- Email: [email protected]
Direct in vitro and in vivo demonstration of muscarinic receptor binding by the novel radioligand, [3H]5-hydroxymethyltolterodine, in the bladder and other tissues of rats
Journal of Pharmacological Sciences xxx (xxxx) xxx
Contents lists available at ScienceDirect
Journal of Pharmacological Sciences journal homepage: www.elsevier.com/locate/jphs
Short Communication
Direct in vitro and in vivo demonstration of muscarinic receptor binding by the novel radioligand, [3H]5-hydroxymethyltolterodine, in the bladder and other tissues of rats Shizuo Yamada a, *, Shiori Kuraoka b, Yoshihiko Ito a, Satomi Kagota c, Kazumasa Shinozuka c, Satomi Onoue b a b c
Center for Pharma-Food Research (CPFR), Shizuoka, Japan Department of Pharmacokinetics and Pharmacodynamics, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Department of Pharmacology II, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2019 Received in revised form 6 September 2019 Accepted 12 September 2019 Available online xxx
In vitro and in vivo binding sites of [3H]-labeled 5-hydroxymethyltolterodine (5-HMT), a new radioligand for labeling muscarinic receptors in rat tissues were characterized. Specific [3H]5-HMT binding in rat tissues was saturable and of high affinity in each tissue. The dissociation constant (Kd) was significantly lower in bladder and heart than in submaxillary gland. Significant levels of in vivo specific [3H]5-HMT binding by intravenous injection of the radioligand were detected in tissues, except for cerebral cortex. Thus, [3H]5-HMT was shown to specifically label muscarinic receptors in rat tissues, suggesting a useful radioligand for labeling muscarinic receptors with high affinity. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: [3H]5-HMT Bladder Muscarinic receptor
1. Introduction Antimuscarinic agents are currently used as a major pharmacological therapy for overactive bladder, however, these agents have been shown to cause a number of adverse effects such as dry mouth, constipation, blurred vision, and cognitive impairment.1 Their therapeutic and adverse effects are mediated by the blockade of muscarinic receptors in bladder and non-target tissues, respectively. Fesoterodine is one of antimuscarinic agents to treat symptoms of overactive bladder. After its oral administration, fesoterodine is rapidly and extensively converted to its active metabolite, 5-hydroxymethyltolterodine (5-HMT).2 We showed that 5-HMT binds muscarinic receptors with markedly higher affinity than fesoterodine in human bladder mucosa and detrusor.3 Previous findings on muscarinic receptor
Abbreviations: [3H]5-HMT, [3H]5-hydroxymethyltolterodine; Bmax, maximal number of binding sites; Kｄ, dissociation constant. * Corresponding author. Center for Pharma-Food Research (CPFR), Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan. Fax: þ81 54 264 5626. E-mail address: [email protected] (S. Yamada). Peer review under responsibility of Japanese Pharmacological Society.
binding of 5-HMT have been indirectly obtained using radioreceptor binding assays with [N-methyl-3H] scopolamine methyl chloride ([3H]NMS), a selective radioligand of muscarinic receptors.4 The present study aimed to directly demonstrate muscarinic receptor binding of 5-HMT in rat bladder and other tissues under in vitro and in vivo conditions, by using newly synthesized tritium ([3H])-labeled 5-HMT ([3H]5-HMT) with high specific activity. 2. Materials and methods 2.1. Materials [3H]5-HMT with high specific activity (370 GBq/mmol) was made from tritium gas by a method developed by Quotient Bioresearch (Radiochemicals) Ltd. (Cardiff, CF24 5JQ). The material was purified by HPLC. All other chemicals were purchased from commercial sources. 2.2. Animals Nine-week-old male SpragueeDawley rats were purchased from Japan SLC Inc. (Shizuoka, Japan). Animal care and experiments
https://doi.org/10.1016/j.jphs.2019.12.004 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Yamada S et al., Direct in vitro and in vivo demonstration of muscarinic receptor binding by the novel radioligand, [3H] 5-hydroxymethyltolterodine, in the bladder and other tissues of rats, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.12.004
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S. Yamada et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx
were performed in accordance with the guidelines for care and use of laboratory animals of University of Shizuoka (registration number:136038). 2.3. Tissue preparation and in vitro radioligand binding assay of [3H]5-HMT Rats were sacrificed by collecting blood from the descending aorta under anesthesia with isoflurane. Each tissue was dissected, carefully minced with scissors, and homogenized using a Kinematica Polytron homogenizer in 19 volumes of ice-cold 30 mM Naþ/HEPES buffer (pH 7.5). The homogenate was centrifuged at 40,000 g for 20 min. The pellet was suspended in same buffer for the binding assay. In saturation binding experiments with [3H]5HMT, tissue homogenates of rats were incubated with various concentrations of [3H]5-HMT in buffer at 25 C for 60 min. The reaction was terminated by rapid filtration (Cell Harvester; Brandel Co, Gaithersburg, MD, USA) through Whatman GF/B glass filters. The filters were then rinsed with ice-cold buffer. Tissue-bound radioactivity was extracted from filters overnight by immersion in scintillation fluid, and radioactivity was determined using liquid scintillation counter (LSC-7400, Hitachi-Aloka Medical, Tokyo, Japan). Specific [3H]5-HMT binding was assessed experimentally as the difference between these counts in the absence and presence of 1 mM atropine. 