Inhibitory Effects of Antipsychotics on the Contractile Response to Acetylcholine in Rat Urinary Bladder Smooth Muscles

Abstract

The clinical applications of antipsychotics for symptoms unrelated to schizophrenia, such as behavioral and psychological symptoms, in patients with Alzheimer’s disease, and the likelihood of doctors prescribing 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 antipsychotics 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 antipsychotics, six (chlorpromazine, levomepromazine (phenothiazines), zotepine (a thiepine), olanzapine, quetiapine, clozapine (multi-acting receptor targeted antipsychotics (MARTAs))) competitively inhibited ACh-induced contractions at concentrations corresponding to clinically significant doses. Further, 11 antipsychotics (perphenazine, fluphenazine, prochlorperazine (phenothiazines), haloperidol, bromperidol, timiperone, spiperone (butyrophenones), pimozide (a diphenylbutylpiperidine), perospirone, blonanserin (serotonin–dopamine antagonists; SDAs), and asenapine (a MARTA)) significantly suppressed ACh-induced contraction; however, suppression occurred at concentrations substantially exceeding clinically achievable blood levels. The remaining nine antipsychotics (pipamperone (a butyrophenone), sulpiride, sultopride, tiapride, nemonapride (benzamides), risperidone, paliperidone (SDAs), aripiprazole, and brexpiprazole (dopamine partial agonists)) did not inhibit ACh-induced contractions at concentrations up to 10⁻⁵ M. These findings suggest that chlorpromazine, levomepromazine, zotepine, olanzapine, quetiapine, and clozapine should be avoided by elderly people with urinary disorders.

INTRODUCTION

Antipsychotics are mainly utilized to treat schizophrenia; however, in recent years, its application has extended to other mental disorders, such as depression, behavioral and psychological symptoms of dementia (BPSD), and gastrointestinal symptoms associated with chemotherapy.^1–5) As a result, the prescription of antipsychotics has been increasing worldwide.^6–8) Owing to the increase in human life span, elderly patients with mental disorders, Alzheimer's dementia, and cancer have also been increasing.^9–11) Thus, the prescription of antipsychotics for elderly patients is expected to rapidly increase in the future. In support of this speculation, the prescription rate of antipsychotics for elderly patients in Japan was found to increase by ≥10% between 2006 and 2012.⁶⁾

In general, elderly patients are more likely to develop adverse effects than middle-age patients are. For example, elderly patients are more prone to antipsychotic-induced movement disorders¹²⁾ and are at an increased risk of adverse events due to the intake of atypical antipsychotics (serotonin–dopamine antagonists (SDAs), multi-acting receptor targeted antipsychotics (MARTAs), dopamine partial agonists (DPAs)); this is because of age-related changes in pharmacokinetics and pharmacodynamics, current medical conditions, polypharmacy, and potential drug interactions.¹³⁾ The U.S. Food and Drug Administration (FDA) issued an advisory and a subsequent black box warning regarding the risks of atypical antipsychotic use among elderly patients with dementia.¹⁴⁾ Owing to the issuance of this black box warning, many clinical studies on the serious side effects of antipsychotics have been carried out.¹³⁾ However, little information is available on the side effects that clearly lead to poorer QOL for patients but are not recognized as severe. Urinary disorders are one of the such side effects induced by antipsychotics.¹⁵⁾ In elderly people, drug-induced as well as aging-associated urinary disorders are likely to occur.¹⁶⁾ Drug-induced urinary disorders affect patient adherence,¹⁷⁾ and thus should be avoided.

The most significant factor for drug-induced urinary disorders is a decrease in urinary bladder smooth muscle (UBSM) contraction due to anticholinergic actions.¹⁸⁾ Among antipsychotics, there is no significant difference in the rate of side effects caused by anticholinergic actions between patients taking typical antipsychotics and those taking atypical antipsychotics.¹⁵⁾ However, little information is available on which antipsychotics inhibit UBSM contractility through their possible anticholinergic actions.

