journal of pharmacology and experimental therapeutics...

JPET #145508 1

Comparison of receptor binding characteristics

of commonly used muscarinic antagonists in

human bladder detrusor and mucosa

Kylie J. Mansfield, Jonathan J Chandran, Kenneth J Vaux,

Richard J Millard, Arthur Christopoulos, Frederick J Mitchelson and

Elizabeth Burcher

Department of Pharmacology, School of Medical Sciences,

University of New South Wales, Sydney, NSW 2052, Australia (KJM, JJC, EB)

Graduate School of Medicine, University of Wollongong, Wollongong, NSW 2522, Australia

(KJM)

Sydney Adventist Hospital, Woollahra, NSW, 2076, Australia (KJV)

Department of Urology, Prince of Wales Hospital, Randwick, NSW 2031, Australia (RJM)

Drug Discovery Biology Laboratory, Dept. of Pharmacology, Monash University, Vic 3800,

Australia (AC)

Department of Pharmacology, University of Melbourne, Parkville, Vic 3010, Australia (FJM)

JPET Fast Forward. Published on November 24, 2008 as DOI:10.1124/jpet.108.145508

Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on November 24, 2008 as DOI: 10.1124/jpet.108.145508

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JPET #145508 2

Running Title Page

Running Title: What’s the target for drugs treating the overactive bladder?

Corresponding Author:

Dr Kylie Mansfield

Graduate School of Medicine

University of Wollongong, Wollongong, NSW 2522, Australia

Phone: +61 2 4221 5851

Fax: +61 2 4221 4341

Email: [email protected]

Number of text pages: 27

Number of tables: 4

Number of figures: 4

Number of references: 36

Number of words in abstract: 250

Number of words in introduction: 495

Number of words in Discussion: 1141

List of non-standard abbreviations:

[3H]QNB, [3H]quinuclidinyl benzilate; OAB, overactive bladder; CHO cells, Chinese hamster

ovary cells; DMEM, Dulbecco’s Modified Eagles Medium; ACh, acetylcholine; UK-148,993,

hydroxylated metabolite of darifenacin; SPM7605, active metabolite of fesoterodine and

tolterodine

Recommended section assignment: Gastrointestinal, Hepatic, Pulmonary and Renal


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Abstract

Recent studies have described muscarinic receptors on the mucosa as well as the detrusor of

the human urinary bladder. Muscarinic receptor antagonists are effective in the treatment of

overactive bladder (OAB), but their site(s) of action and actual therapeutic target are unclear.

Our aim was to compare, in human bladder mucosa and detrusor, the radioligand binding

characteristics of newer, clinically effective agents: darifenacin, its hydroxylated metabolite

UK-148,993, fesoterodine, solifenacin, tolterodine and trospium. Specimens were collected

from asymptomatic patients (aged 50-72years) undergoing open bladder surgery. Radioligand

binding studies with the muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) were

performed separately on detrusor and mucosal membranes. All antagonists displayed high

affinity when competing for [3H]QNB binding in both detrusor and mucosa. Inhibition

constants were also obtained for all antagonists against individual muscarinic receptor

subtypes expressed in CHO cells. Here, fesoterodine showed anomalous binding results,

suggesting that some conversion to its metabolite had occurred. Global nonlinear regression

analysis of bladder binding data with five antagonists demonstrated 82% low affinity sites in

mucosa and 78% low affinity sites in detrusor, probably representing M2/M4 receptors. There

was an excellent correlation (r2=0.99) of low affinity global estimates between detrusor and

mucosa, whereas the corresponding high affinity estimates (~20% of sites) were dissimilar. In

conclusion, commonly used and clinically effective muscarinic receptor antagonists bind to

receptors located on the bladder mucosa as well as the detrusor, providing support for the

hypothesis that muscarinic receptors in the mucosa may represent an important site of action

for these agents in OAB.


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Introduction

Overactive bladder (OAB) is a debilitating disorder where patients experience frequency and

urgency of micturition, with or without urgency incontinence. Of these, the symptom of

“urgency” (urgency due to fear of leakage) is acknowledged to be the most bothersome

(Brubaker, 2004). This condition is experienced by approximately 17% of both men and

women over the age of 40 years, and is more common with increasing age (Milson et al.,

2001, Stewart et al., 2003). Muscarinic receptor antagonists are widely used to treat OAB

(Abrams and Andersson, 2007, Andersson and Yoshida, 2003, Hegde, 2006), but their lack of

organ selectivity has resulted in side effects including dry mouth, constipation and blurred

vision, which often result in patients discontinuing therapy (Colli et al., 2007).

