Comparative Evaluation of Central Muscarinic Receptor Binding Activity by Oxybutynin, Tolterodine and Darifenacin Used to Treat Overactive Bladder

Comparative Evaluation of Central Muscarinic Receptor Binding Activity by Oxybutynin, Tolterodine and Darifenacin Used to Treat Overactive Bladder Tomomi Oki, Aiko Kageyama, Yukiko Takagi, Shinya Uchida and Shizuo Yamada* From the Department of Pharmacokinetics and Pharmacodynamics and Center of Excellence Program in the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan

Purpose: We characterized muscarinic receptor binding in the mouse cerebral cortex after oral administration of anticholinergic agents used to treat overactive bladder. Materials and Methods: Muscarinic receptors in the mouse cerebral cortex and bladder after oral administration of anticholinergic agents were measured using [3H]N-methylscopolamine. Results: In vitro binding affinities of tolterodine and its metabolite 5-hydroxymethyl metabolite in the mouse cerebral cortex and bladder were considerably greater than those of oxybutynin and darifenacin. Also, muscarinic receptor binding affinity of oxybutynin and its metabolite N-desethyl-oxybutynin in the cerebral cortex compared with that in the bladder was 2 to 3 times higher, whereas that of tolterodine and 5-hydroxymethyl metabolite was approximately 2 times lower. Oral administration of oxybutynin (76.1 ␮mol/kg), tolterodine (6.31 ␮mol/kg) and darifenacin (59.1 ␮mol/kg) showed binding activity that was approximately equal to that of bladder muscarinic receptors. Oral administration of oxybutynin (76.1 ␮mol/kg) showed significant binding of cerebral cortical muscarinic receptors in mice, as indicated by about a 2-fold increase in Kd values for specific [3H]N-methylscopolamine binding 0.5 and 2 hours later. On the other hand, tolterodine and darifenacin given at oral doses that would exert a similar extent of bladder receptor binding activity as oxybutynin showed only a low level of binding activity of central muscarinic receptors in mice. Conclusions: Significant binding of brain muscarinic receptors in mice was observed by the oral administration of oxybutynin but not tolterodine and darifenacin. Key Words: bladder; mice; receptors, muscarinic; cholinergic antagonists; cerebral cortex

nitive effects than oxybutynin.6 –11 However, Diokno et al noted that the incidence in the appearance of CNS side effects was similar in a comparative clinical trial of extended release formulations of oxybutynin and tolterodine in 790 women with OAB.12 Thus, it is not clear whether there is a significant difference in CNS effects between oxybutynin and tolterodine at pharmacologically relevant oral doses for OAB. To clarify this issue brain muscarinic receptor occupancy after oral administration of anticholinergic agents should be directly compared but to our knowledge such studies have not yet been done. Our previous studies documented that the time dependent estimation of brain receptor occupancy after systemic administration of centrally acting drugs may be a powerful way to evaluate the permeability of these agents through the BBB.13 Therefore, we quantified the penetration of oxybutynin, tolterodine and darifenacin into brain tissues by comparing muscarinic receptor binding in the cerebral cortex and bladder from mice orally administered these agents. Mice are an appropriate model species because similar metabolic patterns have been observed in rodents and humans for oxybutynin and tolterodine.14,15

veractive bladder exerts a detrimental effect on physical functioning and psychological well-being as well as significantly decreasing quality of life. Several anticholinergic agents are currently available and developmental for the treatment of OAB, including oxybutynin,1 tolterodine2 and darifenacin.3 While anticholinergic agents have proven efficacy in patients with OAB, they are also associated with the troublesome anticholinergic side effects of dry mouth, constipation, somnolence and blurred vision.1 Agents that cross the BBB and bind to muscarinic receptors in the brain carry a risk of CNS dysfunction, including cognitive impairment. In particular in elderly patients CNS adverse effects are an important concern with anticholinergic therapy because of the increased BBB permeability associated with normal aging.4 In fact, case reports of CNS effects with oxybutynin have documented cognitive dysfunction and neuropsychiatric adverse reactions during clinical use for OAB.5 On the other hand, it was shown that tolterodine and darifenacin may be associated with fewer cog-

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Submitted for publication January 31, 2006. Supported by Grant-in-Aid for Scientific Research (C)2 15591703 from the Ministry of Education, Science, Sports and Culture of Japan. * Correspondence: Department of Pharmacokinetics and Pharmacodynamics and Center of Excellence Program in the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan (telephone: ⫹81-54264-5631; FAX: ⫹81-54-264-5635; e-mail: [email protected]).