In this study, we determined the potential inhibitory effects of 26 clinically available antipsychotics on acetylcholine (ACh)-induced contractions in rat UBSM. We then compared the drug concentrations required to produce inhibitory effects against ACh-induced contractions with their clinically achievable concentration ranges to predict the antipsychotics that should be avoided in elderly patients with urinary disorders. We considered that rat UBSM is suitable for evaluating the anticholinergic potencies of drugs for the following reasons: 1) ACh-induced contractions in both rat and human UBSMs are mediated through M₃ receptors, although the expression level of M₂ receptors is higher than that of M₃ receptors in their UBSMs¹⁹⁾; 2) The binding properties of various muscarinic receptor antagonists (such as M₁ receptor antagonist, pirenzepine; M₂ receptor antagonist, AF-DX 116; and M₃ receptor antagonist, darifenacin) to rat UBSM are almost identical to their binding properties to human UBSM, although the binding properties of β₃-adrenoceptor agonists/antagonists to rat UBSM differ from their binding properties to human UBSM.²⁰⁾

MATERIALS AND METHODS

Animals

Male Wistar rats (age, 8–10 weeks old; weight, 175–280 g; Japan SLC, Hamamatsu, Japan) were housed under controlled conditions (21–22°C, relative air humidity 50 ± 5%) and a fixed 12–12 h light–dark cycle (08 : 00–20 : 00), with food and water available ad libitum. This study was approved by the Toho University Animal Care and Use Committee (Approval Nos. 17-53-294, 18-54-294, 19-55-294) and was conducted in accordance with the guidelines of the Laboratory Animal Center of Faculty of Pharmaceutical Sciences, Toho University.

Assessment of the Effects of Antipsychotics on ACh-Induced UBSM Contraction

The effects of antipsychotics on UBSM contraction were assessed as previously described.^21,22) Briefly, isolated rat UBSM strips were equilibrated under a 0.5 g resting tone for 20 min in a 20 mL organ bath containing Locke–Ringer solution equilibrated with 95% O₂ and 5% CO₂ at 32 ± 1°C; the solution was comprised of the following (mM): NaCl, 154; KCl, 5.6; CaCl₂, 2.2; MgCl₂, 2.1; NaHCO₃, 5.9; and glucose, 2.8. The UBSM preparation was contracted using 10⁻⁴ M ACh at least three times at 20 min intervals (preliminary procedures). After a 30 min equilibration period, ACh was incrementally applied to the bath medium until a maximum response was obtained; this contractile response was recorded twice at 30 min intervals. Following this procedure, the concentration–response curves (CRCs) for ACh were plotted after pre-incubation with different concentrations (3 × 10⁻⁷ to 10⁻⁵ M) of each tested antipsychotic or verapamil (10⁻⁵ M) for 30 min. When dimethyl sulfoxide (DMSO) was used as a drug solvent, the experiment was conducted with a DMSO concentration of 0.5% in the bath solution, including the control experiment. All experiments were carried out in the presence of indomethacin (3 × 10⁻⁶ M).

Assessment of the Effects of Antipsychotics on UBSM Contraction Induced by High-KCl Locke–Ringer Solution

After the preliminary procedures, atropine (10⁻⁶ M), phentolamine (10⁻⁶ M), and propranolol (10⁻⁷ M) were added to the bath medium. After a 30 min equilibration period, to produce sustained contractions, the strip was contracted with 80 mM high-KCl solution (containing atropine, prazosin, and propranolol) comprising (mM): NaCl, 79.6; KCl, 80; CaCl₂, 2.2; MgCl₂, 2.1; NaHCO₃, 5.9; and glucose, 2.8. When the contractile response reached a steady state, each antipsychotic was incrementally applied to the bath medium. At the end of the experiment, the UBSM preparations were treated with verapamil (10⁻⁵ M).

