Effect of NS-21, an Anticholinergic Drug with Calcium Antagonistic Activity,
`on Lower Urinary Tract Function in a Rat Model of Urinary Frequency
`
`Yasuo Sasaki, Kozo Hamada, Chiemi Yamazaki, Toshie Seto, Yutaka Kimura,*
`Yojiro Ukai, Yoshiaki Yoshikuni, and Kiyoshi Kimura
`
`Research Laboratories, Nippon Shinyaku Co. Ltd., Kyoto, Japan
`
`is under development for the treatment of urinary frequency and urinary inconti-
`Background: NS—2'l
`nence. The purpose of this study was to investigate the effects of NS-21 and its active metabolite, RCC-
`36, on lower urinary tract function in an experirnental rat model of urinary frequency.
`Methods: Cystometrograms were recorded in anesthetized rats with bilaterally transected hypogastric
`nerves. All drugs were administered intraduodenally.
`Results: In sham-operated rats, NS—21 (2 50 mg/kg} significantly increased the bladder capacity without
`significantly decreasing micturition pressure, while RCC-36 (100 mg/kg) significantly increased bladder
`capacity, and at a dose of 2‘: Tltlnig/kg, also caused a decrease in micturition pressure. This increase in
`bladder capacity appeared at
`lower doses of both NS-21 and RCC36 in the hypogastric nerve-
`transected rats. Propiverine (I00 rug/kg)
`increased bladder Capacity and at 2 30mg/kg, decreased
`micturition pressure in both sham-operated and nerve-transected rats. Oxybutynin (l{JOmg/kg) and
`atropine (30mg/kg) decreased the micturition pressure in both sham-operated and nerve—transected rats
`without increasing the bladder capacity, while a similar anticholinergic calcium antagonist, terodiline
`(100mg/kg) had no effect on bladder capacity in either sham-operated or nervewtransected rats.
`Flavoxate (5{iOrng/kg)
`significantly increased bladder capacity without
`significantly decreasing
`micturition pressure in both sham-operated and nerve-transected rats, while 50 mg/kg of verapamil
`significantly increased bladder capacity without significantly decreasing the micturition pressure in
`nerve-transected rats.
`lower doses in hypogastric nerve-
`Conclusions: NS—21 and RCC-36 increased bladder capacity at
`transected rats than in sham—operated rats. Furtherniorc, NS-LE1 increased the bladder capacity without
`suppressing rnicturition pressure, suggesting that NS-21 may be a more effective therapeutic. drug than
`propiverine, oxybutynin or flavoxate for the treatment of urinary frequency and urinary incontinence.
`Int I Urol 1‘3‘}7;4:401—40fi
`
`Key words: NS-21, bladder capacity, hypogastric nerve transection, rat
`
`INTRODUCTION
`
`2-
`(:)~4-diethylamino-1,1-dimethylbut-2-yn-1-yl
`cyclohexyl—2—hydroxy-2-phenylacetate monohydro-
`chloride monohydratc (NS—21; Fig. 1)
`is a novel
`compound intended for the treatment of urinary fre-
`quency and urinary incontinence. This compound
`was designed to possess both calcium antagonistic
`and anticholinergic activities, and is predicted to have
`a beneficial effect on bladder dysfunction caused by
`hypcrexcitability of the bladder smooth muscle.‘ The
`effects of drugs that have been clinically used to treat
`urinary frequency and urinary incontinence on uri-
`
`Received Oct. 21, 1996:; at't'epted for public.ition in revised form
`Jan. 27, T9197. *Correspondence and requests for reprints to: Re-
`Search Lalmralories Co. Ltd., Nippon Sltinyaim, Nisliioji Hacltiio,
`Minami—ku, Kyoto 601, Japan.
`
`including propiverine and
`nary bladder function,
`oxybutynin, have been evaluated in various species
`such as rats, rabbits, cats and dogs.“ However, there
`are very few studies using these drugs in animal uri-
`nary frequency models that reflect the clinical symp-
`toms of impaired bladder function.
