July 2007 Biol. Pharm. Bull. 30(7) 1297—1300 (2007) 1297

Analysis of Pharmacological Effects of Drugs Used for Treatment of Urinary Disturbance Based on and Smooth Muscle- Relaxing Effects

a a a a b Risa TAKAYANAGI, Haruhi MIZUSHIMA, Takeshi OZEKI, Haruko YOKOYAMA, Tatsuji IGA, and ,a Yasuhiko YAMADA* a Department of Clinical Evaluation of Drug Efficacy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences; 1432–1 Horinouchi, Hachioji, Tokyo 192–0392, Japan: and b International University of Health and Welfare; Kitakanemaru, Ohtawara, Tochigi 324–8501, Japan. Received November 21, 2006; accepted April 2, 2007

Propiverine hydrochloride, hydrochloride and hydrochloride have both anticholiner- gic and effects, and are used for the management of urinary frequency and incontinence. The av- erage standard therapeutic doses of these drugs differ greatly. We retrospectively analyzed their pharmacological effects with consideration given to muscarinic receptor binding affinities, anticholinergic activities, and inhibitory effects on KCl-induced contraction. Muscarinic occupancies and the in- hibitory ratios of the drugs for both acetylcholine-induced and KCl-induced contraction in a steady state after oral administration of standard doses were calculated based on pharmacokinetics and the receptor occupancy theory. The average muscarinic acetylcholine receptor occupancy and inhibitory ratio of acetylcholine-induced contraction were estimated to be 12.61.06% and 3.270.74%, respectively, with no significant differences found between the drugs for those parameters. A significant linear relationship was found between muscarinic acetylcholine receptor occupancy and the maximum ratio of increase in bladder urinary capacity. On the other hand, the inhibitory ratios of KCl-induced contraction varied from 0.01 to 0.48%. The present results suggest that muscarinic acetylcholine receptor occupancy is a principal determinant of the therapeutic effect of a drug used for treatment of urinary disturbance. Key words muscarinic acetylcholine receptor; receptor occupancy; bladder urinary capacity; propiverine; oxybutynin; terodiline

Drugs for the treatment of urinary disturbance with anti- Values for the primary pharmacokinetic parameters, i.e., total and smooth muscle-relaxing effects are used for body clearance (CLtot), bioavailability (F), area under the the management of urinary frequency and incontinence. plasma concentration time curve (AUC0—), and plasma un- 3—8) Oxybutynin hydrochloride, propiverine hydrochloride, bound fraction (fu), were obtained from previous reports. terodiline hydrochloride, and flavoxate hydrochloride have The receptor dissociation constant (KI) and anticholinergic been marketed in Japan as drugs for treatment of urinary dis- activity (KB) values were also obtained as data for the mus- turbance, and tartrate and succinate carinic acetylcholine receptor binding affinity of the drugs 1,9—15) were put on the market in 2006. Sales of terodiline hy- from literature. KI values were determined by in vitro drochloride, however, have been stopped because of its ad- radiolabeled ligand binding inhibition experiments using verse arrhythmic effect. The average standard therapeutic guinea pig bladders, while KB values were determined by in doses of the remaining available drugs differ greatly. In re- vitro pharmacological experiments with guinea pig bladders. gard to anticholinergic and smooth muscle-relaxing effects of The inhibitory effects of KCl-induced contraction (IC50) val- oxybutynin, propiverine, and terodiline, it has been reported ues were also obtained as data regarding the direct effects of 16) in studies using extracted rabbit bladders that in order to the drugs on smooth muscle from a previous study. IC50 nearly completely inhibit contraction of the musculus detru- values were determined by in vitro pharmacological experi- sor urinae, each of those effects independently was insuffi- ments using rat bladders. cient and both were required.1,2) Those results were from in Analysis of Muscarinic Acetylcholine Receptor Occu- vitro pharmacological experiments, while there is no known pancy and Inhibitory Ratio of Acetylcholine-Induced report that considered the inhibitory actions toward musculus Contraction The muscarinic acetylcholine receptor occu- detrusor urinae contraction using oral administration of the pancy and inhibitory ratio of acetylcholine-induced contrac- drugs with standard doses. tion of each drug for urinary disturbance following oral ad- We investigated the anticholinergic and antispasmodic ef- ministration with a standard dose were calculated using data fects of oxybutynin hydrochloride, propiverine hydrochlo- obtained as noted above. We assumed that the drugs used for ride, and terodiline hydrochloride to inhibit bladder smooth urinary disturbance bound competitively to muscarinic muscle contraction, by calculating the muscarinic acetyl- acetylcholine receptors and expressed the concentration (nM) receptor occupancies and inhibitory ratios of both of bound receptors (R) using the following equation: acetylcholine-induced and KCl-induced contractions in a RC⋅⋅RC R max U max M (1) steady state after oral administrations of standard doses. CKdCKdUU(/)11 MMCKdCKdMM (/) UU

