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Noninvasive evaluation of brain muscarinic receptor occupancy of oxybutynin, darifenacin and imidafenacin in rats by positron emission tomography
Life Sciences 87 (2010) 175–180
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Noninvasive evaluation of brain muscarinic receptor occupancy of oxybutynin, darifenacin and imidafenacin in rats by positron emission tomography Akira Yoshida a, Shuji Maruyama a, Dai Fukumoto b, Hideo Tsukada b, Yoshihiko Ito a, Shizuo Yamada a,⁎ a Department of Pharmacokinetics and Pharmacodynamics and Global Center of Excellence (COE) Program, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan b Central Research Laboratory, Hamamatsu Photonics K. K., Hamamatsu, Shizuoka 434-8601, Japan
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Article history: Received 4 April 2010 Accepted 9 June 2010 Keywords: Overactive bladder Antimuscarinics Brain receptor occupancy Positron emission tomography Quantitative autoradiography
a b s t r a c t Aims: The current study was conducted to evaluate, by the noninvasive positron emission tomography (PET), the binding of antimuscarinic agents used to treat overactive bladder (OAB) to muscarinic receptors in rat brain. Main methods: Muscarinic receptor occupancy in the rat brain after the intravenous (i.v.) injection of oxybutynin, darifenacin and imidafenacin was evaluated by using a small animal PET system, and compared with the results by ex vivo autoradiographic and ex vivo radioligand binding experiments. Key findings: In PET study, the i.v. injection of oxybutynin but not darifenacin or imidafenacin at pharmacological doses decreased significantly binding potential (BP) of (+)N-[11C]methyl-3-piperidyl benzilate ([11C](+)3-MPB) in the rat cerebral cortex and corpus striatum in a dose-dependent manner. Similarly, in the in vivo autoradiographic experiment, oxybutynin dose-dependently reduced binding of [11C] (+)3-MPB in the brain, whereas darifenacin and imidafenacin did not. Following the i.v. injection of oxybutynin, darifenacin and imidafenacin, there was a similar degree of binding to muscarinic receptors in the bladder as demonstrated by a significant increase in apparent dissociation constant (Kd) values for specific [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) binding. Significant binding of muscarinic receptors in the brain was observed after the injection of oxybutynin but not darifenacin or imidafenacin. Significance: Oxybutynin but not darifenacin or imidafenacin has potential side effects on the central nervous system (CNS) in patients with OAB. The results reveal the noninvasive characterization of brain receptor occupancy by PET to be a powerful tool for precise evaluation of adverse CNS effects of antimuscarinic agents in pre-clinical and clinical evaluations. © 2010 Elsevier Inc. All rights reserved.
Introduction An overactive bladder (OAB) with symptoms of frequency, urgency and urge incontinence is very common in the geriatric population, a group rapidly increasing in number (Wein and Rovner 2002). Antimuscarinic agents are widely used for the treatment of OAB, because parasympathetic innervation is the predominant stimulus for bladder contraction (Anderson 1993). While antimuscarinic agents have proven effective in patients with OAB, they are also associated with anticholinergic side effects, namely, dry mouth, constipation and blurred vision (Yarker et al. 1995). Notably, agents that can cross the blood brain barrier (BBB) and bind to muscarinic receptors in the brain presented a risk of causing central nervous system (CNS) dysfunction including cognitive impairment. Such side
⁎ Corresponding author. Department of Pharmacokinetics and Pharmacodynamics and Global Center of Excellence (COE) Program, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. Tel.: +81 54 264 5631; fax: +81 54 264 5635. E-mail address: [email protected] (S. Yamada). 0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2010.06.008
effects are of great concern in elderly patients due to the increase in the BBB permeability with age (Pakulski et al. 2000; Ouslander 2004). The usefulness of in vivo receptor binding in predicting the potency, organ selectivity and duration of action of drugs in relation to their pharmacokinetic and pharmacodynamic profiles has been documented (Beauchamp et al. 1995; Yamada et al. 2003; Oki et al. 2007; Maruyama et al. 2008). Oki et al. (2007) have shown that an oral administration of oxybutynin but not darifenacin bound significantly to muscarinic receptors in the mouse brain. Also, Maruyama et al. (2008) have recently demonstrated the dosedependent occupancy of brain muscarinic receptors by oxybutynin using an in vivo autoradiographic analysis. These findings agree with clinical studies demonstrating that short-term and chronic administration of oxybutynin in elderly subjects resulted in mild cognitive dysfunction (Katz et al. 1998; Ancelin et al. 2006). Imidafenacin, a recently developed antimuscarinic agent, has been shown to act in the bladder without influencing the CNS (Kobayashi et al. 2007a,b). Furthermore, clinical observations have indicated a favorable efficacyto-side effect ratio of imidafenacin in patients with OAB (Homma and Yamaguchi 2008, 2009). However the uptake in the brain and the
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binding to muscarinic receptors in the CNS of this agent have not been examined. Positron emission tomography (PET) is a powerful noninvasive technique that can examine the uptake and receptor binding of drugs in the CNS. Moreover, the spatial resolution of PET devices used for humans and other primates is inadequate for small animals such as rodents. A higher resolution PET (microPET) system was applied for the in vivo imaging of neuronal activation and plasticity in the rodent brain by Kornblum et al. (2000). The current study was undertaken to characterize noninvasively muscarinic receptor occupancy in the rat brain after the i.v. injection of oxybutynin, darifenacin and imidafenacin using a small animal PET (ClairviovoPET, Shimadzu Corporation, Kyoto) (Mizuta et al. 2008). The results were also compared with those obtained by in vivo autoradiographic and ex vivo radioligand receptor binding experiments. Materials and methods Materials Positron-emitting carbon-11 (11C) was produced by a 14N (p, α) C nuclear reaction using the cyclotron (HM-18, Sumitomo Heavy Industry, Osaka, Japan) at Hamamatsu Photonics PET center and obtained as [11C]CO2. [11C](+)3-MPB (120.6 GBq/μmol) was labeled by N-methylation of nor-compound with [11C]methyl iodide as described previously (Tsukada et al. 2001). [N-methyl-3H]scopolamine methyl chloride ([3H]NMS, 3.03 TBq/mmol) was purchased from PerkinElmer Life Science, Inc. (Boston, MA). Oxybutynin hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO). Darifenacin hydrobromide and imidafenacin were donated from Pfizer Co., Ltd. (Tokyo, Japan) and Kyorin Pharmaceutical Co., Ltd. (Tokyo, Japan), respectively. All other chemicals were purchased from commercial sources.