`In this
`study,
`the effects of NS-21 and its
`active metabolite, RCC-36
`[(i)-4—ethylamino-
`1,1—dirncthylbut—2—yn—l—yl
`2—cyclohexyl—2-hydroxy—
`2-phenylacetate
`monohydrochloride] ,
`were
`cystomctrically examined in a rat model of urinary
`frequency produced by bilateral
`transaction of thc
`hypogastric nerves on lower urinary tract function,
`and their effects compared with those of propiverine,
`oxybutynin and tcrodiline (all of which have both
`anticholinergic and calcium antagonistic activity),
`flavoxate (a drug which acts on the micturition
`center), atropine (an anticholinergic agent)
`and
`verapamil (a calcium antagonist).
`
`0919-8172/97/0404-0401/US$03.00 © IUA/CL] 1997
`
`401
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2044 - 0001
`
`

`
`Int I Urol 1997;4:401—4()6
`
`lntravesicol pressure
`
`_._
`
`
`
`Micturilion pressure
`
`i .........._. Bjadde, capacity ........... "4
`
`Fig. 2. Schematic cystometrogram. \, threshold pressure.
`
`Drugs
`NS-21, RCC-36, propiverine (propiverine hydrochlo-
`ride), oxybutynin (oxybutynin hydrochloride) and
`terodiline (terodiline hydrochloride) were synthesized
`in our laboratories. Flavoxate (flavoxate hydrochlo-
`ride) was obtained from Recordati
`(Milan, Italy).
`Atropine
`(atropine
`sulfate)
`and urethane were
`purchased from Sigma Chemical
`(St. Louis, MO,
`USA), verapamil
`(verapamil hydrochloride)
`from
`Nacalai Tesque (Kyoto, Japan), and ketamine (kera-
`mine hydrochloride) from Sankyo (Tokyo, Japan).
`The test drugs were suspended in 0.5% methyl cellu-
`lose (Shinetsu Chemical, Tokyo,
`Iapan) at con-
`centrations appropriate for an injection volume of
`1mL/kg.
`
`Statistical Analysis
`Data for the bladder capacity and micturition pressure
`are expressed as the mean : SEM. The significance
`of differences between the values before and after
`
`drug administration was determined by a paired t test
`(data for animals that died during the experiments
`were excluded from analysis).
`
`RESULTS
`
`The bladder capacity of the hypogastric nerve-
`transected rats was significantly less than that of the
`sham—operated rats, but there was no significant dif-
`ference in micturition pressure between the 2 groups
`(Table 1). Figures 3 and 4 show representative re-
`cordings before and after treatment with NS-21 or
`RCC-36 in sham-operated rats and hypogastric
`nerve—transected rats, and the time courses for the
`effects of these drugs on the bladder capacity are
`shown in Figs. 5 and 6. Figures 7 (sham-operated
`rats) and 8 (hypogastric nerve-transected rats) show
`the changes in the bladder capacity and micturition
`pressure at the time when the mean increases in blad-
`der capacity was maximized for. each group after drug
`administration. Although the micturition pressure
`varied among individual rats, it was fairly constant in
`the same animal after injection of vehicle (data not
`shown).
`In sham-operated rats, NS-21 at a dose of 30 mg/kg
`
`0
`
`H0-
`
`t|:H,
`--0—(: —CEC-CH2N(CH2CH3)2 -HCI -H20
`
`lC
`
`H3
`
`Fig. 1. Chemical structure of NS—2l.
`
`MATERIALS AND METHODS
`
`Animals
`
`Male Sprague-Dawley rats 9-12 weeks old and weigh-
`ing 300—380 grams (Japan SLC, Shizuoka, Iapan)
`were housed in groups of4 to 6 in a room maintained
`at 21—25°C and 45-65% humidity with an alternating
`12-hour lightfdark cycle. Food and water were given
`ad libitum.
`
`Transection of the Hypogastric Nerves
`The method of Maggi et al.“ was used to transect the
`hypogastric nerves. Rats under ketamine anesthesia
`(25 mgfkg, im) were placed in a supine position. A
`midline incision was made in the abdomen, both
`hypogastric nerves transected about 5mm from the
`pelvic plexus, and the wound sutured. At the same
`time, a group of rats was subjected to laparotomy
`alone (sham—operated rats). Two to 3 days after sur-
`gery, cystometry was performed.