Drugs

The 26 antipsychotics tested in this study were: levomepromazine maleate, fluphenazine dimaleate, haloperidol, sultopride hydrochloride, and perospirone hydrochloride dihydrate (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); chlorpromazine hydrochloride, prochlorperazine dimaleate, pipamperone, (±)-sulpiride, tiapride hydrochloride, paliperidone, blonanserin, olanzapine, and aripiprazole (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan); perphenazine, spiperone hydrochloride, quetiapine hemifumarate, clozapine, brexpiprazole, and pimozide (Cayman Chemical, Ann Arbor, MI, U.S.A.); bromperidol (MedChemExpress Co., Ltd., Monmouth Junction, NJ, U.S.A.); timiperone (Toronto Research Chemicals, Toronto, ON, Canada); nemonapride, and zotepine (Santa Cruz Biotechnology Inc., Dallas, TX, U.S.A.); risperidone (Acros Organics, Geel, Belgium); and asenapine maleate (AdooQ BioScience LLC, Irvine, CA, U.S.A.). ACh chloride was purchased from Daiichi Sankyo Co., Ltd. (Tokyo, Japan). Atropine sulfate, indomethacin, propranolol hydrochloride, and (±)-verapamil were purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). All other chemicals were commercially available and of reagent grade.

Fluphenazine and clozapine were dissolved in 0.1 N HCl to create a stock solution of 2 × 10⁻² M. Thereafter, the stock solutions were further diluted with distilled water to the desired concentrations. The other antipsychotics were dissolved and diluted in pure DMSO. Indomethacin was dissolved in ethanol to create a stock solution of 10⁻² M. All other drugs were prepared as aqueous stock solutions and diluted with distilled water.

Data Analysis and Statistics

CRCs for ACh-induced contractions and Schild plot analysis of anxiolytics versus ACh concentrations were performed using GraphPad Prism™ (GraphPad Software Inc., San Diego, CA, U.S.A.), as previously described.^21,22) All values are presented as mean ± standard error of the mean (S.E.M.) or mean with 95% confidence intervals (CIs) for different numbers (n) of preparations. GraphPad Prism™ was used for the statistical analyses. Differences among the CRCs were evaluated using post hoc Šidák’s test after two-way ANOVA. Statistical significance was set at p < 0.05.

RESULTS

Effects of Phenothiazine Antipsychotics on ACh-Induced Contractions

Figure 1 shows the effects of phenothiazine antipsychotics (3 × 10⁻⁷ to 10⁻⁵ M) on the CRCs of ACh. All tested phenothiazine antipsychotics (chlorpromazine, Fig. 1Aa; levomepromazine, Fig. 1Ba; perphenazine, Fig. 1Ca; fluphenazine, Fig. 1Da; and prochlorperazine, Fig. 1Ea) were found to inhibit ACh-induced contractions. In the concentration range of 3 × 10⁻⁷ to 10⁻⁵ M, the slopes of the regression lines in the Schild plot of chlorpromazine (Fig. 1Ab) and levomepromazine (Fig. 1Bb) vs. ACh were 1.09 (95% CIs: 0.88–1.31, n = 5, chlorpromazine) and 0.91 (95% CI: 0.72–1.10, n = 5, levomepromazine); these values were not significantly different from unity. In the concentration range of 10⁻⁶ to 10⁻⁵ M, the slopes of the regression lines in the Schild plot of perphenazine (Fig. 1Cb), fluphenazine (Fig. 1Db), and prochlorperazine (Fig. 1Eb) vs. ACh were 0.92 (95% CIs: 0.57–1.27, n = 5, perphenazine), 1.01 (95% CIs: 0.69–1.33, n = 5; fluphenazine), and 0.91 (95% CIs: 0.62–1.19, n = 5, prochlorperazine); these values were not significantly different from unity. Therefore, these phenothiazine antipsychotics were found to competitively antagonize ACh in the above concentration ranges. The pA₂ values of chlorpromazine, levomepromazine, perphenazine, fluphenazine, and prochlorperazine were 6.43 (95% CIs: 6.28–6.63, n = 5), 6.48 (95% CIs: 6.32–6.72, n = 5), 6.18 (95% CIs: 5.95–6.63, n = 5), 5.82 (95% CIs: 5.68–6.04, n = 5), and 6.17 (95% CIs: 5.97–6.50, n = 5), respectively.

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 concentration–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.