Autoradiography An autoradiographic analysis of muscarinic receptors in the brain was performed using [11C](+)3-MPB as reported previously (Maruyama et al. 2008). The anesthetized rats received an i.v. injection of saline, oxybutynin, darifenacin or imidafenacin, and 10 min later, an i.v. injection of [11C](+)3-MPB (75–150 MBq). Then 30 min later, brain tissue was rapidly removed and cut into 2-mmthick coronal sections. An imaging plate exposed to the sections for 10 min was scanned with Fuji Bass system (FLA-7000, Fuji Film, Tokyo). A standard [11C](+)3-MPB solution of known concentration was placed on Advantec filter paper no. 2 (Toyo Roshi Kaisha, Ltd., Tokyo) and exposed simultaneously with the brain sections for the quantitative analysis. ROIs were placed in each area of the brain using a Macintosh computer (Image Reader version 1.2, Fuji Film). The results were described as a standardized uptake value (SUV), calculated from the number of photostimulated luminescence units per millimeter squared. The specific distribution of [11C](+)3-MPB into brain regions was defined as the ratio of SUV values between each region and the cerebellum, since the cerebellum has been shown to have few muscarinic receptor binding sites (Maruyama et al. 2008).
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Animals Male Sprague–Dawley rats at 8 weeks of age were purchased from SLC (Shizuoka). They were housed in the laboratory with free access to food and water, and maintained on a 12-h dark/light cycle in a room with controlled temperature (24 ± 1 °C) and humidity (55 ± 5%). This study was conducted in accordance with the guide for care and use of laboratory animals as adopted by the United States National Institutes of Health.
Tissue preparation and radioligand binding assay The radioligand binding assay for muscarinic receptor was performed using [3H]NMS (Ehlert and Tran 1990). At 40 min after the i.v. injection of saline, oxybutynin, darifenacin or imidafenacin, rats were anesthetized with ethylether and euthanized by taking the blood from the descending aorta, and then the bladder and brain were dissected. The tissues were homogenized by a Kinematica Polytron homogenizer in ice-cold 30 mM Na+/HEPES buffer (pH 7.5). The homogenates were centrifuged at 40,000 ×g for 20 min at 4 °C. The resulting pellet was finally resuspended in the same buffer. All steps were performed at 4 °C. The homogenates of bladder and brain were incubated with various concentrations of [3H]NMS (0.06 to 1.5 nM) in 30 mM Na+/HEPES buffer (pH 7.5) for 60 min at 25 °C. The reaction was terminated by rapid filtration (Cell harvester; Brandel Co, Gaithersburg, MD) through Whatman GF/B glass filters, and the filters were then rinsed three times with 3 mL of ice-cold buffer. The filter containing tissue-bound radioactivity was placed overnight in presence of the scintillation fluid then it was measured with a liquid scintillation counter. Specific binding of [3H]NMS was determined experimentally from the difference between counts in the absence and presence of 1 μM atropine.
PET study
Data analysis
An ultrahigh spatial resolution, small animal PET system (Clairvivo, Shimadzu Corporation, Kyoto) was used to image the rat brain. Each rat was initially anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in the scanner bed in the prone position. After transmission measurement with an external [137Cs]-point source (22 MBq) for attenuation correction, they received an i.v. injection of saline, oxybutynin, darifenacin or imidafenacin at a pharmacological dose. At 10 min after the injection of each agent, [11C](+)3-MPB (25 MBq) was injected intravenously. A PET scan was performed for 60 min. PET images were generated by the summation of image data from 40 to 60 min after the injection of [11C](+)3-MPB. Regions of interest (ROIs) were placed on the cerebral cortex, corpus striatum and cerebellum and time-activity curves in the ROIs were obtained. The time–activity curves in each ROI were fitted to a Simplified Reference Tissue Model using a pixel-wise kinetic modeling (PMOD) software (PMOD group, Zurich, Switzerland) (Innis et al. 2007), the reference region of which was the cerebellum, and the binding potential (BP) of [11C](+)3-MPB for muscarinic receptors in the cerebral cortex and corpus striatum was calculated.
The apparent dissociation constant (Kd) and maximal number of binding sites (Bmax) for specific [3H]NMS binding were estimated by a non-linear regression analysis of the saturation data. Results are expressed as the mean ± SE. Statistical analyses of the data were performed with Student's t test or William's test. Differences with P b 0.05 were considered statistically significant. Results PET study Fig. 1A shows typical PET images in the brain of anesthetized rats which were generated by summing the data from 40 to 60 min postinjection of [11C](+)3-MPB. The regional distribution of [11C](+)3MPB was greatest in the corpus striatum, intermediate in the cerebral cortex and lowest in the cerebellum. Autoradiographic images also illustrated a similar distribution of [11C](+)3-MPB (Fig. 3A). As shown in Fig. 1B, the accumulation of [11C](+)3-MPB in the cerebral cortex and corpus striatum was dose-dependently decreased by the i.v.