`
`Cystometry
`Under urethane anesthesia (900 mg/kg, sc), rats were
`placed in a supine position. The bladder was exposed
`through a midline incision in the abdomen, and
`a
`polyethylene
`catheter
`(PE50; Clay Adams,
`Parsippany, NJ, USA) inserted into the apex of the
`bladder dome and connected to a three—way stopcock.
`One outlet was connected to a pressure transducer
`(TP—200T; Nihon Kohden, Tokyo, Japan) for record-
`ing the intravesical pressure. The other outlet was
`connected to an infusion pump (STC-521; Terumo,
`Tokyo, Japan) for infusion of physiological saline.
`After the bladder was emptied, reflex micturition was
`induced by filling the bladder with warm saline at a
`rate of 2.8mI_./h with the infusion pump. The flow of
`saline was terminated when reflex micturition oc-
`
`curred. The bladder capacity and the rnicturition
`pressure were read from the cystometrogram (Fig. 2).
`After the bladder capacity and micturition pressure
`stabilized, drugs were injected into the duodenum
`and cystometric recordings were taken at 30, 60, 120,
`and 180 minutes after injection in order to evaluate
`the effects of the drugs on bladder capacity and
`micturition pressure.
`
`402
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2044 - 0002
`
`

`
`Effect of N5~21 (in Rat Bladder Function
`
`Y. Sasaki et al.
`
`Table 1. Etfcct of biiaterai transcction of the hypogastric
`nerves on bladder capacity and rT‘IiC‘.iLli'iiiOl‘l pressure in
`urethane anesthesized rats.
`
`Treatnieni
`
`_
`.
`_
`Sham °"’e’a"°“
`Hypogastric n€?rV(‘.ti':lI1St¥('.iit'J11
`
`Bladder
`Cdflatiiy
`_ mil‘)
`__
`“'44 3 ‘m4
`£121’) 1 0.02“
`
`Micturition
`
`Pmsbute
`.’ "I7" H191.
`‘I33 i 3-9
`26.2 "' 2.0
`
`Dale‘ “W presmmd as the mean : SEM OT 6 ‘m'ma|5‘
`‘P < 0.01 between treatment groups.
`
`had no effect on the bladder capacity. However, at 50
`and 100 mg/kg, it caused a significant increase in blad~
`der
`capacity without
`significantly affecting the
`micturition pressure, with the increase in bladder Ca-
`pacity reaching a maximum by 60 or 120 minutes
`after dosing. In hypogastric nc:rve—transected rats, NS-
`21 at 30 mg/kg or more increased the bladder capacity
`in a dose—dependent manner. At 30 and 50mg/kg, it
`causcd small decreases in micturition pressure which
`
`(A)
`
`M
`
`30 mrnfig
`
`(B)
`
`M
`
`30 mmfls
`
`BCTOIB ?’_______,d\_)i:‘
`
`0
`
`30 mg/kg
`1 h after
`
`i
`
`A
`
`M
`
`30 mmiag
`
`0
`
`M
`
`30 mmHg
`
`33°F“ ‘_____,_.Ji__
`
`0
`
`-i
`
`M
`30 mg,/kg
`1)‘: aftcr
`i
`
`3” "“’“”3
`
`0
`
`Before .___—_—__’__r/L.‘
`
`0
`
`Before
`
`50 mg/kg
`
`1 h after
`
`l
`
`1
`
`M
`i
`
`5 min
`
`30 mmHg
`
`O
`
`50 mg/kg
`
`in
`M
`1 h aftcr
`
`A
`
`2 min
`
`M
`
`an mmitg
`
`0
`
`30 ml]
`m
`
`B
`
`0
`
`Fig. 3. Representative cystometric recordings showing the effect of intraduodenally administered NS—21 on the urodynamics
`in .3 (A) sham-operated rat or (B) rat with transected hypogastric nerves.
`I
`, start of saiine infusion; M, micturition.