MATERIALS AND METHODS where Rmax is the maximum concentration (nM) at the recep- tors; CU and CM are the concentrations (nM) of the unchanged Pharmacokinetic and Pharmacodynamic Parameters drug and its metabolite, respectively, at the juxtareceptors;

∗ To whom correspondence should be addressed. e-mail: [email protected] © 2007 Pharmaceutical Society of Japan 1298 Vol. 30, No. 7

Table1. Pharmacokinetic and Pharmacodynamic Parameters of Drugs Used for Urinary Disturbance

Smooth Anticholinergic effect muscle Standard dose AUC Cf Drug 0— f ss relaxing (mg/d) (ng/ml·h)u (nM) KI KB effect (nM)(nM)IC50 (nM)

Propiverine 20 Unchanged propiverine 9753) 0.096) 9.9 3729) 43714) 390016) Metabolite 2163) 0.666) 15.9 1059) 135014) — Oxybutynin 7.5 26a),4) 0.177) 0.5 410) 2015) 450016) Terodiline 24 4960a),5) 0.048) 31.6 203b),11—13) 7591) 660016)

—, no activity. a) Calculated based on the relationship between dosage and AUC. b)Average data.

and KdU and KdM are the receptor dissociation constants, cor- ratio and pharmacodynamic parameters were then examined. responding to KB or KI, of the drug. We assumed that the Analysis of Relationships of Receptor Occupancy and substances acted as full antagonists toward muscarinic Inhibitory Ratio of Contraction with Clinical Effect The acetylcholine receptors and expressed the receptor occu- muscarinic acetylcholine receptor occupancy and inhibitory pancy, F (%), according to the receptor occupation theory ratio of contraction of each of the drugs following oral ad- using the following equation: ministration of a dose found acceptable in clinical trials were calculated using Eqs. 1—3. We then examined the relation- R Φ ⋅100 ships of receptor occupancy and inhibitory ratio with clinical Rmax (2) effect, which was defined as the maximum ratio of increase  C C   U M  ⋅100 in bladder urinary capacity, data for which were obtained 17—27)  CKdCKdUU(/)11 MMCKdCKdMM (/) UU from clinical trial reports.

After the drug has been absorbed into the bloodstream RESULTS from the gastrointestinal tract, it passes across blood capil- lary cell walls into extracellular space and binds to mus- Pharmacokinetic and Pharmacodynamic Parameters f carinic acetylcholine receptors on the plasma membranes of Table 1 shows the values of AUC0—, fu, and Css following bladder cells. If the process of transport of the drug to en- oral administration with standard doses, as well as the values dothelial cells is not active, but rather passive diffusion, or it of KI and KB for the anticholinergic effects, and IC50 for the 1,3—16) is not a rate-limiting step because of fenestration on the muscle-relaxing effects. The values of IC50 did not vary blood capillary cell walls, we assumed that the concentration greatly, whereas the differences between the minimum and of the drug in the extracellular space in the bladder would not maximum values of both KI and KB were nearly 100-fold. be significantly different from the unbound drug concentra- Receptor Occupancy and Inhibitory Ratio of Contrac- tions in plasma. Therefore, we used the unbound drug con- tion in a Steady-State Condition Receptor occupancies of f f centrations (CU and C M) as the concentrations for the recep- propiverine, oxybutynin, and terodiline were 12.9%, 11.4%, tor-binding drugs (CU and CM), respectively. The steady-state and 13.5%. Inhibitory ratios of acetylcholine-induced con- drug concentration in plasma was calculated from CLtot/F or traction were 3.29%, 2.52%, and 3.99%, and inhibitory ratios AUC0—, as follows: of KCl-induced contraction were 0.25%, 0.01%, and 0.48%. Figure 1 shows the relationships of the pharmacodynamic Dose⋅ F AUC ∞ Cff ⋅ f ⋅ 0— (3) parameters with muscarinic acetylcholine receptor occupan- ss u ⋅ττu CLtot cies, and inhibitory ratios for both acetylcholine-induced and KCl-induced contractions in a steady state after oral adminis- where t represents the dosing interval. In order to estimate tration of standard doses of the three drugs. The average the receptor occupancy of the drug in a steady state, we cal- muscarinic acetylcholine receptor occupancy and average in- f culated Css from CLtot/F or AUC0— using Eq. 3. We deter- hibitory ratio of acetylcholine-induced contraction were esti- mined the receptor occupancy of each drug by using Eq. 2. mated to be 12.61.06% and 3.270.74%, respectively, Then, we examined the relationships of receptor occupancy while the inhibitory ratios of KCl-induced contraction varied and inhibitory ratio using the pharmacodynamic parameters. from 0.01 to 0.48%. It is known that oxybutynin and propiverine each have one Relationships of Receptor Occupancy and Inhibitory pharmacologically active metabolite.7,9) However, there are Ratio of Contractions with Clinical Effect Figure 2 no available data on the metabolites of oxybutynin, thus only shows the relationships of receptor occupancy and inhibitory data on the unchanged drug were used for the analysis of ratio of contraction with the maximum ratio of increase in oxybutynin. Terodiline has no pharmacologically active bladder urinary capacity. The relationship between mus- metabolites. carinic acetylcholine receptor occupancy and maximum ratio Analysis of Inhibitory Ratio of KCl-Induced Contrac- of increase in bladder urinary capacity was statistically sig- tion The inhibitory ratio of KCl-induced contraction for nificant (p0.05). A correlation (p0.1) was also found be- each of the drugs following oral administration with the stan- tween acetylcholine-induced contraction and maximum ratio dard dose was calculated using Eqs. 1—3. Kd was calculated of increase in bladder urinary capacity. However, no signifi- from the IC50 value. The relationships between inhibitory cant relationship was found between the inhibitory ratio of July 2007 1299