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Fig. 1. (A) Typical PET images fused with CT images in the brain of rats injected i.v. with [11C](+)3-MPB. The images were generated by summing the data 40–60 min after the [11C] (+)3-MPB injection. Each coronal section was different at 2.1 mm intervals. Upper left section: frontal lobe region, lower right section: cerebellum region. Third section in the upper panel was Bregma. (B) Effects of different doses of oxybutynin (0.1–1.0 mg/kg), darifenacin (0.1–1.0 mg/kg) and imidafenacin (0.01–0.1 mg/kg) on PET images of [11C](+)3-MPB in the rat brain. Rats received an i.v. injection of each agent 10 min prior to the [11C](+)3-MPB injection. Each section represents the typical one for Bregma – 2.1 mm region (fourth section in the upper panel (A)).
injection of oxybutynin (0.1–1.0 mg/kg), but in the cerebellum, a slight decrease in [11C](+)3-MPB accumulation was observed. In contrast, after the i.v. injection of darifenacin (0.1–1.0 mg/kg) and imidafenacin (0.01–0.1 mg/kg), there was little or no decrease in the accumulation of [11C](+)3-MPB in the brain.
A kinetic analysis of [11C](+)3-MPB using the Simplified Reference Tissue Model with cerebellum as a reference demonstrated a significant and dose-dependent decrease in the BP of [11C](+)3MPB in the cerebral cortex (43.7–68.2%) and corpus striatum (43.0– 71.0%) after the i.v. injection of oxybutynin (0.3, 1.0 mg/kg) (Fig. 2).
Fig. 2. Effects of the i.v. injection of oxybutynin (Oxy), darifenacin (Dar) and imidafenacin (Imi) on the binding potential (BP) of [11C](+)3-MPB in the cerebral cortex and corpus striatum of rats. The BP of [11C](+)3-MPB was estimated by a kinetic analysis using the Simplified Reference Tissue Model with the cerebellum as a reference region. Each column shows the mean ± SE (n = 3–4). Asterisks show a significant difference from the control (vehicle), *P b 0.05, **P b 0.01.
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On the other hand, the i.v. injection of darifenacin (0.1–1.0 mg/kg) and imidafenacin (0.01–0.1 mg/kg) did not affect the BP of [11C](+)3MPB in the rat cerebral cortex and corpus striatum. Autoradiography and radioligand binding By a quantitative autoradiographic analysis, we estimated the in vivo distribution of [11C](+)3-MPB in each brain region after the i.v. injection of antimuscarinic agents (Figs. 3B and 4). After the i.v. injection of oxybutynin (0.1–1.0 mg/kg), the distribution of [11C](+) 3-MPB was significantly decreased in the cerebral cortex (23.6– 47.1%), corpus striatum (17.0–46.7%), hippocampus (19.0–41.6%), amygdala (18.4–45.0%), thalamus (22.0–39.4%), hypothalamus (24.6– 38.3%) and pons (15.2–29.4%) in a dose-dependent manner. Darifenacin at 0.1 to 1.0 mg/kg exerted little effect on the distribution of [11C](+)3-MPB in each region, except for a slight but significant decrease (8.6–16.1%) in the thalamus, hypothalamus and pons at relatively high doses (Fig. 4). Similarly, the i.v. injection of imidafenacin (0.01–0.1 mg/kg) had no effect on the distribution of [11C](+)3-MPB in the brain. At 40 min after the i.v. injection of oxybutynin (1.0 mg/kg), darifenacin (1.0 mg/kg) and imidafenacin (0.1 mg/kg), there were significant increases (1.47, 1.49 and 1.58 times, respectively) in Kd values for specific [3H]NMS binding in the bladder (Table 1). A significant increase in the Kd value (1.82 time) in the brain was observed after the i.v. injection of oxybutynin but not darifenacin or imidafenacin. These antimuscarinic agents had no effect on Bmax values for specific [3H]NMS binding in either tissue.
Discussion The testing of antimuscarinic agents used to treat OAB for side effect on the CNS is important. The current study is the first to characterize noninvasively muscarinic receptor occupancy in the rat brain after the systemic injection of oxybutynin, darifenacin and imidafenacin by PET, comparing the results with those of in vivo autoradiographic and ex vivo radioreceptor assays. The i.v. doses of oxybutynin (0.1–1.0 mg/kg), darifenacin (0.1–1.0 mg/kg) and imidafenacin (0.01–0.1 mg/kg) used herein were pharmacologically relevant (Suzuki et al. 2007; Ohno et al. 2008). The plasma concentration of imidafenacin at these doses was similar to (0.01 mg/kg) or 10 fold higher (0.1 mg/kg) than that in patients receiving an oral dose of 0.1 mg of imidafenacin (Ohno et al. 2008). In the PET experiment in rats, the i.v. injection of oxybutynin but not darifenacin or imidafenacin at pharmacological doses decreased the BP of [11C](+)3-MPB in the cerebral cortex and corpus striatum in a dose-dependent manner. Similarly, in the autoradiographic analysis, oxybutynin dose-dependently decreased the binding of [11C](+)3MPB in each region of the brain, whereas darifenacin and imidafenacin had little effect. These results confirm our previous ex vivo binding and ex vivo autoradiographic data on muscarinic receptors (Oki et al. 2007; Maruyama et al. 2008), and seem to be responsible for clinical observations indicating that darifenacin and imidafenacin might have fewer CNS adverse effects than oxybutynin (Ancelin et al. 2006; Kay et al. 2006; Homma and Yamaguchi 2008, 2009). Following the i.v. injection of oxybutynin (1.0 mg/kg), darifenacin (1.0 mg/kg) and imidafenacin (0.1 mg/kg), there was a similar degree
Fig. 3. (A) Typical ex vivo autoradiographic images in the brain of rats injected i.v. with [11C](+)3-MPB. At 30 min, brain tissue was removed, cut into 2-mm-thick coronal sections, and prepared for autoradiography. (B) Effects of different doses of oxybutynin (0.1–1.0 mg/kg), darifenacin (0.1–1.0 mg/kg) and imidafenacin (0.01–0.1 mg/kg) on autoradiographic images of [11C](+)3-MPB in the rat brain. Rats received an i.v. injection of each agent 10 min prior to the [11C](+)3-MPB injection.
