypes. Based on inhibition constant values the inhibitory effects of tolterodine, 5-HM and DPr-P-4(N-->O) were 1.4 to 1.7 times greater in the bladder than in the parotid gland, whereas the inhibitory effects of oxybutynin, DEOB, propiverine and darifenacin were 2 to 10 times greater in the parotid gland. Consequently tolterodine, 5-HM and DPr-P-4(N-->O) compared with oxybutynin, DEOB, propiverine and darifenacin were found to show 3 to 4 times greater affinity to muscarinic receptors in the human bladder than in the parotid gland. Tolterodine and 5-HM were 2-fold more potent for inhibiting specific [(3)H]NMS binding at cell membranes expressing the M(2) vs the M(3) subtype. Conversely oxybutynin, DEOB, propiverine, DPr-P-4(N-->O) and darifenacin showed 2 to 22 times higher affinity to the M(3) than to the M(2) subtype. CONCLUSIONS: Compared with oxybutynin, tolterodine, 5-HM and DPr-P-4(N-->O) may bind more selectively to muscarinic receptors in the human bladder than in the parotid gland.     

Antimuscarinics for the treatment of overactive bladder: understanding the role of muscarinic subtype selectivity

Introduction and hypothesis

Antimuscarinic agents appear to exert their therapeutic activity in overactive bladder (OAB) via blockade of the M₃ muscarinic receptor subtype. Antimuscarinics are broadly similar in efficacy, but their safety and tolerability profiles vary, which may reflect differences in muscarinic receptor selectivity profiles.

Methods

This review of available literature aims to determine whether antimuscarinic agents with greater M₃ selectivity have clinical advantages over less selective drugs.

Results

Antimuscarinic agents differ widely in their propensity to cause cognitive and cardiovascular (CV) effects, which appear mainly to be related to differences in their relative selectivity for binding to non-M₃ receptors, including M₁ receptors in the brain and cardiac M₂ receptors.

Conclusions

Cognitive and CV effects are especially pertinent for the OAB patient who tends to be older with various comorbidities and is often taking multiple medications. Hence, it is important to consider the risk/benefit balance of antimuscarinic agents when selecting OAB treatment.     

Radioligand binding of 3H-ATP to membranes derived from rabbit urinary bladder: A preliminary report

Utilizing radioligand-filtration methodology, we have studied the binding characteristics of ³H-ATP to membrane preparations of rabbit urinary bladder. In order to avoid ATP hydrolysis, the binding assays had to be carried out at 0°C and utilize tissue concentrations between 1 and 3 mg/ml. Under these conditions, ³H-ATP binding to bladder homogenates was rapid, reaching equilibrium within 1 minute. The binding showed both divalent ion-specific (75% of total binding) and independent binding. The specific binding was saturable whereas the divalent ion-independent binding was not saturable at the concentrations studied.     

Action of pirenzepine on the human urinary bladder in vitro

The novel compound pirenzepine was tested for its antimuscarinic effect on the human urinary bladder "in vitro." Its behavior towards the contractions induced by acetylcholine or bethanechol and towards electrically induced contractions was identical to that of atropine. However, its potency was 100 to 300 times lower than that of atropine. Results obtained with ganglion blocking agents, tetrodotoxin and cooled preparations of urinary bladder seem to indicate the virtually total absence of ganglionic cells. On the other hand they point out the fundamental role of post-synaptic muscarinic M2 receptors as the most important component of the cholinergic system in the bladder. Of course the existence of other transmitters released at the cholinergic nerve endings after electrical field stimulation cannot be excluded on the basis of our experiments.     

The effect of tolterodine 4 and 8 mg on the heart rate variability in healthy subjects

Purpose  

To investigate the potential effect of tolterodine on the human heart rate variability (HRV). Oral antimuscarinic treatment for overactive bladder might significantly alter HRV, which is an important predictor for cardiac and all-cause mortality. Yet, little information exists regarding the influence of oral antimuscarinics on the HRV.     