2.4. Measurement of in vivo total radioactivity and specific binding of [3H]5-HMT Total radioactivity and in vivo specific binding of [3H]5-HMT in rat tissues after intravenous injection of the radioligand were measured as described previously for [3H]quinuclidinyl benzilate (QNB)5 and [3H]imidafenacin.6 Briefly, [3H]5-HMT (3.7 MBq: 10.0 nmol/kg) was injected into the tail vein and rats were sacrificed under isoflurane anesthesia 10e180 min after the injection. A blood sample was collected from descending aorta and tissues were rapidly removed. Each tissue specimen was homogenized in icecold 50 mM Naþ/Kþ phosphate buffer (pH 7.5) with a Kinematica Polytron homogenizer. Particulate-bound radioactivity was assessed by rapid filtration of 0.5 mL of the tissue homogenate through a Whatman CF/C filter, followed by washing with 1 mL of ice-cold buffer. The radioactivity in tissue particulate fraction was measured by using a liquid scintillation counter. In vivo specific binding of [3H]5-HMT was estimated as the difference in particulate-bound radioactivity between vehicle- and atropine (14.8 mmol/kg i.p.)-pretreated rats, reflecting total and nonspecific binding, respectively.
[3H]5-HMT (Fig. 1); however, the nonspecific binding increased linearly with an increase in [3H]5-HMT concentrations. Kd values of [3H]5-HMT in tissues were in the nanomolar range (Table 1). Kd value was significantly lower in the bladder, heart and cerebral cortex than in the submaxillary gland. Bmax value of [3H]5-HMT was the highest in the cerebral cortex, followed by the colon, bladder, heart and submaxillary gland. Bmax value of specific [3H]5-HMT binding was significantly higher in the bladder, colon and cerebral cortex than in the submaxillary gland. 3.2. In vivo identification of specific [3H]5-HMT binding sites Total radioactivity in tissue particulate fraction of rats after the intravenous injection of [3H]5-HMT was the highest after 10 min, and then decreased with the time. The pretreatment with atropine (14.8 mmol/kg, i.p.) significantly (62e84%) reduced total binding of [3H]5-HMT in particulate fractions of the bladder, submaxillary gland, heart and colon. Thus, the difference in tissue particulatebound [3H]5-HMT radioactivity between vehicle-pretreated and atropine-pretreated rats represented the in vivo specific binding of the ligand. The in vivo specific binding of [3H]5-HMT varied among tissues (Fig. 2), and it was the highest in the heart after 10 min, followed by the submaxillary gland, bladder, and colon. Furthermore, in the time course of specific [3H]5-HMT binding in the particulate fraction of each tissue of rats harvested 10e180 min after the intravenous injection of [3H]5-HMT, the maximum level of specific [3H]5-HMT binding was noted after 10 min, and then significantly decreased over time (Fig. 2). Specific [3H]5-HMT binding was negligible in the particulate fraction of the cerebral cortex (data not shown). 4. Discussion The present study directly identified specific binding sites of novel tritium ([3H])-labeled 5-HMT ([3H]5-HMT) with highly specific activity in the bladder, submaxillary gland, heart, colon and cerebral cortex of rats. The Kd (0.87 nM, pKd: 9.06) value for specific [3H]5-HMT binding in the rat bladder approximately corresponded to its pharmacological potency for the competitive blockade by 5HMT of carbachol-induced contractions in the rat bladder with pA2 values of 8.8,7 indicating that [3H]5-HMT selectively binds to pharmacologically relevant muscarinic receptors in the rat bladder. Previous studies reported that 5-HMT exhibited higher affinity for the M2 subtype than the M3 subtype8 in the membranes of Chinese hamster ovary cells expressing human muscarinic receptor
2.5. Data analysis [3H]5-HMT binding data were subjected to non-linear regression analysis using Graph Pad PRISM (ver. 4, Graph Pad Software, San Diego, CA). The apparent dissociation constant (Kd) and maximal number of binding sites (Bmax) for [3H]5-HMT were estimated. Statistical analyses of receptor binding data were performed with Student's t-test and a one-way analysis of variance (ANOVA), followed by Dunnett's test for multiple comparisons. All data were expressed as mean ± S.E. 3. Results 3.1. In vitro [3H]5-HMT binding Specific binding of [3H]5-HMT (0.25e5.0 nM) in each tissue was saturable, forming a plateau at approximately 2.0 nM (bladder) of
Fig. 1. Specific binding of [3H]5-HMT with increases in the ligand concentration in the bladder of rats, and its Scatchard plot (insert). Specific [3H]5-HMT binding ( )in the rat bladder was assessed based on the difference in the total and nonspecific ( ) binding of [3H]5-HMT (0.25e5.0 nM). Each point represents the mean of 6 rats.
