`RCC-36, on Isolated Human Urinary Bladder Muscle
`
`Hiroaki Kil<ul<awa,* Masaki Yoshida, Yoshihiro Wada, Kazuhiko Nishi,
`and Shoichi Ueda
`
`Department of Urology, Kurnamoto University School of Medicine, Kumamoto, Japan
`
`Background: Temiverinc tp-INN) is rt newly synthesized drug, that is expected to have anticholinergic
`action. We investigated the pharmacologic actions oftemiverine and its active metabolite, RCC36, on
`isolated human bladder.
`
`Methods: tffects of temiverine and RCC-36 on the detrusor contractions induced by acetylcholine,
`potassium chloride (KCJ), calcium chloride tCaCl1), and electric field stimulation were evaluated using
`the muscle-bath technique, and compared with the effects of atropine and rixybiitynin.
`Results: Atropine (1043 to10“’ moi/L), oxybutynin (10 ll to10‘i’ moi/L), temiverine (1 0‘5 to T0 5 moi/L),
`and RCC-36 (1 0'8 to 3 X 10*’ mol/L] caused a parallel shift to the right of the concentratiomresponse
`curves to acetylcholine stimulation. The rank order of pAL. value was atropine > oxybutynin = RCC-36
`> terniverine. Atropine did not suppress the maximum contractile response to acetylcholine, but the
`other drugs significantly suppressed this at the higher concentrations. Each drug caused a concentration-
`dependent inhibition of KC] (80 mmol/L)-, and CaC|-2 (5 mmol/L)-induced contractile responses. Rank
`order of maximum inhibition was RCC-36 = lemiverine > oxybutynin > atropine. Each drug caused a
`concentration—dependent inhibition of electric field-induced contraction with or without 10"‘ mol/L
`atropine pretreatment. Maximum inhibitions of temiverine and RCC—36 were significantly greater than
`that of oxybutynin.
`Conclusion: Atropine, oxybutynin, temiverinc, and RCC—36 have different efficacies and potencies of
`anticholinergic and calcium antagonistic activity on isolated human detrusor muscles. Furthermore,
`temiverine and RCC-3t’: have significant
`inhibitory actions toward the atropine-resistant part of
`contractions, which maybe related to the calcium antagonistic actions of these compounds.
`Int] Urol1998;5:26El—275
`
`Key words: temiverine p-INN, RCC-36, anticholinergic, calcium antagonist, detrusor muscle
`
`INTRODUCTION
`
`The parasympathetic nervous system plays an impor-
`tant role in the functional regulation of urinary blad-
`der.” Since a major portion of the ncurohormonal
`stimulus for physiologic bladder contractions is acetyl-
`Choline-induced stimulation of postganglionic, para-
`sympathetic, cholinergic, and muscarinic receptor sites,
`the blockade of the muscarinic receptor would be a
`therapeutic approach to treating patients with unstable
`detrusor contractions.3 Accordingly, atropinerlike drugs
`(i.e., anticholinergic drugs) have been used for treatment
`of urinary frequency or incontinence resulting from
`unstable detrusor contractions. Some patients do not
`respond to anticholincrgic medication sufficiently or
`cannot tolerate the administration of these drugs because
`of their adverse effects, such as tachycardia, dry mouth,
`and constipation. Moreover, it has been reported that
`
`R("C_(.‘IV(‘(l Feb. 5, 1997; accepted for publication in revised form Dec.
`I2, l997.‘Correspondence and requests for reprints to: Department
`of Urology, Kumamoto University School of Medicine, l-l -l
`l ionio,
`Kumamoto 860-8556, Japan.
`
`part of the detrusor contractions evoked by nerve
`stimulation was resistant to atropine,“““’ and that, in the
`abnormal condition such as detrusor overactivity, the
`atropine-resistant portion of the contractions was in-
`creased? '’
`Several anticholinergic drugs that are used to treat
`detrusor overactivity have been reported to have anti-
`spasmodic or calcium antagonistic actions in addition
`to antimuscarinic actions in animal experiments. 10 One
`such new drug, tcmiverinc (p—INN),
`is synthesized by
`means of chemical modification of oxybutynin. The
`chemical formula is (i)—4—diethy1amino—l , l-dimer.hylbut-
`2—yn—l—y1 2—cyc1ohcxy1-2—hydroxy—2—phenylacetate
`monohydrochloride monohydrate (Fig. 1). After oral
`administration, temiverine is extensively metabolized in
`the liver, and the metabolites were excreted into urine and
`bile. Seven metabolites were isolated from rat urine.
`
`RCC-36, which is a N—dccthylatcd metabolite (('_r)-4-
`ethylamino—1,l—dimethy1hut-2-yn—l—yl2-cyclohexy1-2-
`hydroxy-2-phenylacetate monohydrochloride),
`is a
`biologically active compound.“ Both temiverine and
`RCC-36 have anticholincrgic and caicium antagonistic
`actions on the rat urinary bladder.”
