Composition for BK.SUB.Ca .channel activation

Abstract

The present invention relates to novel compounds capable of activating BKCa channels. The use of a composition of the present invention can effectively activate the BKCa channels, and can be used for prevention or treatment of various diseases caused by the deactivation of BKCa channels or the degradation of BKCa channel activity.

Claims

1. A method for preventing or treating BK.sub.Ca channel activity degradation-related condition, disease or disorder comprising administering a composition containing a flavanone derivative represented by a following chemical formula 1, or a pharmaceutically acceptable salt thereof to a subject whose activity of the BK.sub.Ca channel is lowered compared to a normal level, or the BK.sub.Ca channel is inactivated: ##STR00002## wherein, in the chemical formula 1, R.sub.1 is a hydrophobic substituent selected from a group consisting of hydrogen and straight-chain or branched-chain C.sub.1-C.sub.15 is alkyl or alkenyl; wherein when R.sub.1 is C.sub.6-C.sub.15 alkyl or alkenyl, R.sub.2 is C.sub.1-C.sub.4 alkoxy, wherein when R.sub.1 is hydrogen or C.sub.1-C.sub.5 alkyl or alkenyl, R.sub.2 is hydroxy; wherein each of R.sub.3, R.sub.4 and R.sub.5 is independently hydrogen or hydroxy, wherein at least one of R.sub.3, R.sub.4 and R.sub.5 is hydroxy, wherein the BK.sub.Ca channel activity degradation-related condition, disease or disorder is a lower urinary tract disorder, and, wherein the lower urinary tract disorder is not the disorder caused by infection, and the lower urinary tract disorder is characterized by at least one selected from the group consisting of: overactive bladder having or without having leaking urine, urinary frequency, urgency to urinate, or nocturia; urinary bladder symptoms including overactive bladder, overactive detrusor muscle, unstable bladder, detrusor hyperreflexia, or sensory urgency to urinate or detrusor overactivity; urinary incontinence or urge incontinence, urinary stress incontinence, slow voiding, terminal dribbling, or anuria or obstructive voiding symptom requiring allowable pressure to squeeze urine out; irritating symptoms of urinary frequency or urge to urinate; neurogenic bladder resulting from neurological injury including stroke, Parkinson's disease, diabetes, multiple sclerosis, peripheral neuropathy, or spinal cord injury prostatic hyperplasia, or spastic bladder in spinal cord injury patients; and unstable bladder, overactive detrusor muscle, detrusor instability, detrusor hyperreflexia, sensory urge to urinate, urinary incontinence, urinary urge incontinence, urinary stress incontinence, neurogenic (reflex) urinary incontinence, slow voiding, terminal dribbling, dysuria, or spastic bladder.

2. The method of claim 1, wherein when R.sub.1 is C.sub.6-C.sub.15 alkyl or alkenyl, R.sub.2 is methoxy.

3. The method of claim 1, wherein R.sub.1 is hydrogen, 3-methyl-2-buten-1-yl, or 2-isopropenyl-5-methyl-4-hexen-1-yl.

4. The method of claim 3, wherein when R.sub.1 is hydrogen, R.sub.2 is hydroxy.

5. The method of claim 3, wherein when R.sub.1 is 3-methyl-2-butene-1-yl, R.sub.2 is hydroxy.

6. The method of claim 3, wherein when R.sub.1 is 2-isopropenyl-5-methyl-4-hexen-1-yl, R.sub.2 is methoxy.

7. The method of claim 1, wherein R.sub.4 is hydroxy, and, each of R.sub.3 and R.sub.5 is independently hydrogen or hydroxy.

8. The method of claim 7, wherein at least one of R.sub.3 and R.sub.5 is hydrogen.

9. The method of claim 1, wherein the flavanone derivative represented by the chemical formula 1 is a compound selected from a group consisting of kurarinone, naringenin and leachianone G.

10. The method of claim 1, wherein the composition shifts a conductance-voltage (G-V) correlation of the BK.sub.Ca channel toward a negative voltage.

11. The method of claim 1, wherein one or more physiological functions selected from the group consisting of neuronal excitability of channel neurons, secretion of neurotransmitters, contraction of smooth muscle cells, and frequency tuning of hair cells of said the subject are lowered compared to the normal level.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A shows screening results of BK.sub.Ca channel activators using a cell-based fluorescence assay. A library of natural compounds was screened for the development of new BK.sub.Ca channel activators. AD293 cells stably expressing overactive mutant BK.sub.Ca channel (G803D/N806K) were used for the TI+-based fluorescence (FluxOR™) assay. Representative traces of fluorescence changes are shown using a relative fluorescence unit (RFU) for five different compounds. Each compound was transferred to each test-well at a final concentration of 5 μM before the experiment. Then, stimulus buffer was added thereto at 120 seconds. DMSO (.square-solid., 1%, vehicle) and kurarinone (.circle-solid.) were highlighted as filled symbols.