Fig. 1. Relationships between Pharmacodynamic Parameters and Muscarinic Acetylcholine Receptor Occupancy (a), and Inhibitory Ratios of Both Acetyl- choline-Induced (b) and KCl-Induced (c) Contractions under a Steady-State after Oral Administration of Standard Doses of the Three Drugs Hatched region indicates meanS.D. Symbols: , propiverine; , oxybutynin; , terodiline.

Fig. 2. Relationships between Maximum Ratio of Increase in Capacity of Urinary Bladder and Muscarinic Acetylcholine Receptor Occupancy (a), and In- hibitory Ratios of Acetylcholine-Induced (b) and KCl-Induced (c) Contractions Symbols: , propiverine; , oxybutynin; , terodiline.

Fig. 3. Relationships between KI Values in Guinea Pigs and Humans (a), and between KI Values in Rabbits and Humans (b)

KCl-induced contraction and maximum ratio of increase in used KI and KB values obtained from experiments with bladder urinary capacity. guinea pigs and IC50 values from those with rats as data for muscarinic acetylcholine receptor binding affinity and the in- DISCUSSION hibitory ratio of KCl-induced contraction, respectively. The

relationships between KI values obtained from guinea pigs In the present study, we investigated the contributions of and humans, and between KI values obtained from rabbits anticholinergic and antispasmodic effects of three drugs used and humans are shown in Fig. 3.10—13) Nevertheless, addi- for management of urinary frequency and incontinence in re- tional assessments of the correlations between these values gard to their inhibitory actions toward bladder smooth mus- obtained from animals and humans is needed to confirm their cle contraction. Although these drugs were put on the market validity. about 15—20 years ago, the quantity and quality of informa- The mean values for muscarinic acetylcholine receptor oc- tion for each drug was different. cupancy and inhibitory ratio of acetylcholine-induced con-

It is more convenient to use values for KI, KB, and IC50 ob- traction under a steady state after oral administration of stan- tained from animal studies as compared to human data. We dard doses of the drugs examined were estimated to be 1300 Vol. 30, No. 7