`
`263
`
`0919-8172/98/0503-0268/US$3.00 © IUA/CL] 1998
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2045 - 0001
`
`
`
`Effect of Temiverine and RCC-36 on the Bladder
`
`H. Kilcukawa et at].
`
`ll
`
`(IZH3
` O
` CH-3
`
`HO-C"“ C—()--?—CEC —CH, NlCH, CH,l3 HCI H,O
`
`lemivcrine
`
`(P—|NN)
`
`II
`
`"mi
`©0
` (TH.
`
`H()— C — C —~O-Cf — CEC-' CH, NHCH3 CH, HCF
`
`REC-36
`
`Fig. 1. The chemical structures of temiverine and RCC—36.
`
`There is, however, little information yet available
`on the actions of these drugs on the human urinary
`bladder, and the exact mechanism of the inhibitory
`actions on the human urinary bladder has not been
`clearly determined. Therefore, we investigated the ef-
`fects ofterniveririe and RCC—36 on the isolated human
`
`detrusor muscles using the muscle—bath technique.
`
`MATERIALS AND METHODS
`
`Tissues
`
`Human urinary bladders were obtained from 25 men
`and 3 women (mean age, 69 years), who were undergo-
`ing total cystectomy due to bladder carcinoma. We
`obtained permission for using human bladder tissue
`from the ethics committee at Kumamoto University
`School of Medicine. We also obtained informed con-
`
`sent from the patient or patient’s family before surgery.
`Preoperative external radiation therapy and Cherno-
`therapy were not given to all patients. The preparations
`were taken from the intact part of the dome region of
`urinary bladder, and immediately placed in modified
`cold Krebs-Henseleit solution. After removal ofserosal
`
`and mucosal layers, detrusor smooth muscle was cut
`into strips (approximately 4 mm wide and 15 mm long).
`
`Functional Experiments
`The functional experiments were performed as previ-
`ously described.” The preparation was mounted in a
`20—mL organ bath filled with modified Krebs—I-lenseleit
`solution at 37°C and bubbled with 95% oxygen gas
`(02) and 5% carbon dioxide gas (CO2), resulting in a
`pH of 7.4. Each tissue preparation was connected to a
`force—displaccment
`transducer (TB-61 1T; Nihon
`Kohden, Tokyo, Japan), and the isometric tension de-
`velopment of the preparation was recorded on an ink-
`
`writing recticorder (RJG 4004; Nihon Kohden). Rest-
`ing tension was adjusted to about 1.5 g, and the
`preparations were allowed to equilibrate for 90 minutes
`before starting the experiments. Each experiment was
`repeated on 20 tissue samples, with the exception of the
`testing for the atropine-resistant portion of the contrac-
`tion (n = 8, each drug).
`The concentration-response curves to acetylcho-
`line were obtained by adding acetylcholine directly to
`the bathing media in a cumulative fashion, whereas the
`responses to high potassium ion (K*) solution (80
`mmol/L potassium chloride, KCI) were obtained by
`replacement ofmodified Krebs—I-Ienseleit solution. Con-
`tractile responses to 5 mmol/L of calcium chloride
`(CaCl2) were measured in a calcium ion (Ca2+)-free
`solution including 20 mmoi/L KCl, after incubating the
`preparation in this medium for 20 minutes (washed
`every 5 minutes with Ca2*—free solution).
`The effects of various drugs (atropine, oxybutynin,
`temiverine, and RCC—3-6) on the contractions induced
`by acetylcholine, KCI, CaCl2, and electric field stimula-
`tion were measured at least 30 minutes after the study
`drug was given, to allow for accommodation. When
`electrical field stimulation was performed, the prepara-
`tion was mounted between 2 platinum electrodes (10
`mm long and 8 mm apart) in the organ bath. The
`intramural stimulation of nerves was performed using
`an electric stimulator {SEN-3301; Nihon Kohden)
`delivering rectangular shock wave pulses with a dura-
`tion of 0.3 milliseconds at supramaximum voltage, at
`a stimulation frequency of 20 Hz. Train duration was
`3 seconds, and the stimulation interval was 120 sec-
`onds.
`
`In the preliminary study to evaluate the frequency-
`response curves in human detrusor smooth muscle, the
`atropine—resistant contraction was more pronounced
`during low-frequency,
`than during high-frequency,
`stimulation (44% of control contraction in 2 Hz and
`14% in 60 Hz). However, low-frequency stimulation
`did not produce a sufficient contractile response to
`enable evaluation of the inhibitory effects of the com-
`pounds used in this experiment. Therefore, we chose a
`stimulation frequency of 20 Hz. During 20-Hz stimu-
`lation, the contractile response was about 75% of the
`maximum contraction induced by 60-Hz stimulation,
`and atropine-resistant portion was about 28% of the
`control contraction induced by 20-Hz stimulation.