0022-5347/07/1772-0766/0 THE JOURNAL OF UROLOGY® Copyright © 2007 by AMERICAN UROLOGICAL ASSOCIATION

MATERIALS AND METHODS Materials [3H]NMS (3.03 TBq/mmol) was obtained from PerkinElmer Life Sciences, Boston, Massachusetts. Oxybutynin hydro-

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Vol. 177, 766-770, February 2007 Printed in U.S.A. DOI:10.1016/j.juro.2006.09.079

CENTRAL MUSCARINIC RECEPTOR BINDING ACTIVITY AND OVERACTIVE BLADDER chloride and DEOB were provided by Meiji Milk Products, Odawara, Japan. Tolterodine tartrate and 5-HM were provided by Pharmacia, Kalamazoo, Michigan. Darifenacin hydrobromide was provided by Pfizer, Tokyo, Japan. Animals Male ddY strain mice (Japan SLC, Shizuoka, Japan) at ages 9 to 13 weeks were used in this study. They were housed with a 12-hour light-dark cycle and had free access to laboratory food and water. Administration of Anticholinergic Agents Mice were fasted for 16 hours and then orally administered oxybutynin (76.1 ␮mol/kg), tolterodine (6.31 and 21.0 ␮mol/ kg) or darifenacin (59.1 ␮mol/kg) dissolved in distilled water. The doses of tolterodine and darifenacin were based on the difference relative to the in vitro muscarinic receptor binding potency of oxybutynin (table 1). Control animals received vehicle alone. The study was done in accordance with the guidelines of the Experimental Animal Ethical Committee of the University of Shizuoka. Tissue Preparation and Muscarinic Receptor Binding Assay At 0.5 to 6 hours after drug administration the mice were exsanguinated by taking the blood from the descending aorta under light anesthesia. The tissues were then perfused with cold saline from the aorta. The bladder and cerebral cortex were removed and each tissue was homogenized in a Kinematica Polytron® homogenizer in 19 volumes of ice-cold 30 mM Na⫹/HEPES buffer (pH 7.5). The homogenates were centrifuged at 40,000 ⫻ gravity for 20 minutes. The resulting pellet was resuspended in buffer for the binding assay. All steps for the preparation were performed at 4C. The mouse tissue homogenates (70 to 370 ␮g protein) were incubated with different concentrations (0.03 to 1.0 nM) of [3H]NMS in 30 mM Na⫹/HEPES buffer. Incubation was done for 60 minutes at 25C. The reaction was terminated by rapid filtration using a cell harvester (Brandel, Gaithersburg, Maryland) through a Whatman® GF/B glass filter. The filters were then rinsed 3 times with 3 ml ice-cold buffer. Tissue bound radioactivity was extracted from the filters overnight in scintillation fluid and 0.3 gm 1,4-bis[2-(5-phenyloxazolyl)]benzene). Radioactivity was determined by a liquid scintillation counter. Specific [3H]NMS binding was determined experimentally from the difference between counts in the absence and presence of 1 ␮M atropine. All assays were done in duplicate.