Muscarinic cholinergic receptors in human gastric mucosa

Muscarinic cholinergic receptor sites in human gastric mucosa were analyzed directly by using radioligand binding techniques with the specific muscarinic antagonist³H-quinuclidinyl benzilate (QNB) as ligand. Specific binding of³H-QNB to membrane preparations from human gastric mucosa was saturable, of high affinity (Kd=4.17±1.94 nM, Bmax=0.37±0.04 pmol/mg protein) and selectively inhibited by muscarinic antagonists (atropine, scopolamine) and agonists (acetylcholine, pilocarpine). These findings provide direct evidence for the existence of muscarinic cholinergic receptors in human gastric mucosa. The specific³H-QNB binding to its receptor was blocked by atropine but not by histamine, cimetidine, pentagastrin, or synthetic human gastrin. The muscarine and histamine H₂-receptor, or muscarine and gastrin receptor, probably do not share the same locus.     

Case series data to encourage randomized trials of bladder retraining compared to antimuscarinic agents

PURPOSE: This study was designed to test the power potential of a number of different clinical trial designs that could be deployed to test the efficacy of an antimuscarinic drug against bladder retraining. MATERIALS AND METHODS: This was an observational cohort study. Data were collected prospectively from patients treated for an overactive bladder by antimuscarinic agents with bladder retraining, or by bladder retraining alone. At initiation and at followup data on frequency, incontinence, urgency and urge incontinence were collected. Data from visits up to 16 weeks of treatment were analyzed using the parametric methods.708 patients were studied, 44 males and 664 females, and their mean age was 54 (sd 22). 52 patients used pure bladder retraining and 656 used bladder retraining and an antimuscarinic agent. The drug was oxybutynin, tolterodine or imipramine combined with oxybutynin or tolterodine as combination therapy. RESULTS: A between groups analysis demonstrated that bladder retraining was associated with a greater improvement in urinary frequency compared to antimuscarinic therapy (Z = -4.6, 95% CI of difference -3.3, -1.4, p <0.001) whereas antimuscarinic therapy was associated with a greater improvement in incontinence compared to bladder retraining (Z = -2.6, 95% CI of difference -0.93, -0.27, p = 0.024). The within group change in incontinence episodes in the bladder retraining group did not appear to show an effect (95% CI of change -0.19, 0.43). A subgroup showing greatest DeltaInc was sought. Boxplots of DeltaInc against age group, sex and the grading of symptoms were examined for maximum effect. Female sex, age group of 50 or greater and patients describing urge incontinence demonstrated the greatest DeltaInc. Their mean daily frequency was 11.45 (sd 6.1) and incontinence 1.6 (sd 2.1). A sample with such characteristics would be most sensitive to treatment effect. The bladder retraining group had a higher daily frequency (Z = -3.2, p = 0.001, 95% CI for bladder retraining 10 to 11, 95% CI for antimuscarinic group 10 to 12) and a lower daily incontinence compared to the antimuscarinic group (Z = -3.4, p <0.001, 95% CI of median for bladder retraining 0.75, 0.85, 95% CI of median for antimuscarinic group 0.75, 1.75). CONCLUSIONS: Change in frequency is a poor outcome measure, DeltaInc is significantly superior. An antimuscarinic tested against bladder retraining, using DeltaInc for outcome, would probably compare favorably.     

Which muscarinic receptor is important in the bladder?