Please cite this article as: Yamada S et al., Direct in vitro and in vivo demonstration of muscarinic receptor binding by the novel radioligand, [3H] 5-hydroxymethyltolterodine, in the bladder and other tissues of rats, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.12.004
S. Yamada et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx Table 1 Dissociation constant (Kd) and maximum number of binding sites. (Bmax) for specific binding of [3H]5-HMT in rat tissues. [3H]5-HMT Bmax (fmol/mg protein)
Kd (nM) Bladder Submaxillary gland Heart Colon Cerebral cortex
0.87 1.30 0.74 0.97 0.77
± ± ± ± ±
0.15* 0.17 0.18* 0.23 0.20*
248 ± 16* 167 ± 14 193 ± 15 377 ± 32* 1188 ± 105*
Specific [3H]5-HMT binding in crude membrane fractions from rat tissues was determined from the difference of total binding and nonspecific binding of [3H]5HMT (0.25e5.0 nM). The dissociation constant (Kd) and maximum number of binding sites (Bmax) were estimated. Each value represents mean ± S.E. of 6 rats. Asterisks show significant difference from the value in the submaxillary gland. *P < 0.05.
3
the administration of fesoterodine at 4 and 8 mg once daily had no significant effects on any cognitive function including memory in healthy older adults. Fesoterodine and 5-HMT are substrates of glycoprotein (P-gp) and, thus, are actively transported away from the brain.13 LogD values, a determinant of lipophilicity and permeability across the BBB, were previously shown to be very low for fesoterodine and 5-HMT.14,15 Therefore, their brain penetration is considered to be low. Fesoterodine has favorable CNS tolerability in the elderly.14,15 [3H]Quinuclidinyl benzylate ([3H]QNB) and [N-methyl-3H] scopolamine methyl chloride ([3H]NMS) have commonly been used as radioligands to measure muscarinic receptor binding in a number of tissues, and these ligands are nonselective for each subtypes or slightly M3 selective. The advantage of [3H]5-HMT over these radioligands may be due to its greater selectivity to the M2 subtype of muscarinic receptors. 5. Conclusion The present results have revealed, for the first time, that [3H]5HMT binds muscarinic receptors with high affinity in the bladder under in vitro and in vivo conditions, which supports a rationale for the pharmacological usefulness of fesoterodine (prodrug of 5-HMT) as a therapeutic agent of overactive bladder. Thus, [3H]5-HMT may also be a useful radioligand for labeling muscarinic receptors with high affinity. Declaration of Competing Interest
Fig. 2. Time course of the in vivo specific binding of [3H]5-HMT in rat tissues after an intravenous injection of the ligand. [3H]5-HMT (10.2 MBq, 10.0 nmol/kg) was injected into the tail vein, and rats were sacrificed 10, 30, 90 and 180 min later. Specific [3H]5HMT binding was experimentally defined as the difference in binding in particulate fractions of each tissue between saline (total binding)- and atropine (14.8 mmol/kg, i.p.) (nonspecific binding)-pretreated rats. Each column represents the mean ± S.E. of 6 rats.