Traditionally, muscarinic receptor antagonists were thought to mediate their clinical

effects by blocking muscarinic receptors on the detrusor muscle, thus inhibiting bladder

contraction due to acetylcholine released from parasympathetic nerves. However, at

therapeutic doses, muscarinic antagonists do not appear to inhibit bladder contractility

(Andersson and Yoshida, 2003; Finney et al., 2006), and it is now considered that muscarinic

antagonists act mainly during bladder filling to increase bladder capacity and to decrease

urgency (Andersson and Yoshida, 2003). Clinical trials with some of the newer antagonists

have demonstrated a beneficial effect on urgency (Zinner et al., 2004, Wagg et al, 2006,

Freeman et al, 2003, Chapple et al, 2004, Millard and Halaska, 2006): this symptom is not

attributable to activation of muscarinic receptors on the detrusor. Furthermore, the efficacy of

intravesically applied antimuscarinic agents strengthens the case for a role for muscarinic

receptors associated with the bladder mucosa (Madersbacher and Jilg, 1991, Frohlich et al.,

1998, Walter et al., 1999, Enzelsberger et al., 1995, Frohlich et al., 1998).

The existence of muscarinic receptors on the human bladder mucosa is now well

established. Using molecular techniques, consistent expression of M1, M2, M3 and M5


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muscarinic receptors in the bladder mucosa (urothelium and lamina propria) was found

(Mansfield et al., 2005, Bschleipfer et al., 2007). Localization studies using

immunohistochemistry are limited, due to problems with muscarinic receptor antibodies.

However, recent studies in human bladder have reported receptor immunoreactivity on the

bladder urothelium: M2 and M3 (Tyagi et al., 2006; Mukerji et al., 2006) and M1-5:

(Bschleipfer et al., 2007). Muscarinic (M2 and M3) receptor immunoreactivity also occurs on

suburothelial myofibroblasts, and this was increased in patients with OAB (Mukerji et al.,

2006). These mucosal receptors may thus represent sites of action for the muscarinic receptor

antagonists used to treat this disorder.

The aim of the current study was to target the muscarinic receptor proteins in the human

bladder mucosa and the detrusor, to compare the binding characteristics of six newer,

clinically relevant muscarinic receptor antagonists. The agents examined were darifenacin and

its hydroxylated metabolite UK-148,993, fesoterodine, solifenacin, tolterodine and trospium.

Fesoterodine is a pro-drug in that it is rapidly metabolized by non-specific tissue and plasma

esterases to produce the more active metabolite SPM7605 (Ney et al., 2008).


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Methods

Patients and specimens. Collection of human bladder specimens was obtained with

informed consent with approval from the Human Research Ethics Committee of the

University of New South Wales (HREC 03175). Whole wall segments of macroscopically

normal bladder (body) were collected from 28 males and 5 females (age range 50 - 75 years,

median age 65 years). Patients were asymptomatic for OAB, and were undergoing open

bladder surgery (17 radical prostatectomy, 15 cystectomy and one ileal conduit).

Bladder segments were placed immediately into ice-cold Krebs-Henseleit solution

(composition in mM: NaCl 118, KCl 4.7, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5

and D-glucose 11.7), pre-gassed with carbogen (95% O2, 5% CO2). Specimens were

transported to the laboratory and dissected into detrusor muscle and mucosa (containing

urothelium and lamina propria), then stored at -70oC until used.

Radioligand binding studies. Radioligand binding with the muscarinic receptor ligand [3H]

QNB (specific activity; 37 Ci mmol-1; NEN, Boston, MA, USA) was performed as described

previously using membranes from the human detrusor muscle and mucosa (Mansfield et al.,

2005) and from CHO cells (Lanzafame et al., 2006).

Human bladder detrusor or mucosa tissue (500 mg) was finely minced in ice-cold sodium

phosphate buffer (10 ml, 50 mM Na2HPO4, pH 7.4) and homogenized then centrifuged at

1000g for 15 minutes. The pellet was discarded and the supernatant re-centrifuged at 40,000g

for 20 minutes. The final pellet was resuspended in 10 ml of 50 mM sodium phosphate

buffer, pH 7.4.