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Table 1 Kd and Bmax for specific [3H]NMS binding in the bladder and brain of rats after the i.v. injection of oxybutynin (1.0 mg/kg), darifenacin (1.0 mg/kg) and imidafenacin (0.1 mg/kg). Tissues
Drugs
Kd (nM)
Bladder
Vehicle Oxybutynin Darifenacin Imidafenacin Vehicle Oxybutynin Darifenacin Imidafenacin
332 ± 16 489 ± 19 496 ± 42 525 ± 51 258 ± 4 471 ± 13 249 ± 9 273 ± 9
Brain
Bmax (fmol/mg tissue) (1.47)* (1.49)* (1.58)* (1.82)*
174 ± 20 186 ± 13 177 ± 3 174 ± 17 1167 ± 26 1137 ± 44 1081 ± 48 1167 ± 38
The values in parentheses represent the ratio of Kd relative to the control (vehicle). Values are expressed as the mean ± SE. (n = 5–8). Asterisks show a significant difference, *P b 0.01.
Fig. 4. Effects of the i.v. injection of oxybutynin, darifenacin and imidafenacin on the distribution of [11C](+)3-MPB in the cerebral cortex, corpus striatum, hippocampus, amygdala, thalamus, hypothalamus and pons of rats. The distribution of [11C](+)3-MPB (Y-axis) was defined as the ratio of SUV values between each region and the cerebellum. Each column shows the mean ± SE (n = 3–12). Asterisks show a significant difference from the control (vehicle), *P b 0.05, **P b 0.01.
of increases in Kd values for specific [3H]NMS binding in the bladder (Table 1). With the view that an increase in Kd values for radioligand binding in agent-pretreated tissues in this type of assay is generally considered as competition between the agent and radioligand for the same binding sites (Yamada et al. 2003; Oki et al. 2007), these data indicate that antimuscarinic agents undergo significant binding to
muscarinic receptor of the bladder which is the target organ. The binding of muscarinic receptors in the brain was significantly different after the injection of oxybutynin but not darifenacin or imidafenacin. These findings, with the results of PET and autoradiography, confirm that oxybutynin orally administered at pharmacological doses in the bladder, binds significantly to muscarinic receptors in the brain, while darifenacin and imidafenacin exert little such effect. Generally, agents must first cross the BBB to distribute into the brain and to occupy the CNS receptors. The observed difference among these antimuscarinic agents in the degree of uptake and binding to muscarinic receptors in the brain may depend on their ability to permeate the BBB. The passive penetration of the BBB is dependent principally on physicochemical properties (Scheife and Takeda 2005), and thus its small size and molecular characteristics (lipophilicity, Log Ko/w: 4.68; neutral polarity, pKa: 6.44) make oxybutynin likely to cross the BBB. In contrast, both darifenacin and imidafenacin have moderate polarity and low lipophilicity, suggestive of lower permeability. In addition, darifenacin is considered as a substrate of P-glycoprotein, an active-transport system that carries this agent back across the BBB (Skerjanec 2006). The muscarinic receptor subtype selectivity of antimuscarinic agents may also be involved in the risk of side effects. Although all five muscarinic receptor subtypes are expressed in the CNS, the M1 receptor, in particular, is considered to play a crucial role in modulating cognitive function (Kay et al. 2005). Oxybutynin and imidafenacin were selective of both M3 and M1 receptors, while darifenacin was selective of M3 receptors in vitro (Maruyama et al. 2006; Kobayashi et al. 2007a). Thus the side effect of oxybutynin on the CNS during OAB treatment may be attributed to selectivity for the M1 receptor in addition to the ability to permeate the BBB. In contrast, no influence of imidafenacin on cognitive function was reported by Kobayashi et al. (2007b) possibly due to insignificant distribution of imidafenacin in the CNS. Because muscarinic receptors mediate the excitatory and inhibitory actions of acetylcholine in the central and peripheral nervous systems, the clinical use of antimuscarinic agents may bring on not only central but also peripheral adverse effects. In fact, dry mouth commonly occurs with the blockade of muscarinic receptors in the salivary gland and thereby decreases the quality of life of patients. Recently, pharmacological studies have shown that forebrain lesions or intraventricular injection of atropine reduced pilocarpine-induced salivation, indicating central mechanism of salivary secretion (Takakakura et al. 2003; Lopes de Almeida et al. 2006). In addition Borella et al. (2008) have demonstrated central M3 receptors to be involved in pilocalpine-induced salivation. Therefore, the extensive distribution of oxybutynin in the CNS might lead to peripheral adverse effects such as dry mouth. The current results are consistent with clinical findings that darifenacin and imidafenacin caused dry mouth less frequently than oxybutynin (Abrams and Andersson 2007; Homma et al. 2008; Homma and Yamaguchi 2009).
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This is the first PET-based study to demonstrate noninvasively that oxybutynin but not darifenacin or imidafenacin may have potential side effects on the CNS in patients with OAB. These results also reveal the in vivo characterization of brain receptor occupancy by PET to be a powerful tool for the precise evaluation of side effects of antimuscarinic agents in pre-clinical and clinical evaluations. Conflict of interest statement The authors state no conflict of interest.