subtypes. The salivary gland predominantly contains M3 muscarinic receptors,9,10 while the bladder contains both the M2 and M3 subtypes with a predominance of the M2 receptor.9,11 Furthermore, the expression of the M3 subtype was stronger in the salivary gland than in the bladder.9 In the present study, the Kd value of [3H]5HMT was significantly lower in the bladder and heart of rats with a predominance of the M2 subtype versus the M3 subtypedominated submaxillary gland (Table 1), which may confirm a higher affinity of 5-HMT for the M2 subtype over the M3 subtype, as reported by Maruyama et al.8 and Yoshida et al.3 High level of in vivo [3H]5-HMT binding in the heart (Fig. 2) may be due to the predominance of M2 subtype.9 A markedly high level of specific [3H]5-HMT binding in the rat cerebral cortex was detected in the in vitro binding assay (Table 1), whereas specific [3H]5-HMT binding was negligible in this tissue after the intravenous injection of the radioligand in rats. Very recently, the oral administration of fesoterodine and intravenous injection of 5-HMT in rats was shown to exert low levels of muscarinic receptor binding activity in the cerebral cortex,4 which is consistent with the present results. These findings indicate the low occupancy of brain muscarinic receptors by fesoterodine and 5HMT. In agreement with these findings, Kay et al.12 reported that
The authors declare that they have no conflicts of interest to disclose. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. References 1. Yamada S, Ito Y, Nishijima S, Kadekawa K, Sugaya K. Basic and clinical aspects of antimuscarinic agents used to treat overactive bladder. Pharmacol Ther. 2018;189:130e148. 2. Michel MC. Fesoterodine: a novel muscarinic receptor antagonist for the treatment of overactive bladder syndrome. Expert Opin Pharmacother. 2008;9: 1787e1796. 3. Yoshida A, Fuchihata Y, Kuraoka S, et al. Fesoterodine, its active metabolite, and tolterodine bind selectively to muscarinic receptors in human bladder mucosa and detrusor muscle. Urology. 2013;81:920.e1e920.e5. 4. Yamada S, Kuraoka S, Ito Y, Kato Y, Onoue S. Muscarinic receptor binding of fesoterodine, 5-hydroxymethyl tolterodine, and tolterodine in rat tissues after the oral, intravenous, or intravesical administration. J Pharmacol Sci. 2019;140: 73e78. 5. Maruyama S, Hasuike N, Suzuki K, Yamada S. In vivo characterization of muscarinic receptors in peripheral tissues: evaluation of bladder selectivity of anticholinergic agents to treat overactive bladder. Naunyn-Schmiedeberg’s Arch Pharmacol. 2008;377:463e471. 6. Ito Y, Kuraoka S, Endo S, Takahashi A, Onoue S, Yamada S. Urinary excretion contributes to long-lasting blockade of bladder muscarinic receptors by imidafenacin: effect of bilateral ureteral ligation. J Pharmacol Exp Ther. 2017;360: 69e74. 7. Ney P, Pandita RK, Newgreen DT, et al. Pharmacological characterization of a novel investigational antimuscarinic drug, fesoterodine, in vitro and in vivo. BJU Int. 2008;101:1036e1042. 8. Maruyama S, Oki T, Otsuka A, et al. Human muscarinic receptor binding characteristics of antimuscarinic agents to treat overactive bladder. J Urol. 2006;175:365e369. 9. Ito Y, Oyunzul L, Seki M, et al. Quantitative analysis of the loss of muscarinic receptors in various peripheral tissues in M1-M5 single subtype knockout mice. Br J Pharmacol. 2009;156:1147e1153. 10. Yoshida A, Fujino T, Maruyama S, et al. The forefront for novel therapeutic agents based on the pathophysiology of lower urinary tract dysfunction: bladder selectivity based on in vivo drug-receptor binding characteristics of
Please cite this article as: Yamada S et al., Direct in vitro and in vivo demonstration of muscarinic receptor binding by the novel radioligand, [3H] 5-hydroxymethyltolterodine, in the bladder and other tissues of rats, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.12.004
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antimuscarinic agents for treatment of overactive bladder. J Pharmacol Sci. 2010;112:142e150. 11. Wang P, Luthin GR, Ruggieri MR. Muscarinic acetylcholine receptor subtypes mediating urinary bladder contractility and coupling to GTP binding proteins. J Pharmacol Exp Ther. 1995;273:959e966. 12. Kay GG, Maruff P, Scholfield D, et al. Evaluation of cognitive function in healthy older subjects treated with fesoterodine. Postgrad Med. 2012;124:7e15. 13. Chancellor MB, Staskin DR, Kay GG, et al. Blood-brain barrier permeation and efflux exclusion of anticholinergics used in the treatment of overactive bladder. Drugs Aging. 2012;29:259e273.