`After reaching a stable baseline, the first 5 contrac-
`tions induced by the shock wave pulses were used as
`controls. Then each drug was added in a cumulative
`fashion, and the pharmacologic action was evaluated.
`The response to electric field stimulation was completely
`abolished by pretreatment with 10"“ molfl. tetrodotoxin.
`To evaluate the effects of various drugs on atropine-
`resistant contraction, various drugs were administered
`during the electric field stimulation after pretreatment
`
`269
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2045 - 0002
`
`
`
`Int I Urol 1998;5:26ti-275
`
`with 10*“ mol/L atropine for 20 minutes. Before and
`after performing the experiments, the KCI-induced
`contractions were measured in each preparation to
`evaluate the contractility. There were no significant
`differences between the 2 contractile responses in each
`preparation.
`In preliminary experiments, the concentration»
`response curve to acetylcholine, or the contractile re-
`sponses to KCI or CaCl2, without any antagonist, were
`measured once every hour in t_he same strip. The con-
`tractile responses were similar for 9 to 10 hours. In
`further experiments, the contractile response to one
`agonist was studied in the absence of an antagonist, and
`thereafter, in the same strip, in the presence of increas-
`ing concentrations of one antagonist.
`To assess the potency of anticholinergic action, the
`dose-ratio was obtained from the ratio of EDS” values
`(the concentration of an agonist that produced 50% of
`the maximum contraction) for acetylcho1ine—iriduced
`contractions in the presence and absence of each an-
`tagonist. Antagonist dissociation constants (KB) were
`determined from the following equation: KB = antago-
`nist (mol/L)/ (dose ratio — 1). The affinity constant (pA2)
`values were then expressed as the negative logarithm of
`K3. In addition, the Schild plots were constructed by
`plotting the log of (close ratio — 1) against the log of the
`molar concentration of the antagonist.”
`
`Drugs and Solution
`Drugs used were: atropine sulfate, acetylcholine chlo-
`ride, tetrodotoxin (Sigma Chemical, Tokyo, japan).
`Ten-iiverine and RCC—36 were donated by Nippon
`Shinyaku (Kyoto, Japan). Oxybutynin hydrochloride
`and other chemicals and materials were of analytical
`grades and were obtained from commercial sources.
`The modified Krebs-Henseleit solution had t_he follow-
`
`ing composition (mmol/L): sodium chloride (NaCl),
`117.7; KCl, 4.69; CaC12, 2.16; magnesium sulfate
`(MgSO4), 1.20; sodium bicarbonate (Nat-{CO3 ), 24.39;
`potassium dihydrogenphosphate (Kl-IZPO4), 1.20; and
`glucose, 9.99. Ahigh K‘ solution (80 mmol/L KCI) was
`made by substituting NaCl with equimolar KCI in the
`modified K.rebs—Henseleit solution. Ca3+-free solution
`was made by removing CaCl3 from modified Krebs—
`Henseleit solution and adding 0.1 mmol/L ethylene
`glycol—bis ()8-aminoethyl ether) tetraacetic acid (EGTA).
`Drugs were dissolved in distilled water and volumes of
`0.2 mL were added to the bath. Concentrations were
`
`expressed as the final bath concentrations.
`
`Data Analysis
`Contractile data were expressed in terms of active force
`(grams) divided by the cross—sectional area (mm2) . 1 '5 The
`cross—sectional area was calculated according to the
`following formula: cross—sectional area = weight
`in
`grams/(length in centimeters X 1.05), where 1.05 is the
`
`270
`
`assumed density (g/mm‘) of the muscle. The Ema, (the
`maximum Contractile response) value was obtained
`from the maximum stress developed and the ED50 and
`IC50 (the concentration of an antagonist that produced
`50% of the maximum inhibition) values were calculated
`from a sernilogarithrnic plot of the percentage of maxi-
`mum response vs drug concentration. The Em.“ values
`were calculated as arithmetic means, whereas the ED50
`and IQ,” and values were calculated as geometric
`means.” The analysis of variance and the multiple com-
`parison Fisher’s test were used for statistical analyses
`between groups and concentration response curves.
`
`RESULTS
`
`Acetylcholine-Induced Contractions
`Acetylcholine (10 5 to 2 x 10' 2 mol/L) caused a concen-
`tration-dependent contraction in human detrusor
`muscles. The Em, and ED50 values were 8.42 i 0 .75
`g/rnmz and 13.2 i 1.5 ,urnolfL, respectively. Atropine
`(10 9 to 10‘5 mol/L), oxybutynin (lO‘8 to 10‘5 mol/L),
`temiverine (10-8 to 10-5 mol/L), and RCC—36 (10-3 to
`3 X 10"" moi/L) caused inhibitory actions of the acetyl-
`choline response, and shifted the concentration-re-
`sponse curves to the right (Fig. 2). Atropine did not
`suppress the maximum contractile response to acetyl-
`choline, but the other drugs significantly suppressed
`the maximum contractile response to acetylcholine at
`the concentration of 105 mol/L or 3 x 10'“ mol/L.