(2) FIG. 1B shows screening results of BK.sub.Ca channel activators using a cell-based fluorescence assay. A library of natural compounds was screened for the development of new BK.sub.Ca channel activators. AD293 cells stably expressing overactive mutant BK.sub.Ca channel (G803D/N806K) were used for the TI+-based fluorescence (FluxOR™) assay. Representative traces of fluorescence changes are shown using a relative fluorescence unit (RFU) for five different compounds. Each compound was transferred to each test-well at a final concentration of 5 μM before the experiment. Then, stimulus buffer was added thereto at 120 seconds. RFU values as obtained at 240 seconds were shown for 14 different compounds. Kurarinone was highlighted in gray.

(3) FIG. 1C shows screening results of BK.sub.Ca channel activators using a cell-based fluorescence assay. A library of natural compounds was screened for the development of new BK.sub.Ca channel activators. AD293 cells stably expressing overactive mutant BK.sub.Ca channel (G803D/N806K) were used for the TI+-based fluorescence (FluxOR™) assay. Representative traces of fluorescence changes are shown using a relative fluorescence unit (RFU) for five different compounds. Each compound was transferred to each test-well at a final concentration of 5 μM before the experiment. Then, stimulus buffer was added thereto at 120 seconds. Fold-increase values compared with DMSO as obtained at 240 seconds were shown for 14 different compounds. Kurarinone was highlighted in gray.

(4) FIG. 2A shows a concentration-dependent increase of a fluorescence signal after treatment with kurarinone. AD293 cells stably expressing mutant BK channels were treated with different concentrations of kurarinone. FIG. 2A represents a representative fluorescent trace. After obtaining a baseline for 20 seconds, a TI+-containing stimulation buffer was treated. Cells were cultured in the presence of 1% DMSO (□) as vehicle or other concentrations of kurarinone (.square-solid.: 3 μM, .circle-solid.: 5 μM, .box-tangle-solidup.: 10 μM, .Math.: 30 μM). Cells were further incubated with 1 μM of paxillin, a BKCa channel blocker, along with kurarinone (5 μM) (+).

(5) FIG. 2B shows a concentration-dependent increase of a fluorescence signal after treatment with kurarinone. AD293 cells stably expressing mutant BK channels were treated with different concentrations of kurarinone. FIG. 2B shows an initial RFU increase at different concentrations of kurarinone. After obtaining a baseline for 20 seconds, a TI+-containing stimulation buffer was treated. Cells were cultured in the presence of 1% DMSO (□) as vehicle or other concentrations of kurarinone (.square-solid.: 3 μM, .circle-solid.: 5 μM, .box-tangle-solidup.: 10 μM, .Math.: 30 μM). Cells were further incubated with 1 μM of paxillin, a BKCa channel blocker, along with kurarinone (5 μM) (*). An error bar (S.E.M.) is shown. An inset represents a chemical structure of kurarinone.

(6) FIG. 3A and FIG. 3B show results of a structure-activity relationship study of flavonoid derivatives. FIG. 3A shows a structure of the flavonoid used in the present disclosure. FIG. 3B shows a representative trace of RFU for each compound treated at 10 μM. FIG. 3C shows an initial RFU increase in a presence of 10 μM of each compound.

(7) FIG. 4 shows a reversible enhancement of a macroscopic BK.sub.Ca channel current by kurarinone. A representative diary-plot of a tail current induced by the BK.sub.Ca channel was shown as a continuous record. An ion current was recorded every second with a 50-ms step-pulse of 100 mV from a holding voltage of −100 mV. The current was obtained at a specific point (0.8 ms after a 100 mV voltage pulse). Each representative current trace (a-d) represents a current at a point indicated by an arrow.

(8) FIG. 5A, FIG. 5B and FIG. 5C show an effect of kurarinone on current-voltage and conductance-voltage relationships of a macroscopic BK.sub.Ca channel current. FIG. 5A shows representative traces of the BK.sub.Ca channel current at 3 μM[Ca2+]i at different kurarinone concentrations. The ion current was induced with a 100-ms voltage step-pulse. The current was recorded in 10 mV increments from −80 mV to 200 mV. The holding voltage was −100 mV. FIG. 5B shows an effect of kurarinone on the conductance-voltage (G-V) relationship. The conductance was obtained from a peak-tail current. The current was normalized by a maximum current of a vehicle trace. Each symbol represents a conductance at each of different kurarinone concentrations: vehicle (.square-solid., n=12), 3 μM (.circle-solid., n=8), 5 μM (.box-tangle-solidup., n=12), 10 μM (.Math., n=8) and 20 μM (.diamond-solid., n=4). FIG. 5C shows an effect of kurarinone on a half-maximum voltage (V1/2). Each symbol on the graph represents each of S.E.M and average of V1/2. Results were obtained by fitting all of independent data sets using a Boltzmann function (P.sub.0=[1/(1+exp{(V.sub.1/2−V)/k}]).