`
`(A)
`
`M
`
`30 mmHg
`
`Before LL B f
`
`A
`
`”
`3U rn.inHg
`
`50 mg/kg
`1h aftcr
`
`M
`i
`
`0
`
`(B)
`
`M
`
`I
`
`3UmmHg
`
`“
`M
`
`30 rrtrnHg
`
`0
`
`e are
`
`50 mg/kg
`111 after
`
`‘
`
`i
`
`i
`
`*
`
`Before
`
`M
`
`30 mmflg
`
`0
`
`M
`
`Before Aéflfk ’
`
`30 mmHg
`
`0
`
`30 mmlig
`
`‘
`
`30 rnmllg
`
`:00 mgfkg
`M
`I h after 0
`
`100 mgfkg
`1 h after
`
`
`M
` ‘? 0
`
`*
`
`i
`
`5 min
`
`2 mm
`
`Fig. 4. Representative cystonietric recordings showing the effect of intracluodenaliy administered RCC-.36 on the urodynamics
`of (A) sham-operated rats and (B) rats with transected hypogastric nerves.
`i, start of saline infusion; M, niicturition.
`
`403
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2044 - 0003
`
`

`
`In! J Urol 1997;4:401—406
`
`1.0
`
`O.8
`
`0.6
`
`(l. 4
`
`(mL) 0.2 no
`
`Bladdercapacity
`
`Bcfure
`
`an
`
`50
`
`90
`
`120
`
`150
`
`130
`
`
`
`
`
`Bladdercapacity(rnI.}
`
`Time after administration (min)
`
`Fig. 5. Effect of intraduedenally administered NS—',:"l on the
`h|.trl(ler c.1p.1i'ity of sham-<')per;tted rats (solid lines) and rats
`with transected hypogastric nerves (brol<en lines). <=,
`l(]mg/
`kg; c, ._%()n1g/kg;
`.1, 5Urni_.r,/kg; I, l00mg/kg. Eacli puint rep-
`resents the mean i SIEM for 6 animals. *P< 0.05; **P< 0.01;
`sig,nifican(:e determined between pre— and postdrug adminis-
`tration (paired treat).
`
`1 .0
`
`
`
`
`
`Micturitienpressure(mmHg)
`
`U. 8
`
`,1‘?ca
`
`(mL) 0.2 0.0
`
`Bladdercapacity
`
`Before
`
`an
`
`on
`
`90
`
`no
`
`150
`
`,
`I so
`
`Time after administration (min)
`
`Fig. 6. Effet t of inlradundenally administered RCC-.i€a on the
`bladder capacity of sham-operated rats [solid lines) and rats
`with transected hypogastric nerves (broken lines}.
`30 mg/
`kg; 0, Sflrng/kg," "‘,
`i00rnp,/kg. Each point represents the
`mean i SEM for 6 animals. *1” < 0.05; **P < 0.0]; signifi-
`CdI‘IC(.‘ Cleterniinetl between pre— and postdrug adminislrettinn
`(paired ttest).
`
`were not statistically significant, but caused a signifi-
`cant decrease at 100 mg/kg.
`In sham-operated rats, RCC—36 at 100mg/kg
`caused a significant increase in bladder capacity, and
`at 30—100mg/kg, a significant decrease in rnicturition
`pressure. In hypogastric nerve—transeeted rats, RCC-
`36 at ‘$0 and 100mg/kg caused significant increases in
`bladder capacity and at 30-100 mg./kg, caused a sig-
`nificant decrease in mieturition pressure.
`Pmpiverine caused a significant increase in bladder
`capacity at 100mgfkg in both sham-operated and
`hypogastric nerve-transected rats. The rnieturitien
`
`404
`
`{.2
`
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`
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`50 W0
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`
`50 L09
`
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`30 50 I00
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`100 100 500 m 30
`
`an
`
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`016
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`l
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`
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`r
`
`r
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`l
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`
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`SU I00 1003005“)
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`F
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`I
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`10
`
`30
`
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`
`(5!
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`
`(fa)
`( 1
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`F-‘laverale Atrngnnc Vuu mil
`
`Fig. 7. Effect of NS-ll, RCC-3b and reference drugs on the
`bladder capacity and rnicturitinn pressure in shan1—c>peratetl
`rats. :1, before drug administration; I, after. Each value rep-
`resents the mertn i SEM, with the nurnber of animals used for
`each experiment shown in parentheses. *P < 8.05; **P <
`(1.01; significance determined between pre- and pnstclrug
`adrninistmtion (pnirerl tlesl).
`
`pressure was decreased using a dose of at least 30 mg!