`
`The pA2 values and slopes ofthe Schild plots for each
`drug are shown in Table l.All slopes of the regression line
`were close to unity. The pA2 value for atropine was
`significantly greater those that for the other drugs, and
`the pA2 values for oxybutynin and RCC-36 were sig-
`nificantly greater than that for temiverine.
`
`Potassium Chloride-Induced Contractions
`
`The Em, value for the KCl—induced contractions was
`6.85 i 0.41 g/tnmz. Atropine (10“‘ to 10*?‘ mol/L), oxy-
`butynin (1043 to 10-3 mol/L), temiverine (1043 to 104
`mol/L), and RCC-36 (10“’ to 10*’ rnol/I.) caused con-
`Cen[rati0n—dependent inhibition of KCl—indyced con-
`tractions (Fig. 3A). The maximum inhibition and IC50
`values are shown in Table 2. The maximum inhibitions
`oftemiverine and RCC-36 were not significantly differ-
`ent from that of oxybutynin. The maximum inhibition
`of atropine was significantly smaller than that of the
`other drugs. The IC50 value of atropine was statistically
`greater than that of the other drugs, and the IC50 value
`of oxybutynin and RCC-36 were significantly greater
`than that of temiverine.
`
`Calcium Chloride-Induced Contractions
`
`The Ema,‘ value for the CaCl2—induced contractions in
`human detrusor smooth muscle was 5.65 t 0.53 g/mmz.
`Atropine (l0"7 to 10'?’ mo]/L), oxybutynin (104 to 104
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2045 - 0003
`
`
`
`Effect of Temiverine and RCC-36 on the Bladder
`
`H. Kikukawa et al.
`
`l 00
`
`0 fiftmtrul
`
`
`—Q~ Uxylitityliin, 0.0l pmol/L
`
`{- Oxybutynirt, 0.1 ,umo|r'L
`—A.— Oxybutynin. l.O,umol;'L
` 80
`Al} Ovcyliutyiiiri, I0 wnoi/I,
`
`60
`
`40
`
`20
`
`-8.0
`
`--6.0
`
`100
`
`rO- Control
`
`
`.9. RCC—36, 0.0? ,umolr’L
`
`-- RCC-36, 0.1 ttmolft
`—A.~ RCC-"$6. t.0,umol/L
` 80
`-i:l'“ RCC.-36, 3.0;trn0l/L
`€30
`-
`
`
`
`40
`
`20
`
`O
`-8.0
`
`
`
`
`v2.0
`
`Log lacetylcllolirtej, moi/L
`
`Fig. 2. Effects ofseveral drugs on acetylcholine concentration-response curves in h u man detrusor muscles. Each point
`represents the mean i SE; if not shown, SE bars fall within the size of the symbols used.
`
`Table 1. Affinity constant (pA2) values and Slopes of Schild
`plots for various drugs applied to isolated human bladder
`detrusor muscle.
`
`was significantly greater than those of the other drugs,
`and that of oxybutynin was significantly greater than
`those of temiverine and RCC—36.
`
`Drugs
`
`Atropine
`Oxybutynin
`Temivcrinc‘
`RCC—36
`
`pr’-\2 values
`
`8.35 :0.l3-3
`7.42 i 0.15”
`6.65 .‘l_’ 0.10
`7.20 i 034"
`
`Slope
`
`1.03
`0.97
`0.94
`0.82
`
`Values are means : SEM. “P< 0.01 vs. oxybutynin, temiverine,
`and R('.C-36; "P< 0.05 vs. temiverine.
`
`tnolfL), temiverine (10 '7 to 10“ mol/L}, and RCC-36
`(10’7 to 105 mol/L) caused concentratiomdependent
`inhibitions of CaCl2-induced contractions (Fig. 3B).
`The maximum inhibitions of each drug are shown in
`Table 2. The maximum inhibition of atropine was
`significantly smaller than that of the other drugs, and
`that of oxybutynin was significantly smaller than those
`oftemiverine and RCC-36. The IC50 value ofatropinc
`
`Contractions Induced by Electric Field Stimulation
`The Ema,‘ value for the electric field stimulation in-
`duced contractions in the present study was 3.24 i
`0 .27 g/mm? Atropine (1 0*” to 10"‘ mol/L), oxybutynin
`(10’’‘’ to 10'“ mol/L), temiverine (109 to 2 X lO“‘ rnol/L)
`and RCC-36 (1049 to 2 x 10*‘ mol/L) caused concentra-
`tion~dependent inhibitions of the contraction induced
`by electric field stimulation without atropine pretreat-
`ment (Fig. 3C). The maximum inhibitions of each
`drug in atropine, oxybutynin, terniverine, and RCC—
`36 were 63.4 t 6.1%, 51.3 i 3.6%, 84.5 i 3.9%, and
`81.1 1- 2.9%, respectively. The maximum inhibitions
`of RCC—36 and remiverine were significantly greater
`than those of atropine and oxybutynin. The IC50 value
`of atropine was statistically different from those of the
`other drugs (Table 2).