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Data Analysis Kd and Bmax for [3H]NMS (0.03 to 1.0 nM) were estimated by Rosenthal analysis of the saturation data. The ability of muscarinic receptor antagonists to inhibit specific [3H]NMS binding (125 pM) was estimated from IC50 values and Ki was calculated using the equation, Ki ⫽ IC50/(1⫹L/Kd), where L represents the concentration of [3H]NMS. Statistical analysis of data was performed by 1-way ANOVA, followed by Dunnett’s test for multiple comparison. Statistical significance was considered at p ⬍0.05. RESULTS In Vitro Muscarinic Receptor Binding Each anticholinergic agent inhibited specific [3H]NMS binding in the mouse bladder and cerebral cortex in a concentration dependent manner (fig. 1). The Ki value for oxybutynin in the cerebral cortex was significantly lower than that in the bladder (1/2.6), while the Ki value for DEOB was not different between these tissues (table 1). On the other hand, Ki values for tolterodine and 5-HM in the cerebral cortex were approximately 2 times higher than those in the bladder. The Ki value for oxybutynin in the bladder was significantly higher than the values for DEOB, tolterodine and 5-HM (2.8, 12 and 18 times, respectively) and it was equivalent to the value for darifenacin. The Ki value for oxybutynin in the cerebral cortex was significantly higher than the values for tolterodine and 5-HM (2.1 and 3.4 times, respectively), and significantly lower than that for darifenacin (1/5.1). Thus, Ki value ratios in the cerebral cortex relative to the bladder were 0.39 for oxybutynin, 0.68 for DEOB, 2.13 for tolterodine, 1.99 for 5-HM and 1.31 for darifenacin (table 1). Effects of Oral Administration of Anticholinergic Agents At 0.5 and 2 hours after oral administration of oxybutynin (76.1 ␮mol/kg) there was a significant increase in Kd values for specific [3H]NMS binding in mouse bladder compared with the corresponding control value (54.7% and 40.6%, respectively, table 2). Similarly at 0.5 or 2 to 6 hours after oral administration of tolterodine (6.31 and 21.0 ␮mol/kg) there were significant and dose related increases in Kd values for specific [3H]NMS binding in the bladder. The increases at 2 and 6 hours were 21.4% and 37.0% (6.31 ␮mol/kg), and the increases at 0.5, 2 and 6 hours were 99.5%, 309% and 308% (21.0 ␮mol/kg), respectively. The maximal enhancement in Kd values by oxybutynin was seen

TABLE 1. Ki for in vitro inhibition by oxybutynin, DEOB, tolterodine, 5-HM and darifenacin of specific [3H]NMS binding in mouse bladder and cerebral cortex Mean ⫾ SE Ki (nM) Drugs

Bladder

Oxybutynin DEOB Tolterodine 5-HM Darifenacin

14.3 ⫾ 1.9 5.20 ⫾ 1.08 1.22 ⫾ 0.14 0.81 ⫾ 0.12 21.6 ⫾ 4.9

Values represent 3 to 5 mice.

p Value

p Value vs Oxybutynin

Cerebral Cortex

Vs Bladder

⬍0.05 ⬍0.01 ⬍0.01

5.55 ⫾ 0.57 3.25 ⫾ 0.21 2.60 ⫾ 0.25 1.61 ⫾ 0.13 28.4 ⫾ 1.5

⬍0.01 ⬍0.01 ⬍0.01

Vs Oxybutynin

⬍0.05 ⬍0.01 ⬍0.01

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CENTRAL MUSCARINIC RECEPTOR BINDING ACTIVITY AND OVERACTIVE BLADDER TABLE 2. Kd and Bmax for specific [3H]NMS binding in mouse bladder 0.5 to 6 hours after oral administration of anticholinergic agents Kd Drugs (hrs after administration) Control 76.1 ␮mol/kg 0.5 2 6 6.31 ␮mol/kg 0.5 2 6 21.0 ␮mol/kg 0.5 2 6 59.1 ␮mol/kg 0.5 2 6

Mean ⫾ SE pM (fold increase vs control) 192 ⫾ 5

Oxybutynin:

Tolterodine:

Tolterodine:

Darifenacin:

p Value vs Control

139 ⫾ 5

(1.0)