`kg. Oxybutynin caused a significant decrease in
`micturitien pressure using at least 50mg/kg in sham-
`eperated rats, and at
`least 30mg/kg in hypogastric
`nerve—transected rats but had no effect on the bladder
`
`capacity at any close tested. In sham-operated rats,
`terocliline had no effect on the bladder capacity at 30
`and 50mg/kg, but caused a significant decrease in
`rnicturition pressure at 50mg/kg. At 100 mg/kg,
`it
`showed a tendency to increase bladder capacity, how-
`ever, 3 of 6 rats died within 180 minutes after receiv-
`ing this dose. In hypogastric nerve—transected rats,
`terodiline had no effect on bladder capacity at 30 or
`50mg/kg, but at 100mgfkg, it showed the same ten-
`dency to increase bladder capacity as it did in sham-
`nperated
`rats. The micturition
`pressure was
`significantly decreased with 30 mg/kg terodiline, but
`this effect was not significant at 50 or 100 mg/
`kg. As with sham—operated rats, 3 of 6 rats died
`within 180 minutes after receiving a dose of 100mg.’
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2044 - 0004
`
`

`
`Effect of NS-21 on Rat Bladder Function
`
`Y. Sasaki et al.
`
`05
`
`04
`
`0.3
`
`U1
`'
`
`G‘
`
`0.0
`
`
`
`
`
`Bladdercapacity(ml.}
`
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`
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`
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`."\|.t(!}')lI'I: Vcrapemll
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`
`
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`
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`(6)
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`is:
`is:
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`
`tory control of the urinary bladder and induces a
`decrease in the bladder capacity of rats. In this study,
`we confirmed the decrease in bladder capacity in rats
`with bilateral transaction of the hypogastric nerves
`and, using this model, performed a cystometrical
`evaluation ofthe effects of NS-21, RCC—36 and refer-
`encc drugs on bladder function.
`acetylcholine
`Atropine,
`a
`potent muscarinic
`receptor antagonist, and oxybutynin, which has po-
`tent anticholinergic activity and direct, but weak,
`muscle relaxant activity,” significantly decreased the
`micturition pressure in both sham-operated and
`hypogastric netve—transcctcd rats, but had no effect
`on the bladder capacity in rats from either group,
`which is similar to results reported by N01-onha—
`Blob et al.
`in anesthetized guinea pigs.” In a study
`using conscious rats, Guarneri et al. reported that
`oxybutynin decreased the micturition pressure in a
`dosc—dependent manner, but increased the bladder
`capacity significantly only at low doses.“ Therefore,
`the major effect of anticholinergic agents on bladder
`function in rats and guinea pigs is thought to be a
`decrease in micturition pressure.
`In this study, verapamil, a calcium antagonist, sig-
`nificantly increased the bladder capacity at 50mg/kg
`without decreasing the micturition pressure in hypo-
`gastric nerve—transected rats, but had no effect
`in
`sham-operated rats. In sympathectomized rats,
`the
`intravcsical pressure during the urine storage phase
`was shown to be significantly higher than in sham-
`opcrated rats, and consequently the bladder capacity
`was reduced.” Taken together with the report that
`calcium antagonists decrease the basal
`tension of
`bladder smooth muscle,” the findings of this study
`suggest that a decrease in the basal tension of bladder
`smooth muscle induced by calcium antagonists in
`hypogastric nerve—transectcd rats, which is more pro-
`nounced than the decrease obscrved in sham—oper—
`ated rats, and which leads to a depression of afferent
`neural activity in the pelvic nerves that contributes to
`the inhibition of the micturition reflex and a conse-
`
`quent increase in bladder capacity.
