Note: Descriptions are shown in the official language in which they were submitted.
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
SPECIFICATION
AGENT FOR THE TREATMENT OF OVERACTIVE BLADDER
FIELD OF THE INVENTION
The present invention relates to agents for the
treatment of overactive bladder.
BACKGROUND OF THE INVENTION
Overactive bladder is a medical condition referring
to the symptoms of urinary urgency and frequency, with or
without urge urinary incontinence, when appearing in the
absence of local pathologic or metabolic factors that would
account for these symptoms. Storage and voiding of urine
are physiologically controlled by complex reflex pathways
including peripheral and central nervous systems (Urology,
50 Suppl. 6A: 36-52 (1997)). The urinary urgency refers to
urgent and strong desire to void, and the urge urinary
incontinence refers to involuntary urine leakage due to the
urinary urgency.
In patients suffering from the symptoms such as
urinary urgency and urge urinary incontinence due to
overactive bladder, involuntary (uninhibited) contraction
of the detrusor muscle is frequently observed in a
cystometric measurement, and is called detrusor
ov'eractivity. This detrusor overactivity is considered to
- 1 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
be a cause of urinary urgency and urge urinary incontinence.
Moreover, urinary urgency can lead to urinary frequency.
The detrusor overactivity is divided into 2 categories:
neurogenic bladder (detrusor hyperreflex) when a
neurological. problem is found in a patient, and unstable,
bladder (detrusor instability) when a neurological problem
is not found. It is considered that the cause of unstable
bladder is potentially neurogenic bladder or disorder of
detrusor smooth muscle per se (or both of them). Examples
of the neurological problem relating to neurogenic bladder
include Parkinson's disease, stroke, diabetes, multiple
sclerosis, neuropathy and spinal cord injury.
Feeling of the filled bladder is transferred to the
central nervous system via two bladder afferent neurons,
the Ab-fiber and the C-fiber; however, under the normal
condition, the. C-fiber is not involved (silent). On the
other hand, sensitivity of the C-fiber is known to be
increased under the condition of bladder hypersensitivity
and the like (Clinical J. Pain, 16: S86--89 (2000)).
Furthermore, it is known that a spinal cord reflex
mechanism via the C-fiber bladder afferents is involved in
the overactive bladder in patients with supranuclear spinal
cord injury (J.~Urol., 157: 585-589 (1997)).
The potassium (K*) channel is present on cell
membranes of various tissues and is involved in various
physiological activities via the control of membrane
- 2 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
potential. The K~ channel is classified into various types
depending on the voltage-dependency, Ca+*-sensitivity, and
other properties of the channel. The slowly-inactivating
A-type K~ channel is expressed in capsaicin-sensitive
dorsal root ganglion (DRG) neuronal cells (J. Neurophysiol'.,
75: 2629-2646 (1996)) and controls excitability of the C-
fiber (J. Phys.zol., 494: 1-16 (1996)). The action
potential of bladder afferent C-fiber of normal rats keeps
a high threshold value by the effect of the slowly-
inactivating A-type K+ channel. In contrast, in the rats
with chronic cystitis, the K+ current is attenuated due to
the changes in this ion channel characteristic. Thus, it
has been supposed that at the time of cystitis excitability
of the C-fiber increases, resulting in the overactive
bladder (,T. Neurosci., l9: 4644-4653 0 999)). In addition,
in the rats with overactive bladder following spinal cord
injury, density of the slowly-inactivating A-type Kt
channel is reduced and excitability of the C-fiber is
increased.
DISCLOSURE OF THE INVENTION
According to the above observations, we have made
the hypothesis that the overactive bladder, resulting from
various diseases such as neurogenic bladder like spinal
cord injury or bladder cystitis, can be treated by reducing
excitability of the C-fiber through opening the slowly-
_ 3 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
inactivating A-type K+ channel. We have found a compound
having a slowly-inactivating A-type K+ channel opening
activity or a pharmaceutically acceptable salt thereof is
useful for the treatment of overactive bladder, and we have
achieved the present invention.
An object of the present invention is to provide an
excellent agent for the treatment of overactive bladder.
The present invention relates to
(1) an agent for the~treatment of overactive bladder,
comprising, as an active ingredient, a compound having a
slowly-inactivating A-type K~ channel opening activity or a
pharmaceutically acceptable salt thereof, and
(2) the agent for the treatment of overactive bladder
according to (1), wherein the compound having a slowly-
inactivating A-type K+ channel opening activity is N-(5,5-
dioxido-10-oxo-4,10-dihydrothieno[3,2-c][1]benzothiepin-9-
'yl)-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide.
The present invention relates to
(3) a method for the treatment of overactive bladder,
which comprises administering a therapeutically effective
amount of a compound having a slowly-inactivating A-type K+
channel opening activity, or a pharmaceutically acceptable
salt thereof, and
(4) use of a compound having a slowly-inactivating A-
type K+ channel opening activity, or a pharmaceutically
- 4 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
acceptable salt thereof for the manufacture of the agent
for the treatment of overactive bladder.
