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1140 Biol. Pharm. Bull. 44, 1140–1150 (2021) Vol. 44, No. 8 Regular Article

Inhibitory Effects of on the Contractile Response to Acetylcholine in Rat Urinary Bladder Smooth Muscles Keisuke Obara,*,a Yuka Matsuoka,a Naoya Iwata,a Yukako Abe, a Yohei Ikegami,a Nanako Shioda,a Yume Hattori,a Shoko Hamamatsu,a Kento Yoshioka, a Fumiko Yamaki,a,b Kazuhiro Matsuo,c Takashi Yoshio,c and Yoshio Tanakaa a Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Toho University; 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan: b Department of Pharmacy, Faculty of Pharmacy, Musashino University; 1–1–20 Shinmachi, Nishitokyo, Tokyo 202–8585, Japan: and c Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Toho University; 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan. Received April 24, 2021; accepted May 27, 2021

The clinical applications of antipsychotics for symptoms unrelated to , such as behavioral and psychological symptoms, in patients with Alzheimer’s disease, and the likelihood of doctors prescrib- ing antipsychotics for elderly people are increasing. In elderly people, drug-induced and aging-associated urinary disorders are likely to occur. The most significant factor causing drug-induced urinary disorders is a decrease in urinary bladder smooth muscle (UBSM) contraction induced by the anticholinergic action of therapeutics. However, the anticholinergic action-associated inhibitory effects of antipsychotics on UBSM contraction have not been sufficiently assessed. In this study, we examined 26 clinically available antipsychot- ics to determine the extent to which they inhibit acetylcholine (ACh)-induced contraction in rat UBSM to predict the drugs that should not be used by elderly people to avoid urinary disorders. Of the 26 antipsychot- ics, six (, (), (a thiepine), , , (multi-acting receptor targeted antipsychotics (MARTAs))) competitively inhibited ACh-induced contractions at concentrations corresponding to clinically significant doses. Further, 11 antipsychotics (per- phenazine, fluphenazine, (phenothiazines), , , , (), (a ), , (– antagonists; SDAs), and (a MARTA)) significantly suppressed ACh-induced contraction; however, suppression occurred at concentrations substantially exceeding clinically achievable blood levels. The re- maining nine antipsychotics ( (a ), , , , (), , (SDAs), , and (dopamine partial agonists)) did not inhibit ACh-induced contractions at concentrations up to 10 5 M. These findings suggest that chlor- , levomepromazine, zotepine, olanzapine, quetiapine, and clozapine should be avoided by elderly people with urinary disorders. Key words antipsychotics; rat urinary bladder smooth muscle; anticholinergic effect; urinary disorder

INTRODUCTION cause of age-related changes in and pharma- codynamics, current medical conditions, polypharmacy, and Antipsychotics are mainly utilized to treat schizophrenia; potential drug interactions.13) The U.S. Food and Drug Ad- however, in recent years, its application has extended to other ministration (FDA) issued an advisory and a subsequent black mental disorders, such as depression, behavioral and psycho- box warning regarding the risks of atypical use logical symptoms of dementia (BPSD), and gastrointestinal among elderly patients with dementia.14) Owing to the issu- symptoms associated with chemotherapy.1–5) As a result, the ance of this black box warning, many clinical studies on the prescription of antipsychotics has been increasing world- serious side effects of antipsychotics have been carried out.13) wide.6–8) Owing to the increase in human life span, elderly However, little information is available on the side effects that patients with mental disorders, Alzheimer's dementia, and clearly lead to poorer QOL for patients but are not recognized cancer have also been increasing.9–11) Thus, the prescription as severe. Urinary disorders are one of the such side effects of antipsychotics for elderly patients is expected to rapidly induced by antipsychotics.15) In elderly people, drug-induced increase in the future. In support of this speculation, the pre- as well as aging-associated urinary disorders are likely to scription rate of antipsychotics for elderly patients in Japan occur.16) Drug-induced urinary disorders affect patient adher- was found to increase by ≥10% between 2006 and 2012.6) ence,17) and thus should be avoided. In general, elderly patients are more likely to develop ad- The most significant factor for drug-induced urinary disor- verse effects than middle-age patients are. For example, elder- ders is a decrease in urinary bladder smooth muscle (UBSM) ly patients are more prone to antipsychotic-induced movement contraction due to anticholinergic actions.18) Among antipsy- disorders12) and are at an increased risk of adverse events due chotics, there is no significant difference in the rate of side to the intake of atypical antipsychotics (serotonin–dopamine effects caused by anticholinergic actions between patients antagonists (SDAs), multi-acting receptor targeted antipsychot- taking typical antipsychotics and those taking atypical anti- ics (MARTAs), dopamine partial agonists (DPAs)); this is be- psychotics.15) However, little information is available on which