14. Malhotra B, Gandelman K, Sachese R, Wood N, Michel MC. The design and development of fesoterodine as a prodrug of 5-hydroxymethyl tolterodine (5HMT), the active metabolite of tolterodine. Curr Med Chem. 2009;16: 4481e4489. 15. Kerdraon J, Robain G, Jeandel C, et al. Impact on cognitive function of anticholinergic drugs used for the treatment of overactive bladder in the elderly. Prog Urol. 2014;24:672e681.
Please cite this article as: Yamada S et al., Direct in vitro and in vivo demonstration of muscarinic receptor binding by the novel radioligand, [3H] 5-hydroxymethyltolterodine, in the bladder and other tissues of rats, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.12.004
Contents lists available at ScienceDirect
Journal of Pharmacological Sciences journal homepage: www.elsevier.com/locate/jphs
Short Communication
Direct in vitro and in vivo demonstration of muscarinic receptor binding by the novel radioligand, [3H]5-hydroxymethyltolterodine, in the bladder and other tissues of rats Shizuo Yamada a, *, Shiori Kuraoka b, Yoshihiko Ito a, Satomi Kagota c, Kazumasa Shinozuka c, Satomi Onoue b a b c
Center for Pharma-Food Research (CPFR), Shizuoka, Japan Department of Pharmacokinetics and Pharmacodynamics, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Department of Pharmacology II, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2019 Received in revised form 6 September 2019 Accepted 12 September 2019 Available online xxx
In vitro and in vivo binding sites of [3H]-labeled 5-hydroxymethyltolterodine (5-HMT), a new radioligand for labeling muscarinic receptors in rat tissues were characterized. Specific [3H]5-HMT binding in rat tissues was saturable and of high affinity in each tissue. The dissociation constant (Kd) was significantly lower in bladder and heart than in submaxillary gland. Significant levels of in vivo specific [3H]5-HMT binding by intravenous injection of the radioligand were detected in tissues, except for cerebral cortex. Thus, [3H]5-HMT was shown to specifically label muscarinic receptors in rat tissues, suggesting a useful radioligand for labeling muscarinic receptors with high affinity. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: [3H]5-HMT Bladder Muscarinic receptor
1. Introduction Antimuscarinic agents are currently used as a major pharmacological therapy for overactive bladder, however, these agents have been shown to cause a number of adverse effects such as dry mouth, constipation, blurred vision, and cognitive impairment.1 Their therapeutic and adverse effects are mediated by the blockade of muscarinic receptors in bladder and non-target tissues, respectively. Fesoterodine is one of antimuscarinic agents to treat symptoms of overactive bladder. After its oral administration, fesoterodine is rapidly and extensively converted to its active metabolite, 5-hydroxymethyltolterodine (5-HMT).2 We showed that 5-HMT binds muscarinic receptors with markedly higher affinity than fesoterodine in human bladder mucosa and detrusor.3 Previous findings on muscarinic receptor
Abbreviations: [3H]5-HMT, [3H]5-hydroxymethyltolterodine; Bmax, maximal number of binding sites; Kｄ, dissociation constant. * Corresponding author. Center for Pharma-Food Research (CPFR), Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan. Fax: þ81 54 264 5626. E-mail address: [email protected] (S. Yamada). Peer review under responsibility of Japanese Pharmacological Society.