Furthermore, the present invention relates to
(5) a method for screening agents for the treatment of
overactive bladder, comprising measuring a ~ slowly-
inactivating A-type K+ channel opening activity as an index.
The term "compound having a slowly-inactivating
A-type K+ channel opening activity" as used herein means
all compounds having a~ slowly-inactivating A-type K+
channel opening activity regardless of a novel compound or
a known Compound and without limitation to the structure of
compounds, so long as they have the slowly-inactivating A-
type K+ channel opening activity as one of their properties.
The compounds having a slowly-inactivating A-type
K~" channel opening activity used in the present invention
include (S)-(+)-N-(5,5-dioxido-10-oxo-4,10-
dihydrothieno[3,2-c][1]benzothiepin-9-yl)-3,3,3-trifluoro-
2-hydroxy-2-methylpropanamide (Compound 1). Compound 1 is
the same as Compound 1-25 described in WO 98/46587.
- 5 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
O
CF3
ow
CH3
Compound 1
In the screening method of the present invention,
the method for measuring a slowly-inactivating A-type K+
channel opening activity is not particularly limited, but
examples thereof include methods descried in Test Examples
land 2 described below.
The pharmacological activities of the compound used
in the present invention are described below based. on Test
Examples.
Test Example 1: Facilitatory effects on slowly-
inactivating K+ currents in DRG cells
Materials and methods
Animal preparation:
Experiments were performed on adult female Sprague
Dawley rats. First and second series of the experiments
were performed, respectively, in unidentified DRG neurons
and a specific population of DRG neurons innervating the
urinary bladder. The population of DRG neurons that
innervate the urinary bladder were labeled by retrograde
- 6 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
axonal transport of the fluorescent dye, Fast Blue (4~ w/v)
(Polyloy, Gross Umstadt, Germany), injected into the wall
of the bladder in halothane-anesthetized animals 7 days
before the dissociation. The dye was injected with a 28
gauge needle at three to six sites on the dorsal surface of
the organ (5-6 ~L per site, total volume of 20-30 ~,L).
Each injection site was washed with saline to minimize
contamination of adjacent organs with the dye.
Cell dissociation:
Freshly dissociated neurons from DRG were prepared
from halothane-anesthetized animals. h6 and S1 DRG were
dissected from animals and then dissociated in a shaking
bath for 25 minutes at' 35°C with 5 mL DMEM (Sigma)
containing 0.3 mg/mL trypsin (Type 3, Sigma), 1 mg/mL
collagenase (Type 1, Sigma), and 0.1 mg/mL
deoxyribonuclease (Type 4, Sigma). Trypsin inhibitor (Type
2a, Sigma) was then added thereto to neutralize the
activity of trypsin. Individual DRG cell bodies were
isolated by trituration and then plated on a poly-L-lysine-
coated 35 mm Petri dishes.
Electrical recordings:
Dye-labeled primary afferent neurons that innervate
the urinary bladder were identified using an inverted
phase-contrast microscope (Nikon, Tokyo, Japan) with
- 7 _
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
fluorescent attachments (W-lA filter; excitation
wavelength, 365 nm). Gigaohm-seal whole-cell recordings
were performed at room temperature' (20-22°C) on each
labeled neuron in a culture dish that. usually contained
three to seven labeled cells among a few hundred unlabeled
neurons. The internal' solution contained (in mmol/L): KC1
140, CaCl2 1, MgClz 2, EGTA 11, HEPES 10, Mg-ATP 2, and
Tris-GTP 0.4 adjusted to pH 7.4 with KOH. Patch electrodes
had resistances of 1-4 MS2 when filled with the internal
solution. Neurons were superfused at a flow rate of 1.5
mL/minutes with an external solution containing (in
mmol/L): NaCl 150, KCl 5, CaCl2 2.5, MgCla 1, HEPES 10, and
D-glucose 10, adjusted to pH 7.4 with NaOH. All recordings
were made with an Axopatch-1D patch-clamp amplifier (Axon
Instruments, Foster City, CA), and data were acquired and
analyzed by PCLAMP software (Axon Instruments).
In voltage-clamp recordings, outward K+ currents
and inward Na+ currents were measured. For the isolation
of K~' currents, the external solution was changed to one
containing (in mmol/L): choline-C1 150, KOH 5,~CaCl2 0.03,
HEPES 10 , Mg ( OH ) z 3 , and D-glucos a 10 , adj usted to pH 7 ..4
with HC1.
In first series of experiments using unidentified
DRG neurons, outward K''' currents were evoked by voltage
steps to +60 mV, 800 ms long from a holding potentials of -
_ g -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
90 mV, this was followed by a 1-second conditioning pre-
pulse to -20 mV followed by a second pulse to +60 mV,
identical to the first in the sequence. Tn the second
series of experiments using Fast Hlue-labeled bladder
afferent neurons, slowly-inactivating A-type K+ currents
were isolated, by subtraction of outward K'~ currents
activated from a holding potential of -40 mV (on the
condition of inactivation of the majority of slowly-
inactivating A-type K+ currents) from those activated from
a holding potential of -120 mV (on the condition of full
activation of slowly-inactivating A-type K+ currents). A
compound was added cumulatively, starting with a lower
concentration. Currents were measured at the maximum
(peak) and normalized to control (before addition of the
compound).