`
`271
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2045 - 0004
`
`100
`
`80
`
`{)0
`
`40
`
`20
`
`
`
`-—O—- Cuntml
`-.# Atropine, 0.00] ,umt)l/L
`-‘- Atropine, 0.01 ,un1nlfL
`—A— Atropine, 0.l ,umo|/L
`"E" Atropine,
`I .U_t.trtt(tl/l
`
`
`
`s<1
`Q
`-8.0
`
`-6.0
`
`~4.0
`
`f‘+"
`-2.0
`
`—Q— Control
`
`r.~ Ternivvrirte, 0.0T ,umol'/L
`—I— Ternlverine, 0.1 ;.tm0l,’L
`—L— Temiverine, 3.0,.'.rrnol/l.
`Ttrniiverinn,
`l(l_utnnl/‘i_
`
`
`100
`
`80
`
`EU
`
`5%
`
`
`
`Int] Urul ‘.|998;5:26B—275
`
`A
`
`100
`
`80
`
`““
`4.3
`
`3111
`
`U
`-6.0
`
`8
`15’
`"8
`"T -
`
`E
`g E
`'v‘- 8
`}§
`_E
`e
`
`B
`
`100 5
`
`§
`:3
`E
`‘Nu.
`
`“U
`so
`
`1
`
`C
`
`100
`
`_>
`'3 (Ii 80
`-E E
`U “
`
`60
`
`2 3
`o 1: 40
`*3
`3‘
`:9 .3 20
`E
`
`
`
`E
`O ‘E 40
`CC: 8
`E
`
`.
`20
`0 _
`-7.0
`
`-5.0
`
`_. ._
`-4 0
`
`-3.0
`
`5
`-2 0
`
`'5“
`
`50
`
`_5_..__u___+
`--5.0
`-4.0
`-3.0
`
`5*
`
`<1
`-11
`
`.
`
`_#J_..+_.__-
`-7
`-5
`-1
`
`Log [drug], mol/L
`
`Log {drug}, mol/L
`
`LDE; [drug]. W101/L
`
`Fig. 3. Effects ofseverai drugs on induced contractions in isolated human detrusor muscles. (A) Potassium chloride (80 n1rn0l/Ll-
`induced contractions; (8) calcium chloride (5 mrnol/L)-induced contractions in Caz’-free buffer including 20 mmol/L potassium
`chloride; (C) electric field stimulation-induced contractions [0.3—mi|lisecond duration; 20 Hz frequency; 3-second train; 120-
`second interval). —.—, atropine; —p—- , oxybutynin; —o— , temiverine; -4:-—, RCC-36. Each point represents the mean 1 SE;
`il not shown, SE bars fall within the size of the symbols used.
`
`Table 2. Maximum inhibition and lC.,,, values of various drugs for induced contractions in isolated
`human detrusor muscles.
`
`Stimulant
`Potassium chloride
`Max inhibition [‘’/o)
`|C._,,(;1mol/L)
`Calcium chloride
`Max inhibition (%)
`lC5O(p_mol/L)
`
`Electric field
`Max inhibition ('79)
`ic,“ (pmol/LJ
`
`Atropine
`
`Oxybutynin
`
`Temiverine
`
`RCC-3Ei—
`
`69.2 1 4.2“
`> 250'
`
`80.5 i 4.2
`11.2 : 2.0
`
`90.5 i 5.6
`5.52 1: 1.3ob
`
`87.1 i 4.5
`13.0 1 22
`
`59.61 5.3“
`20412.04
`
`74.2 i 4.6‘
`8.34i1.81‘-
`
`91.3 i 5.2
`2.14 :0.34
`
`98.5 i 5.9
`1.34 $0.28
`
`63.4 1 6.1
`8.42 11.315“
`
`51.3 :t 3.6
`1845 :154
`
`84.5 i 3.9“
`4275 1 355
`
`81.1 i 2.9“
`25.53 : 356
`
`lC.,.,, the concentration of an antagonist that produced 50% ofthe maximum (max) inhibition; “P< 0.05
`vs. oxybutynin, temiverine, and RCC-3b; "P< 0.05 vs. oxybutynin and RCC-36; ‘-P< 0.05 vs.temiverir1e
`and I-{CC-36; "Pct 0.05 vs. atropine and oxybutynin; "P< 0.01 vs. oxybutynin, temiverine, and RCC-36.
`
`A1|'0PinE'R€SiS13M C0m1’3Cti0I‘|5
`After treatment with 10'° moi/L atropine, contraction
`of atropine—resistant portion of the Contraction was
`27.8 i 4.8% of the control contraction. Oxybutynin
`(10'“ to 3 X 10“" mol/L), temiverine (l0"“ to 3 x 10"“
`rnol/L}, and RCC-36 (10 8 to 3 x 10" mol/L) showed
`
`concentration—dependent inhibitory actions on the
`atropine—resistant contractions. The maximum in-
`hibitions induced by terniverinc and RCC—36 were
`significantly greater than that by oxybuty1'1in.ThcIC5D
`values induced by temiverine and RCC—36 were sig-
`nificantly smaller than that induced by oxybutynin
`(Fig. 4, Table 3).