Antimuscarinic agents are the most widely used therapy for urge incontinence, but have side effects such as constipation, tachycardia and dry mouth, resulting from a lack of selectivity for the bladder. M₂ receptors are the predominant cholinoceptors present in urinary bladder, but mainly the minor population of M₃ receptors mediate its contraction. M₂ receptors modulate detrusor contraction by several mechanisms, and may contribute more to contraction of the bladder in pathological states such as bladder denervation or spinal cord injury. Prejunctional inhibitory M₂ or M₄ receptors and prejunctional facilitatory muscarinic M₁ receptors in the bladder have all been reported. In clinical studies, tolterodine, a non-selective muscarinic antagonist, has been reported to be as effective as oxybutynin but inducing less dry mouth. Thus, although it is not certain which antimuscarinic drugs have the better efficacy and tolerability, the non-selective antimuscarinic drugs seem to be better than M₃-selective antagonists in their clinical efficacies. However, controlled release, or intravesical, intravaginal, or rectal administrations of oxybutynin have been reported to cause fewer side effects. Darifenacin, a new M₃ selective antagonist, has been reported to have selectivity for the bladder over the salivary gland in vivo. To verify which antimuscarinic drugs selective for the muscarinic subtypes have the best efficacy and tolerability, comparative clinical trials between M₃ selective antagonists and non-selective compounds, such as olterodine, are required in the future.     

Comparative effects of five tricyclic compounds on the rabbit urinary bladder

The tricyclic antidepressant irniprarnine is widely utilized in treating a number of the disorders of micturition. Its effect on urinary tract smooth muscle is believed to occur through several mechanisms including direct antimuscarinic activity, direct smooth muscle relaxation, and indirectly by the inhibition of noradrenaline uptake into adrenergic neurons. The present study compared the potency of five tricyclic compounds on the contractile response of the rabbit urinary bladder to bethanechol. In addition, the direct antimuscarinic potency of these agents on radioligand binding to muscarinic receptors isolated from rabbit urinary bladder was determined. Of the five agents tested (imipramine, desrnethylirnipramine, amitriptyline, nortripty-line, and doxapine), doxapine was found to be significantly more potent than the other agents with respect to both antimuscarinic and musculotropic relaxant activity.     

Comparisons of the responses of anterior and posterior human adult male bladder neck smooth muscle to in vitro stimulation

OBJECTIVE

To evaluate differing methods of stimulation on strips of human bladder neck smooth muscle and compare muscle taken from the anterior and posterior aspects.

MATERIALS AND METHODS

Samples of adult human male bladder neck muscle were obtained from patients undergoing open radical prostatectomy. Muscle was taken from either the anterior or posterior (nine and six patients, respectively) aspects of the bladder neck. Muscle strips dissected from these samples were suspended in the Brading‐Sibley organ bath. The strips were superfused with 100 mm KCl‐enriched Krebs’ solution for 4 min to determine viability. This allowed experimentation on 17 strips from the anterior aspect of the bladder neck and 13 from the posterior bladder neck. These remaining strips were then superfused either with various concentrations (×10^?7 to ×10^?3m ) of carbachol or noradrenaline in Krebs’ solution, for 15 s. A further set of strips (eight from anterior, six from posterior) was suspended and responses to electrical field stimulation (EFS) with varying parameters were measured. Each EFS experiment was repeated after a 15 min exposure to 10^?3m atropine, and again after a 15 min exposure 10^?7m tetrodotoxin (TTX). Tension responses produced in these series of experiments were measured using strain gauges and analysed using data acquisition software. Student’s t‐test was used for the statistical analysis.

RESULTS

All muscle strips included in the study responded to EFS. The magnitude of this contraction is frequency dependent. The contractions were abolished by superfusion of the muscle strips with atropine. There was no further suppression of the contractile response on addition of TTX. Posterior bladder neck samples had a greater mean contractile response per unit mass than anterior strips at all frequencies of >1 Hz, and significantly more at 20 and 30 Hz. There was a concentration‐dependent response in bladder neck contraction to carbachol but only in the strips from the anterior bladder neck at concentrations of <10^?3m . Posterior bladder neck strips did not significantly contract upon application of carbachol. Similarly, there was a concentration‐dependent response to noradrenaline. Responses to noradrenaline were not uniform around the bladder neck, but not significantly different. Carbachol was the more ‘potent’ stimulator in anterior smooth muscle strips, but again the differences between agonists were not statistically significant.