The muscarinic receptor antagonists used were darifenacin (darifenacin hydrobromide,

Pfizer, Sandwich, UK), UK-148,993 (Novartis, Cork, Ireland), solifenacin (solifenacin

succinate, Astellas, Osaka, Japan), tolterodine (tolterodine tartrate, Pfizer, Sandwich, UK),

trospium (trospium chloride, Dr. R. Pfleger GmbH, Bamberg, Germany) and fesoterodine


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(fesoterodine fumarate, Schwarz Pharma AG, Monheim, Germany). Increasing concentrations

(10-12 M - 10-4 M) were incubated with matched detrusor or mucosal membranes (2% wet

weight final tissue concentration) or CHO cell membranes (10 µg membrane protein) and 200

pM [3H]QNB in 50 mM sodium phosphate buffer (pH 7.4) at 37oC for 2 hours. Non-specific

binding was determined using 10 μM atropine.

Incubations were terminated by addition of 3 ml ice-cold 50 mM sodium phosphate buffer

(pH 7.4). Membranes were filtered onto GF/B filters (Whatman, Maidstone, UK) pre-soaked

in sodium phosphate buffer containing 0.5% polyethyleneimine (PEI) and 10 μM atropine,

and then placed into scintillation vials containing 2 mL scintillant (Beckman Ready Safe,

Fullerton, CA, USA). Vials were left overnight before measurement of radioactivity using

liquid scintillation spectrometry (TriCarb Model 1900TR, Packard, Meriden, CT, USA).

CHO cell culture and homogenate preparation. CHO-FlPin cells, stably transfected with

human M1 - M5 receptors, were generated as described previously (May et al., 2007). They

were grown and maintained in a humidified incubator (37oC, 5% CO2/ 95% air) in DMEM

containing 20 mM HEPES, 10% foetal bovine serum and antibiotics. Cells were passaged by

trypsinization. Cell cultures between passages 8 and 15 were used for membrane preparations.

CHO cells were grown until confluence in T75 flasks. Cells were mechanically removed from

the flask into ice-cold sodium phosphate buffer (10 mL, 50 mM Na2HPO4, pH 7.4),

homogenized and centrifuged as above. The final pellet was resuspended in 50 mM sodium

phosphate buffer and the protein concentration determined by the Lowry method, using

bovine serum albumen as standard. Membranes were stored at -80oC. Prior to use, membranes

were diluted to 100 µg protein mL-1.


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Data analysis. Competition binding data from the studies in CHO cell membranes

expressing M1 to M5 muscarinic receptors were analyzed using the non-linear regression

analysis program GraphPad Prism (version 5.0, GraphPad Software Inc., San Diego, CA,

USA) and tested to determine if a one-site or 2-site model was statistically preferred (F test, p

< 0.05). Inhibition constants (mean pKi values with S.E.M.) of competitors for [3H]QNB

binding sites were calculated according to the formula Ki = IC50 / (1+L/KD), where L is the

concentration of radioligand, KD is the dissociation constant of the radioligand and IC50

denotes the concentration producing 50% inhibition of specific binding by the competitor.

The KD of [3H]QNB for cloned M1 to M5 muscarinic receptors was determined in parallel

saturation studies carried out as previously described (Mansfield et al., 2005). Data from

saturation studies in CHO cell membranes were fitted via nonlinear regression to a one site

binding model using GraphPad Prism. Determination of whether the Hill slope (competition

binding studies) was significantly different to 1.0 was carried out using the formula t = (mean

Hill slope – 1) / S.E.M. The t value obtained was then compared to tabulated values with n-1

degrees of freedom and a significance level of p < 0.025.

Competition binding data in detrusor and mucosa membranes were also analyzed using

GraphPad Prism and tested to determine if a one-site or 2-site model was statistically

preferred (F test, p < 0.05). Inhibition constants (mean pKi values with S.E.M.) of competitors

for [3H]QNB binding sites were calculated as described above. The KD for [3H]-QNB for

detrusor and mucosa membranes was calculated based on the proportions of muscarinic

receptors previously determined in detrusor and mucosal membranes (Mansfield et al., 2005)

together with the KD determined for the individual muscarinic receptor subtypes in the CHO

cell membranes. The KD values used were 68.1 pM for detrusor and 66.9 pM for mucosa. Hill

slopes determined from this analysis were compared to a slope of 1.0 as described above.