* To whom correspondence should be addressed. e-mail: [email protected] © 2021 The Pharmaceutical Society of Japan Vol. 44, No. 8 (2021) Biol. Pharm. Bull. 1141 antipsychotics inhibit UBSM contractility through their pos- to the bath medium. After a 30 min equilibration period, to sible anticholinergic actions. produce sustained contractions, the strip was contracted with In this study, we determined the potential inhibitory effects 80 mM high-KCl solution (containing atropine, , and of 26 clinically available antipsychotics on acetylcholine ) comprising (mM): NaCl, 79.6; KCl, 80; CaCl2, (ACh)-induced contractions in rat UBSM. We then compared 2.2; MgCl2, 2.1; NaHCO3, 5.9; and , 2.8. When the the drug concentrations required to produce inhibitory effects contractile response reached a steady state, each antipsychotic against ACh-induced contractions with their clinically achiev- was incrementally applied to the bath medium. At the end of able concentration ranges to predict the antipsychotics that the experiment, the UBSM preparations were treated with should be avoided in elderly patients with urinary disorders. verapamil (10−5 M). We considered that rat UBSM is suitable for evaluating the Drugs The 26 antipsychotics tested in this study were: le- anticholinergic potencies of drugs for the following reasons: 1) vomepromazine maleate, fluphenazine dimaleate, haloperidol, ACh-induced contractions in both rat and human UBSMs are sultopride hydrochloride, and perospirone hydrochloride dihy- mediated through M3 receptors, although the expression level drate (FUJIFILM Wako Pure Chemical Corporation, Osaka, of M2 receptors is higher than that of M3 receptors in their Japan); chlorpromazine hydrochloride, prochlorperazine di- UBSMs19); 2) The binding properties of various muscarinic maleate, pipamperone, (±)-sulpiride, tiapride hydrochloride, receptor antagonists (such as M1 , pirenz- paliperidone, blonanserin, olanzapine, and aripiprazole (Tokyo epine; M2 receptor antagonist, AF-DX 116; and M3 receptor Chemical Industry Co., Ltd., Tokyo, Japan); , antagonist, darifenacin) to rat UBSM are almost identical spiperone hydrochloride, quetiapine hemifumarate, clozapine, to their binding properties to human UBSM, although the brexpiprazole, and pimozide (Cayman Chemical, Ann Arbor, binding properties of β3-adrenoceptor agonists/antagonists MI, U.S.A.); bromperidol (MedChemExpress Co., Ltd., Mon- to rat UBSM differ from their binding properties to human mouth Junction, NJ, U.S.A.); timiperone (Toronto Research UBSM.20) Chemicals, Toronto, ON, Canada); nemonapride, and zotepine (Santa Cruz Biotechnology Inc., Dallas, TX, U.S.A.); risperi- MATERIALS AND METHODS done (Acros Organics, Geel, Belgium); and asenapine maleate (AdooQ BioScience LLC, Irvine, CA, U.S.A.). ACh chloride Animals Male Wistar rats (age, 8–10 weeks old; weight, was purchased from Daiichi Sankyo Co., Ltd. (Tokyo, Japan). 175–280 g; Japan SLC, Hamamatsu, Japan) were housed under Atropine sulfate, indomethacin, propranolol hydrochloride, controlled conditions (21–22°C, relative air humidity 50 ± 5%) and (±)-verapamil were purchased from Sigma-Aldrich Co. and a fixed 12–12 h light–dark cycle (08 : 00–20 : 00), with food (St. Louis, MO, U.S.A.). All other chemicals were commer- and water available ad libitum. This study was approved by cially available and of reagent grade. the Toho University Animal Care and Use Committee (Ap- and clozapine were dissolved in 0.1 N HCl to proval Nos. 17-53-294, 18-54-294, 19-55-294) and was con- create a stock solution of 2 × 10−2 M. Thereafter, the stock so- ducted in accordance with the guidelines of the Laboratory lutions were further diluted with distilled water to the desired Animal Center of Faculty of Pharmaceutical Sciences, Toho concentrations. The other antipsychotics were dissolved and University. diluted in pure DMSO. Indomethacin was dissolved in etha- Assessment of the Effects of Antipsychotics on ACh-In- nol to create a stock solution of 10−2 M. All other drugs were duced UBSM Contraction The effects of antipsychotics on prepared as aqueous stock solutions and diluted with distilled UBSM contraction were assessed as previously described.21,22) water. Briefly, isolated rat UBSM strips were equilibrated under a Data Analysis and Statistics CRCs for ACh-induced 0.5 g resting tone for 20 min in a 20 mL organ bath contain- contractions and Schild plot analysis of anxiolytics versus ™ ing Locke–Ringer solution equilibrated with 95% O2 and 5% ACh concentrations were performed using GraphPad Prism CO2 at 32 ± 1°C; the solution was comprised of the following (GraphPad Software Inc., San Diego, CA, U.S.A.), as previ- 21,22) (mM): NaCl, 154; KCl, 5.6; CaCl2, 2.2; MgCl2, 2.1; NaHCO3, ously described. All values are presented as mean ± stan- 5.9; and glucose, 2.8. The UBSM preparation was contracted dard error of the mean (S.E.M.) or mean with 95% confidence using 10−4 M ACh at least three times at 20 min intervals intervals (CIs) for different numbers (n) of preparations. (preliminary procedures). After a 30 min equilibration period, GraphPad Prism™ was used for the statistical analyses. Differ- ACh was incrementally applied to the bath medium until a ences among the CRCs were evaluated using post hoc Šidák’s maximum response was obtained; this contractile response test after two-way ANOVA. Statistical significance was set at was recorded twice at 30 min intervals. Following this pro- p < 0.05. cedure, the concentration–response curves (CRCs) for ACh were plotted after pre-incubation with different concentrations RESULTS (3 × 10−7 to 10−5 M) of each tested antipsychotic or verapamil (10−5 M) for 30 min. When dimethyl sulfoxide (DMSO) was Effects of Antipsychotics on ACh-Induced used as a drug solvent, the experiment was conducted with a Contractions Figure 1 shows the effects of phenothiazine DMSO concentration of 0.5% in the bath solution, including antipsychotics (3 × 10−7 to 10−5 M) on the CRCs of ACh. All the control experiment. All experiments were carried out in tested phenothiazine antipsychotics (chlorpromazine, Fig. 1Aa; the presence of indomethacin (3 × 10−6 M). levomepromazine, Fig. 1Ba; perphenazine, Fig. 1Ca; fluphen- Assessment of the Effects of Antipsychotics on UBSM azine, Fig. 1Da; and prochlorperazine, Fig. 1Ea) were found to Contraction Induced by High-KCl Locke–Ringer Solu- inhibit ACh-induced contractions. In the concentration range tion After the preliminary procedures, atropine (10−6 M), of 3 × 10−7 to 10−5 M, the slopes of the regression lines in the (10−6 M), and propranolol (10−7 M) were added Schild plot of chlorpromazine (Fig. 1Ab) and levomeproma- 1142 Biol. Pharm. Bull. Vol. 44, No. 8 (2021)