Inward Na+~currents were evoked by voltage steps to
+60 mV, 800 ms long from a holding potential of -90 mV.
Currents were measured at the maximum (peak) and normalized
to control (before addition of the compound).
The results obtained in unidentified DRG neurons
are shown in Tables 1 to 5, and the results obtained in
capsaicin-sensitive bladder afferent neurons are shown in
Table 6. Table 1 shows the activity of Compound 1 upon
changes in currents when the holding potential is -90 mV
(on the condition of activation of slowly-inactivating A-
- g _
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
type K~ currents) or -20 mV (ow the condition of
inactivation of slowly-inactivating A-type K+ currents).
Table 2 shows the activity of Compound 1 in the presence of
20 mmol/L tetraethylammonium, Table 3 shows the activity of
Compound 1 in the presence of 5 ~u,mol/L verapamil, and Table
4 shows the activity of Compound 1 in the presence of 60
mmol/L tetraethylammonium. Table 5 shows the activity of
Compound 1 upon Na+ currents. In Tables 1 to 5, n means
the number of cases.
Table 1
Compound 1 (mol/L) HP: -90 n HP: -20 n~
mV mV
5.0x10'9 1.01 0..03 7 ~ 0.98 0.02 7
l.OxlO'B 0.99 0.08 3 0.98 0.04 3
2.5x10'8 1.07 0.03 5 1.01 0.03 5
5.0x10'8 . 1.07 0.02 16 1.00 0.01 16
1.0x10'' 1.12 0.03 10 1.02 0.01 10
5.0x10'' 1.15 0.03 7 1.01 0.02 7
1.0x10'6' 1.06 0.02 13 1.00 0.02 13
1.0x10'5 0.96 0.05 8 0.88 0.07 8
5.0x10'5 0.83 0.09 3 0.81 0.08 3
Table 1 shows the activity of Compound 1 on
currents measured by a voltage clamp using unidentified DRG
cells. The currents at a holding potential (HP) of -90 mV
are results of the measurement of slowly--inactivating A-
- 10 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
type K~ currents, and the currents at an HP value of -20 mV
are results of the measurement of delayed rectifier K+
currents. Table 1 shows that Compound 1 increases slowly-
inactivating A=type K+ currents with a peak compound
concentration of from 1x10-' to 5x10-' mol/L, but does not
have no influences (little influences) upon delayed
rectifier K+ currents. Also, effects (values) of Compound
1 are shown by relative values when the values before the
compound application are defined as I.
Table 2
Compound 1 (mol/L) HP: -90 mV N HP: -20 mV n
5.0x10-9 1.02 0.02 8 0.99 0.00 8
5.0x10'8 1.08 0.02 10 0.99 0.01 10
1.0x10-' 1.10 0.01 3 0.99 0.02 3
5.0x10-' 1.15 0.04 14 1.01 0.01 I4
I.OxlO-6 1.19 0.05 4 1.00 0.00 4
5.0x10-6 1.16 0.03 10 0.98 0.01 10
1.0x10'5 1.19 0.01 3 1.01 0.02 3
5.0x10-5 0.91 0.04 8 0.84 0.06 8
5.0x10-4 0.29 0.02 4 0.25 0.04 4
Table 2 shows the activity of Compound 1 in the
presence of tetraethylammonium as a blocker of delayed
rectifier K+ currents. Since Compound 1 shows the same
results in the presence of the blocker of delayed rectifier
- 11 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
K* currents, it is suggested that the K* currents
increasing effect of 'Compound 1 is not mediated by the
delayed rectifier K+ channel.
Table 3
Compound 1 (mol/L) HP: -90 N HP: -20
mV mV
n
5.0x10-a 1.03 0.02 3 1.00 0.00 3
1.0x10-~ 1.13 0.02 4 1.00 0.01 4
5.0x10-~ 1.17 0.03 8 1.00 0.01 8
1.0x10-6 1.11 0.03 5 1.00 0.01 5
5.0x10-6 1.02 0.03 6 1.01 0.01 6
1.0x10-5 0.99 0.02 6 1.00 0.02 6
5.0x10-S 0.94 0.02 9 0.98 0.01 9
5.0x10'4 0.61 0.05 6 0.83 0.06 6
Table 3 shows the activity of Compound 1 in the
presence of verapamil as a blocker of delayed rectifier K*
currents. Since the results obtained in Table' 3 are
similar to those in Table 2, it is suggested that the K+
currents increasing effect of Compound 1 is not mediated by
the delayed rectifier K* channel.
- 12 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
Table 4
Compound 1 (mol/L) HP: -90 mV N HP: -20 mV n
5.0x10'8 1.08 0.02 12 1.00 0.01 12
5.Ox10'~ 1.11 0.03 12 0.97 0.03 12.