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Noninvasive evaluation of brain muscarinic receptor occupancy of oxybutynin, darifenacin and imidafenacin in rats by positron emission tomography Akira Yoshida a, Shuji Maruyama a, Dai Fukumoto b, Hideo Tsukada b, Yoshihiko Ito a, Shizuo Yamada a,⁎ a Department of Pharmacokinetics and Pharmacodynamics and Global Center of Excellence (COE) Program, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan b Central Research Laboratory, Hamamatsu Photonics K. K., Hamamatsu, Shizuoka 434-8601, Japan
a r t i c l e
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Article history: Received 4 April 2010 Accepted 9 June 2010 Keywords: Overactive bladder Antimuscarinics Brain receptor occupancy Positron emission tomography Quantitative autoradiography
a b s t r a c t Aims: The current study was conducted to evaluate, by the noninvasive positron emission tomography (PET), the binding of antimuscarinic agents used to treat overactive bladder (OAB) to muscarinic receptors in rat brain. Main methods: Muscarinic receptor occupancy in the rat brain after the intravenous (i.v.) injection of oxybutynin, darifenacin and imidafenacin was evaluated by using a small animal PET system, and compared with the results by ex vivo autoradiographic and ex vivo radioligand binding experiments. Key findings: In PET study, the i.v. injection of oxybutynin but not darifenacin or imidafenacin at pharmacological doses decreased significantly binding potential (BP) of (+)N-[11C]methyl-3-piperidyl benzilate ([11C](+)3-MPB) in the rat cerebral cortex and corpus striatum in a dose-dependent manner. Similarly, in the in vivo autoradiographic experiment, oxybutynin dose-dependently reduced binding of [11C] (+)3-MPB in the brain, whereas darifenacin and imidafenacin did not. Following the i.v. injection of oxybutynin, darifenacin and imidafenacin, there was a similar degree of binding to muscarinic receptors in the bladder as demonstrated by a significant increase in apparent dissociation constant (Kd) values for specific [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) binding. Significant binding of muscarinic receptors in the brain was observed after the injection of oxybutynin but not darifenacin or imidafenacin. Significance: Oxybutynin but not darifenacin or imidafenacin has potential side effects on the central nervous system (CNS) in patients with OAB. The results reveal the noninvasive characterization of brain receptor occupancy by PET to be a powerful tool for precise evaluation of adverse CNS effects of antimuscarinic agents in pre-clinical and clinical evaluations. © 2010 Elsevier Inc. All rights reserved.
Introduction An overactive bladder (OAB) with symptoms of frequency, urgency and urge incontinence is very common in the geriatric population, a group rapidly increasing in number (Wein and Rovner 2002). Antimuscarinic agents are widely used for the treatment of OAB, because parasympathetic innervation is the predominant stimulus for bladder contraction (Anderson 1993). While antimuscarinic agents have proven effective in patients with OAB, they are also associated with anticholinergic side effects, namely, dry mouth, constipation and blurred vision (Yarker et al. 1995). Notably, agents that can cross the blood brain barrier (BBB) and bind to muscarinic receptors in the brain presented a risk of causing central nervous system (CNS) dysfunction including cognitive impairment. Such side
⁎ Corresponding author. Department of Pharmacokinetics and Pharmacodynamics and Global Center of Excellence (COE) Program, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. Tel.: +81 54 264 5631; fax: +81 54 264 5635. E-mail address: [email protected] (S. Yamada). 0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2010.06.008
effects are of great concern in elderly patients due to the increase in the BBB permeability with age (Pakulski et al. 2000; Ouslander 2004). The usefulness of in vivo receptor binding in predicting the potency, organ selectivity and duration of action of drugs in relation to their pharmacokinetic and pharmacodynamic profiles has been documented (Beauchamp et al. 1995; Yamada et al. 2003; Oki et al. 2007; Maruyama et al. 2008). Oki et al. (2007) have shown that an oral administration of oxybutynin but not darifenacin bound significantly to muscarinic receptors in the mouse brain. Also, Maruyama et al. (2008) have recently demonstrated the dosedependent occupancy of brain muscarinic receptors by oxybutynin using an in vivo autoradiographic analysis. These findings agree with clinical studies demonstrating that short-term and chronic administration of oxybutynin in elderly subjects resulted in mild cognitive dysfunction (Katz et al. 1998; Ancelin et al. 2006). Imidafenacin, a recently developed antimuscarinic agent, has been shown to act in the bladder without influencing the CNS (Kobayashi et al. 2007a,b). Furthermore, clinical observations have indicated a favorable efficacyto-side effect ratio of imidafenacin in patients with OAB (Homma and Yamaguchi 2008, 2009). However the uptake in the brain and the
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binding to muscarinic receptors in the CNS of this agent have not been examined. Positron emission tomography (PET) is a powerful noninvasive technique that can examine the uptake and receptor binding of drugs in the CNS. Moreover, the spatial resolution of PET devices used for humans and other primates is inadequate for small animals such as rodents. A higher resolution PET (microPET) system was applied for the in vivo imaging of neuronal activation and plasticity in the rodent brain by Kornblum et al. (2000). The current study was undertaken to characterize noninvasively muscarinic receptor occupancy in the rat brain after the i.v. injection of oxybutynin, darifenacin and imidafenacin using a small animal PET (ClairviovoPET, Shimadzu Corporation, Kyoto) (Mizuta et al. 2008). The results were also compared with those obtained by in vivo autoradiographic and ex vivo radioligand receptor binding experiments. Materials and methods Materials Positron-emitting carbon-11 (11C) was produced by a 14N (p, α) C nuclear reaction using the cyclotron (HM-18, Sumitomo Heavy Industry, Osaka, Japan) at Hamamatsu Photonics PET center and obtained as [11C]CO2. [11C](+)3-MPB (120.6 GBq/μmol) was labeled by N-methylation of nor-compound with [11C]methyl iodide as described previously (Tsukada et al. 2001). [N-methyl-3H]scopolamine methyl chloride ([3H]NMS, 3.03 TBq/mmol) was purchased from PerkinElmer Life Science, Inc. (Boston, MA). Oxybutynin hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO). Darifenacin hydrobromide and imidafenacin were donated from Pfizer Co., Ltd. (Tokyo, Japan) and Kyorin Pharmaceutical Co., Ltd. (Tokyo, Japan), respectively. All other chemicals were purchased from commercial sources.