Fig. 1. Effects of Phenothiazine Antipsychotics (3 × 10−7 to 10−5 M) on the ACh-Induced Contractions in Rat UBSM Aa–Ea: Effects of chlorpromazine (Chl, Aa), levomepromazine (Lev, Ba), perphenazine (Perp, Ca), fluphenazine (Flu, Da), and prochlorperazine (Pro, Ea) on the con- centration–response curves of ACh-induced contractions. Data are presented as mean ± S.E.M. (n = 5). Ab–Eb: Schild plot analysis of Chl (Ab), Lev, (Bb), Prep (Cb), Flu (Db), and Pro (Eb) vs. ACh. The slope and pA2 values are presented as means with 95% confidence intervals (CIs). ACh, acetylcholine; UBSM, urinary bladder smooth muscle. zine (Fig. 1Bb) vs. ACh were 1.09 (95% CIs: 0.88–1.31, 2Aa), bromoperidol (Fig. 2Ba), timiperone (Fig. 2Ca), and spi- n = 5, chlorpromazine) and 0.91 (95% CI: 0.72–1.10, n = 5, perone (Fig. 2Da) inhibited ACh-induced contractions. In the levomepromazine); these values were not significantly differ- concentration range of 3 × 10−6 to 10−5 M, the slopes of the re- ent from unity. In the concentration range of 10−6 to 10−5 M, gression lines in the Schild plot of haloperidol (Fig. 2Ab) and the slopes of the regression lines in the Schild plot of perphen- timiperone (Fig. 2Cb) vs. ACh were 1.14 (95% CIs: 0.18–2.10, azine (Fig. 1Cb), fluphenazine (Fig. 1Db), and prochlorpera- n = 5, haloperidol) and 0.96 (95% CIs: 0.28–1.63, n = 5, timi- zine (Fig. 1Eb) vs. ACh were 0.92 (95% CIs: 0.57–1.27, n = 5, perone), respectively; these values were not significantly dif- perphenazine), 1.01 (95% CIs: 0.69–1.33, n = 5; fluphenazine), ferent from unity. In the concentration range of 10−6–10−5 M, and 0.91 (95% CIs: 0.62–1.19, n = 5, prochlorperazine); these the slope of the regression lines in the Schild plot of brom- values were not significantly different from unity. Therefore, peridol (Fig. 2Bb) vs. ACh was 0.96 (95% CI: 0.33–1.60, these phenothiazine antipsychotics were found to competi- n = 5); this value was not significantly different from unity. tively antagonize ACh in the above concentration ranges. The Therefore, these butyrophenone antipsychotics competitively pA2 values of chlorpromazine, levomepromazine, perphen- antagonized ACh in the above concentration ranges. The pA2 azine, fluphenazine, and prochlorperazine were 6.43 (95% CIs: values of haloperidol, timiperone, and bromperidol were 5.43 6.28–6.63, n = 5), 6.48 (95% CIs: 6.32–6.72, n = 5), 6.18 (95% (95% CIs: 5.21–6.51, n = 5), 5.88 (95% CIs: 5.59–7.44, n = 5), CIs: 5.95–6.63, n = 5), 5.82 (95% CIs: 5.68–6.04, n = 5), and and 5.83 (95% CIs: 5.56–6.62, n = 5), respectively. 6.17 (95% CIs: 5.97–6.50, n = 5), respectively. In contrast, the slope of the regression line in the Schild Effects of Phenothiazine Antipsychotics on ACh-Induced plot of spiperone (Fig. 2Db) vs. ACh was 0.47 (95% CIs: Contractions Figure 2 shows the effects of butyrophenone 0.24–0.70, n = 5); this value was significantly less than unity. antipsychotics (3 × 10−7 to 10−5 M) on the CRCs of ACh. Of Therefore, spiperone did not competitively antagonize ACh in the tested butyrophenone antipsychotics, haloperidol (Fig. the above concentration ranges. Vol. 44, No. 8 (2021) Biol. Pharm. Bull. 1143