`
`27.?
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2045 - 0005
`
`
`
`Effect of Temiverine and RCC-36 on the Bladder
`
`H. Kikukawa et al.
`
`l 00
`
`
`
`03 C
`
`C‘9
`
`£._.
`
`?\J 0
`
`contractions
`
`
`
`
`
`‘.7/1:»inhibitionofelectricallyinduced
`
`Log [drug], rnol/L
`
`Fig. 4. Effects of several drugs on atropine-resistant part of
`contraction induced by electric field stimulation (0.3-millisec-
`ond duration; 20 Hz frequency; _3—seconcl train; 120-second
`interval)
`in human rletrusor muscles. —u¥, oxybutynin;
`-4- , temiverine; ~—u-—, RCC—36. Each point represents the
`mean t SE;
`if not shown, SE bars fall within the size of the
`symbols used. Each drug was applied after pretreatment with
`it] “ moi/L atropine.
`
`Table 3. Maximum inhibition and IC.,., values ofvarious drugs
`for atropine—resistant contraction in isolated human detrusor
`muscle.
`
`|C,0 l,urnol/L)
`Max inhibition (9%)
`Drugs
`20.1 : 4.4“
`18.3 i 4.2‘
`Oxybutynin
`8.43 i 2.10
`85.3 .i_- 8.9
`Temiverine
`
`RCC-36
`82.3 “t 8.5 2.65 i 0.38
`
`Values are means : SEM; effects measured during electric field
`stimulation induced contractions, and after pretreatment with
`10"‘? mol/L of atropine; [CW the concentration of an antagonist
`thatproduced 50% ofthe maximum (max) inhihition;=‘P < 0.01
`vs.
`temiverine and RCC-36; ”P< 0.05 vs.
`temiverine and
`RCC36.
`
`DISCUSSION
`
`In this study, atropine (10"9 to 10"“ mol/L), oxybutynin
`(1 0’3 to 10'“ mol/L), temiverinc (1045 to 3 X 10'“ mol/L),
`and RCC—36 (10‘3 to 10"“ mol/L) caused parallel shifts in
`the concentratiomresponse curves without significant
`suppression in the maximum contractile response, indi-
`cating that all drugs have an anticholinergic action on
`human dctrusor muscles at these concentrations. The
`
`rank order ofanticholinergic potency, from the pA2 value,
`was atropine > oxybutynin = RCC-36 > temiverine. The
`other drugs, except for atropine, suppressed the maxi-
`mum contraction to acctylcholine at the concentration of
`
`10*‘ mol/L or 3 x 10 *5 mol/L. This finding indicates that
`these drugs have nonspecific blocking actions on the
`acetylcholinc—induced contractions in human dctrusor
`muscles, in addition to an anticholinergic action.
`A rapid inward flow of Ca“ through potential-
`operatcd and receptor-operated Ca” channels is prob-
`ably one ofthe most important triggers for smooth-muscle
`contractions.” It has been suggested that inhibitions of
`cholinergic receptor—independent Ca” entry into cells
`(calcium antagonistic actions) may attribute to the
`actions of these drugs. The present study showed that
`the contractions induced by KCl and CaCl2 were
`inhibited by all the drugs we tested on human detrusor
`muscles. In regard to calcium antagonistic actions, the
`inhibitory action of temiverine was equal to that of
`RCC—36, but it was greater than that of oxybutynin and
`atropine. These results suggest that the efficacy of the
`calcium antagonistic actions is different among the 4
`drugs used in our study. As temiverine was synthesized
`by the chemical modification of oxybutynin, the struc-
`tural difference may contribute to the differences in
`anticholinergic and calcium antagonistic activities be-
`tween Icmiverinc and oxybutynin.
`All drugs have concentration—dependent inhibitory
`actions on electrical field stimulation induced-con-
`tractions in our study. The maximum inhibitions of
`temiverine and RCC—36 were significantly greater than
`those of atropine and oxybutynin. Furthermore, com-
`pared to oxybutynin, temiverine and RCC-36 showed
`significantly greater inhibitory actions on contractions
`after treatment with 10*‘ rnol/L atropine. The data
`suggest that temiverine and RCC-36 significantly in-
`hibited the atropine-resistant part of the contractions,
`which were thought to be noncholinergic, nonadrenergic
`ncrvc—mediated contractions.