(9) FIG. 6A to FIG. 6D show an effect of kurarinone on activation and deactivation of a macroscopic BK.sub.Ca current. FIG. 6A shows a representative trace of activation when treated with vehicle (black) or 20 μM of kurarinone (gray). FIG. 6B shows a representative trace of deactivation when treated with vehicle (black) or 20 μM of kurarinone (gray). Current traces obtained at 100 mV were compared with each other. FIG. 6C shows activation time-constant values (τ) at different concentrations of kurarinone. FIG. 6D shows deactivation time-constant values (τ) at different concentrations of kurarinone. Symbols indicate vehicle (□, n=12), 3 μM (.square-solid., n=8), 5 μM (.circle-solid., n=12), 10 μM (.box-tangle-solidup., n=8) and 20 μM (.diamond-solid., n=4). The time-constant values were obtained from fitting all independent data sets using an exponential standard function (y(t)=A.sub.1exp(−t/τ.sub.1)+C) using the Clampfit program.

(10) FIG. 7A to FIG. 7E illustrates an effect of kurarinone on a single BK.sub.Ca channel. Each graph of FIG. 7A represents a typical single-channel current recording of the BK.sub.Ca channel at different membrane voltages. A concentration of Ca.sup.2+ in a cell (pipette Ca.sup.2+ concentration) was fixed to 10 μM. The current was recorded for the first time in the absence of kurarinone at different voltages, and then recorded in the presence of kurarinone (5 μM). A kurarinone solution was sprayed on an outer cell side. A solid line represents a closed level of a single BK.sub.Ca channel, while a dotted line represents an open level thereof. FIG. 7B shows an effect of kurarinone on a single-channel conductance. A urine current-amplitude of the channel was measured at a 10 μM intracellular Ca.sup.2+ solution. In the absence of kurarinone (∘), a channel current was first recorded and then 5 μM of kurarinone (.circle-solid.) was applied. Each membrane voltage was 75 mV(n=4), 50 mV(n=5), 25 mV(n=5), −25 mV(n=3), and −50 mV(n=2). Each data preset on the graph was obtained from all-points amplitude histograms fitted using a Gaussian function. A conductance of a single channel was estimated using a slope fitted with a linear function. FIG. 7C shows an effect of kurarinone on a voltage-dependent open-probability (P.sub.0) of a single BK.sub.Ca channel. The open-probability was measured using the same trace as in FIG. 7B. An increase in a cell membrane-dependent open-probability of a single BK.sub.Ca channel was measured under the conditions without kurarinone (∘) and with 5 μM of kurarinone treatment (.circle-solid.). Then, data points thereof were fitted by a Boltzmann function (P.sub.0=[1/(1+exp{(V.sub.1/2−V)/k}]). FIG. 7D represents a representative single-channel current of the BK.sub.Ca channel at the absence (upper trace) and presence (lower trace) of 5 μM kurarinone at 50 mV. FIG. 7E represents an effect of kurarinone on an average open-time and average closed-time of a single BK.sub.Ca channel at 50 mV in the absence (empty bar) and presence (filled bar) of 5 μM kurarinone. Each bar graph represents average±SEM (n=5).

(11) FIG. 8A and FIG. 8B show an effect of kurarinone on contraction induced by ACh in detached rat bladder strips. FIG. 8A represents a representative contraction trace induced by ACh with or without preincubation of kurarinone. FIG. 8B represents a percentage of relaxation induced by kurarinone on the ACh-induced contraction. Each bar graph represents an average±SEM of 6 experiments. Black show a control and white shows a kurarinone treatment experiment.

(12) FIG. 9A and FIG. 9B shows an effect of kurarinone on a voiding behavior of rats. FIG. 9A shows an effect of kurarinone on a voiding frequency of WKY and SHR after intraperitoneal injection of kurarinone (0.5 and 5 mg/kg). The voiding frequency was monitored for 3 hours. FIG. 9B represents a total number of voidings. Each symbol or bar represents average±SEM of 5 (WKY) or 7 (SHR) animals (*** P<0.001).

DETAILED DESCRIPTIONS

(13) Hereinafter, examples will illustrate the present disclosure in more detail. These examples are intended to illustrate the present disclosure. It will be apparent to those skilled in the art that the scope of the present disclosure in accordance with the spirit of the present disclosure is not limited by these examples.