Fig. 2. Effects of Butyrophenone Antipsychotics (3 × 10−7 to 10−5 M) on the ACh-Induced Contraction of Rat UBSM Aa–E: Effects of haloperidol (Hal, Aa), bromperidol (Bro, Ba), timiperone (Tim, Ca), spiperone (Spi, Da), and pipamperone (Pip, E) on the concentration–response curves of ACh-induced contractions. Data are presented as mean ± S.E.M. for n = 5. Ab–Db: Schild plot analysis of Hal (Ab), Bro, (Bb), Tim (Cb), and Spi (Db) vs. ACh. The slope and pA2 values are presented as means with 95% confidence intervals (CIs). ACh, acetylcholine; UBSM, urinary bladder smooth muscle.

Fig. 3. Effects of Antipsychotics (A–D), a Thiepine Antipsychotic (E), and a Diphenylbutylpiperidine Antipsychotic (F) (3 × 10−7 to 10−5 M) on the ACh-Induced Contraction of Rat UBSM A–Fa: Effects of sulpiride (Sulp, A), sultopride (Sult, B), tiapride (Tia, C), nemonapride (Nem, D), zotepine (Zot, Ea), and pimozide (Pim, Fa) on the concentration– response curves of ACh-induced contractions. Data are presented as mean ± S.E.M. (n = 5). Eb, Fb: Schild plot analysis of Zot (Eb) and Pim (Fb) vs. ACh. The slope and pA2 values are presented as means with 95% confidence intervals (CIs). ACh, acetylcholine; UBSM, urinary bladder smooth muscle.

Pipamperone (Fig. 2E) was not found to affect the CRCs of fect the CRCs of ACh in the concentration range of 3 × 10−7 ACh in the concentration range of 3 × 10−7 to 10−5 M. to 10−5 M. Effects of Benzamide Antipsychotics on ACh-Induced Effects of Zotepine and Pimozide on ACh-Induced Con- Contractions Figures 3A–D show the effects of benzamide tractions Figures 3E and 3F show the effects of zotepine (a antipsychotics (3 × 10−7 to 10−5 M) on the CRCs of ACh. The thiepine antipsychotic) and pimozide (a diphenylbutylpiperi- tested benzamide antipsychotics (sulpiride, Fig. 3A; sultopride, dine antipsychotic) (3 × 10−7 to 10−5 M) on the CRCs of ACh. Fig. 3B; thiapride, Fig. 3C; nemonapride, Fig. 3D) did not af- Both zotepine (Fig. 3Ea) and pimozide (Fig. 3Fa) inhibited 1144 Biol. Pharm. Bull. Vol. 44, No. 8 (2021)

Fig. 4. Effects of Serotonin–Dopamine Antagonists (SDAs; A–D) and Dopamine Partial Agonists (DPAs; E, F) (3 × 10−7 to 10−5 M) on ACh-Induced Contraction of Rat UBSM A–F: Effects of risperidone (Ris, A), paliperidone (Pal, B), perospirone (Pero, Ca), blonanserin (Blo, Da), aripiprazole (Ari, E), and brexpiprazole (Bre, F) on the concen- tration–response curves of ACh-induced contractions. Data are presented as mean ± S.E.M. (n = 5). Cb, Db: Schild plot analysis of Pero (Cb) and Blo (Db) vs. ACh. The slope and pA2 values are presented as means with 95% confidence intervals (CIs). ACh, acetylcholine; UBSM, urinary bladder smooth muscle.