CONCLUSION

These experiments show physiological variability around the circumference of the human male bladder neck. The posterior bladder neck shows significantly stronger contraction to α‐adrenergic agonists compared with cholinergic agonists; the anterior bladder neck does not have a similarly significant differential response. The uniform response to noradrenaline may underlie the bladder neck’s role in the prevention of retrograde ejaculation. The differential responses to carbachol may reflect differences in the embryological derivation of the anterior and posterior bladder neck fibres or in their innervation. Some of these differences may have clinical importance through the action of therapeutic agents.     

β3-Adrenoceptor agonist mirabegron is effective for overactive bladder that is unresponsive to antimuscarinic treatment or is related to benign prostatic hyperplasia in men

Purpose

To investigate the safety and efficacy of mirabegron for patients with overactive bladder (OAB) that is unresponsive to antimuscarinic agents or is related to benign prostatic hyperplasia (BPH).

Methods

Fifty-two newly diagnosed OAB patients (M group) and 45 patients with OAB that was unresponsive to antimuscarinics (S group) received mirabegron 50 mg once daily and were evaluated by OAB symptom score (OABSS), IPSS-QOL index, and IPSS at the time of baseline, 4 and 8 weeks. Newly diagnosed OAB patients treated with antimuscarinic agents were compared as controls.

Results

Mirabegron was effective for 85.2 % in M group. Significant improvements were seen in each domain of OABSS, and there was no significant difference with antimuscarinic therapy. Mirabegron was efficacious for 61.6 % of S group, and significant decreases of OABSS and IPSS-QOL index were observed. Significant improvements were also seen in voiding symptoms in men. Post-void residual urine volumes before and after treatment were 32.1 and 34.8 ml, and 26.2 and 31.3 ml in M and S group, respectively, and there was no significant difference. The incidence of adverse events was 8.4 %, although none were serious, and the patients recovered spontaneously after mirabegron was discontinued.

Conclusion

The present study suggests mirabegron is as effective as antimuscarinics for OAB. It improves OAB symptoms in patients with OAB for which antimuscarinic agents are insufficient. This study revealed that mirabegron improves not only OAB symptoms related to BPH, but also voiding symptoms in men. Low and mild incidences of side effects support the safe utility of mirabegron.     

Characterization of insulin-like growth factor I binding sites in human bladder cancer cell lines

Summary The role of insulin-like growth factor I (IGF-I) in the growth and development of bladder cancer cells was investigated using cultured human cell lines representing differentiated (RT-4, 5637) or undifferentiated (T-24, J-82, TCC-SUP) transitional cell carcinoma (TCC). In the presence of 2% serum, IGF-I significantly stimulated the growth of all cell lines. The proliferation of T-24, 5637, and RT-4 cells was more sensitive to IGF-I than that of J-82 and TCC-SUP cells. [¹²⁵I]IGF-I binding to 5637 and J-82 cells was significantly higher than that to T-24 and TCC-SUP cells (P<0.001). RT-4 cells possessed the lowest binding capacity among the cell lines tested. Scatchard analysis of [¹²⁵I]IGF-I binding to four of the five cell lines indicated a single binding site for IGF-I, with apparent dissociation constants (K _d) of 1.27, 1.18, 1.34, and 1.39 nmol/l for TCC-SUP, J-82, 5637, and T-24, respectively. Therefore, the difference observed in [¹²⁵I]IGF-I binding among the bladder cancer cell lines was attributed to the difference of IGF-I binding sites and not to a change in receptor binding affinity. Cross-linking studies supported the suggestion that [¹²⁵I]IGF-I was bound to a receptor on these cells. The results indicate that cultured human bladder cancer cells contain functional IGF-I receptors. A differentiated cell line, RT-4, possesses significantly fewer IGF-I receptors than other cell lines. This suggests that the overexpression of IGF-I receptor may reflect the malignant potential of bladder cancer cells.     

Is the beneficial effect of antimuscarinics related to motor or sensory changes in the bladder?

Introduction and hypothesis  

The aim of the study was to assess the sensory and motor effects of antimuscarinic treatment on the bladder in women with overactive bladder, detrusor overactivity demonstrated on urodynamics and a mean bladder wall thickness (BWT) greater than 5 mm.