BEST MODE FOR CARRYING OUT THE INVENTION

Examples

Example 1: Experimental Material

(14) A chemical library containing 794 natural compounds was obtained from Korea Research Institute of Chemical Technology (KRICT; www.chembank.org). Additional kurarinone and its derivatives were purified from dried root of Sophora flavescens, that is, Kushen (Jung et al. 2008). Kurarinone and other compounds were dissolved in DMSO (dimethyl sulfoxide) (Sigma-Aldrich) as a stock solution. 4-chloro-7-(trifluoromethyl)-10H-benzofuro[3,2-b]indole-1-carboxylic acid (CTBIC) was further dissolved in DMSO.

Example 2: Cell Culture

(15) AD-293 cells (Lee et al., 2013), as a modified HEK293 cell for expressing a mutant BKCa channel, were placed in DMEM (Dulbecco's Modified Eagle's medium) supplemented with 10% fetal bovine serum and 1 mg/ml geneticin (Gibco) as an antibiotic. The cells were cultured under a humidified condition of 5% CO.sub.2 and 37° C.

Example 3: Fluorescence Assay and Data Analysis

(16) AD-293 cells stably expressing the mutant BK.sub.Ca channel (G803D/N806K) were used for cell-based assays (Lee et al., 2013). Approximately 20,000 cells/well were inoculated on a black-wall assay plate (Corning Incorporated) coated with poly-D-lysine (Sigma-Aldrich), which is 96-well clear-bottom. A FluxOR™ calcium channel assay (Invitrogen) was used for initial screening of compound libraries, and for further assay of candidate compounds. Experiments were performed according to the manufacturer's following guidelines: growth medium was replaced with 80 μl/well of loading buffer containing FluxOR™ fluorescent dye, and incubation was carried out for 1 hour under a light-free condition. After the incubation, the loading buffer was replaced with 100 μl/well of assay buffer containing various concentrations of compounds of interest, followed by incubation for 20 minutes to 30 minutes. DMSO (1%) was vehicle and the vehicle was used for all test compounds. CTBIC (Cormemis et al. 2005; Lee et al. 2012), previously identified as an activator of the BK.sub.Ca channel, was used as a positive control. For fluorescence measurements, synergy TM H1 hybrid multi-mode microplate reader (BioTek Instrument Inc., Winnoski, Vt.) and Cen5 software was used in initial screening. For additional assays, Flexstation 3 multi-mode microplate reader (Molecular Devices) and SoftMax®Pro software were used, respectively. A fluorescence signal was obtained at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Membrane polarization was induced by a stimulus buffer containing thallium ion. The fluorescence signal was measured under two conditions: every 10 seconds for 2 minutes before treating the stimulation buffer and then every 10 seconds for 3 minutes after addition of stimulation buffer for synergy H1, and every 2 seconds for 20 seconds before stimulus buffer treatment, and then every 2 seconds for 160 seconds after adding the stimulus buffer for FlexStation 3.

(17) A fluorescence signal change was measured based on a relative fluorescence unit (RFU) or F/F0 where F0 is ta minimum fluorescence value of each fluorescence trace. To quantitatively compare activation effects of kurarinone and its derivatives, an initial fluorescence increase was calculated using first three points after treatment with the stimulation buffer, and a linear slope was predicted using OriginPro 9.1 (OriginLab Corp., Northampton, Mass.).

Example 4: Functional Expression of Cloned BK.SUB.Ca .Channel in Xenopus Oocyte

(18) Xenopus laevis oocytes heterologously expressing a BK.sub.Ca channel α-subunit (Slo1) were used for electrophysiological recording. Subcloning and functional expression of the rat BK.sub.Ca channel α-subunit using the oocyte expression vector pNBC1.0 has been reported (Ha et al., 2000). Sequence information of Slo1 used in the present disclosure is published in GenBank as expression number AF135265. Plasmid DNA was linearized using Notl restriction enzyme, and complementary RNA (cRNA) was synthesized using T7 RNA polymerase in the presence of nucleoside triphosphate and cap analog m7G(5′)ppp(5′)G from a linear form of DNA using mMessage Machine (Ambion).