297 ⫾ 30 (1.55) 270 ⫾ 25 (1.41) 231 ⫾ 10

Mean ⫾ SE Bmax (fmol/ mg protein)

⬍0.01 ⬍0.01

112 ⫾ 12 125 ⫾ 17 124 ⫾ 17

200 ⫾ 16 231 ⫾ 23 (1.21) 263 ⫾ 22 (1.37)

⬍0.05 ⬍0.01

137 ⫾ 16 130 ⫾ 23 142 ⫾ 20

383 ⫾ 25 (1.99) 593 ⫾ 112 (3.09) 592 ⫾ 42 (3.08)

⬍0.01 ⬍0.01 ⬍0.01

124 ⫾ 15 137 ⫾ 9 132 ⫾ 4

313 ⫾ 29 (1.63) 321 ⫾ 15 (1.67) 338 ⫾ 46 (1.76)

⬍0.01 ⬍0.01 ⬍0.01

114 ⫾ 8 138 ⫾ 7 138 ⫾ 11

Values in 23 control mice, 4 with oxybutynin, 3 to 5 with tolterodine at 0.5, 2 and 6 hours (at 6.31 ␮mol/kg 4, 5 and 4, and at 21.0 ␮mol/kg 3, 4 and 3, respectively) and 4 with darifenacin.

ratios for [3H]NMS in the cerebral cortex relative to the bladder at 0.5 and 2 hours were 1.42 and 1.21 for oxybutynin, 0.89 and 1.18 for 6.31 ␮mol/kg tolterodine, 0.33 and 0.56 for 21.0 ␮mol/kg tolterodine, and 0.60 and 0.71 for darifenacin (fig. 2). FIG. 1. Competitive inhibition by oxybutynin, DEOB, tolterodine, 5-HM and darifenacin of specific [3H]NMS binding in homogenates of mouse bladder (A) and cerebral cortex (B). Ordinate represents specific [3H]NMS binding as percent of control value. Abscissa represents logarithmic molar concentration of oxybutynin (1 to 300 nM), DEOB (1 to 100 nM), tolterodine (0.3 to 30 nM), 5-HM (0.1 to 30 nM) and darifenacin (3 nM to 1 ␮M). Points represent mean ⫾ SE of 3 to 5 mice.

0.5 hours later, while that by tolterodine was seen 2 or 6 hours later. In addition, a significant (63% to 76%) increment of Kd values in the bladder was observed 0.5 to 6 hours after oral darifenacin (59.1 ␮mol/kg). On the other hand, each anticholinergic agent had little significant effect on Bmax values in the bladder. At 0.5 and 2 hours after oral administration of oxybutynin (76.1 ␮mol/kg) there was a significant (120% and 71.2%, respectively) increase in Kd values for specific [3H]NMS binding in mouse cerebral cortex compared with the corresponding control value (table 3). However, such significant increases in Kd values for cerebral [3H]NMS binding were not seen by oral administration of tolterodine (6.31 and 21.0 ␮mol/kg) and darifenacin (59.1 ␮mol/kg). In these anticholinergic agent treated mice Bmax values for cerebral [3H]NMS binding were little altered. Mean ⫾ SE nonspecific binding of [3H]NMS in mouse bladder and cerebral cortex tissues was extremely low (4.14% ⫾ 1.04% and 1.42% ⫾ 0.11% of total binding). It was little altered by oral administration of anticholinergic agents. Based on Kd increases for [3H]NMS binding the relative occupancy of muscarinic receptors in the cerebral cortex relative to the bladder was estimated. Average Kd increase

DISCUSSION The muscarinic receptor binding affinity of tolterodine and 5-HM in the mouse cerebral cortex and bladder was considerably greater than those of oxybutynin and darifenacin. Also, the receptor binding affinity of oxybutynin and DEOB in the cerebral cortex compared with the bladder was 2 to 3 times higher, whereas that of tolterodine and 5-HM was approximately 2 times lower. It is known that M1 muscarinic