5.0x10'6 1.14 0.05 10 0.97 0.02 10
5.0x10'5 1.15 0.08 7 0.95 0.03 7
5.0x10'4 0.56 0.05 5 0.54 0.02 5
Table 4 shows the activity of Compound 1 in the
presence of high concentration tetraethylaminonium (TEA).
The high concentration tetraethylammonium (TEA) acts as a
blocker of delayed rectifier K+ currents.. Since the
results obtained in Table 4 are also similar to those in
Table 2, it is suggested that the K~ currents increasing
effect of Compound 1 is not mediated by the delayed
rectifier K+ channel.
Table 5
Compound 1 (mol/Z) Na+ ion currents n
5.0x10'8 0.99 0.01 9
5.0x19"' 0.99 0.01 9
1.0x10'6 0.99 0.02 4
5.0x10'6 1.00 0.00 6
5.0x10'5 1.00 0.01 6
5.0x10-4 1.00 0.01 5
- 13 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
Table 5 shows the activity of Compound 1 upon Na+
currents in DRG cells. It is evident from Table 5 that
Compound 1 does not exert influence upon Na+ currents.
Table 6
Compound 1 A-type IK n Delayed n
( mol /L ) rectif ier
(HP: -120 mV)-
(HP: -40 mV) HP: -40 mV
1.0x10-6 1.26 ~ 0.03 ~ 6 1.10 ~ 0.01 6
Table 6 shows the activity of Compound 1 upon
slowly-inactivating A-type K+ currents (Ix) and delayed
rectifier K'" currents in Fast Hlue-labeled bladder afferent
neurons that were sensitive to capsaicin (presumed C-fiber
neurons). Slowly-inactivating A-type K+ currents were
isolated by subtraction of outward K+ currents activated
from a holding potential of -40 mV (on the condition of
inactivation of the majority of slowly-inactivating A-type
K''~ currents) from those activated from a.holding potential
of -120 mV (on the condition of full activation of slowly-
inactivating A-type K+ currents). As demonstrated in
unidentified DRG neurons, Table 6 shows that Compound 1
increases slowly-inactivating A-type K+ currents, but have
smaller influences upon delayed rectifier K~' currents.
- 14 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
Test Example 2: Changes in membrane potential in DRG cells
Material and methods
Animal preparation:
Experiments were performed on adult female Sprague
Dawley rats. A population of unidentified DRG cells and a
population of DRG neurons that innervate the urinary
bladder were labeled by retrograde axonal transport of the
fluorescent dye, Fast Blue (4~ w/v) (Polyloy, Gross Umstadt,
Germany), injected into the wall of the bladder in
halothane-anesthetized animals 7 days before the
dissociation. The dye was injected with a 28 gauge needle
at three to six sites on the dorsal surface of the organ
(5-6 ~uL , per ' site, total volume of 20-30 pL) . Each
injection site was washed with saline to minimize
contamination of adjacent organs with the dye.
Cell dissociation:
Freshly dissociated neurons from DRG were prepared
from halothane-anesthetized animals. L6 and S1 DRG were
dissected from animals and then dissociated in a shaking
bath for 25 minutes at 35°C with 5 mL DMEM (Sigma)
containing 0.3 mg/mL trypsin (Type 3, Sigma), 1 mg/mL
collagenase (Type 1, Sigma), and 0.1 mg/mL
deoxyribonuclease (Type 4, Sigma). Trypsin inhibitor (Type
2a, Sigma) was then added thereto to neutralize the
activity of trypsin. Individual DRG cell bodies were
- 15 .-
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
isolated by trituration and then plated on a poly-L-lysine-
coated 35 mm Petri dishes.
Electrical recordings:
Dye-labeled primary afferent neurons that innervate
the urinary bladder were identified using an inverted
phase-contrast microscope (Nikon, Tokyo, Japan) with
fluorescent attachments (UV-1A filter; excitation
wavelength, 365 nm). Gigaohm-seal whole-cell recordings
were performed within 6-8 hours after cell dissociation at
room temperature (20-22°C) on each labeled neuron in a
culture dish that usually contained three to seven labeled
cells among a few hundred unlabeled neurons. The internal
solution contained (in mmol/L) : KC1 140, CaCl2 1, MgCl2 2,
EGTA~ 11, HEPES 10 , Mg-ATP 2 , and Tris-GTP 0 . 4 adjusted to
pH 7.4 with KOH. Patch electrodes had resistances of 1-4 M
S~ when filled with the internal solution. Neurons were
superfused at a flow rate of 1.5 mL/minutes with an
external solution containing (in mmol/L): NaCI 150, KC1 5,
CaCl2 2.5, MgCl2 1, HEPES 10, and D-glucose 10, adjusted to
pH 7.4 with NaOH. All recordings were made with an
Axopatch-1D patch-clamp amplifier (Axon Instruments, Foster
City, CA), and data were acquired and analyzed by PCLAMP
software (Axon Instruments).