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Our previous binding studies (Mansfield et al., 2005) had identified two major muscarinic

receptor subtype populations (a large population of M2 receptors and a lesser population M3

receptors) in both detrusor and mucosa membranes. Thus, competition binding data obtained

for darifenacin, UK-148,993, solifenacin, tolterodine and trospium were further analyzed

using global curve fitting (version 5.0, GraphPad Software Inc., San Diego, CA, USA).

Fesoterodine was excluded from the global fit analysis (see Results section). Global curve

fitting is a powerful approach that permits the sharing of model parameters across multiple

families of curves, and directly addressed the hypothesis that the observed binding profiles of

these antagonists were adequately explained by interaction with a common pool of two

receptor subtypes for all antagonists fitted to the model. This method thus allowed for the

determination of a single estimate for the proportion of high affinity sites, in each of the

tissues examined (see Results), that accommodated the binding data of all antagonists fitted to

the global model. Since trospium had the same Ki value at M2 and M3 receptors in the CHO

cell studies, it was set to have the same -log IC50 at each site. Inhibition constants (mean pKi

values with S.E.M.) for all five antagonists were then calculated, as above, using the –logEC50

values generated from the simultaneous fit to two sites.


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Results

Studies in CHO cell membranes. All antagonists showed high affinity competitive binding

in membranes from CHO cells expressing each of the five muscarinic receptor subtypes

(Table 1). For all agents except fesoterodine, the Hill slopes calculated from competition

binding to CHO cell membranes were not significantly different from unity. Trospium,

solifenacin and tolterodine were non-selective, with inhibition constants (Ki) differing by less

than one order of magnitude. As expected, darifenacin, its metabolite UK 148,993 and

solifenacin showed a degree of selectivity for M3 over M2 receptors (31-, 35- and 5-fold

respectively). It was notable that all antagonists showed high affinity for M4 and M5

receptors.

Studies in CHO cell membranes: fesoterodine. In contrast to the other agents, fesoterodine

appeared to bind to more than one site in CHO cell membranes. The competition binding data

for fesoterodine were further analyzed (Table 2). In membranes expressing M1 and M5

muscarinic receptors, the Hill slopes were significantly (p < 0.05) lower than 1. At M2 or M4

membranes, 2-site binding was preferred (p < 0.001).

One possible explanation for the anomalous binding results in the CHO cell membranes

may be the generation of the high affinity fesoterodine metabolite (SPM7605) during the

incubation. Fesoterodine 2-site binding data were compared with the data from Ney and

associates (2008), for fesoterodine and SPM7605 (Table 2). The pKi values for the “high

affinity site” in M1 to M5 CHO cell membranes appeared to correspond closely to the pKi

values reported for SPM7605 at M1 to M5 receptor subtypes, whereas the pKi values at the

“low affinity binding site” in M1 to M5 CHO cell corresponded closely to pKi values reported

for fesoterodine binding to M1 to M5 receptor subtypes (Table 2). Therefore our fesoterodine

“high affinity binding site” could represent binding by the metabolite (SPM7605), generated


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during the incubation period. We calculated that approximately 32% of the parent compound

is metabolized in 2 h, as this is the average fraction of “high affinity” binding sites determined

in CHO cell membranes (Table 2).

Studies in human bladder membranes. All antagonists displayed high affinity competition

for [3H]QNB in membranes from detrusor and mucosa (Fig. 1, Table 3). When the

competition binding curves in detrusor and mucosal membranes were compared, most agents

demonstrated indistinguishable binding curves.

Competition binding was initially analyzed for one site binding. In this analysis, the order

of potency in detrusor membranes was tolterodine ≥ trospium ≥ fesoterodine >> darifenacin ≥

UK-148,993 ≥ solifenacin. In mucosal membranes, the order of potency was trospium ≥

fesoterodine ≥ tolterodine >> darifenacin ≥ UK-148,993 ≥ solifenacin. There was an excellent

correlation for one site values from detrusor membranes with corresponding values from

mucosal membranes (Fig. 2A, r2 = 0.99).

In detrusor membranes, Hill slopes were significantly lower than unity for darifenacin,

UK-148,993, solifenacin and fesoterodine; this was also the case for darifenacin, solifenacin,

trospium and fesoterodine in mucosal membranes. Furthermore, comparison of one- and 2-

site binding indicated that 2-site binding was preferred for darifenacin and fesoterodine in

both detrusor and mucosa membrane preparations and for UK-148,993 in mucosal

membranes.