binding of 5-HMT have been indirectly obtained using radioreceptor binding assays with [N-methyl-3H] scopolamine methyl chloride ([3H]NMS), a selective radioligand of muscarinic receptors.4 The present study aimed to directly demonstrate muscarinic receptor binding of 5-HMT in rat bladder and other tissues under in vitro and in vivo conditions, by using newly synthesized tritium ([3H])-labeled 5-HMT ([3H]5-HMT) with high specific activity. 2. Materials and methods 2.1. Materials [3H]5-HMT with high specific activity (370 GBq/mmol) was made from tritium gas by a method developed by Quotient Bioresearch (Radiochemicals) Ltd. (Cardiff, CF24 5JQ). The material was purified by HPLC. All other chemicals were purchased from commercial sources. 2.2. Animals Nine-week-old male SpragueeDawley rats were purchased from Japan SLC Inc. (Shizuoka, Japan). Animal care and experiments
https://doi.org/10.1016/j.jphs.2019.12.004 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Yamada S et al., Direct in vitro and in vivo demonstration of muscarinic receptor binding by the novel radioligand, [3H] 5-hydroxymethyltolterodine, in the bladder and other tissues of rats, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.12.004
2
S. Yamada et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx
were performed in accordance with the guidelines for care and use of laboratory animals of University of Shizuoka (registration number:136038). 2.3. Tissue preparation and in vitro radioligand binding assay of [3H]5-HMT Rats were sacrificed by collecting blood from the descending aorta under anesthesia with isoflurane. Each tissue was dissected, carefully minced with scissors, and homogenized using a Kinematica Polytron homogenizer in 19 volumes of ice-cold 30 mM Naþ/HEPES buffer (pH 7.5). The homogenate was centrifuged at 40,000 g for 20 min. The pellet was suspended in same buffer for the binding assay. In saturation binding experiments with [3H]5HMT, tissue homogenates of rats were incubated with various concentrations of [3H]5-HMT in buffer at 25 C for 60 min. The reaction was terminated by rapid filtration (Cell Harvester; Brandel Co, Gaithersburg, MD, USA) through Whatman GF/B glass filters. The filters were then rinsed with ice-cold buffer. Tissue-bound radioactivity was extracted from filters overnight by immersion in scintillation fluid, and radioactivity was determined using liquid scintillation counter (LSC-7400, Hitachi-Aloka Medical, Tokyo, Japan). Specific [3H]5-HMT binding was assessed experimentally as the difference between these counts in the absence and presence of 1 mM atropine. 2.4. Measurement of in vivo total radioactivity and specific binding of [3H]5-HMT Total radioactivity and in vivo specific binding of [3H]5-HMT in rat tissues after intravenous injection of the radioligand were measured as described previously for [3H]quinuclidinyl benzilate (QNB)5 and [3H]imidafenacin.6 Briefly, [3H]5-HMT (3.7 MBq: 10.0 nmol/kg) was injected into the tail vein and rats were sacrificed under isoflurane anesthesia 10e180 min after the injection. A blood sample was collected from descending aorta and tissues were rapidly removed. Each tissue specimen was homogenized in icecold 50 mM Naþ/Kþ phosphate buffer (pH 7.5) with a Kinematica Polytron homogenizer. Particulate-bound radioactivity was assessed by rapid filtration of 0.5 mL of the tissue homogenate through a Whatman CF/C filter, followed by washing with 1 mL of ice-cold buffer. The radioactivity in tissue particulate fraction was measured by using a liquid scintillation counter. In vivo specific binding of [3H]5-HMT was estimated as the difference in particulate-bound radioactivity between vehicle- and atropine (14.8 mmol/kg i.p.)-pretreated rats, reflecting total and nonspecific binding, respectively.
[3H]5-HMT (Fig. 1); however, the nonspecific binding increased linearly with an increase in [3H]5-HMT concentrations. Kd values of [3H]5-HMT in tissues were in the nanomolar range (Table 1). Kd value was significantly lower in the bladder, heart and cerebral cortex than in the submaxillary gland. Bmax value of [3H]5-HMT was the highest in the cerebral cortex, followed by the colon, bladder, heart and submaxillary gland. Bmax value of specific [3H]5-HMT binding was significantly higher in the bladder, colon and cerebral cortex than in the submaxillary gland. 3.2. In vivo identification of specific [3H]5-HMT binding sites Total radioactivity in tissue particulate fraction of rats after the intravenous injection of [3H]5-HMT was the highest after 10 min, and then decreased with the time. The pretreatment with atropine (14.8 mmol/kg, i.p.) significantly (62e84%) reduced total binding of [3H]5-HMT in particulate fractions of the bladder, submaxillary gland, heart and colon. Thus, the difference in tissue particulatebound [3H]5-HMT radioactivity between vehicle-pretreated and atropine-pretreated rats represented the in vivo specific binding of the ligand. The in vivo specific binding of [3H]5-HMT varied among tissues (Fig. 2), and it was the highest in the heart after 10 min, followed by the submaxillary gland, bladder, and colon. Furthermore, in the time course of specific [3H]5-HMT binding in the particulate fraction of each tissue of rats harvested 10e180 min after the intravenous injection of [3H]5-HMT, the maximum level of specific [3H]5-HMT binding was noted after 10 min, and then significantly decreased over time (Fig. 2). Specific [3H]5-HMT binding was negligible in the particulate fraction of the cerebral cortex (data not shown). 