Effects of Phenothiazine Antipsychotics on ACh-Induced Contractions

Figure 2 shows the effects of butyrophenone antipsychotics (3 × 10⁻⁷ to 10⁻⁵ M) on the CRCs of ACh. Of the tested butyrophenone antipsychotics, haloperidol (Fig. 2Aa), bromoperidol (Fig. 2Ba), timiperone (Fig. 2Ca), and spiperone (Fig. 2Da) inhibited ACh-induced contractions. In the concentration range of 3 × 10⁻⁶ to 10⁻⁵ M, the slopes of the regression lines in the Schild plot of haloperidol (Fig. 2Ab) and timiperone (Fig. 2Cb) vs. ACh were 1.14 (95% CIs: 0.18–2.10, n = 5, haloperidol) and 0.96 (95% CIs: 0.28–1.63, n = 5, timiperone), respectively; these values were not significantly different from unity. In the concentration range of 10⁻⁶–10⁻⁵ M, the slope of the regression lines in the Schild plot of bromperidol (Fig. 2Bb) vs. ACh was 0.96 (95% CI: 0.33–1.60, n = 5); this value was not significantly different from unity. Therefore, these butyrophenone antipsychotics competitively antagonized ACh in the above concentration ranges. The pA₂ values of haloperidol, timiperone, and bromperidol were 5.43 (95% CIs: 5.21–6.51, n = 5), 5.88 (95% CIs: 5.59–7.44, n = 5), and 5.83 (95% CIs: 5.56–6.62, n = 5), respectively.

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.

In contrast, the slope of the regression line in the Schild plot of spiperone (Fig. 2Db) vs. ACh was 0.47 (95% CIs: 0.24–0.70, n = 5); this value was significantly less than unity. Therefore, spiperone did not competitively antagonize ACh in the above concentration ranges.

Pipamperone (Fig. 2E) was not found to affect the CRCs of ACh in the concentration range of 3 × 10⁻⁷ to 10⁻⁵ M.

Effects of Benzamide Antipsychotics on ACh-Induced Contractions

Figures 3A–D show the effects of benzamide antipsychotics (3 × 10⁻⁷ to 10⁻⁵ M) on the CRCs of ACh. The tested benzamide antipsychotics (sulpiride, Fig. 3A; sultopride, Fig. 3B; thiapride, Fig. 3C; nemonapride, Fig. 3D) did not affect the CRCs of ACh in the concentration range of 3 × 10⁻⁷ to 10⁻⁵ M.

Fig. 3. Effects of Benzamide 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.

Effects of Zotepine and Pimozide on ACh-Induced Contractions

Figures 3E and 3F show the effects of zotepine (a thiepine antipsychotic) and pimozide (a diphenylbutylpiperidine antipsychotic) (3 × 10⁻⁷ to 10⁻⁵ M) on the CRCs of ACh. Both zotepine (Fig. 3Ea) and pimozide (Fig. 3Fa) inhibited ACh-induced contractions. The slopes of the regression lines in the Schild plot of zotepine (Fig. 3Eb, 3 × 10⁻⁷ to 10⁻⁵ M) and pimozide (Fig. 3Eb, 3 × 10⁻⁷ to 3 × 10⁻⁶ M) vs. ACh were 0.98 (95% CIs: 0.76–1.19, n = 5, zotepine) and 1.01 (95% CIs: 0.70–1.32, n = 5, pimozide), respectively; these values were not significantly different from unity. Therefore, both zotepine and pimozide competitively antagonized ACh in the above concentration ranges. The pA₂ values of zotepine and pimozide were 6.23 (95% CIs: 6.09–6.42, n = 5) and 7.04 (95% CIs: 6.77–7.52, n = 5), respectively.

Effects of SDAs on ACh-Induced UBSM Contractions

Figures 4A–D show the effects of SDAs (3 × 10⁻⁷ to 10⁻⁵ M) on the CRCs of ACh. Of the tested SDAs, perospirone (Fig. 4Ca) and blonanserin (Fig. 4Da) inhibited ACh-induced contractions. The slopes of the regression lines in the Schild plot of perospirone (Fig. 4Cb, 3 × 10⁻⁶ to 10⁻⁵ M) and blonanserin (Fig. 4Db, 3 × 10⁻⁷ to 10⁻⁵ M) vs. ACh were 1.05 (95% CIs: 0.26–1.84, n = 5, perospirone) and 1.08 (95% CIs: 0.81–1.34, n = 5, blonanserin), respectively; these values were not significantly different from unity. Therefore, these SDAs were found to competitively antagonize ACh in the above concentration range. The pA₂ values of perospirone and blonanserin were 5.51 (95% CIs: 5.32–6.38, n = 5) and 6.48% (95% CIs: 6.29–6.77, n = 5), respectively.