`
`Atropine—resistant contractions in the urinary blad-
`der are well recognized in many mammalian species? "
`.In the human urinary bladder, some reports have
`indicated almost complete inhibition of field stimula-
`tion-induced contractions by atropine or atropinic
`agents.13“9° However, in our study, atropine—rcsistant
`contractions (about 30% of control) still remained. It
`has been reported that atropine—resistant contractions
`were increased about 30% to 50% in obstructed blad-
`
`ders, such as may occur with benign prostatic hypertro-
`phy and in cases of neurogenic b1adder.7"9
`In the previous reports,” the human bladder prepa-
`rations were taken from younger patients (mean age, 45
`years old), in contrast, those in this study came from
`older patients (mean age, 69 years old). It is possible
`that the isolated human urinary bladder tissue used in
`our study might have come from patients with some
`bladder dysfunction, such as benign prostatic hypertro-
`phy and neurogenic bladder. This may be one of the
`reasons for the differences in atropine—resistant contrac-
`tions between our data and data of previous repons.”-2°
`
`273
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2045 - 0006
`
`
`
`Int ] Urol 1998;5:268-275
`
`it may be possible to explain the
`Furthermore,
`difference in atropine-resistant contraction as due to
`the difference in stimulation parameters used. Luheshi
`and Zar“ demonstrated the presence ofa noncholinergic
`component in the motor transmission of the isolated
`human urinary bladder under the electrical field stimu-
`lated condition, which is short trains of stimuli at long
`intervals. Our stimulation conditions were consistent
`
`with those used in Luheshi and Zar’s report.
`It is well established that part ofthe contraction of
`the urinary bladder muscle is due to neurotransmitters
`that are not cholinergic, but are associated with adeno-
`sine triphosphate (ATP) or a related nucleotide.‘-22'“
`Thus, it may be possible that temiverine and RCC-36
`inhibit such neurotransmitter-induced contractions in
`
`the human urinary bladder. Zar et al.‘‘ have shown that
`an atropine-resistant component of the response to
`nerve stimulation is more sensitive to calcium influx
`
`than is a cholinergic component. Furthermore, it has
`been demonstrated in the rat bladder that decreasing
`the calcium influx by using a calcium channel blocking
`drug was more effective in inhibiting the atropine-resis-
`
`tam component of the contraction induced by electri-
`cal field stimulation, than was using 05, 3- methylene
`ATP.°-25 This may imply that the calcium antagonistic
`actions of temiverine and RCC—36 contribute to the
`
`inhibition of the atropine—resistant part of the contrac-
`tions, which may be partly mediated by purinoceptor
`stimulation. However, the fact that Of, 13- methylene ATP
`desensitization did not completely abolish the noncho—
`linergic, nonadrenergic contractions in field stimulation
`responses in the rat bladder“ suggests that ATP is not the
`sole neurotransmitter for noncholinergic, nonadrenergic
`contractions. Therefore, the exact mechanism of atro-
`pine—resistant contractions of the urinary bladder re-
`mains to be elucidated.
`
`Temiverine is extensively metabolized, and only a
`trace amount of unchanged form was excreted into
`urine and bile in rats and dogs. ‘
`1 The concentration of
`unchanged temiverine and the biologically active me-
`tabolite, RCC-36, in the plasma, kidney, lung, liver,
`and bladder were determined in the rat after oral
`administration of temiverine labeled with "‘C.“ The
`
`concentration of the unchanged drug and RCC-36
`were higher in the bladder than in the plasma. This
`result may indicate that both temiverine and RC C-36
`easily distribute to the bladder, and contribute to the
`pharmacologic effects on the urinary bladder function.
`In conclusion, the present study showed that atro-
`pine, oxybutynin, temiverine, and RCC-36 had dif-
`ferent efficacies and potencies of anticholinergic and
`calcium antagonistic actions on human detrusor muscles.
`As compared to atropine and oxybutynin, temivcrine
`and RCC—36 effectively inhibited the isolated human
`detrusor—contractions induced by acetylcholinc, KCl,
`
`274
`
`CaCl2, and electric field stimulation. Thus, it is sug-
`gested that tcmiverine and its active metabolite, RCC-
`36, will be promising drugs for treatment of bladder
`overactivity.
`
`REFERENCES
`
`1. Khanna OP, Barbieri E], Altamura M, McMichael R.
`Vcsicourethral smooth muscle: function and relation to
`structure. Urology 1981;18:211-218.
`Z. Weiss RM. Physiology of ureter, bladder, and urethra.
`In: Massry SG, Glassock RJ (eds) Textbook of nephrol-
`ogy. Vol. 1. Baltimore:Williams &Wilkins, l983:79—84.
`3. Andersson KE, Fovaeus M, Hedlund H. Urogenital mus-
`carinic receptors and drug effects. Ann Clin Res 1988;
`20:356—366.
`4. Ambache N, Zar MA. Non-cholincrgic transmission by
`post-ganglionic motor neurones in the mammalian blad-
`der.J Physiol 1970;210:761--783.
`‘3. Johns A, Paton DM. Evidence for a role Ufprnstaglandins
`in atropine-resistant transmission in the mammalian uri-
`nary bladder. Prostaglandins 1976;11:595-597.