ACh-induced contractions. The slopes of the regression lines Effects of MARTAs on ACh-Induced UBSM Contrac- in the Schild plot of zotepine (Fig. 3Eb, 3 × 10−7 to 10−5 M) tions Figure 5 shows the effects of MARTAs (3 × 10−7 to and pimozide (Fig. 3Eb, 3 × 10−7 to 3 × 10−6 M) vs. ACh were 10−5 M) on the CRCs of ACh. All tested MARTAs (olanzap- 0.98 (95% CIs: 0.76–1.19, n = 5, zotepine) and 1.01 (95% CIs: ine, Fig. 5Aa; quetiapine, Fig. 5Ba; clozapine, Fig. 5Ca; and 0.70–1.32, n = 5, pimozide), respectively; these values were asenapine, Fig. 5D) inhibited ACh-induced contractions. In not significantly different from unity. Therefore, both zotepine the concentration range of 3 × 10−6–10−5 M, the slopes of the and pimozide competitively antagonized ACh in the above regression lines in the Schild plot of olanzapine (Fig. 5Ab) and concentration ranges. The pA2 values of zotepine and pimo- clozapine (Fig. 5Cb) vs. ACh were 0.97 (95% CIs: 0.67–1.28, zide were 6.23 (95% CIs: 6.09–6.42, n = 5) and 7.04 (95% CIs: n = 5, olanzapine) and 0.93 (95% CIs: 0.33–1.53, n = 5, clo- 6.77–7.52, n = 5), respectively. zapine), respectively; these values were not significantly dif- Effects of SDAs on ACh-Induced UBSM Contractions ferent from unity. In the concentration range of 3 × 10−6 to Figures 4A–D show the effects of SDAs (3 × 10−7 to 10−5 M) 10−5 M, the slope of the regression lines in the Schild plot of on the CRCs of ACh. Of the tested SDAs, perospirone (Fig. quetiapine (Fig. 5Bb) vs. ACh was 0.90 (95% CIs: 0.82–1.30, 4Ca) and blonanserin (Fig. 4Da) inhibited ACh-induced con- n = 5); this value was not significantly different from unity. tractions. The slopes of the regression lines in the Schild plot Therefore, these MARTAs competitively antagonize ACh in −6 −5 of perospirone (Fig. 4Cb, 3 × 10 to 10 M) and blonanserin the above concentration range. The pA2 values of olanzap- (Fig. 4Db, 3 × 10−7 to 10−5 M) vs. ACh were 1.05 (95% CIs: ine, quetiapine, and clozapine were 7.13 (95% CIs: 6.91–7.54, 0.26–1.84, n = 5, perospirone) and 1.08 (95% CIs: 0.81–1.34, n = 5), 6.29 (95% CIs: 5.94–7.26, n = 5), and 7.19 (95% CIs: n = 5, blonanserin), respectively; these values were not sig- 6.80–8.87, n = 5), respectively. nificantly different from unity. Therefore, these SDAs were At ≥10−6 M, the slopes of the regression line in the Schild found to competitively antagonize ACh in the above concen- plot of olanzapine (Fig. 5Ab) and clozapine (Fig. 5Cb) vs. ACh tration range. The pA2 values of perospirone and blonanserin were 0.67 (95% CIs: 0.48–0.86, n = 5, olanzapine) and 0.51 were 5.51 (95% CIs: 5.32–6.38, n = 5) and 6.48% (95% CIs: (95% CIs: −0.14–1.17, n = 5, clozapine), respectively; these 6.29–6.77, n = 5), respectively. values were less than unity. Further, in the concentration Risperidone (Fig. 4A) and paliperidone (Fig. 4B) were not range of 3 × 10−7 to 3 × 10−6 M, the slope of the regression found to affect the CRCs of ACh in the concentration range of line in the Schild plot of quetiapine (Fig. 5Bb) vs. ACh was 3 × 10−7–10−5 M. 0.64 (95% CIs: 0.43–0.84, n = 5); this value was significantly Effects of DPAs on ACh-Induced UBSM Contrac- less than unity. tions Figures 4E and F show the effects of DPAs (3 × 10−7 In contrast, asenapine (Fig. 5D) inhibited ACh-induced con- to 10−5 M) on the CRCs of ACh. Aripiprazole (Fig. 4E) and tractions only at 10−5 M. However, asenapine did not shift the brexpiprazole (Fig. 4F) did not affect the CRCs of ACh in the CRCs of ACh to the right. Instead, it inhibited the maximum concentration range of 3 × 10−7 to 10−5 M. response to ACh. The maximum response of CRCs of ACh Vol. 44, No. 8 (2021) Biol. Pharm. Bull. 1145

Fig. 5. Effects of Multi-Acting Receptor Targeted Antipsychotics (MARTAs) (3 × 10−7 to 10−5 M) on the ACh-Induced Contraction of Rat UBSM Aa–D: Effects of olanzapine (Ola, Aa), quetiapine (Que, Ba), clozapine (Clo, Ca), and asenapine (Ase, D) on the concentration–response curves of ACh-induced contrac- tions. Data are presented as mean ± S.E.M. (n = 5). Ab–Cb: Schild plot analysis of Ola (Ab), Que (Bb), and Clo (Cb) vs. ACh. The slope and pA2 values are presented as means with 95% confidence intervals (CIs). ACh, acetylcholine; UBSM, urinary bladder smooth muscle.