- 16 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
In current-clamp recordings, membrane potential of
DRG cells were measured before and after compound
applications. The membrane potentials were normalized to
control (before addition of the compound).
Effect of Compound 1 on membrane potential is'shown
in Table 7.
Table 7
. Compound 1 (mol/L) Membrane potential
(mV) n
0 -47.95 0.22 20
1.0x10'9 -48.10 0.48 10
1.0x10'8 -49.80 0.65 5
1.0x10'' -57.17 0.53 6.
' 1.0x10'6 -53.67 0.74 6
5.0x10'6 -50.50 0.88 4
5.0x10'5 -48.50 1.28 4
5.0x10'4 -42.67 1.46 3
Table 7 shows the activity of Compound 1 upon
membrane potential in DRG cells. It showed that Compound 1
increases slowly-inactivating A-type K+ currents, namely
increases outward currents to effect hyperpolarization (a
negative change of membrane potential). This activity
suggests reduction of excitability of DRG cells.
- 17 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
Based on the results of Test Examples 1 and 2, it
was revealed that Compound 1 has an activity of increasing
slowly-inactivating A-type K+ currents.
Test Example 3: Activity of inhibiting detrusor
hyperreflexia
The test was carried out in accordance with the
method of Cheng et a1. (Brain Res., 678: 40-48 (I995)).
Female SD rats of 8 to 10 weeks of age (supplied by
Japan SLC) were used in the test. Five to seven animals of
these rats were put in each metal cage and reared by
allowing them to freely take commercially available chow
and water, in a rearing room at a room temperature of from
19 to 25°C and a humidity ~of from 30 to 70~ under
illumination for 12 hours (from 7 a.m. to 7 p.m.) per day.
Spinal cord injury was induced in rats. Each rat
was anesthetized with diethyl ether and the skin of the
backside thoracic cord part was incised. Vertebral arch
around the 7th to 8th thoracic vertebrae was excised in a
length of about 5 mm, and the wound cavity of the excised
part was filled with cellulose oxide for blood stanching.
The incised part was sutured with a surgical silk thread.
After the spinal cord injury operation, forced-pressure
urination was manually carried out for about 3 weeks twice
a day (between 8 and 9 o'clock and 18 and 19 o'clock) until
complete automatic micturition developed. Also,
~, 8 _
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
intramuscular injection of the antibiotic ampicillin
(manufactured by Sigma, 150 mg/kg) was carried out once or
twice a day for about 2 weeks.
Four to five weeks after the spinal cord injury,
each rat was subjected to bladder catheter operation. The
bladder was exposed by midline incision of the abdomen
under diethyl ether anesthesia. A polyethylene tube (PE-
50; Becton Dickinson) having a blunt end to protect tissues
from injury was filled with physiological saline (Otsuka
Pharmaceutical Industries, Tokushima, Japan) and inserted
from the bladder top. This bladder catheter was fixed with
a surgical silk thread and indwelled. Also, the other end
was exposed subcutaneously from the back neck, plugged and
then fixed to the skin with the surgical thread.
Four to six days after the bladder catheter
operation, a cystometry test was carried out. The rat was
put in a Ballman cage (Natsume Seisakusho), a three way
cock was connected to the bladder catheter, one end of the
cock was connected to a pressure transducer (Nikon Kohden)
and the other end was connected to a 50 mL capacity syringe
(Terumo) arranged to an infusion pump (Harvard Apparatus)
for physiological saline injection. The intravesical
pressure signal from the pressure transducer was amplified
by a strain pressure amplifier (AP-6016; Nikon Kohden)
connected thereto and recorded on a thermal array recorder
(RTA-1200; Nikon Kohden) via a polygraph system (RPM-6008;
- 19 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
Nihon Kohden) contained therein. Sixty to ninety minutes
after the completion of the preparation, physiological
saline kept at room temperature was continuously injected
into the bladder at a flow rate of 10 mL/h for 30 minutes,
and the occurrence of micturition contraction was confirmed.
Thirty minutes after the. treatment, physiological saline
was injected again over 30 minutes, and the intravesical
pressure was measured to be used as a pre-drug
administration value. The test compound (Compound 1) was
suspended in 0.5 w/v~ aqueous methyl cellulose at a
concentration of 1 mg/mL. This suspension was further
diluted with 0.5 w/v~ aqueous methyl cellulose to prepare a
suspension or solution for the administration at the
intended concentration, and orally administered at a volume
of 1 mL/kg. The period of 1, 3 or 5 hours after the
administration was.°used as the measuring time after the
administration of the solvent or the drug tested, and the
intravesical injection of physiological saline was carried
out during a duration of 15 minutes around each measuring
time (45 to 75 minutes, 165 to 195 minutes and 285 to 315
minutes after the administration of the drug).