4. Discussion The present study directly identified specific binding sites of novel tritium ([3H])-labeled 5-HMT ([3H]5-HMT) with highly specific activity in the bladder, submaxillary gland, heart, colon and cerebral cortex of rats. The Kd (0.87 nM, pKd: 9.06) value for specific [3H]5-HMT binding in the rat bladder approximately corresponded to its pharmacological potency for the competitive blockade by 5HMT of carbachol-induced contractions in the rat bladder with pA2 values of 8.8,7 indicating that [3H]5-HMT selectively binds to pharmacologically relevant muscarinic receptors in the rat bladder. Previous studies reported that 5-HMT exhibited higher affinity for the M2 subtype than the M3 subtype8 in the membranes of Chinese hamster ovary cells expressing human muscarinic receptor
2.5. Data analysis [3H]5-HMT binding data were subjected to non-linear regression analysis using Graph Pad PRISM (ver. 4, Graph Pad Software, San Diego, CA). The apparent dissociation constant (Kd) and maximal number of binding sites (Bmax) for [3H]5-HMT were estimated. Statistical analyses of receptor binding data were performed with Student's t-test and a one-way analysis of variance (ANOVA), followed by Dunnett's test for multiple comparisons. All data were expressed as mean ± S.E. 3. Results 3.1. In vitro [3H]5-HMT binding Specific binding of [3H]5-HMT (0.25e5.0 nM) in each tissue was saturable, forming a plateau at approximately 2.0 nM (bladder) of
Fig. 1. Specific binding of [3H]5-HMT with increases in the ligand concentration in the bladder of rats, and its Scatchard plot (insert). Specific [3H]5-HMT binding ( )in the rat bladder was assessed based on the difference in the total and nonspecific ( ) binding of [3H]5-HMT (0.25e5.0 nM). Each point represents the mean of 6 rats.
Please cite this article as: Yamada S et al., Direct in vitro and in vivo demonstration of muscarinic receptor binding by the novel radioligand, [3H] 5-hydroxymethyltolterodine, in the bladder and other tissues of rats, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.12.004
S. Yamada et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx Table 1 Dissociation constant (Kd) and maximum number of binding sites. (Bmax) for specific binding of [3H]5-HMT in rat tissues. [3H]5-HMT Bmax (fmol/mg protein)
Kd (nM) Bladder Submaxillary gland Heart Colon Cerebral cortex
0.87 1.30 0.74 0.97 0.77
± ± ± ± ±
0.15* 0.17 0.18* 0.23 0.20*
248 ± 16* 167 ± 14 193 ± 15 377 ± 32* 1188 ± 105*
Specific [3H]5-HMT binding in crude membrane fractions from rat tissues was determined from the difference of total binding and nonspecific binding of [3H]5HMT (0.25e5.0 nM). The dissociation constant (Kd) and maximum number of binding sites (Bmax) were estimated. Each value represents mean ± S.E. of 6 rats. Asterisks show significant difference from the value in the submaxillary gland. *P < 0.05.
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the administration of fesoterodine at 4 and 8 mg once daily had no significant effects on any cognitive function including memory in healthy older adults. Fesoterodine and 5-HMT are substrates of glycoprotein (P-gp) and, thus, are actively transported away from the brain.13 LogD values, a determinant of lipophilicity and permeability across the BBB, were previously shown to be very low for fesoterodine and 5-HMT.14,15 Therefore, their brain penetration is considered to be low. Fesoterodine has favorable CNS tolerability in the elderly.14,15 [3H]Quinuclidinyl benzylate ([3H]QNB) and [N-methyl-3H] scopolamine methyl chloride ([3H]NMS) have commonly been used as radioligands to measure muscarinic receptor binding in a number of tissues, and these ligands are nonselective for each subtypes or slightly M3 selective. The advantage of [3H]5-HMT over these radioligands may be due to its greater selectivity to the M2 subtype of muscarinic receptors. 5. Conclusion The present results have revealed, for the first time, that [3H]5HMT binds muscarinic receptors with high affinity in the bladder under in vitro and in vivo conditions, which supports a rationale for the pharmacological usefulness of fesoterodine (prodrug of 5-HMT) as a therapeutic agent of overactive bladder. Thus, [3H]5-HMT may also be a useful radioligand for labeling muscarinic receptors with high affinity. Declaration of Competing Interest
Fig. 2. Time course of the in vivo specific binding of [3H]5-HMT in rat tissues after an intravenous injection of the ligand. [3H]5-HMT (10.2 MBq, 10.0 nmol/kg) was injected into the tail vein, and rats were sacrificed 10, 30, 90 and 180 min later. Specific [3H]5HMT binding was experimentally defined as the difference in binding in particulate fractions of each tissue between saline (total binding)- and atropine (14.8 mmol/kg, i.p.) (nonspecific binding)-pretreated rats. Each column represents the mean ± S.E. of 6 rats.