(control = 100%, 3 × 10−3 M) was suppressed to 68.0 ± 7.3% (n = 5) by pretreatment with asenapine (10−5 M). Effect of Antipsychotics on UBSM Contraction Induced by High-KCl Locke–Ringer Solution Among the tested an- tipsychotics, 18 significantly inhibited ACh-induced contrac- tions. However, some of these antipsychotics did not display competitive antagonism with ACh. A possible mechanism underlying the non-competitive inhibition of ACh-induced contraction could be the inhibition of voltage-dependent L- type Ca2+ channels (VDCCs). Accordingly, this possibility was pharmacologically examined. First, we investigated the effects of verapamil on ACh- induced contractions to elucidate whether the contractions were mediated via VDCCs. The following results were ob- tained from Fig. 6: the maximum response of the CRC of ACh (100%, 3 × 10−3 M ACh) was significantly inhibited to Fig. 6. Effect of Verapamil (10−5 M) on ACh-Induced Contraction of 52.0 ± 9.1% by verapamil (10−5 M) (n = 5). Thus, Ca2 + influx Rat UBSM through VDCCs was demonstrated to be responsible for ACh- Data are presented as mean ± S.E.M. (n = 5). * p < 0.05, ** p < 0.01 vs. the control (post hoc Šidák’s test after two-way ANOVA). ACh, acetylcholine; UBSM, urinary induced contractions in smooth muscle preparations. bladder smooth muscle. We proceeded to determine the effects of 18 antipsychot- ics on 80 mM KCl-induced contraction to elucidate whether DISCUSSION these antipsychotics could inhibit VDCC-mediated contraction (Table 1). Of the tested drugs, olanzapine hardly suppressed In the present study, we sought to investigate the possible the contractions induced by the 80 mM KCl solution, whereas inhibitory effects of 26 clinically available antipsychotics on 17 antipsychotics, besides olanzapine, suppressed these ACh-induced contractions in rat UBSM to predict the drugs contractions at 10−5 M by approximately 20–50% (Table 1). that should not be used by the elderly population to avoid Pimozide suppressed 80 mM KCl-induced contraction by ap- urinary disorders. Of the tested antipsychotics, two typical an- proximately 10%, even at a concentration of 10−6 M (Table 1). tipsychotics (chlorpromazine and levomepromazine) and four As the remaining contractions after each drug treatment atypical antipsychotics (zotepine, olanzapine, quetiapine, and were completely suppressed by verapamil (10−5 M) (data not clozapine) significantly inhibited ACh-induced contractions at shown), the contractions induced by 80 mM KCl solution were clinically significant doses. Such findings suggest that these elicited via VDCCs. six antipsychotics should be avoided in elderly people with 1146 Biol. Pharm. Bull. Vol. 44, No. 8 (2021)

Table 1. Effects of Antipsychotics (10−7 to 10−5 M) on 80 mM KCl Solution-Induced Contractions in Rat Urinary Bladder Smooth Muscle

Inhibition of high-K-induced contraction (%) Category/Generic name 10−7 M 10−6 M 10−5 M

Phenothiazine antipsychotics Chlorpromazine 0.1 ± 0.2 3.1 ± 1.7 38.0 ± 8.5 Levomepromazine 1.3 ± 1.9 4.3 ± 1.8 40.8 ± 2.5 Perphenazine 0.7 ± 0.9 2.2 ± 0.7 20.2 ± 3.8 Fluphenazine 0.6 ± 0.8 4.2 ± 2.8 27.7 ± 5.8 Prochlorperazine 0.7 ± 0.7 2.0 ± 0.7 25.2 ± 2.0 Butyrophenone antipsychotics Haloperidol 1.1 ± 1.6 2.4 ± 1.1 46.3 ± 6.0 Bromperidol 0.7 ± 0.4 2.1 ± 0.6 41.9 ± 5.3 Timiperone 0.0 ± 0.1 2.1 ± 0.7 42.7 ± 10.7 Spiperone 2.7 ± 2.8 4.4 ± 1.6 26.1 ± 4.4 Thiepin antipsychotic Zotepine 1.0 ± 0.9 2.1 ± 0.5 22.3 ± 4.4 Diphenylbutylpiperidine antipsychotic Pimozide 2.2 ± 1.9 10.9 ± 2.4 49.6 ± 4.7 Serotonin–dopamine antagonists (SDAs) Perospirone 1.5 ± 2.2 3.5 ± 1.0 25.5 ± 4.9 Blonanserin 1.8 ± 1.8 8.5 ± 6.1 36.8 ± 10.7 Multi-acting receptor-targeted antipsychotics (MARTAs) Olanzapine 0.4 ± 1.1 1.1 ± 0.7 7.3 ± 2.7 Quetiapine 1.1 ± 0.9 1.7 ± 0.7 18.9 ± 4.6 Clozapine 0.4 ± 1.1 0.7 ± 0.7 32.2 ± 3.7 Asenapine 1.2 ± 1.1 3.8 ± 0.7 56.4 ± 6.3

Data are presented as mean ± S.E.M. (n = 5).