(19) Oocytes from stages V to VI were surgically removed from ovarian lobes of anesthetized X. laevis (Xenopus I, Dexter, Mich.). The removed oocytes were transferred to a Ca.sup.2+-free oocyte ringer's (OR) culture medium (86 mM NaCl, 1.5 mM KCl, 2 mM MgCl.sub.2 and 10 mM HEPES, pH 7.6). The oocytes were cultured in Ca.sup.2+-free OR medium containing 3 mg/ml collagenase (Worthington Biochemicals) for 1 hour and 30 minutes to 2 hours. This removed the follicular cell layer of the oocyte. Then, the oocytes were widely washed with Ca.sup.2+-free OR culture medium and ND-96 medium (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl.sub.2, 5 mM HEPES, and 50 g/ml gentamycin, pH 7.6). The washed oocytes were stored at 18° C. in ND-96 medium. Before oocyte was used, the oocyte was stabilized for at least one day. After the stabilization, approximately 50 ng of synthesized cRNA in 50 nl of nuclease-free water was injected into each oocyte for macroscopic current recording, and approximately 1 ng of synthesized cRNA in 50 nl of nuclease-free water was injected into each oocyte for single-channel recording using a micro-dispenser (Drummond Scientific, Broomall, Pa.). The cRNA-injected oocytes were cultured in ND-96 medium for 1 to 3 days at 18 degree C. Immediately prior to the patch-clamp experiment, the oocyte's vitelline membrane was manually removed with fine forceps.

Example 5: Electrophysiological Recording and Data Analysis

(20) All macroscopic current recordings and single-channel recording were performed using a gigaohm-seal patch-clamp method in an outside-out arrangement as conventionally known (Ha et al., 2000). Patch pipettes were prepared from borosilicate and glass (WPI, Sarasota, Fla.). The pipettes were fire-polished with a resistance of 2 to 4 MΩ for patch recording. For single-channel recording, the patch pipettes were fire-polished with 4 to 8 MΩ resistors. To reduce electrical noise, the pipettes were coated with beeswax. Using an Axopatch 200B amplifier (Axon Instruments), the channel current was amplified. Then, the current was low-pass filtered at 1 kHz using a four-pole Bessel filter. Then, the current was digitized at a rate of 10 points/ms using a Digidata 1200A (Axon Instruments).

(21) A single BKCa channel was readily activated by a membrane potential simply transferred at 100 mV at a high concentration of intracellular Ca.sup.2+. For the single-channel assay, the switching between the closed and open conditions was determined by setting a potency at a half of a unitary current amplitude. To determine the single-channel conductance of the expressed channel, the average amplitude of the channel current was obtained from the histograms fitted with Gaussian distributions. An average current for a membrane-pass voltage is shown. A slope-conductance values were obtained from linear regression. The macroscopic current of the expressed BK.sub.Ca channel was activated by a voltage-clamp pulse delivered from a holding potential of −100 mV at 10 mV increment to a membrane potential of typically −80 to 200 mV, A dwell-time of recorded open and close events for the single BK.sub.Ca channel was analyzed using a linear histogram method. The dwell-time distribution was fitted into a single index using simplex-least-squares fitting methods (Clampfit, Axon Instruments). A peak in the dwell-time distribution was located at a time-constant of an exponential component.

(22) To prevent activation of endogenous calcium-activated chloride channels, solutions for single and macroscopic channel recording contained gluconate as a non-permeant negative ion. Intracellular and extracellular solutions contained following ingredients unless otherwise specified: 120 mM calcium gluconate, 10 mM MHEPES, 4 mM KCl, and 5 mM MEGTA, pH 7.2. To provide the required free-[Ca.sup.2+] i, an appropriate amount of total Ca.sup.2+ to be added to the intracellular solution was calculated using a program MaxChelator (Patton et al., 2004; http://maxchelator.stanford.edu/). To accurately compare channel characteristics, the same set of intracellular solutions was used throughout the experiments. Commercial software packages such as Clampex 8.0 or 8.1 (Axon Instruments) and Origin 9.1 (Origin Lab Corp., Northampton, Mass.) were used to obtain and analyze both single-channel and macroscopic recording data. The data were summarized into average±SE (n=number of independent recordings). The data were compared using a paired Student's t-test. A p-value smaller than 0.05 was considered statistically significant.