Global fit analysis of binding data in human bladder. In order to further investigate

multiple binding sites in human bladder, the data from binding of darifenacin, UK-148,993,

solifenacin, tolterodine and trospium to detrusor and mucosa membranes were analyzed using

a global fit (Fig. 3) Results of this analysis demonstrated 21.7 ± 1.5% high affinity sites in

detrusor membranes and 18.2 ± 2.4% high affinity sites in mucosal membranes.


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When mucosal and detrusor data for each agent were compared, there was an excellent

correlation for the low affinity values (Fig. 2B, r2 = 0.99) whereas that for the high affinity

values was not significant (Fig. 2C, r2 = 0.70, p = 0.078).

Correlations of human bladder data with CHO cell data. In table 4, pKi values from

one site binding analyses, and from global fit analyses (high and low affinity binding sites,

Table 3) were compared with pKi values determined in CHO cells (Table 1) for five

antagonists. Fesoterodine data are excluded.

The one site data and the low affinity components of the global fit analysis obtained from

human detrusor and mucosa membranes showed significant correlations with affinity of these

five antagonists in M2 and M4 CHO cell membranes (Table 4). Correlations of the high

affinity components with CHO cell data were not significant.

Two site analysis of binding in human bladder membranes: fesoterodine. Table 2 shows

the 2-site binding analysis for fesoterodine to detrusor and mucosal membranes. Our pKi

values at the low affinity binding sites in detrusor and mucosal membranes (7.68 and 7.87

respectively) correspond well with the pKi value reported by Ney et al (2008) for fesoterodine

at the M2 receptor (pKi 7.7). Furthermore, our pKi values for the high affinity binding site in

detrusor and mucosa membranes (8.78 and 9.95 respectively) correspond well with the pKi

value reported by Ney et al (2008) for SPM7605 binding to M2 receptors (pKi 9.2).


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Discussion

This study was designed to compare binding in detrusor and mucosa using clinically

relevant muscarinic receptor antagonists. The most important finding is that all antagonists

tested showed approximately equal binding affinity for muscarinic receptors in mucosal as

well as detrusor membranes. This confirms that muscarinic receptor proteins from both

regions are able to interact with these therapeutic agents. Data from global fit analysis

demonstrated the existence of 18% high / 82% low affinity sites in mucosal membranes and

22% high / 78% low affinity sites in detrusor membranes. Our study shows a highly

significant correlation between affinity for the low affinity sites in both detrusor and mucosa,

and affinity for M2 and M4 receptors expressed in CHO cell membranes. However, no

conclusion was possible regarding the nature of the receptor subtype(s) corresponding to the

minor high affinity sites. The lack of correlation with CHO data (Table 1) indicates that more

than one subtype may make up this minor site, probably M1 and M3. Moreover, the lack of

correlation between the high affinity binding parameters in detrusor and mucosa suggests that

these tissues express some different subtypes, of potentially important function.

We have previously explored the nature of the receptor subtypes corresponding to these

high and low affinity sites (Mansfield et al., 2005). In this earlier paper, we used several

selective muscarinic receptor antagonists, including methoctramine (M2) and pirenzepine

(M1). It was concluded that the muscarinic receptor subtypes present in the detrusor were 70%

M2, 20% M3 and 10% M1, whereas in mucosa, receptors were predominantly M2 with lower

expression of M3 / M5 (Mansfield et al., 2005).

The present study was not designed to delineate the contribution of all muscarinic

receptor subtypes present in the detrusor and mucosa. It is likely that the low affinity sites

represent M2 receptors. However it is tempting to speculate on a possible role of M4 receptors,

whose potential we discounted due to an apparent absence of M4 receptor mRNA from both


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human bladder regions (Mansfield et al., 2005). If M4 receptors were located on neurons, as

suggested previously (D’Agostino et al., 2000), M4 mRNA would only show low levels of

expression. In addition, M5 receptors cannot be excluded, particularly since all antagonists

used here showed high affinity for this subtype expressed in CHO cell membranes (Table 1).

Muscarinic M5 receptors in the bladder may play an important role that has not yet been

defined. The lack of selective antagonists for M4 and M5 receptors hamper current

investigations, and our findings may point the way for further studies when more selective

antagonists become available.