`6. Zar MA, Iravani MM, Luheshi GN. Effect of nifedipine
`on the contractile responses of the isolated rat bladder.]
`Urol 1000;143:835-839.
`7. Sjéigren C, Andersson KE, Husted S, Mattiasson A,
`Moler-Madsen B. Atropine resistance of transmurally
`stimulated isolated human bladder muscle. J Urol 1982;
`l28:1368—l37l.
`8. Saito M, Kondo A, Kato'1‘, Levin RM. Response ofiso-
`lated human neurogenic cletrusor smooth muscle to in-
`tramural nerve stimulation. Br] Um] I 9913;72:723-727.
`9. Palea S,Artibani\X/, Ostardo E,Trist DG, Pietra C. Evi-
`dence for purinergic neurotransmission in human uri-
`nary bladder affected by interstitial cystitis. J Urol 1993;
`l'30:2007— 2012.
`
`10. AidaY, Kaneko Y, KasamaT. Effect of oxybutynin hydro-
`chloride on isolated smooth muscles. Folia Pharrr1acolJpn
`1986;87:629-639.
`1 1. Hirota T, Nakamura A, Sugihara K, Morino A. Pharma-
`colcinetics of (ir)-4-diethylamino-1 ,1 -dimethylbut-2-yn-1 -
`yl 2-cyclohexyl-2-hydroxy—2—phenylacetate mon0hydro-
`chloride niouohydrate. 4th communication: metabolism
`in rats and dogs. Drug Res 1997;47:175-181.
`12. Harnada K, SasakiY,T-aniguchi N, Fukui H, Miyatsuka
`Y, Kimura Y, Ukai Y, Yoshikuni Y, Kimura K. Anti-
`cholinergic and calcium antagonistic activities of NS-21
`contribute to the inhibition of rat urinary blacfdcr con-
`tractions. Gen Pharmacol 1997;29:771-778.
`13. Yoshida M, Nishi K, Machida J, Sakiyama H, Ikeda K,
`Ueda S. Effects of phorbol ester on lower urinary tract
`smooth muscles in rabbits. Eur J Pharmacol 1992,-222:
`205-21 1 .
`
`141. Arunlakshana O, Schild HO. Some quantitative uses of
`drug antagonists. Brit] Pharmacol 19S9;14:48-58.
`15. Latifpour J, Gousse A, Kondo S, Morita T, Weiss RM.
`Effects of experimental diabetes on biochemical and
`functional characteristics of bladder muscarinic receptors.
`J Pharmacol Exp Ther l989,'248:8l—88.
`16. Fleming \W\W,\X'/estfall DP, De La Lande IS, Jellett LB.
`Log-normal distribution of equieffective doses of note-
`pincphrine and acetylcholine in several tissues. J Pharmacol
`Exp Ther 1972;181:339-345.
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2045 - 0007
`
`
`
`Effect of Temiverine and RCC-30 on the Bladder
`
`H. Kikukawa et al.
`
`17.
`
`I8.
`
`19.
`
`20.
`
`21.
`
`Boltm-i'l‘B. Mechanism of action of transmitters and other
`substances on smooth muscle. Physiol Rev 1979;50:606-
`718.
`
`Nergérdh A, Kinn AC. Neurotransmission in activation
`of the contractile response in the human urinary blad-
`der. Scand J Urol Nephrol 1983;17:153-157.
`Sibley GNA. A comparison of spontaneous and nerve-
`mediated activity in bladder muscle from man, pig and
`rabbit. J Physiol l984;.'5‘34:43l—443.
`Kinder RB, Mundy AR. Atropine blockade of nerve-
`mediated stimulation of the human detrusor. Br J Urol
`19855572418-—-42 1.
`Luheshi GN, Zar MA. Presence of non-cholinergic mo-
`tor transmission in human isolated bladder. J Pharm
`
`23.
`
`Pharmacol 1990;42:223-224.
`. Dumsday B. Atropine-resistance of the urinary bladder
`innervation. J Pharm Pharrnat: 1071;23:222-225.
`Burnstock G, Cocks T, Kasakov L, Wong I-IK. Direct
`evidence for ATP release from non—adrenergic, non-
`cholinergic (purinergic) nerves in the guinea-pig taenia
`colt and bladder. Eur] Pharmacol 1978;49:145-149.
`Chancellor MB, Kaplan SA, Blaivas JG.The cholinergic
`and purinergic components of dctrusor contractility in a
`whole rabbit bladder model. J Urol 1992; 1 48:906—909.
`Luheshi GN, Zar MA. Purinoceptor desensitization im-
`pairs but does not abolish the noncholinergic motor trans-
`mission in rat isolated urinary bladder. EurJ Pharrnacol
`1990;185:203 -208.
`
`24.
`
`25.
`
`275
`
`Patent Owner, UCB Pharma GmbH — Exhibit