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 concentration–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.

Risperidone (Fig. 4A) and paliperidone (Fig. 4B) were not found to affect the CRCs of ACh in the concentration range of 3 × 10⁻⁷–10⁻⁵ M.

Effects of DPAs on ACh-Induced UBSM Contractions

Figures 4E and F show the effects of DPAs (3 × 10⁻⁷ to 10⁻⁵ M) on the CRCs of ACh. Aripiprazole (Fig. 4E) and brexpiprazole (Fig. 4F) did not affect the CRCs of ACh in the concentration range of 3 × 10⁻⁷ to 10⁻⁵ M.

Effects of MARTAs on ACh-Induced UBSM Contractions

Figure 5 shows the effects of MARTAs (3 × 10⁻⁷ to 10⁻⁵ M) on the CRCs of ACh. All tested MARTAs (olanzapine, Fig. 5Aa; quetiapine, Fig. 5Ba; clozapine, Fig. 5Ca; and asenapine, Fig. 5D) inhibited ACh-induced contractions. In the concentration range of 3 × 10⁻⁶–10⁻⁵ M, the slopes of the regression lines in the Schild plot of olanzapine (Fig. 5Ab) and clozapine (Fig. 5Cb) vs. ACh were 0.97 (95% CIs: 0.67–1.28, n = 5, olanzapine) and 0.93 (95% CIs: 0.33–1.53, n = 5, clozapine), respectively; these values were not significantly different from unity. In the concentration range of 3 × 10⁻⁶ to 10⁻⁵ M, the slope of the regression lines in the Schild plot of quetiapine (Fig. 5Bb) vs. ACh was 0.90 (95% CIs: 0.82–1.30, n = 5); this value was not significantly different from unity. Therefore, these MARTAs competitively antagonize ACh in the above concentration range. The pA₂ values of olanzapine, quetiapine, and clozapine were 7.13 (95% CIs: 6.91–7.54, n = 5), 6.29 (95% CIs: 5.94–7.26, n = 5), and 7.19 (95% CIs: 6.80–8.87, n = 5), respectively.

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 contractions. 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.

At ≥10⁻⁶ M, the slopes of the regression line in the Schild plot of olanzapine (Fig. 5Ab) and clozapine (Fig. 5Cb) vs. ACh were 0.67 (95% CIs: 0.48–0.86, n = 5, olanzapine) and 0.51 (95% CIs: −0.14–1.17, n = 5, clozapine), respectively; these values were less than unity. Further, in the concentration range of 3 × 10⁻⁷ to 3 × 10⁻⁶ M, the slope of the regression line in the Schild plot of quetiapine (Fig. 5Bb) vs. ACh was 0.64 (95% CIs: 0.43–0.84, n = 5); this value was significantly less than unity.

In contrast, asenapine (Fig. 5D) inhibited ACh-induced contractions only at 10⁻⁵ M. However, asenapine did not shift the CRCs of ACh to the right. Instead, it inhibited the maximum response to ACh. The maximum response of CRCs of ACh (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 antipsychotics, 18 significantly inhibited ACh-induced contractions. 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 obtained from Fig. 6: the maximum response of the CRC of ACh (100%, 3 × 10⁻³ M ACh) was significantly inhibited to 52.0 ± 9.1% by verapamil (10⁻⁵ M) (n = 5). Thus, Ca^2 + influx through VDCCs was demonstrated to be responsible for ACh-induced contractions in smooth muscle preparations.

Fig. 6. Effect of Verapamil (10⁻⁵ M) on ACh-Induced Contraction of Rat UBSM

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 bladder smooth muscle.