Example 6: Isometric Tension Recording of Bladder Smooth Muscle

(23) Isotropic tension recording of UBSM experiment was performed by a known method (Dela Pena et al. 2009; Kullmann et al. 2014). In short, a male Sprague-Dawley rat (300-350 g) was euthanized by CO.sub.2 choking. Then, the bladder of the rat was excised. The bladder was longitudinally divided into four strips (approximately 2×8 mm). Each separated strip was clipped between a static mount and a force-displacement transducer. Then, the clipped strips were suspended in a temperature-controlled (37° C.) organ bath containing 10 ml of Krebs solution ((mM): 118.4 NaCl, 4.7 KCl, 1.2 KH.sub.2PO.sub.4, 1.2 MgSO.sub.4, 25.0 NaHCO.sub.3, 2.5 CaCl.sub.2, and 12.2 glucose; pH 7.35-7.40). Then, a mixture of 95% 02 and 5% CO.sub.2 was used to continuously generate bubbles in the organ bath. Each UBSM strip was stretched to 1.0 gram of an optimal isometric tension, and was equilibrated for 60 minutes. During the equilibration, the tissue was washed every 15 minutes with fresh Krebs solution, and a baseline tension was adjusted to 1.0 g. After reaching the equilibration, the strips were stabilized via repeated application of acetylcholine (1 μM) until a continuous reaction was recorded. To investigate a mitigating effect of kurarinone, the tissue was pre-incubated with kurarinone for 30 min prior to addition of acetylcholine. Then, in the presence of kurarinone, the acetylcholine-induced contraction reaction was repeated. Relaxation was expressed as a percentage reduction in the tension due to acetylcholine-induced contraction. One strip in each series was assigned as a time control. Changes in the isometric tension were recorded using a Power Lab Data Acquisition System (ADInstruments) associated with a computer with Lab Chart Software (Version 7, AD Instruments) installed therein. The data were summarized into average±SE (n=number of DSM strips). The data were compared using a paired Student's t-test. A p-value smaller than 0.05 was considered statistically significant.

(24) Result

(25) 1. Identification of BK.sub.Ca Channel Activator Using Thallium-Fluorescence Assay

(26) To identify new BK.sub.Ca channel activators, a total of 794 single compounds purified from natural sources were screened using a cell-based assay employing TI+fluorescence. At a final concentration of 5 μM, some compounds with increased TI+fluorescence were significantly compared to vehicle (1% DMSO) (see FIG. 1A). RFU values as obtained at 240 seconds were shown for 14 different compounds (FIG. 1B). Fold-increase values compared with DMSO as obtained at 240 seconds were shown for 14 different compounds (FIG. 1C). Kurarinone was highlighted in gray. Among the compounds, the strongest fluorescence increase was obtained from the treatment of kurarinone as flavanone compound from Sophora flavescens (see FIG. 2A and FIG. 2B). TI+fluorescence for kurarinone increased in a dose-dependent manner (cf. FIG. 2A). The fluorescence enhancement was completely blocked by co-treatment of 1 μM pacilin, a selective BK.sub.Ca channel inhibitor. The initial increase in RFU induced by different concentrations of kurarinone was quantified in FIG. 2B (n=4).

(27) Because kurarinone is a natural flavanone compound, several related compounds of flavonoids have been tested. The several related compounds of flavonoids were treated with 10 μM concentration (n=4). These compounds showed differential effects on TI+-fluorescence enhancement. While kurarinone, leachianone G, and naringenin showed a strong fluorescence increase, other derivatives, including kurarinol showed weaker fluorescence effects. It is noteworthy that kurarinol containing an additional hydroxyl group in an aliphatic chain at a position 8 of the flavanone backbone shows a dramatic decrease in its efficacy. Among the tested ones, kurarinone exhibits the strongest increase for the initial TI+-based fluorescence assay. Thus, subsequent functional studies were carried out using this kurarinone compound.

(28) 2. Effect of Flavanone Derivative on Macroscopic Current of BK.sub.Ca Channel

(29) Although the cell-based TI+-fluorescence assay was adapted for high-throughput screening and provided first BK.sub.Ca channel activator candidates, there was a need to demonstrate and characterize the activity of each of the compound regarding electrophysiological experiments using wild-type BK.sub.Ca channels. Therefore, the present applicants characterized the effect of kurarinone on the a subunit of the rat BK.sub.Ca channel (rSlo1) heterologously expressed on Xenopus oocytes. Time-dependent effects on macroscopic channel currents were investigated using excised membrane patches in an outside-out arrangement in the presence of 3 μM intracellular Ca.sup.2+ (see FIG. 4). While a small tail current is induced by each test pulse (see upper graph in FIG. 4), treatment of 5 μM kurarinone on the extracellular surface strongly potentiates the tail-current in a time-dependent manner (see b and c in FIG. 4). Upon the removal of kurarinone, the channel current was gradually de-potentiated with respect to the base level (see d in FIG. 4). It is noteworthy that both potentiation and de-potentiation of the BK.sub.Ca channel by kurarinone show two phases: the first rapid increase in seconds, and a slow and gradual increase over several minutes. The association time-constant values for the fast phase (τfast) and the low-speed phase (τslow) were estimated to be 1.47±0.34 seconds and 64.9±8.8 seconds, respectively, when fitted with the double-exponential function. Although two different phases are obvious, the de-potentiation of the BK.sub.Ca channel was longer than the potentiation with a dissociation time-constant of 2.78±0.95 and 90.06±12.21 sec, respectively as measured. When treatment of kurarinone from the inside of the cell was carried out using the excised inside-out patch recording, no significant change in BK.sub.Ca channel current was observed. Therefore, these results indicate that kurarinone can directly and reversibly potentiate the activity of the BK.sub.Ca channel from the outside of the cell.