TABLE 3. Kd and Bmax for specific [3H]NMS binding in mouse cerebral cortex 0.5 to 6 hours after oral administration of anticholinergic agents Drugs (hrs after administration) Control 76.1 ␮mol/kg 0.5 2 6 6.31 ␮mol/kg 0.5 2 6 21.0 ␮mol/kg 0.5 2 6 59.1 ␮mol/kg 0.5 2 6

Oxybutynin:

Tolterodine:

Tolterodine:

Darifenacin:

Mean ⫾ SE pM Kd (fold increase vs control)

Mean ⫾ SE Bmax (fmol/mg protein)

153 ⫾ 4 (1.0)

677 ⫾ 32

336 ⫾ 37 (2.20)* 262 ⫾ 27 (1.71)* 195 ⫾ 7

731 ⫾ 15 751 ⫾ 37 693 ⫾ 32

188 ⫾ 12 186 ⫾ 22 186 ⫾ 9

865 ⫾ 63 814 ⫾ 100 805 ⫾ 80

171 ⫾ 9 160 ⫾ 5 155 ⫾ 4

691 ⫾ 21 660 ⫾ 26 665 ⫾ 20

177 ⫾ 18 160 ⫾ 8 160 ⫾ 10

791 ⫾ 37 775 ⫾ 37 788 ⫾ 24

Values in 7 control mice, 3 with oxybutynin, 3 to 5 with tolterodine at 0.5, 2 and 6 hours (at 6.31 ␮mol/kg in 5 and at 21.0 ␮mol/kg in 3, respectively) and 3 with darifenacin. * Significantly different vs controls (p ⬍0.01).

CENTRAL MUSCARINIC RECEPTOR BINDING ACTIVITY AND OVERACTIVE BLADDER

FIG. 2. Ratios of Kd increases for specific [3H]NMS binding in mouse cerebral cortex relative to bladder after oral administration of oxybutynin, tolterodine and darifenacin. Ordinate represents ratio of Kd increases for specific [3H]NMS binding in mouse cerebral cortex relative to bladder 0.5 to 6 hours (h) after oral administration of oxybutynin (76.1 ␮mol/kg), tolterodine (6.31 and 21.0 ␮mol/kg) and darifenacin (59.1 ␮mol/kg). Columns represent mean ⫾ SE of 3 mice for oxybutynin, 3 or 5 for tolterodine (at 0.5, 2 and 6 hours 5 for 6.31 ␮mol/kg and 3 for 21.0 ␮mol/kg) and 3 for darifenacin.

receptor in the cerebral cortex may have an important role in the higher cognitive processes, such as learning and memory.16 Recently we also noted that binding affinities (Ki values) of oxybutynin, DEOB, tolterodine, 5-HM and darifenacin in human M1 recombinant muscarinic subtype were 8.70, 4.49, 1.92, 0.89 and 31.1 nM, respectively.17 These Ki values agree reasonably with the binding affinity of each agent in the cerebral cortical muscarinic receptors (table 1). After oral administration of oxybutynin, tolterodine and darifenacin there were dose and time dependent increases in Kd values for specific [3H]NMS binding in the mouse bladder compared with control values. Given that an increase in Kd values for radioligands in drug pretreated tissues in radio receptor assay generally refers to the competition between the agent and radioligand for the same binding sites,13 these data strongly suggest that orally administered anticholinergic agents undergo significant binding to muscarinic receptors in the mouse bladder. Oral administration of oxybutynin (76.1 ␮mol/kg), tolterodine (6.31 ␮mol/kg) and darifenacin (59.1 ␮mol/kg) showed approximately equal binding activity of bladder muscarinic receptors. This relative potency based on molar doses is in agreement with in vitro binding data (table 1). Oral administration of oxybutynin showed a significant occupancy of cerebral cortical muscarinic receptors in mice (table 3). The time course was similar to that of bladder receptor occupancy, in which maximal occupancy was observed at 0.5 hours after oral oxybutynin. Notably the binding activity of muscarinic receptors was detected only to a small extent in the cerebral cortex after oral administration of tolterodine and darifenacin. Such a difference between anticholinergic agents in the central muscarinic receptor occupancy was demonstrable based on the Kd ratio increase for [3H]NMS binding in the cerebral cortex relative to the bladder. That is, oral oxybutynin exerted relatively greater occupancy of muscarinic receptors in the cerebral cortex than in the bladder, whereas the opposite tissue selectivity was observed by oral tolterodine (21.0 ␮mol/kg) and darifenacin (fig. 2). Thus, to our knowledge we report for the first