25) urinary disorders. value for M5 receptor ; however, we do not have any reason- Chlorpromazine/Levomepromazine (Phenothiazines), able explanations for this. Nonetheless, the pA2 value (6.29) of Zotepine (a Thiepine), Olanzapine/Quetiapine/Clozapine quetiapine was in its plausible blood concentration levels and (MARTAs) Chlorpromazine, levomepromazine, zotepine, was expressed as the minus logarithm (−log M: 5.92–7.95). olanzapine, and clozapine inhibited ACh-induced contrac- This finding suggests that clinically applied quetiapine re- tions in a competitive antagonistic manner. The calculated pA2 duces UBSM contractility and urinary disorders by inhibiting values were consistent with the pKi values obtained from our muscarinic receptors. binding study where [3H]N-methyl scopolamine (NMS) was In the concentration range of ≥ 10−6 M, olanzapine and employed in the mouse cerebral cortex23) (Table 2). Therefore, clozapine did not show competitive inhibitory effects on ACh we concluded that the inhibition of ACh-induced UBSM con- as the slope of the regression line in the Schild plot was sig- tractions by these antipsychotics was due to their anticholiner- nificantly less than unity (0.67 and 0.51, respectively). The ap- gic effects. The pA2 values of the antipsychotics were found to parent non-competitive effects of these antipsychotics against be comparable with their plausible blood concentration levels, ACh could be explained by their different affinities for the which are presented as minus logarithm values, or larger M2/M3 receptors. Although UBSM expresses both the M2/M3 than the plausible blood concentration levels (clozapine). This receptors, the receptor subtype responsible for UBSM con- 26) finding indicates that these antipsychotics can reduce UBSM tractions is M3. Both olanzapine and clozapine have higher contractility by inhibiting muscarinic receptors, thereby in- affinity for M3 than M2. The pKi values for olanzapine were ducing urinary functional disorders within their clinically 7.89/7.32 (M 3/M2) while those for clozapine were 8.15/7.32 25) applied antipsychotic dose ranges. The pA2 values/pKi values/ (M3/M2). Based on these previous results, we infer the fol- plausible blood concentration levels (expressed as minus loga- lowing for the muscarinic receptor subtypes responsible for rithm values) of each drug were (Table 2): 6.43/6.40/5.85–7.55 inducing inhibition vs. ACh-induced contraction by olanzapine (chlorpromazine); 6.48/6.22/6.21–7.35 (levomepromazine); and clozapine: in the low concentration ranges (3 × 10−7 to −6 6.23/6.21/6.14–7.04 (zotepine); 7.13/6.85/6.37–7.38 (olanzapine); 10 M), both antipsychotics selectively bind to M3 and pro- and 7.19/6.83/5.23–6.30 (clozapine). duce competitive antagonistic effects against ACh and inhibi- In the concentration range of 3 × 10−6 to 10−5 M, quetiapine tion of ACh-induced UBSM contraction. In contrast, at higher −6 competitively inhibited ACh-induced contractions, and its pA2 concentrations (≥ 10 M), both antipsychotics could bind value was calculated to be 6.29. This value was one order of to M2, which is not responsible for inducing ACh-induced magnitude larger than the calculated pKi value (5.31) (Table contraction, and thus, did not show apparently competitive 2), but consistent with the pKi value for the muscarinic M3 antagonistic effects against ACh. receptor (6.65) obtained from the binding experiment using In the low concentration range (3 × 10−7 to 3 × 10−6 M), 3 24) 3 [ H] NMS. Our calculated pKi value for [ H] NMS in the quetiapine did not exhibit any competitive inhibitory effects mouse cerebral cortex corresponded to the quetiapine’s pKi against ACh, as the slope of the regression line in the Schild Vol. 44, No. 8 (2021) Biol. Pharm. Bull. 1147

Table 2. Comparison of the pA2 Values of Drugs vs. ACh, Their pKi Values vs. Muscarinic Receptor, and the Clinically Achievable Blood Concentra- tion of Antipsychotics

pK values Clinically achievable blood Category/Generic name pA values vs. ACh i Ref. 2 vs. muscarinic receptor concentration (−log M)

Phenothiazine antipsychotics Chlorpromazine 6.43 (6.28–6.63) 6.40 ± 0.08 5.85–7.55 37) Levomepromazine 6.48 (6.32–6.72) 6.22 ± 0.07 6.21–7.35 37) Perphenazine 6.18 (5.95–6.63) <5.39 7.43–9.00 37) Fluphenazine 5.82 (5.68–6.04) <5.39 7.59–8.87 37) Prochlorperazine 6.17 (5.97–6.50) 5.68 ± 0.14 7.87–9.55 38) Butyrophenone antipsychotics Haloperidol 5.43 (5.21–6.51) <5.39 6.19–7.90 37) Bromperidol 5.83 (5.56–6.62) <5.39 7.25–8.28 39) Timiperone 5.88 (5.59–7.74) 5.39 ± 0.09 7.62–8.06 40) Spiperone — <5.39 7.88* 41) Pipamperone — <5.39 6.22 42) Benzamide antipsychotics Sulpiride — <5.39 5.48–6.69 43) Sultopride — <5.39 5.12–7.04 43) Tiapride — <5.39 5.25–5.55 44) Nemonapride — <5.39 8.44–9.33** 45) Thiepin antipsychotic Zotepine 6.23 (6.09–6.42) 6.21 ± 0.03 6.14–7.04 46) Diphenylbutylpiperidine antipsychotic Pimozide 7.04 (6.77–7.52) 5.64 ± 0.09 7.39–8.97 47) Serotonin–dopamine antagonists (SDA) Risperidone — <5.39 6.53–8.61 47) Paliperidone — <5.39 6.70–7.53 48) Perospirone 5.51 (5.32–6.38) <5.39 7.82–9.70 49) Blonanserin 6.48 (6.32–6.72) 6.09 ± 0.13 8.61–9.26 50) Dopamine partial agonists (DPAs) Aripiprazole — <5.39 5.71–7.65 47) Brexpiprazole — <5.39 6.11–6.83 51) Multi-acting receptor-targeted antipsychotics (MARTA) Olanzapine 7.13 (6.91–7.54) 6.85 ± 0.14 6.37–7.38 48) Quetiapine 6.29 (5.94–7.26) 5.31 ± 0.12 5.92–7.95 52) Clozapine 7.19 (6.80–8.87) 6.83 ± 0.09 5.23–6.30 53) Asenapine — <5.39 7.70–8.53 54)