(30) 3. Concentration-Dependent Effect of Flavanone Derivative on Macroscopic Current of BK.sub.Ca Channel

(31) Next, the mechanism of the flavanone derivative-induced potentiation of the BK.sub.Ca channel was studied. The BK.sub.Ca channel was activated by a series of voltage pulses. Macro-current was recorded under an increased concentration of activated extracellular kurarinone. As the kurarinone concentration increases, it is clear that the channel current is activated at a lower voltage and is deactivated more slowly (see FIG. 5A). In FIG. 5B, the voltage-dependent activation of the macroscopic BK.sub.Ca channel current is shown as a conductance-voltage (G-V) relationship. kurarinone gradually shifts the G-V curve to the left, and increases the maximum conductance (Gmax) in a dose-dependent manner. The kurarinone-dependent shift in the G-V relationship was further quantified. In FIG. 5C, quantification is shown. In the presence of 20 μM kurarinone, the half-active voltage (V1/2) is shifted by approximately 80 mV in the negative direction from 107.4±2.2 mV to 27.7±3.0 mV. Kurarinone gradually increased the maximum conductance (G/Gmax) of the channel. G/Gmax was predicted to be 1.35 for 20 μM kurarinone and 1.8 times higher than vehicle control. Thus, these results demonstrate that kurarinone potentiates the BK.sub.Ca channel by activating the channel at a membrane voltage that is more negative, and, hence, increases the maximum openability of the channel.

(32) 4. Activation and Deactivation Kinetic Effects of Flavanone Derivative on BK.sub.Ca Channel

(33) As shown in the macroscopic current trace of FIG. 5A, the deactivation of the BK.sub.Ca channel was significantly affected by the flavanone derivative of the present disclosure. Therefore, we investigated the effect of kurarinone on opening and closing movement of BK.sub.Ca channel. In FIG. 6A to FIG. 6D, the activation (or open) and deactivation (or closure) procedures of the BK.sub.Ca channel were analyzed. In the presence of 20 μM, the current level was increased, while the activation rate did not vary significantly (FIG. 6A). At four increased concentrations of kurarinone (0, 5, 10, 20 μM), the activation time-constant (τactivation) was not significantly increased (FIG. 6C). On the other hand, the deactivation rate was dramatically decreased by kurarinone. Interestingly, the slowing of deactivation by kurarinone was voltage-dependent, in that when the channel was activated by a higher positive voltage, kurarinone caused the closure of the BK.sub.Ca channel to be noticeably slower. Overall, these results indicate that kurarinone stabilizes the open form of the BK.sub.Ca channel, the binding affinity of kurarinone may be further strengthened with the channel form at higher voltage.

(34) FIG. 6A shows a representative trace of activation when treated with vehicle (black) or 20 μM of kurarinone (gray). FIG. 6B shows a representative trace of deactivation when treated with vehicle (black) or 20 μM of kurarinone (gray). Current traces obtained at 100 mV were compared with each other. FIG. 6C shows activation time-constant values (τ) at different concentrations of kurarinone. FIG. 6D shows deactivation time-constant values (τ) at different concentrations of kurarinone. Symbols indicate vehicle (□, n=12), 3 μM(.square-solid., n=8), 5 μM (.circle-solid., n=12), 10 μM (.box-tangle-solidup., n=8) and 20 μM (.diamond-solid.n=4). The time-constant values were obtained from fitting all independent data sets using an exponential standard function (y(t)=A.sub.1exp(−t/τ.sub.1)+C) using the Clampfit program.

(35) 5. Effect of Flavanone Derivative on Single-Channel Current of BK.sub.Ca Channel