Micturition contraction was measured as the index
of normal voiding function, and pre-micturition contraction
as the index of detrusor hyperreflexia. The average of all
micturition contraction values observed during each of the
30 minutes-measuring periods and the average of maximum
- 20 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
pre-micturition contraction values observed during each
micturition contraction period were respectively defined as
the size of micturition contraction and pre-micturition
contraction at each period. In this case, both of the
contraction values were read out from the intravesical
pressure wave form recorded on the chart paper, using a
digitizer (KD3220; Graphtech) controlled by a computer (PC-
9801NS/R; manufactured by NEC), and stored as a WJ2 type
file on Lotus 1-2-3 R2.5J (manufactured by Lotus). The WJ2
file was.put in Excel far Windows version 7.0 (manufactured
by Microsoft). Sizes of pre-micturition contraction and
micturition contraction were converted to relative values
when,the values before the drug administration was defined
as 100, and average ~ standard error was calculated fox
each group.
The results for Compound 1 are shown in Table 8 on
the value (~) of pre-micturition contraction after the
administration of the solvent or agent, and in Table 9 on
the value (~) of micturition contraction.
- 21 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
Table 8
Compound 1 (mg/kg, p.o.)
Control 0.001 0.01 0.1
Before 100.0 0.0 100.0 t 0.0 100.0 0.0 100.0 0.0
~ t t
administration
After 1 hour115.0 12.183.5 ~ 6.8 59.6 8.1* 52.3 9.1*
~ t t
After 3 hours128.4 21.595.4 ~ 12.051.2 6.7* 33.3 6.3*
t t t
After 5 hours120.2 24.5105.5 20.8 42.2 7.2* 28.5 6.2*
t t
*: p<0.05 (comparison with the control group)
(n = 5-6; Dunnett's test)
Table 9
Compound p.o.)
1
(mg/kg,
Control 0.001 0.01 0.1
Before 100.0 0.0 100.0 t 0.0 100.0 0.0100.0 0.0
t t
administration
After 1 hour 97.5 5.9 106.5 t 11.3114.4 9.5109.3 6.3
t t t
After 3 hours99.3 4.8 101.7 t 9.5 117.6 13.9110.2 5.4
t t t
After 5 hours94.2 6.5 103.1 t 6.5 117.6 12.7, 112.4 7.0
t t t
According to the results shown in Test Example 3,
Compound 1 inhibited pre-micturition contraction (detrusor
overactivity) in spinal cord-injured rats, but had no
influence on micturition (physiological) contraction.
- 22 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
Test Example 4: Activity of inhibiting detrusor instability
The test was carried out in accordance with the
method of Malmgren et al. (J. Urol., 142: 1134-1138 (1989)).
Female SD rats of 8 to 10 weeks of age (supplied by
,Tapan SLC) were used in the test. Five to seven animals of
these rats were put in each metal cage and reared by
allowing them to freely take commercially available solid
chow and water, in a rearing room at a room temperature of
from 19 to 25°C and a humidity of from 30 to 70~ under
illumination for l2 hours (from 7 a.m. to 7~p.m.) per day.
Partial urethra obstruction was induced in rats.
Each rat was anesthetized by intraperitoneal administration
of 50 mg/kg of pentobarbital sodium (Dainippon
Pharmaceutical, Osaka, Japan) and the skin 'and muscle of
the abdominal side were cut by midline incision. A
polyethylene tube (PE-20; Becton Dickinson) was inserted
into urethra. The urethra base was peeled and double-
ligated, and then the polyethylene tube was pulled out to
induce partial obstruction of the urethra. The incised
part was sutured with a surgical silk thread. The
antibiotic ampicillin (manufactured by Sigma, 150 mg/kg)
was intramuscularly injected.
Six weeks after the urethra obstruction operation,
each rat with hypertrophic bladder was subjected to bladder
catheter operation. The bladder was exposed by midline
incision of the abdomen under pentobarbital sodium
- 23 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
anesthesia. A polyethylene tube (PE-50; Becton Dickinson)
having a blunt end to protect tissues from injury was
filled with physiological saline (otsuka Pharmaceutical
Industries, Tokushima, Japan) and inserted from the bladder
top. This bladder catheter was fixed with a surgical silk
thread and indwelled. The other end was exposed
subcutaneously from the back neck, plugged and then fixed
to the skin with the surgical silk thread.
Four to six days after the bladder catheter
operation, a cystometry test was carried out: The rat was
put in a Ballman cage (Natsume Seisakusho), a three way
cock was connected to the bladder catheter, one 'end of the
cock was connected to a pressure transducer (Nihon Kohden)
and the other end was connected to a 50 mL capacity syringe
(Terumo) arranged to an infusion pump (Harvard Apparatus)
for physiological saline injection. The intravesical
pressure signal from the pressure transducer was amplified
by a strain pressure amplifier (AP-6016; Nihon Kohden)
connected thereto and recorded'on a thermal array recorder
(RTA-1200; Nihon Kohden) via a polygraph system (RPM-6008;
Nihon Kohden)~contained therein. Sixty to ninety minutes
after the completion of the preparation, physiological
saline kept at room temperature was continuously injected
into the bladder at a flow rate of 10 mL/h until the
completion of the test, and the occurrence of micturition
contraction and pre-urination contraction were confirmed.