Autoradiography An autoradiographic analysis of muscarinic receptors in the brain was performed using [11C](+)3-MPB as reported previously (Maruyama et al. 2008). The anesthetized rats received an i.v. injection of saline, oxybutynin, darifenacin or imidafenacin, and 10 min later, an i.v. injection of [11C](+)3-MPB (75–150 MBq). Then 30 min later, brain tissue was rapidly removed and cut into 2-mmthick coronal sections. An imaging plate exposed to the sections for 10 min was scanned with Fuji Bass system (FLA-7000, Fuji Film, Tokyo). A standard [11C](+)3-MPB solution of known concentration was placed on Advantec filter paper no. 2 (Toyo Roshi Kaisha, Ltd., Tokyo) and exposed simultaneously with the brain sections for the quantitative analysis. ROIs were placed in each area of the brain using a Macintosh computer (Image Reader version 1.2, Fuji Film). The results were described as a standardized uptake value (SUV), calculated from the number of photostimulated luminescence units per millimeter squared. The specific distribution of [11C](+)3-MPB into brain regions was defined as the ratio of SUV values between each region and the cerebellum, since the cerebellum has been shown to have few muscarinic receptor binding sites (Maruyama et al. 2008).
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Animals Male Sprague–Dawley rats at 8 weeks of age were purchased from SLC (Shizuoka). They were housed in the laboratory with free access to food and water, and maintained on a 12-h dark/light cycle in a room with controlled temperature (24 ± 1 °C) and humidity (55 ± 5%). This study was conducted in accordance with the guide for care and use of laboratory animals as adopted by the United States National Institutes of Health.
Tissue preparation and radioligand binding assay The radioligand binding assay for muscarinic receptor was performed using [3H]NMS (Ehlert and Tran 1990). At 40 min after the i.v. injection of saline, oxybutynin, darifenacin or imidafenacin, rats were anesthetized with ethylether and euthanized by taking the blood from the descending aorta, and then the bladder and brain were dissected. The tissues were homogenized by a Kinematica Polytron homogenizer in ice-cold 30 mM Na+/HEPES buffer (pH 7.5). The homogenates were centrifuged at 40,000 ×g for 20 min at 4 °C. The resulting pellet was finally resuspended in the same buffer. All steps were performed at 4 °C. The homogenates of bladder and brain were incubated with various concentrations of [3H]NMS (0.06 to 1.5 nM) in 30 mM Na+/HEPES buffer (pH 7.5) for 60 min at 25 °C. The reaction was terminated by rapid filtration (Cell harvester; Brandel Co, Gaithersburg, MD) through Whatman GF/B glass filters, and the filters were then rinsed three times with 3 mL of ice-cold buffer. The filter containing tissue-bound radioactivity was placed overnight in presence of the scintillation fluid then it was measured with a liquid scintillation counter. Specific binding of [3H]NMS was determined experimentally from the difference between counts in the absence and presence of 1 μM atropine.
PET study
Data analysis
An ultrahigh spatial resolution, small animal PET system (Clairvivo, Shimadzu Corporation, Kyoto) was used to image the rat brain. Each rat was initially anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in the scanner bed in the prone position. After transmission measurement with an external [137Cs]-point source (22 MBq) for attenuation correction, they received an i.v. injection of saline, oxybutynin, darifenacin or imidafenacin at a pharmacological dose. At 10 min after the injection of each agent, [11C](+)3-MPB (25 MBq) was injected intravenously. A PET scan was performed for 60 min. PET images were generated by the summation of image data from 40 to 60 min after the injection of [11C](+)3-MPB. Regions of interest (ROIs) were placed on the cerebral cortex, corpus striatum and cerebellum and time-activity curves in the ROIs were obtained. The time–activity curves in each ROI were fitted to a Simplified Reference Tissue Model using a pixel-wise kinetic modeling (PMOD) software (PMOD group, Zurich, Switzerland) (Innis et al. 2007), the reference region of which was the cerebellum, and the binding potential (BP) of [11C](+)3-MPB for muscarinic receptors in the cerebral cortex and corpus striatum was calculated.
The apparent dissociation constant (Kd) and maximal number of binding sites (Bmax) for specific [3H]NMS binding were estimated by a non-linear regression analysis of the saturation data. Results are expressed as the mean ± SE. Statistical analyses of the data were performed with Student's t test or William's test. Differences with P b 0.05 were considered statistically significant. Results PET study Fig. 1A shows typical PET images in the brain of anesthetized rats which were generated by summing the data from 40 to 60 min postinjection of [11C](+)3-MPB. The regional distribution of [11C](+)3MPB was greatest in the corpus striatum, intermediate in the cerebral cortex and lowest in the cerebellum. Autoradiographic images also illustrated a similar distribution of [11C](+)3-MPB (Fig. 3A). As shown in Fig. 1B, the accumulation of [11C](+)3-MPB in the cerebral cortex and corpus striatum was dose-dependently decreased by the i.v.