In this study, fesoterodine exhibited anomalous results. Fesoterodine is metabolized by

non-specific tissue and plasma esterases to produce SPM7605, which is also an active

metabolite of tolterodine, produced by a cytochrome P450 dependent enzyme pathway

(Michel and Hedge, 2006). In contrast to all other antagonists, fesoterodine binding in CHO

cell membranes was associated with low slope factors and binding to more than one site

(Table 2). A likely explanation is the generation of the high affinity fesoterodine metabolite

during the incubation with CHO cell membranes at 37oC. Support for this hypothesis can be

found in a recent publication showing that the binding affinity of SPM7605 is more than 10-

fold higher than that of fesoterodine (Ney et al., 2008); a study performed at a lower

temperature, possibly to reduce the breakdown of fesoterodine. Our results suggest that there

may be some conversion of fesoterodine to the metabolite during the incubation with both

CHO cell and bladder membrane preparations in the current study (Table 2). Thus, the

fesoterodine metabolite (SPM7605) appears to contribute to the apparent “high affinity

binding”, whereas the parent compound contributes to the apparent “low affinity binding” in

both CHO cell and tissue membranes.

The demonstration that these commonly used muscarinic antagonists bind to muscarinic

receptors in the mucosa adds further weight to the hypothesis that mucosal receptors may be

the real target of muscarinic antagonists in urge incontinence. Recent speculation has


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suggested that muscarinic antagonists act mainly during bladder filling, a role that may centre

on afferent functions (Andersson and Yoshida, 2003, Masuda et al., 2006), rather than

inhibiting parasympathetic nerve-mediated contractions of the detrusor.

Recent studies in rat bladder indicate that intravesical administration of carbachol can

induce detrusor overactivity, wheras intravesical instillation of muscarinic antagonists

(oxybutynin, trospium and tolterodine) has inhibitory effects on bladder contraction during

the storage phase (Kim et al., 2005). Furthermore, intravesical instillation of oxybutynin (De

Wachter and Wyndaele, 2003), tolterodine (Yokoyama et al, 2005) and darifenacin (Iijima et

al., 2007) reduces distension-evoked activity of C-fibre afferents in rat bladder, an effect also

seen following systemic administration of oxybutynin (De Laet et al, 2006).

In patients with overactive bladder, intravesical administration of muscarinic antagonists

such as oxybutynin and trospium is associated with reduced adverse affects (Madersbacher

and Jilg, 1991; Frohlich et al., 1998; Walter et al., 1999), whereas clinical efficacy remains

(Enzelsberger et al., 1995; Frohlich et al., 1998). Intravesical instillation of muscarinic

antagonists may be a useful approach in neurogenic patients, as these patients are already

undertaking clean intermittent self catheterization.

The mechanism of action of intravesically administered muscarinic antagonists is unclear.

Their exact cellular targets remain to be conclusively identified and could include urothelium,

nerves and/or myofibroblasts. Receptor immunoreactivity for all five muscarinic receptor

subtypes were demonstrated on the urothelium (Bschleipfer et al., 2007). In addition, M2 and

M3 receptor immunoreactivity was associated with suburothelial myofibroblasts (Mukerji et

al., 2006). Acetylcholine probably has several roles in the bladder mucosa, and it should be

noted that acetylcholine is found not only in neurons but also in other cell types including

urothelial cells (Yoshida et al., 2006). In vitro studies have demonstrated that the bladder

epithelial lining (the urothelium) responds to stretch, and agonist stimulation, by releasing

mediators including ATP (Ferguson et al., 1997; Fry et al., 2004) and acetylcholine itself


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(Yoshida et al., 2004; Hanna-Mitchell et al., 2007). These mediators may modulate afferent

activity through actions on suburothelial structures such as nerves and/or myofibroblasts (Fry

et al., 2004), or may induce further mediator release from the urothelium (Fig 4). We

hypothesize that the muscarinic antagonists used to treat bladder overactivity may be acting at

urothelial muscarinic receptors to block the action of non-neuronal acetylcholine on release of

other urothelial mediators such as ATP. This would decrease activation of suburothelial

afferent nerves, leading to a decrease in bladder sensation perceived at central micturition

centres, and therefore a decrease in parasympathetic nerve activity to the detrusor.

In conclusion, this appears to be the first demonstration that antimuscarinic agents

currently being used to treat patients with bladder overactivity bind with high affinity to the

muscarinic receptor proteins in the mucosa as well as the detrusor. This finding reinforces the

concept that antimuscarinic drugs interact with receptors in the human urothelium and/or

lamina propria as well as in detrusor.

Acknowledgements

We thank Dr G Testa and Dr S. Ehsman for kindly providing additional bladder specimens

and Emma Schofield and Neville McDonnell for assistance with tissue collection.