`NS-21, which has both calcium antagonistic and
`anticholinergic activities, significantly increased the
`bladder capacity at 50mg/kg in sham-operated rats
`and at 30 mg/kg in hypogastric nerve—transected rats
`without affecting the micturition pressure. RCC-36,
`which has similar drug activities to NS-21, also in-
`creased the bladder capacity in hypogastric nerve-
`transected rats at lower doses than in sham-operated
`rats. The increase in bladder capacity caused by NS-
`2l is likely to be mediated by its calcium antagonistic
`activity through the mechanism similar to that re-
`ported for verapamil, and its anticholincrgic activity
`may act synergistically with its calcium antagonistic
`activity to potently inhibit the contraction of bladder
`
`405
`
`Fig. 8. Effect of NS—2l, R(fC—'%6 and refermce drugs on
`hlaritier cap‘.1t'ity and lTil(‘IlJI'lll()|‘I presstire in rats with
`llnnscctcd hypogastric nerves. :, hefore drug administration;
`I, after. Each value represents the mean : SEM, with the
`number of animals for each experiment shown in parenthe-
`ses. ”‘P< 0.05, *”‘P< (L01; significance determined between
`pre- and postrlrug aciministralion (paired 1 test).
`
`kg. Flavoxatc had no effect on the bladder capacity
`in
`either
`sharn—operatcd
`or hypogastric nerve-
`transected rats at 100 or 300 mg/kg. However, at
`500mg/kg, it caused a significant increase in bladder
`capacity without affecting micturition pressure.
`In
`both sham—operated and hypogastric ncrve—transected
`rats, atropine had no effect on bladder capacity
`even at 30 mg/kg, but it caused a significant decrease
`in micturition pressure. Verapamil at 30 or 50mg!
`kg had no significant effect on either bladder capacity
`or micturition pressure in sham-operated rats, but
`in hypogastric nerve-transected rats, a dose of 50 mg/
`kg Caused a significant increase in bladder capacity
`without any significant effect on the micturition
`pressure.
`
`DISCUSSION
`
`Maggi et al.3 reported that bilateral transection of the
`hypogastric nerves abolishes the sympathetic inhibi-
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2044 - 0005
`
`

`
`Int I Urol ‘I997;4:401—40E>
`
`leading to an increase in bladder
`smooth muscle,
`capacity. It is possible that the decrease in micturition
`pressure caused by NS-21 at high doses may be medi-
`ated through its anticholinergic action. RCC—36,
`whose anticholinergic activity is exerted at much
`lower concentrations than its calcium antagonistic:
`activity,‘ is likely to produce a decrease in micturition
`pressure at doses lower than those which produce an
`increase in bladder capacity.
`Propiverine,” which has pharmacological proper-
`ties similar to those of NS-21 and RCC-36, has both
`calcium antagonistic and anticholinergic activities.
`However, unlike NS—21 or RCC-36, the increase in
`bladder capacity caused by propiverine was elicited
`only at high doses in the both sham—operated and
`hypogastric nerve-transected rats.
`It has been re-
`ported that
`the calcium antagonistic activity of
`propiverine is about 10 times less than that of NS—21
`in vitro.‘ Its weak calcium antagonistic activity may
`account for the weak effect of this drug on the bladder
`capacity. Although terodiline has both calcium an-
`tagonistic and anticholinergic activity,“ it had no
`effect in either sham-operated or hypogastric nerve-
`transected rats, even at a toxic dose. This drug may
`have pharmacological activities other than its calcium
`antagonistic and anticholinergic activities, which may
`prevent the inhibition of bladder contraction by a
`calcium antagonistic or anticholinergic mechanism,
`and at the same time cause the cardiac side effects that
`
`led to the withdrawal of this drug from clinical use.
`In this study, the effects of NS—21 and RCC—36 on
`lower urinary tract function were investigated in
`sham—operated and hypogastric nerve-transected rats.
`Both compounds significantly increased the bladder
`capacity in hypogastric nerve-transected rats at lower
`doses than in sham—operatcd rats. In particular, NS-
`2l caused an in_crcase in bladder capacity without a
`significant effect on the micturition pressure. High
`doses of propiverine caused an increase in bladder
`capacity and a decrease in micturition pressure in
`both sham—operated and hypogastric nerve—transected
`rats, but terodiline had no effect even at toxic doses.
`Oxybutynin caused a marked decrease in micturition
`pressure but no increase in bladder capacity in
`hypogastric nerve—transected rats. Our findings sug-
`gest that NS-21 may be a useful drug for the treat-
`ment of urinary frequency and urinary incontinence,
`and has fewer side effects than the drugs in current
`clinical use.
`
`406
`
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`Patent Owner, UCB Pharma GmbH — Exhibit 2044 - 0006