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Fig. 1. (A) Typical PET images fused with CT images in the brain of rats injected i.v. with [11C](+)3-MPB. The images were generated by summing the data 40–60 min after the [11C] (+)3-MPB injection. Each coronal section was different at 2.1 mm intervals. Upper left section: frontal lobe region, lower right section: cerebellum region. Third section in the upper panel was Bregma. (B) Effects of different doses of oxybutynin (0.1–1.0 mg/kg), darifenacin (0.1–1.0 mg/kg) and imidafenacin (0.01–0.1 mg/kg) on PET images of [11C](+)3-MPB in the rat brain. Rats received an i.v. injection of each agent 10 min prior to the [11C](+)3-MPB injection. Each section represents the typical one for Bregma – 2.1 mm region (fourth section in the upper panel (A)).
injection of oxybutynin (0.1–1.0 mg/kg), but in the cerebellum, a slight decrease in [11C](+)3-MPB accumulation was observed. In contrast, after the i.v. injection of darifenacin (0.1–1.0 mg/kg) and imidafenacin (0.01–0.1 mg/kg), there was little or no decrease in the accumulation of [11C](+)3-MPB in the brain.
A kinetic analysis of [11C](+)3-MPB using the Simplified Reference Tissue Model with cerebellum as a reference demonstrated a significant and dose-dependent decrease in the BP of [11C](+)3MPB in the cerebral cortex (43.7–68.2%) and corpus striatum (43.0– 71.0%) after the i.v. injection of oxybutynin (0.3, 1.0 mg/kg) (Fig. 2).
Fig. 2. Effects of the i.v. injection of oxybutynin (Oxy), darifenacin (Dar) and imidafenacin (Imi) on the binding potential (BP) of [11C](+)3-MPB in the cerebral cortex and corpus striatum of rats. The BP of [11C](+)3-MPB was estimated by a kinetic analysis using the Simplified Reference Tissue Model with the cerebellum as a reference region. Each column shows the mean ± SE (n = 3–4). Asterisks show a significant difference from the control (vehicle), *P b 0.05, **P b 0.01.
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On the other hand, the i.v. injection of darifenacin (0.1–1.0 mg/kg) and imidafenacin (0.01–0.1 mg/kg) did not affect the BP of [11C](+)3MPB in the rat cerebral cortex and corpus striatum. Autoradiography and radioligand binding By a quantitative autoradiographic analysis, we estimated the in vivo distribution of [11C](+)3-MPB in each brain region after the i.v. injection of antimuscarinic agents (Figs. 3B and 4). After the i.v. injection of oxybutynin (0.1–1.0 mg/kg), the distribution of [11C](+) 3-MPB was significantly decreased in the cerebral cortex (23.6– 47.1%), corpus striatum (17.0–46.7%), hippocampus (19.0–41.6%), amygdala (18.4–45.0%), thalamus (22.0–39.4%), hypothalamus (24.6– 38.3%) and pons (15.2–29.4%) in a dose-dependent manner. Darifenacin at 0.1 to 1.0 mg/kg exerted little effect on the distribution of [11C](+)3-MPB in each region, except for a slight but significant decrease (8.6–16.1%) in the thalamus, hypothalamus and pons at relatively high doses (Fig. 4). Similarly, the i.v. injection of imidafenacin (0.01–0.1 mg/kg) had no effect on the distribution of [11C](+)3-MPB in the brain. At 40 min after the i.v. injection of oxybutynin (1.0 mg/kg), darifenacin (1.0 mg/kg) and imidafenacin (0.1 mg/kg), there were significant increases (1.47, 1.49 and 1.58 times, respectively) in Kd values for specific [3H]NMS binding in the bladder (Table 1). A significant increase in the Kd value (1.82 time) in the brain was observed after the i.v. injection of oxybutynin but not darifenacin or imidafenacin. These antimuscarinic agents had no effect on Bmax values for specific [3H]NMS binding in either tissue.
Discussion The testing of antimuscarinic agents used to treat OAB for side effect on the CNS is important. The current study is the first to characterize noninvasively muscarinic receptor occupancy in the rat brain after the systemic injection of oxybutynin, darifenacin and imidafenacin by PET, comparing the results with those of in vivo autoradiographic and ex vivo radioreceptor assays. The i.v. doses of oxybutynin (0.1–1.0 mg/kg), darifenacin (0.1–1.0 mg/kg) and imidafenacin (0.01–0.1 mg/kg) used herein were pharmacologically relevant (Suzuki et al. 2007; Ohno et al. 2008). The plasma concentration of imidafenacin at these doses was similar to (0.01 mg/kg) or 10 fold higher (0.1 mg/kg) than that in patients receiving an oral dose of 0.1 mg of imidafenacin (Ohno et al. 2008). In the PET experiment in rats, the i.v. injection of oxybutynin but not darifenacin or imidafenacin at pharmacological doses decreased the BP of [11C](+)3-MPB in the cerebral cortex and corpus striatum in a dose-dependent manner. Similarly, in the autoradiographic analysis, oxybutynin dose-dependently decreased the binding of [11C](+)3MPB in each region of the brain, whereas darifenacin and imidafenacin had little effect. These results confirm our previous ex vivo binding and ex vivo autoradiographic data on muscarinic receptors (Oki et al. 2007; Maruyama et al. 2008), and seem to be responsible for clinical observations indicating that darifenacin and imidafenacin might have fewer CNS adverse effects than oxybutynin (Ancelin et al. 2006; Kay et al. 2006; Homma and Yamaguchi 2008, 2009). Following the i.v. injection of oxybutynin (1.0 mg/kg), darifenacin (1.0 mg/kg) and imidafenacin (0.1 mg/kg), there was a similar degree
Fig. 3. (A) Typical ex vivo autoradiographic images in the brain of rats injected i.v. with [11C](+)3-MPB. At 30 min, brain tissue was removed, cut into 2-mm-thick coronal sections, and prepared for autoradiography. (B) Effects of different doses of oxybutynin (0.1–1.0 mg/kg), darifenacin (0.1–1.0 mg/kg) and imidafenacin (0.01–0.1 mg/kg) on autoradiographic images of [11C](+)3-MPB in the rat brain. Rats received an i.v. injection of each agent 10 min prior to the [11C](+)3-MPB injection.