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Footnotes

a) This study was supported by the Faculty of Medicine, University of New South Wales and

Dr. R. Pfleger GmbH (Bamberg, Germany).

b) Please address reprint requests to:

Dr Kylie Mansfield

Graduate School of Medicine

University of Wollongong, Wollongong, NSW 2522, Australia

Phone: +61 2 4221 5851

Fax: +61 2 4221 4341

Email: [email protected]


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Legends for figures

Figure 1. Competition for specific binding of 200 pM [3H]QNB to human detrusor (�) and

mucosal (�) membranes by muscarinic receptor antagonists. A, darifenacin (n = 9-15); B,

tolterodine (n = 5); C, UK-148,993 (n = 5); D, fesoterodine (n = 6-8); E, solifenacin (n = 5); F,

trospium (n = 9-11). Values are mean ± S.E.M.

Figure 2. Linear regression analysis of binding data in detrusor and mucosa membranes.

Fesoterodine has been excluded. A, one site analysis of binding and B, low affinity binding

site from global fit show highly significant correlations (r2 = 0.99, p < 0.001 and r2 = 0.99, p <

0.001 respectively). C, high affinity binding site from global fit shows a trend but does not

show significant correlation (r2 = 0.70, p > 0.05).

Figure 3 Global fit analysis of competition binding to (A) detrusor and (B) mucosa

membranes with 200 pM [3H]QNB and muscarinic receptor antagonists. darifenacin (�);

tolterodine (�);UK-148,993 (�); solifenacin (�); trospium (�). Values are mean ± S.E.M.

Figure 4. Schematic diagram illustrating the potential roles of mucosal muscarinic receptors in

the regulation of bladder function. Stretch during bladder filling induces urothelial cells to

release acetylcholine (ACh), which in turn activates mucosal muscarinic receptors, leading to

release of ATP from the urothelium. ATP then excites purinergic P2X3 receptors on afferent

nerves, signalling bladder fullness. Other mechanisms and possible urothelial mediators have

been omitted for clarity.


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TABLE 1

Binding constants (pKD and pKi) for muscarinic receptor antagonist in membranes prepared

from CHO cells expressing human M1 to M5 receptors

M1 M2 M3 M4 M5

QNB (pKD) a 13.1 ± 0.74 13.2 ± 1.07 13.2 ± 0.46 13.5 ± 0.41 13.1 ± 0.97

Darifenacin b

(n = 3) 7.18 ± 0.59 6.94 ± 0.71 8.43 ± 0.68 7.48 ± 0.92 7.61 ±0.58

UK-148,993 b

(n = 3) 7.75 ± 0.59 7.10 ± 0.74 8.65 ± 0.69 7.44 ± 0.98 8.21 ± 0.61

Solifenacin b

(n = 5 - 6) 7.08 ± 0.55 7.03 ± 0.70 7.72 ± 0.63 7.73 ± 0.92 7.46 ± 0.57

Tolterodine b

(n = 3 - 5) 7.79 ± 0.62 7.99 ± 0.72 8.26 ± 0.67 8.58 ± 0.90 8.62 ± 0.57

Trospium b

(n = 3 - 5) 8.46 ± 0.59 8.94 ± 0.70 8.99 ± 0.67 8.84 ± 0.93 8.22 ± 0.58

Fesoterodine b

(n = 5 - 7) 7.8 ± 0.56 c 8.61 ± 0.77d 7.33 ± 0.67 7.98 ± 0.94 d 8.84 ± 0.57 c

a Data for pKD obtained from saturation studies (n= 4 to 5) for each cloned receptor subtype using

[3H]QNB

b Data represent mean pKi ± s.e. mean. Ki was calculated according to the formula Ki = IC50 /

(1+L/KD)

c Hill slope was significantly different from 1 (see these data in Table 2)

d 2-site binding analysis was preferred (see these data in Table 2)


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TABLE 2

pKi values (± S.E.M.) for “low” and “high” affinity sites based on 2-site binding analysis with fesoterodine compared with published pKi values for

fesoterodine and the fesoterodine metabolite SPM7605.

Fesoterodine SPM7605 a Fesoterodine a

n Hill slope % H pKi values

pKi pKi H L

Bladder detrusor b 8 0.64 ± 0.04 47 ± 24 8.78 ± 0.25 7.68 ± 0.30 N.D. N.D.