subtypes. The salivary gland predominantly contains M3 muscarinic receptors,9,10 while the bladder contains both the M2 and M3 subtypes with a predominance of the M2 receptor.9,11 Furthermore, the expression of the M3 subtype was stronger in the salivary gland than in the bladder.9 In the present study, the Kd value of [3H]5HMT was significantly lower in the bladder and heart of rats with a predominance of the M2 subtype versus the M3 subtypedominated submaxillary gland (Table 1), which may confirm a higher affinity of 5-HMT for the M2 subtype over the M3 subtype, as reported by Maruyama et al.8 and Yoshida et al.3 High level of in vivo [3H]5-HMT binding in the heart (Fig. 2) may be due to the predominance of M2 subtype.9 A markedly high level of specific [3H]5-HMT binding in the rat cerebral cortex was detected in the in vitro binding assay (Table 1), whereas specific [3H]5-HMT binding was negligible in this tissue after the intravenous injection of the radioligand in rats. Very recently, the oral administration of fesoterodine and intravenous injection of 5-HMT in rats was shown to exert low levels of muscarinic receptor binding activity in the cerebral cortex,4 which is consistent with the present results. These findings indicate the low occupancy of brain muscarinic receptors by fesoterodine and 5HMT. In agreement with these findings, Kay et al.12 reported that
The authors declare that they have no conflicts of interest to disclose. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. References 1. Yamada S, Ito Y, Nishijima S, Kadekawa K, Sugaya K. Basic and clinical aspects of antimuscarinic agents used to treat overactive bladder. Pharmacol Ther. 2018;189:130e148. 2. Michel MC. Fesoterodine: a novel muscarinic receptor antagonist for the treatment of overactive bladder syndrome. Expert Opin Pharmacother. 2008;9: 1787e1796. 3. Yoshida A, Fuchihata Y, Kuraoka S, et al. Fesoterodine, its active metabolite, and tolterodine bind selectively to muscarinic receptors in human bladder mucosa and detrusor muscle. Urology. 2013;81:920.e1e920.e5. 4. Yamada S, Kuraoka S, Ito Y, Kato Y, Onoue S. Muscarinic receptor binding of fesoterodine, 5-hydroxymethyl tolterodine, and tolterodine in rat tissues after the oral, intravenous, or intravesical administration. J Pharmacol Sci. 2019;140: 73e78. 5. Maruyama S, Hasuike N, Suzuki K, Yamada S. In vivo characterization of muscarinic receptors in peripheral tissues: evaluation of bladder selectivity of anticholinergic agents to treat overactive bladder. Naunyn-Schmiedeberg’s Arch Pharmacol. 2008;377:463e471. 6. Ito Y, Kuraoka S, Endo S, Takahashi A, Onoue S, Yamada S. Urinary excretion contributes to long-lasting blockade of bladder muscarinic receptors by imidafenacin: effect of bilateral ureteral ligation. J Pharmacol Exp Ther. 2017;360: 69e74. 7. Ney P, Pandita RK, Newgreen DT, et al. Pharmacological characterization of a novel investigational antimuscarinic drug, fesoterodine, in vitro and in vivo. BJU Int. 2008;101:1036e1042. 8. Maruyama S, Oki T, Otsuka A, et al. Human muscarinic receptor binding characteristics of antimuscarinic agents to treat overactive bladder. J Urol. 2006;175:365e369. 9. Ito Y, Oyunzul L, Seki M, et al. Quantitative analysis of the loss of muscarinic receptors in various peripheral tissues in M1-M5 single subtype knockout mice. Br J Pharmacol. 2009;156:1147e1153. 10. Yoshida A, Fujino T, Maruyama S, et al. The forefront for novel therapeutic agents based on the pathophysiology of lower urinary tract dysfunction: bladder selectivity based on in vivo drug-receptor binding characteristics of
Please cite this article as: Yamada S et al., Direct in vitro and in vivo demonstration of muscarinic receptor binding by the novel radioligand, [3H] 5-hydroxymethyltolterodine, in the bladder and other tissues of rats, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.12.004
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Please cite this article as: Yamada S et al., Direct in vitro and in vivo demonstration of muscarinic receptor binding by the novel radioligand, [3H] 5-hydroxymethyltolterodine, in the bladder and other tissues of rats, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.12.004