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Table 1 Kd and Bmax for specific [3H]NMS binding in the bladder and brain of rats after the i.v. injection of oxybutynin (1.0 mg/kg), darifenacin (1.0 mg/kg) and imidafenacin (0.1 mg/kg). Tissues
Drugs
Kd (nM)
Bladder
Vehicle Oxybutynin Darifenacin Imidafenacin Vehicle Oxybutynin Darifenacin Imidafenacin
332 ± 16 489 ± 19 496 ± 42 525 ± 51 258 ± 4 471 ± 13 249 ± 9 273 ± 9
Brain
Bmax (fmol/mg tissue) (1.47)* (1.49)* (1.58)* (1.82)*
174 ± 20 186 ± 13 177 ± 3 174 ± 17 1167 ± 26 1137 ± 44 1081 ± 48 1167 ± 38
The values in parentheses represent the ratio of Kd relative to the control (vehicle). Values are expressed as the mean ± SE. (n = 5–8). Asterisks show a significant difference, *P b 0.01.
Fig. 4. Effects of the i.v. injection of oxybutynin, darifenacin and imidafenacin on the distribution of [11C](+)3-MPB in the cerebral cortex, corpus striatum, hippocampus, amygdala, thalamus, hypothalamus and pons of rats. The distribution of [11C](+)3-MPB (Y-axis) was defined as the ratio of SUV values between each region and the cerebellum. Each column shows the mean ± SE (n = 3–12). Asterisks show a significant difference from the control (vehicle), *P b 0.05, **P b 0.01.
of increases in Kd values for specific [3H]NMS binding in the bladder (Table 1). With the view that an increase in Kd values for radioligand binding in agent-pretreated tissues in this type of assay is generally considered as competition between the agent and radioligand for the same binding sites (Yamada et al. 2003; Oki et al. 2007), these data indicate that antimuscarinic agents undergo significant binding to
muscarinic receptor of the bladder which is the target organ. The binding of muscarinic receptors in the brain was significantly different after the injection of oxybutynin but not darifenacin or imidafenacin. These findings, with the results of PET and autoradiography, confirm that oxybutynin orally administered at pharmacological doses in the bladder, binds significantly to muscarinic receptors in the brain, while darifenacin and imidafenacin exert little such effect. Generally, agents must first cross the BBB to distribute into the brain and to occupy the CNS receptors. The observed difference among these antimuscarinic agents in the degree of uptake and binding to muscarinic receptors in the brain may depend on their ability to permeate the BBB. The passive penetration of the BBB is dependent principally on physicochemical properties (Scheife and Takeda 2005), and thus its small size and molecular characteristics (lipophilicity, Log Ko/w: 4.68; neutral polarity, pKa: 6.44) make oxybutynin likely to cross the BBB. In contrast, both darifenacin and imidafenacin have moderate polarity and low lipophilicity, suggestive of lower permeability. In addition, darifenacin is considered as a substrate of P-glycoprotein, an active-transport system that carries this agent back across the BBB (Skerjanec 2006). The muscarinic receptor subtype selectivity of antimuscarinic agents may also be involved in the risk of side effects. Although all five muscarinic receptor subtypes are expressed in the CNS, the M1 receptor, in particular, is considered to play a crucial role in modulating cognitive function (Kay et al. 2005). Oxybutynin and imidafenacin were selective of both M3 and M1 receptors, while darifenacin was selective of M3 receptors in vitro (Maruyama et al. 2006; Kobayashi et al. 2007a). Thus the side effect of oxybutynin on the CNS during OAB treatment may be attributed to selectivity for the M1 receptor in addition to the ability to permeate the BBB. In contrast, no influence of imidafenacin on cognitive function was reported by Kobayashi et al. (2007b) possibly due to insignificant distribution of imidafenacin in the CNS. Because muscarinic receptors mediate the excitatory and inhibitory actions of acetylcholine in the central and peripheral nervous systems, the clinical use of antimuscarinic agents may bring on not only central but also peripheral adverse effects. In fact, dry mouth commonly occurs with the blockade of muscarinic receptors in the salivary gland and thereby decreases the quality of life of patients. Recently, pharmacological studies have shown that forebrain lesions or intraventricular injection of atropine reduced pilocarpine-induced salivation, indicating central mechanism of salivary secretion (Takakakura et al. 2003; Lopes de Almeida et al. 2006). In addition Borella et al. (2008) have demonstrated central M3 receptors to be involved in pilocalpine-induced salivation. Therefore, the extensive distribution of oxybutynin in the CNS might lead to peripheral adverse effects such as dry mouth. The current results are consistent with clinical findings that darifenacin and imidafenacin caused dry mouth less frequently than oxybutynin (Abrams and Andersson 2007; Homma et al. 2008; Homma and Yamaguchi 2009).
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This is the first PET-based study to demonstrate noninvasively that oxybutynin but not darifenacin or imidafenacin may have potential side effects on the CNS in patients with OAB. These results also reveal the in vivo characterization of brain receptor occupancy by PET to be a powerful tool for the precise evaluation of side effects of antimuscarinic agents in pre-clinical and clinical evaluations. Conflict of interest statement The authors state no conflict of interest.
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