Bladder mucosa b 6 0.53 ± 0.03 27 ± 37 9.95 ± 0.39 7.87 ± 0.53 N.D. N.D.

M1 CHO cells b 5 0.69 ± 0.03 22 ± 4 9.32 ± 0.31 7.49 ±0.47 9.5 8.0

M2 CHO cells c 5 0.55 ± 0.07 30 ± 3 10.8 ± 0.37 8.01 ±0.53 9.2 7.7

M3 CHO cells 7 0.65 ± 0.05 42 ± 10 8.44 ± 0.30 6.92 ± 0.45 8.9 7.4

M4 CHO cells c 7 0.51 ± 0.03 21 ± 2 10.6 ± 0.67 7.50 ± 0.81 8.7 7.3

M5 CHO cells b 6 0.45 ± 0.02 45 ± 3 10.2 ± 0.40 7.77 ± 0.44 9.2 7.5

a pKi values published in Ney et al. (2008)

b Hill slope was significantly different from 1

c 2-site binding analysis was preferred

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

JPET

Fast Forward. Published on N

ovember 24, 2008 as D

OI: 10.1124/jpet.108.145508

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JPET #145508 26

TABLE 3

Radioligand binding characteristics of muscarinic antagonists in human bladder detrusor and

mucosa membranes determined from global fit analysis.

DETRUSOR One site analysis Global fit analysis

Drug n Hill slope pKi ± SEM a H pKi ± SEM a L pKi ± SEM a

Darifenacin 15 0.59 ± 0.04 b 7.14 ± 0.65 c 9.56 ± 0.72 6.94 ± 0.63

UK-148,993 5 0.71 ± 0.05 b 7.11 ± 0.65 9.20 ± 0.77 6.90 ± 0.64

Solifenacin 5 0.78 ± 0.04 b 6.99 ± 0.63 8.201 ± 0.78 6.81 ± 0.65

Tolterodine 5 0.99 ± 0.07 8.50 ± 0.61 9.46 ± 0.79 8.27 ± 0.65

Trospium d 11 0.78 ± 0.06 8.42 ± 0.64 8.37 ± 0.62

Fesoterodine e 8 0.64 ± 0.04 b 8.36 ± 0.55 c N.D. N.D.

MUCOSA One site analysis Global fit analysis

Drug n Hill slope pKi ± SEM a H pKi ± SEM a L pKi ± SEM a

Darifenacin 9 0.57 ± 0.05 b 6.98 ± 0.67 c 8.84 ± 0.80 6.78 ± 0.64

UK-148,993 5 0.90 ± 0.08 6.99 ± 0.65 c 9.16 ± 0.84 6.83 ± 0.65

Solifenacin 5 0.91 ± 0.07 b 6.90 ± 0.64 7.61 ± 0.87 6.78 ± 0.66

Tolterodine 5 0.87 ± 0.10 8.39 ± 0.66 9.51 ± 0.83 8.23 ± 0.65

Trospium d 9 0.87 ± 0.04 b 8.54 ± 0.63 8 53 ± 0.63

Fesoterodine e 6 0.53 ± 0.03 b 8.50 ± 0.55 c N.D. N.D.

a Inhibition constant shown as mean pKi ± S.E.M.

b The Hill slope was significantly different from unity (t-test, P < 0.05)

c 2-site analysis was preferred

d The non-selective agent trospium was set to have same IC50 for both sites in the one tissue in

the global fit analysis.

e Fesoterodine was excluded from the global fit analysis.


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TABLE 4

Linear correlation (r2) of pKi values determined from binding in detrusor and mucosa

membranes with pKi values from binding in CHO cell membranes.

DETRUSOR

M1 a M2

a M3 a M4

a M5 a

One site analysis b 0.601 0.809 * 0.196 0.911 * 0.616

H global fit analysis b 0.028 0.098 0.028 0.074 0.113

L global fit analysis b 0.648 0.868 * 0.227 0.940 ** 0.567

MUCOSA

One site analysis b 0.667 0.885 * 0.230 0.949 ** 0.565

H global fit analysis b 0.129 0.024 0.222 0.023 0.619

L global fit analysis b 0.697 0.921 ** 0.240 0.965 ** 0.528

a pKi values in CHO cells expressing M1 to M5 muscarinic receptors, see Table 1

b pKi values determined for one site analysis and high (H) and low (L) affinity values

determined from global fit analysis, see Table 3

* p < 0.05

** p < 0.01


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