3 *Data from rabbit. **Total concentration of nemonapride and its metabolites. pKi values vs. the muscarinic receptor were obtained from our previous report using [ H]N- methyl scopolamine in the mouse cerebral cortex.23) Ref.: references from which the clinically achievable blood concentrations were obtained. plot was significantly less than unity (0.64). Such finding chotics on ACh-induced contraction were mainly due to their might be explained by the different affinities of quetiapine anticholinergic effects. The pA2/pKi values of each antipsy- between M2 and M3: quetiapine has a higher affinity for M2 chotic were (Table 2): 6.17/5.68 (prochlorperazine), 5.88/5.39 24) (pKi = 6.20) than M3 (pKi = 5.88). Therefore, in the low (timiperone), and 6.48/6.09 (blonanserin). −7 −6 concentration ranges (3 × 10 to 3 × 10 M), quetiapine is In our previous study, the pKi values could not be calculated speculated to bind to non-contractile M2 and M3, and thus for perphenazine, fluphenazine, haloperidol, and bromperidol 23) exert apparently non-competitive effects against ACh. In con- (pKi < 5.39). However, the pA2 values of perphenazine (6.18) trast, at higher concentrations (3 × 10−6 to 10−5 M), where the and fluphenazine (5.82) were found to be approximately com- binding of quetiapine to M2 becomes saturated, quetiapine parable to their pKi values (5.31, perphenazine; and 5.39, flu- displayed competitive antagonism against ACh on contractile phenazine), which were calculated from the binding study for 3 27) M3, and thus, produced inhibitory effects against ACh-induced rat cerebral cortex using [ H] quinuclidinyl benzilate (QNB). contraction in a competitive manner. In addition, the pA2 value of haloperidol (5.43) was almost Prochlorperazine/Perphenazine/Fluphenazine (Phenothi- comparable to its pKi value (5.80) calculated from the bind- azines), Haloperidol/Bromperidol/Timiperone (Butyrophe- ing study for the rat submandibular gland using [3H] NMS.24) nones), Pimozide (a Diphenylbutylpiperidine), and Blonan- Therefore, the inhibitory effects of perphenazine, fluphen- serin (SDA) Prochlorperazine, timiperone, and blonanserin azine, and haloperidol on ACh-induced contraction could be suppressed ACh-induced contraction in a competitive fashion, mainly caused by their anticholinergic effects. We speculated and their calculated pA2 values were close to their pKi values that bromperidol inhibited ACh-induced contraction through obtained from our binding experiments23) (Table 2). As a re- the same mechanism (anticholinergic effects) as haloperidol sult, we concluded that the inhibitory effects of these antipsy- because bromperidol has a bromine atom that replaces the 1148 Biol. Pharm. Bull. Vol. 44, No. 8 (2021)

−5 chlorine atom possessed by haloperidol in its structure. induced contractions up to 10 M. However, their pKi values The pA2 value of pimozide (7.04) was one order of mag- (pKi<5.39) could not be obtained because of their marginal 3 23) nitude larger than the pKi value (5.64) obtained from our effects on [ H]NMS in the cerebral cortex. Sulpiride and binding experiments23) (Table 2) and the value (6.10) obtained aripiprazole did not suppress ACh-induced contractions in from binding experiments in the human brain using [3H]QNB guinea pig UBSM.36) Additionally, the plausible blood con- (6.10).28) These findings imply that pimozide inhibited ACh- centration levels of all drugs were found to be markedly lower induced contractions primarily through anticholinergic action- than 10−5 M (Table 2). Therefore, these antipsychotics were not unrelated mechanisms, despite exhibiting anticholinergic ac- found to cause anticholinergic action-mediated urinary disor- tions at higher concentrations. ders within the doses administered clinically.

In addition to pimozide, the pA2 values of most other anti- psychotics were slightly higher than their pKi values (Table 2). Acknowledgments The authors would like to thank Mr. Therefore, mechanisms besides those used to elucidate their Yunfeng Ban for his expert technical assistance. This study anticholinergic effects might be responsible for their inhibi- was partly supported by the Joint Research Grants of the Toho tory effects against ACh-induced contractions. One plausible University Faculty of Pharmaceutical Sciences (K.O.). mechanism could be the inhibition of VDCCs, which was supported by the evidence that these antipsychotics suppressed Conflict of Interest The authors declare no conflict of the depolarizing contraction induced by 80 mM KCl (Table interest. 1). 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