We proceeded to determine the effects of 18 antipsychotics on 80 mM KCl-induced contraction to elucidate whether these antipsychotics could inhibit VDCC-mediated contraction (Table 1). Of the tested drugs, olanzapine hardly suppressed the contractions induced by the 80 mM KCl solution, whereas 17 antipsychotics, besides olanzapine, suppressed these contractions at 10⁻⁵ M by approximately 20–50% (Table 1). Pimozide suppressed 80 mM KCl-induced contraction by approximately 10%, even at a concentration of 10⁻⁶ M (Table 1).

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

Category/Generic name	Inhibition of high-K-induced contraction (%)
	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).

As the remaining contractions after each drug treatment were completely suppressed by verapamil (10⁻⁵ M) (data not shown), the contractions induced by 80 mM KCl solution were elicited via VDCCs.

DISCUSSION

In the present study, we sought to investigate the possible inhibitory effects of 26 clinically available antipsychotics on ACh-induced contractions in rat UBSM to predict the drugs that should not be used by the elderly population to avoid urinary disorders. Of the tested antipsychotics, two typical antipsychotics (chlorpromazine and levomepromazine) and four atypical antipsychotics (zotepine, olanzapine, quetiapine, and clozapine) significantly inhibited ACh-induced contractions at clinically significant doses. Such findings suggest that these six antipsychotics should be avoided in elderly people with urinary disorders.

Chlorpromazine/Levomepromazine (Phenothiazines), Zotepine (a Thiepine), Olanzapine/Quetiapine/Clozapine (MARTAs)

Chlorpromazine, levomepromazine, zotepine, olanzapine, and clozapine inhibited ACh-induced contractions in a competitive antagonistic manner. The calculated pA₂ values were consistent with the pK_i values obtained from our binding study where [³H]N-methyl scopolamine (NMS) was employed in the mouse cerebral cortex²³⁾ (Table 2). Therefore, we concluded that the inhibition of ACh-induced UBSM contractions by these antipsychotics was due to their anticholinergic effects. The pA₂ values of the antipsychotics were found to be comparable with their plausible blood concentration levels, which are presented as minus logarithm values, or larger than the plausible blood concentration levels (clozapine). This finding indicates that these antipsychotics can reduce UBSM contractility by inhibiting muscarinic receptors, thereby inducing urinary functional disorders within their clinically applied antipsychotic dose ranges. The pA₂ values/pK_i values/plausible blood concentration levels (expressed as minus logarithm values) of each drug were (Table 2): 6.43/6.40/5.85–7.55 (chlorpromazine); 6.48/6.22/6.21–7.35 (levomepromazine); 6.23/6.21/6.14–7.04 (zotepine); 7.13/6.85/6.37–7.38 (olanzapine); and 7.19/6.83/5.23–6.30 (clozapine).

Table 2. Comparison of the pA₂ Values of Drugs vs. ACh, Their pK_i Values vs. Muscarinic Receptor, and the Clinically Achievable Blood Concentration of Antipsychotics

Category/Generic name	pA2 values vs. ACh	pKi values vs. muscarinic receptor	Clinically achievable blood concentration (−log M)	Ref.
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)

*Data from rabbit. **Total concentration of nemonapride and its metabolites. pK_i values vs. the muscarinic receptor were obtained from our previous report using [³H]N-methyl scopolamine in the mouse cerebral cortex.²³⁾ Ref.: references from which the clinically achievable blood concentrations were obtained.

In the concentration range of 3 × 10⁻⁶ to 10⁻⁵ M, quetiapine competitively inhibited ACh-induced contractions, and its pA₂ value was calculated to be 6.29. This value was one order of magnitude larger than the calculated pK_i value (5.31) (Table 2), but consistent with the pK_i value for the muscarinic M₃ receptor (6.65) obtained from the binding experiment using [³H]NMS.²⁴⁾ Our calculated pK_i value for [³H]NMS in the mouse cerebral cortex corresponded to the quetiapine’s pK_i value for M₅ receptor²⁵⁾; however, we do not have any reasonable explanations for this. Nonetheless, the pA₂ value (6.29) of quetiapine was in its plausible blood concentration levels and was expressed as the minus logarithm (−log M: 5.92–7.95). This finding suggests that clinically applied quetiapine reduces UBSM contractility and urinary disorders by inhibiting muscarinic receptors.