- 24 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
Charts for 30 minutes after 3 hours from the commencement
of the physiological saline injection were used as the
values before the drug administration. The test. compound
was suspended in 0.5 w/v~ aqueous methyl cellulose at a
concentration of 1 mg/mL. This suspension was further
diluted with 0.5 w/v~ aqueous methyl cellulose to prepare a
suspension or solution for the administration at the
intended concentration. This was orally administered at a
volume of 1 mL/kg. The period of 1, 3 or 5.hours after the
administration was used as the measuring time after the
administration of the drug tested, and a duration of 15
minutes around each measuring time (45 to 75 minutes, 165
to 195 minutes and 285 to 315 minutes after the
administration of the drug) was used as the measuring
period. .
Micturition contraction was measured as the index
of normal voiding function, and pre-micturition contraction
as the index of detrusor instability. The average of all
micturition contraction values observed during each of the
30 minutes-measuring periods and the average of maximum
pre-micturition contraction values observed during each
micturition contraction period were respectively defined as
the size of micturition contraction and pre-micturition
contraction at each period. In this case, both of the
contraction values were read out from the intravesical
pressure wave form recorded on the chart paper, using a
- 25 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
digitizer (KD3220; Graphtech) controlled by a computer (PC-
9801NS/R; manufactured by NEC), and stored as a WJ2 type
file on Lotus 1-2-3 R2.5J (manufactured by Lotus). The WJ2
file was put in Excel for Windows version 7.0 (manufactured
by Microsoft). Sizes of pre-micturition contraction and
micturition contraction were converted to relative values
when the values before the drug administration was defined
as 100, and average ~ standard error was calculated for
each group.
Table 10 shows the value (~) of pre-micturition
contraction after the administration of the solvent or
Compound 1, and Table 11 shows the value (~) of micturition
contraction after the administration of the solvent or
Compound 1.
Table 10
Compound 1 (mg/kg, p.o.)
Control 0.001 0.01 0.1
Before 100.0 t 0.0 100.0 m 0.0 100.0 t 0.0 100.0 t 0.0
administration
After 1 hour 105.5 m 6.5 10S.9 m 7.8 59.5 m 4.1*** 55.8 m 8.2***
After 3 hours 109.4 m 14.8 100.3 x 10.0 69 5 ~ 4.4* 66.2 * 4.7*
After 5 hours 103.3 t 3.6 104.2 * 10.2 67.8 t 5.5** 69.2 m 7.4**
*: p<0.05, ** p<0.01, *** p<0.001 {comparison with the control group)
{n = 5-6, Dunnett~s test)
- 26 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
Table 11
Compound (mg/kg,p.o.)
1
Control 0.001 0.01 0.1
Hefore 100.0 ~ 0.0 100.0 * 0.0 100.0* 0.0 100.0 0.0
*
administration .
After 1 hour 102.5 * 3.5 99.3 * 2.3 99.6 * 1.2 106.2 9.2
*
After 3 hours104.4 * 5.1 101.5 * 3.3 92.6 * 3.2 105.2 9.6
*
After 5 hours96.6 * 3.2 98.7 * 5.2 90.1 * 4.3 100.1 8.6
*
According to the results shown in Test Example 4,
Compound 1 had no influence on micturition contraction
which is the contraction at micturition in rats with
hypertrophic bladder (no influence on normal voiding), but
inhibited pre-micturition contraction which is irregular,
detrusor instability at the time other than normal voiding.
Test Examples 3 and 4 show that the compound used
in the present invention inhibits the pre-urination
contraction (detrusor overactivity) and are useful as an
agent for the treatment of overactive bladder. Thus, it is
considered that the compound having a slowly-inactivating
A-type K* channel opening activity or a pharmaceutically
acceptable salt thereof is useful as an agent for the
treatment of overactive bladder.
- 27 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
Test Example 5: Acute toxicity test:
The test compound was administered orally or
intraperitoneally to 3 animals per group of dd male mice
(body weight, 20 ~ 1 g). Minimum lethal dose (MLD) value
was obtained by observing mortality on the 7th day after
the administration.
As a result, MLD of Compound 1 was >1,000 mg/kg by
oral administration.
Based on the results of Test Examples 1 to 5, the
compound having a slowly-inactivating A-type K+ channel
opening activity or a pharmaceutically acceptable salt
thereof is useful as an agent for the treatment of
overactive bladder.
The compound having a slowly-inactivating A-type K~
channel opening activity or a pharmaceutically acceptable
salt thereof can be used as it is or in various dose forms.
Pharmaceutical compositions~of the present invention can be
produced by uniformly mixing an effective amount of the
compound having a slowly-inactivating A-type K+ channel
opening activity or a pharmaceutically acceptable salt
thereof as an active ingredient with a pharmacologically
acceptable carrier. It is preferred that these
pharmaceutical compositions are in a unit dose form
suitable for oral or parenteral (including intravenous)
administration or the like.