12.6% and 3.27%, respectively. It is known that oxybutynin 4) Kishimoto T., Takimoto S., Morita H., Kasama T., Shimizu S., Shi- and propiverine each have one pharmacologically active nozaki Y., Kiso to Rinsho, 20, 1—9 (1986). 7,9) 5) Mictrol Interview Form, 1988, p. 26. metabolite. However, there are no available data on the 6) Bup-4 Interview Form, 2003, p. 40. metabolite of oxybutynin. To explain why the values for 7) Pollakisu Interview Form, 2004, pp. 14—16. muscarinic acetylcholine receptor occupancy and inhibitory 8) Hallen B., Guilbaud O., Stromberg S., Lindeke B., Biopharm. Drug ratio of acetylcholine-induced contraction of oxybutynin Dispos., 9, 229—250 (1988). were low, we considered that only data regarding the un- 9) Isogai M., Shimizu K., Kaneko S., Kitazato K., “The Collected Papers of Bup-4,” Taiho Pharmaceutical Co., Ltd., Tokyo, 1993, pp. 121— changed drug were used in the present analysis. On the other 130. hand, the inhibitory ratios of KCl-induced contraction varied 10) Nilvebrant L., Sparf B., Acta Pharmacol. Toxicol., 53, 304—313 from 0.01 to 0.48%. Further, though a significant linear rela- (1983). tionship was found between muscarinic acetylcholine recep- 11) Batra S., Eur. J. Pharmacol., 138, 83—88 (1987). tor occupancy and the maximum ratio of increase in bladder 12) Batra S., Biorklund A., Hedlund H., Andersson K. E., Bjorklund A., J. Auton. Nerv. Syst., 20, 129—135 (1987). urinary capacity, no significant relationship was found be- 13) Nilvebrant L., Andersson K. E., Mattiasson A., J. Urol., 134, 418— tween the ratio of KCl-induced contraction and maximum 423 (1985). ratio of increase in bladder urinary capacity. In order to as- 14) Haruno A., Yamasaki Y., Miyoshi K., Miyake H., Tsuchiya K., Kosaka certain the inhibitory ratios of KCl-induced contraction, we M., Nagai M., Iriki M., Folia Pharmacol. Jpn., 94, 145—150 (1989). determined the parameters of flavoxate which had only 15) Aida Y., Kaneko Y., Kasama T., Folia Pharmacol. Jpn., 87, 629—639 (1986). smooth muscle-relaxing effects, there are no data, however, 16) Kaneko S., Miyake S., Nakano D., Ohara M., Kobayasi F., Nishimori, on the inhibitory effects of KCl-induced contraction (IC50) “The Collected Papers of Bup-4,” Taiho Pharmaceutical Co., Ltd., values. Tokyo, 1993, pp. 162—181. Together, these results indicate that the pharmacotherapeu- 17) Takayasu H., Ueno A., Tsuchida S., Koiso K., Kurita T., Kawabe K., Hanaoka K., Rinsho Iyaku, 6, 761—776 (1990). tic activities of the three drugs for treatment of urinary dis- 18) Takayasu H., Ueno A., Tsuchida S., Koiso K., Kurita T., Kawabe K., turbance are elicited under a certain rate of muscarinic Hanaoka K., Igaku no Ayumi, 153, 459—471 (1990). acetylcholine receptor occupancy (ca. 13%) and that the re- 19) Iwatsubo E., Yamashita H., Takei M., Kumazawa J., Komine S., ceptor occupancy of the drugs administered at standard doses Masaki Z., Ito K., Masuda S., Naoto I., Iwatsubo E., Nishinihon J. is about 13%, regardless of the binding affinity to muscarinic Urol., 52, 233—240 (1990). 20) Koyanagi T., Maru A., Taniguchi K., Shinno Y., Takamatsu T., Morita acetylcholine receptors. H., Kumamoto Y., Okayama S., Ohmura K., Yokoyama E., Orikasa S., Receptor occupancy of a drug is considered to be a ra- Imabayashi K., Sohma F., Nakano N., Ohmura T., Nishimura Y., Shi- tional index of curative effectiveness. In the case of drug ac- raiwa Y., Yamaguchi O., Fukaya Y., Sato S., Uehara T., Okada K., Hi- tion elicited by specific receptors, it is important to clarify ki- raga S., Shimazaki J., Yasuda K., Murayama N., Endo H., Yamashiro netically the relationship between drug concentration in the Y., Kamura K., Namiki T., Takano M., Kawabe K., Matsumura T., Matsuki K., Kishimoto T., Takimoto Y., Okada K., Kawazoe K., Ki- region of the site of action of the drug and binding of the yotaki S., Fuse T., Miyazaki K., Ishido T., Hisazumi H., Nagano K., drug to receptors. We have reported on the utility of the eval- Ueno A., Kobayashi K., Mitsuya H., Kondo A., Kobayashi M., Yama- 28) uation of the effect of dopamine D2 receptor antagonists, mura H., Nishinihon J. Urol., 48, 1051—1072 (1986). 29) 21) Kawabe K., Abe S., Kanda T., Kyuchun C., Nihon Saigai Igakukai b-adrenergic receptor antagonists, and 5-HT3 receptor an- tagonists.30) In this study, the predicted average receptor oc- Kaishi, 34, 237—242 (1986). 22) Hattori T., Yasuda K., Hirayama K., Shinkei Naika Chiryou, 2, 335— cupancy (ca. 13%) was lower than those antagonists. But it is 343 (1985). reported that the receptor occupancy of oxybutynin using 23) Toma H., Nakamura R., Hinyou Kiyou, 32, 907—911 (1986). free plasma drug levels and binding affinity constants at the 24) Iwatsubo E., Kitada S., Kumazawa J., Komine S., Masaki Z., Ito K., Kuroda N., Yamashita H., Minoda K., Iwakawa A., Koga H., Iwatsubo human muscarinic receptors (M2/M3) appears to be low 31) E., Nishinihon J. Urol., 48, 697—705 (1986). (about 4%/25%). 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