In the concentration range of ≥ 10⁻⁶ M, olanzapine and clozapine did not show competitive inhibitory effects on ACh as the slope of the regression line in the Schild plot was significantly less than unity (0.67 and 0.51, respectively). The apparent non-competitive effects of these antipsychotics against ACh could be explained by their different affinities for the M₂/M₃ receptors. Although UBSM expresses both the M₂/M₃ receptors, the receptor subtype responsible for UBSM contractions is M₃.²⁶⁾ Both olanzapine and clozapine have higher affinity for M₃ than M₂. The pK_i values for olanzapine were 7.89/7.32 (M₃/M₂) while those for clozapine were 8.15/7.32 (M₃/M₂).²⁵⁾ Based on these previous results, we infer the following for the muscarinic receptor subtypes responsible for inducing inhibition vs. ACh-induced contraction by olanzapine and clozapine: in the low concentration ranges (3 × 10⁻⁷ to 10⁻⁶ M), both antipsychotics selectively bind to M₃ and produce competitive antagonistic effects against ACh and inhibition of ACh-induced UBSM contraction. In contrast, at higher concentrations (≥ 10⁻⁶ M), both antipsychotics could bind to M₂, which is not responsible for inducing ACh-induced contraction, and thus, did not show apparently competitive antagonistic effects against ACh.

In the low concentration range (3 × 10⁻⁷ to 3 × 10⁻⁶ M), quetiapine did not exhibit any competitive inhibitory effects against ACh, as the slope of the regression line in the Schild plot was significantly less than unity (0.64). Such finding might be explained by the different affinities of quetiapine between M₂ and M₃: quetiapine has a higher affinity for M₂ (pK_i = 6.20) than M₃ (pK_i = 5.88).²⁴⁾ Therefore, in the low concentration ranges (3 × 10⁻⁷ to 3 × 10⁻⁶ M), quetiapine is speculated to bind to non-contractile M₂ and M₃, and thus exert apparently non-competitive effects against ACh. In contrast, at higher concentrations (3 × 10⁻⁶ to 10⁻⁵ M), where the binding of quetiapine to M₂ becomes saturated, quetiapine displayed competitive antagonism against ACh on contractile M₃, and thus, produced inhibitory effects against ACh-induced contraction in a competitive manner.

Prochlorperazine/Perphenazine/Fluphenazine (Phenothiazines), Haloperidol/Bromperidol/Timiperone (Butyrophenones), Pimozide (a Diphenylbutylpiperidine), and Blonanserin (SDA)

Prochlorperazine, timiperone, and blonanserin suppressed ACh-induced contraction in a competitive fashion, and their calculated pA₂ values were close to their pK_i values obtained from our binding experiments²³⁾ (Table 2). As a result, we concluded that the inhibitory effects of these antipsychotics on ACh-induced contraction were mainly due to their anticholinergic effects. The pA₂/pK_i values of each antipsychotic were (Table 2): 6.17/5.68 (prochlorperazine), 5.88/5.39 (timiperone), and 6.48/6.09 (blonanserin).

In our previous study, the pK_i values could not be calculated for perphenazine, fluphenazine, haloperidol, and bromperidol (pK_i < 5.39).²³⁾ However, the pA₂ values of perphenazine (6.18) and fluphenazine (5.82) were found to be approximately comparable to their pK_i values (5.31, perphenazine; and 5.39, fluphenazine), which were calculated from the binding study for rat cerebral cortex using [³H]quinuclidinyl benzilate (QNB).²⁷⁾ In addition, the pA₂ value of haloperidol (5.43) was almost comparable to its pK_i value (5.80) calculated from the binding study for the rat submandibular gland using [³H]NMS.²⁴⁾ Therefore, the inhibitory effects of perphenazine, fluphenazine, and haloperidol on ACh-induced contraction could be mainly caused by their anticholinergic effects. We speculated that bromperidol inhibited ACh-induced contraction through the same mechanism (anticholinergic effects) as haloperidol because bromperidol has a bromine atom that replaces the chlorine atom possessed by haloperidol in its structure.