- 28 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
In preparing a composition in the oral dose form,
certain~useful pharmacologically acceptable carriers can be
used. For example, oral liquid preparations such as
suspensions or syrups can be produced using water;
saccharides, such as sucrose, sorbitol, fructose, or the
Like; glycols, such as polyethylene glycol, propylene
glycol, or the like; oils, such as sesame oil, olive oil,
soybean oil, or the like; antiseptics, such as p-
hydroxybenzoic acid esters or the like; flavors, such as
strawberry flavor, peppermint, or the like; or the like.
Capsules, tablets, powders and granules can be produced
using fillers, such as lactose, glucose, sucrose, mannitol,
or the like; disintegrators, such as starch, sodium
alginate, or the like; lubricants, such as magnesium
stearate, talc, or the like; binders, such as polyvinyl
alcohol, hydroxypropylcellulose, gelatin, or the like;
surfactants, such as fatty acid .esters or the like;
plasticizers, such as glycerine or the like; or the like.
Tablets and capsules are the most useful unit oral
administration preparations because of their easy
administration. In producing tablets or capsules, solid
pharmaceutical carriers are used.
In addition, injections can be prepared using a
carrier comprising distilled water, a salt solution, a
glucose solution or a mixture of salt water and a glucose
solution. In this case, they are prepared as solutions,
9 _
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
suspensions or dispersions' using suitable auxiliaries in
the conventional way.
The compound having a slowly-inactivating A-type K+
channel opening activity or a pharmaceutically acceptable
salt thereof can be administered orally in the above dose
forms or parenterally as injections, and, although its
effective dose and administration frequency may vary
depending, for example, on the dose form,.the age and body
weight of each patient, and symptoms of the disease, from 1
to 900 mg/60 kg/day, preferably from 1 to.200 mg/60 kg/day,
is suitable.
The embodiments of the present invention are
described below based on Examples.
BEST MODE FOR CARRYING OUT THE INVENTION
Example 1: Tablets
Tablets having the following composition were
prepared in the conventional way.
Compound 1 (250 g) was mixed with 1598.5 g of
mannitol; 100 g of sodium starch glycollate, 10 g of light
anhydrous silicic acid, 40 g of magnesium stearate and 1.5
of yellow ferric oxide in the conventional way. The
resulting mixture was applied to a tablet making machine
having a punch and die of 8 mm in diameter (Purepress
Correct-12, manufactured by Kikusui) to obtain tablets
(containing 25 mg of the active component per one tablet).
- 30 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
The prescription is shown in Table 12.
Table 12
Prescription Compound 1 25 mg
Mannitol 159.85 mg
Sodium starch glycollate 10 mg
Light anhydrous silicic acid 1 mg
Magnesium stearate 4 mg
Yellow ferric oxide 0.15 mg
200 mg
Example 2: Capsules
Capsules having the following composition were
prepared in the conventional way.
Compound 1 (500 g) was mixed with 300 g of lactose,
100 g of light anhydrous silicic acid and 100 g of sodium
lauryl sulfate in the conventional way. The resulting
mixture was packed in hard capsules No. 1 (100 mg per one
capsule) using an encapsulation machine (LZ-64,
manufactured by Zanasi) to obtain capsules (containing 50
mg of the active component per one capsule).
The prescription~is shown in Table 13.
- 31 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
Table 13
Prescription Compound 1 50 mg
Lactose 30 mg
Light anhydrous silicic acid 10 mg
Sodium lauryl sulfate 10 mg
100 mg
Example 3: Injections
Injections having the following composition are
prepared in the conventional way.
Compound 1 ( 1 g) is dissolved in 100 g of purified
soybean oil, and 12 g of purified yolk lecithin and 25 g of
glycerol for injection are added thereto. The resulting
mixture is kneaded with distilled water for injection
(total: 1,000 mL) and emulsified therein in the
conventional way. The obtained dispersion is aseptically
filtered using a 0.2 ~,m disposable membrane filter and then
aseptically dispensed into glass vials in 2 ml portions to
obtain injections (containing 2 mg of the active component
per one vial).
The prescription is shown in Table 14.
- 32 -
SUBSTITUTE SHEET (RULE 26)
CA 02442437 2003-09-26
WO 02/078523 PCT/US02/09577
Table 14
Prescription Compound 1 2 mg
Purified soybean oil 200 mg
Purified yolk lecithin 24 mg
Glycerol for injection 50 mg
Distilled water for injection 1.72 ml
2.00 m1
INDUSTRIAL APPhICABILITY
The present invention provides an agent for the
treatment of overactive bladder, comprising, as an active
ingredient, a compound having a slowly-inactivating A-type
K+ channel opening activity or a pharmaceutically
acceptable salt thereof, and a method for screening agents
for the treatment of overactive bladder, comprising
measuring a slowly-inactivating A-type K+ channel opening
activity as an index.
- 33 -
SUBSTITUTE SHEET (RULE 26)