(36) To further understand the mechanism of the flavanone derivative action, the effect was investigated at the single-channel level. The single BK.sub.Ca channel was recorded in an outstand-out patch in the presence of 10 μM intracellular Ca.sup.2+. To activate the BK.sub.Ca channel, the membrane voltage was first depolarized until it was greater than 80 mV. The number of channels in the patch membrane was counted. Only patches containing the single channel were used in subsequent experiments. A representative trace of a single channel in the absence or presence of 5 μM of kurarinone is shown in FIG. 7A. The opening of the channel was highly dependent on the membrane voltage as expected. However, the opening and closing behavior was dramatically altered by the application of 5 μM kurarinone to the outside of the cell. While the BK.sub.Ca channel was not nearly open in the control solution above −25 mV, the more frequent opening of the channel was evident in application of kurarinone. At 50 mV, the channel remained open for an extended period of time in the presence of kurarinone. To test the effect of kurarinone on the channel conductance, the unitary current amplitude of each BK.sub.Ca channel was measured at various membrane voltages in the presence or absence of compounds. A channel current-voltage (I-V) relationship (see FIG. 7B) was shown. Single-channel conductance was estimated to be 221.1±16.4 for the control and 238.3±8.7 pS for kurarinone. This indicates that the compound does not significantly modify the single-channel conductance of the channel. After that, the effect of kurarinone on P0 of the single channel was analyzed. P0 was measured at several different voltages in the presence and absence of kurarinone. Measurements were fitted using the Boltzmann function (see FIG. 7C). The half-activation voltage (V1/2) as a voltage at which 1/2 of a full openness is required was found to be 68.7±3.7 mV in the control and 43.7±2.1 mV in the presence of 5 μM kurarinone (see FIG. 7D). These results are in good agreement with the already mentioned discovery for the macroscopic channel currents. This demonstrates that without affecting single channel conductance, kurarinone increases the openness of the channel and thus potentiates the BK.sub.Ca channel. Then, we analyzed the opening and closing behavior of single BK.sub.Ca channels in the presence of kurarinone. Because the kurarinone dramatically increases the P0 of the single BK.sub.Ca channel, the assays used herein are limited to single-channel recordings at 50 mV where open-close switching may be compared in a reasonable time-scale (see: FIG. 7D). The average closing time was 3.69±0.80 ms in the absence of kurarinone and was 3.60±4.1 ms in the presence of 5 μM of kurarinone, while the average opening-time was measured as 2.98+/−0.34 ms and 5.77+/−0.90 ms in the absence of kurarinone and the presence of 5 μM of kurarinone respectively (FIG. 7E). These results suggest that the binding of kurarinone stabilizes the open form, and, thus, the channel closing rate is reduced without affecting the open switching of the channel, and, the results of the macroscopic current recording are validated.

(37) 6. Effect of Flavanone Derivative on Rat Bladder Tissue

(38) Because the flavanone derivative strongly potentiates the cloned BK.sub.Ca channel expressed on the heterologous system, we determined whether flavanone derivative compounds could relax the bladder smooth muscle in vivo. To confirm the efficacy of kurarinone on the contraction of acetylcholine (ACh)-induced UBSM, the isometric tension of rat voiding muscle strips was recorded. While 1 μM ACh leads to reduction of the peak tension and, consequently, reduction of a relatively stable plateau level (see FIG. 8A), pretreatment of tissue with kurarinone significantly inhibited ACh-induced contraction. The relaxation effect was 58.2±6.2% at 100 μM kurarinone compared to vehicle treatment (p<0.05, n=6) (see FIG. 8B). In contrast, there was no significant change in contraction response in a vehicle-treated time-matched control tissues.

(39) 7. Effect of Flavanone Derivative on Voiding Behavior of WKY and SHR

(40) To further demonstrate the effect of the flavanone derivative on bladder relaxation and voiding behavior, we investigated the voiding behavior of Wistar Kyoto rat (WKY) and spontaneous hypertensive rats (SHR). The cumulative voiding frequency is shown in FIG. 9A for WKY and SHR orally administered with kurarinone. The administration of kurarinone may allow distinct differences in voiding frequency to be evident between WKY and SHR. While the voiding frequency of control WKY was not affected by kurarinone over 5 mg/kg dose, the compound reduced the voiding frequency for SHR in a dose-dependent manner. The total voiding frequency for 3 hours is shown in FIG. 9B. When 5 mg/kg of kurarinone was administered, a significant decrease in the voiding frequency was observed in SHR (10.9±1.4 for vehicle and 6.9±0.8 for kurarinone). In the WKY control rat, the decrease of the voiding frequency was not observed. Together with the ex vivo isometric tension recording, these results further indicate that kurarinone potentiates the voiding muscle BK.sub.Ca channel, thereby to activate the bladder relaxation. In this way, it shows that kurarinone is an effective candidate compound to improve OAB syndrome.

(41) Having described specific portions of the present disclosure in detail, those skilled in the art will appreciate that these specific portions are merely preferred embodiments. It is evident that the scope of the present disclosure is not limited thereto. Accordingly, the actual scope of the present disclosure is to be defined by the appended claims and their equivalents.

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INDUSTRIAL AVAILABILITY

(43) The present disclosure relates to novel compounds that can activate the BKCa channel. Using the composition of the present disclosure may allow the BK.sub.Ca channel to be effectively activated. Thus, the composition of the present disclosure may be used to prevent or treat various diseases caused by BK.sub.Ca channel deactivation or activity-degradation.