The pA₂ value of pimozide (7.04) was one order of magnitude larger than the pK_i value (5.64) obtained from our binding experiments²³⁾ (Table 2) and the value (6.10) obtained from binding experiments in the human brain using [³H]QNB (6.10).²⁸⁾ These findings imply that pimozide inhibited ACh-induced contractions primarily through anticholinergic action-unrelated mechanisms, despite exhibiting anticholinergic actions at higher concentrations.

In addition to pimozide, the pA₂ values of most other antipsychotics were slightly higher than their pK_i values (Table 2). Therefore, mechanisms besides those used to elucidate their anticholinergic effects might be responsible for their inhibitory effects against ACh-induced contractions. One plausible mechanism could be the inhibition of VDCCs, which was supported by the evidence that these antipsychotics suppressed the depolarizing contraction induced by 80 mM KCl (Table 1). Consistently, fluphenazine was reported to exhibit Ca²⁺ channel blocking effects²⁹⁾; haloperidol and pimozide were reported to suppress Ca²⁺ currents³⁰⁾; and phenothiazines³¹⁾ and butyrophenones³²⁾ were reported to exert calmodulin inhibitory effects. Thus, in addition to anticholinergic actions, the antipsychotic suppression of ACh-induced contractions might be mediated via the VDCC-inhibitory actions and/or calmodulin inhibitory actions. However, the concentrations required to inhibit ACh-induced contractions were markedly higher than the clinically achieved blood concentrations (pA₂ values<−log [clinical blood concentrations]) of these antipsychotics (Table 2). Therefore, these antipsychotics do not induce or worsen urinary disorders as long as their clinically applied doses are administered. However, it should be noted that the higher levels of unmetabolized drugs and metabolites of these drugs in urine than in the blood might cause urinary disorders by acting from within the urinary bladder.

Spiperone (a Butyrophenone), Perospirone (SDA), and Asenapine (MARTA)

Spiperone, perospirone, and asenapine apparent-competitively or non-competitively suppressed ACh-induced contractions. However, the results of our previous study and others revealed that their pK_i values for the muscarinic receptor were lower than their blood concentrations, presented as minus logarithm, or lower than experimentally detectable levels: spiperone, pK_i<5.39²³⁾; perospirone, pK_i<5.39,²³⁾ pK_i<6³³⁾; asenapine, pK_i<5.39,²³⁾ pK_i≤5.³⁴⁾

Nonetheless, these drugs were found to inhibit depolarizing contractions in the 80 mM KCl solutions (Table 1). Therefore, we concluded that the inhibition of ACh-induced contractions by these drugs was mediated by their non-anticholinergic actions, such as the inhibition of VDCC/calmodulin. Based on prior reports, these drugs possess the following effects: spiperone, Ca²⁺ current inhibitory effect²⁹⁾; asenapine, action potential duration-reducing effects on Purkinje fibers³⁵⁾; and butyrophenones, calmodulin inhibitory effects.³²⁾ However, the clinically achievable blood concentrations of these drugs are markedly lower than the concentrations needed to suppress ACh-induced contractions (Table 2). Therefore, these antipsychotics are unlikely to induce urinary disorders at the clinical doses.

Pipamperone (a Butyrophenone), Sulpiride/Sultopride/Tiapride/Nemonapride (Benzamides), Risperidone/Paliperidone (SDAs), and Aripiprazole/Brexpiprazole

Pipamperone, sulpiride/sultopride/tiapride/nemonapride, risperidone/paliperidone, and aripiprazole/brexpiprazole suppressed ACh-induced contractions up to 10⁻⁵ M. However, their pK_i values (pK_i<5.39) could not be obtained because of their marginal effects on [³H]NMS in the cerebral cortex.²³⁾ Sulpiride and aripiprazole did not suppress ACh-induced contractions in guinea pig UBSM.³⁶⁾ Additionally, the plausible blood concentration levels of all drugs were found to be markedly lower than 10⁻⁵ M (Table 2). Therefore, these antipsychotics were not found to cause anticholinergic action-mediated urinary disorders within the doses administered clinically.

Acknowledgments

The authors would like to thank Mr. Yunfeng Ban for his expert technical assistance. This study was partly supported by the Joint Research Grants of the Toho University Faculty of Pharmaceutical Sciences (K.O.).

Conflict of Interest

The authors declare no conflict of interest.

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