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time that tolterodine and darifenacin given at oral doses that would exert a similar extent of bladder receptor binding activity as oxybutynin showed only a low level of binding activity of central muscarinic receptors in mice. The current findings in mice may represent the fundamental process for the apparent difference in the CNS effects of oxybutynin, tolterodine and darifenacin in clinical settings. Todorova et al comparatively evaluated the electrophysiological effects of anticholinergic agents on the CNS in healthy male volunteers using QEEG.6 They found that oxybutynin significantly altered QEEG activity, while tolterodine and trospium chloride induced only a slight effect on QEEG activity. Similar electrophysiological data with oxybutynin in healthy males were observed by Pietzko et al.18 Also, treatment with darifenacin had no effect on cognitive function in elderly volunteers10 and on QEEG in young volunteers.8 Anticholinergic agents that are orally administered must first cross the BBB to occupy central muscarinic receptors. It was reported that BBB permeability may increase with aging in humans.4 Passive penetration of anticholinergic agents through this physiological barrier is generally promoted by physicochemical factors such as high lipophilicity, a low degree of ionization (neutral charge) and small molecular size.9,19 The chemical properties of oxybutynin (lipophilicity, small size and neutral polarity) make it the most likely of anticholinergic agents to cross the BBB.19 Tolterodine has extremely lower lipophilicity (greater than 30 times),6,19,20 indicating a decreased likelihood that this agent would enter the CNS. In fact, Pahlman et al reported that the distribution of radioactivity in the brain was lowest in tissues from mice that received oral [14C]tolterodine.7 This is consistent with little binding activity of muscarinic receptors in the cerebral cortex of mice that received oral tolterodine (table 3). Darifenacin also has molecular characteristics that make it unlikely to cross the BBB.19 In addition, it is a substrate for P-glycoprotein, an active transporter system that can carry darifenacin back across the BBB.9,19 In addition to BBB permeability, muscarinic receptor subtype selectivity of anticholinergic agents may be also implicated in the appearance of CNS effects. The cognitive adverse effect of anticholinergic agents may be due to the antagonism of M1 and M2 receptors in the CNS.9 Oxybutynin shows high selectivity for M3 and M1 receptors, while tolterodine and 5-HM are nonselective with respect to 5 muscarinic subtypes.2,17 Darifenacin shows high selectivity of M3 receptor with negligible M1 receptor antagonism.11,17 Thus, due to M1 selectivity oxybutynin may be more apt to cause significant cognitive adverse effects compared with other agents.

CONCLUSIONS Significant binding of mouse brain muscarinic receptors was observed by oral administration of oxybutynin but not tolterodine and darifenacin. Thus, novel anticholinergic agents with a different propensity to cross the BBB may have a theoretical advantage over oxybutynin and they may be expected to have fewer CNS adverse effects when used as oral treatment for OAB.

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CENTRAL MUSCARINIC RECEPTOR BINDING ACTIVITY AND OVERACTIVE BLADDER 9.

Abbreviations and Acronyms BBB Bmax CNS DEOB 5-HM [3H]NMS

⫽ ⫽ ⫽ ⫽ ⫽ ⫽

Kd Ki OAB QEEG

⫽ ⫽ ⫽ ⫽

blood-brain barrier maximal number of binding sites central nervous system N-desethyl-oxybutynin 5-hydroxymethyl metabolite [N-methyl-3H]scopolamine methyl chloride apparent dissociation constant inhibition constant overactive bladder quantitative electrocephalography

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