JP4530335B2 - Inhibitors of epithelial sodium channels expressed in the bladder - Google Patents

Description

  The present invention relates to a target substance used for screening a therapeutic agent for overactive bladder syndrome, and a therapeutic agent for overactive bladder syndrome.

  Overactive bladder syndrome (hereinafter also referred to as overactive bladder) is a very frequent disease as a result of recent epidemiological studies. Usually, symptoms of overactive bladder such as frequent urination, urgency and urge incontinence appear based on abnormal detrusor function (detrusor overactivity), but the pathophysiology is unknown. Currently, effective treatment is pharmacotherapy using an antimuscarinic agent such as oxybutynin or propiverine described in Non-Patent Document 1, but this drug suppresses detrusor contractile force in overactive bladder. Although it has an improved mechanism of action, it has side effects such as dry mouth, constipation, and poor memory.

On the other hand, as one of the pathophysiology of overactive bladder, an increase in sensory nerve input from the bladder to the bladder afferent and the accompanying overactivity of the bladder is being considered. Capsaicin described in Non-Patent Document 2, Alternatively, although the effectiveness of vanilloid receptor stimulants such as resiniferatoxin has been reported, it has a problem of large side effects. Therefore, the development of a new overactive bladder therapeutic agent that acts on the bladder afferent is awaited.
Yoshimura N, Chancellor MB (Journal of Urology Vol 168, p1897-1913, 2002.) Geirsson G, Fall M, Sullivan L (Journal of Urology Vol 154, p1825-1829, 1995.)

  Therefore, an object of the present invention is to provide a novel target substance that can be used in the development of a new overactive bladder therapeutic agent that acts on the bladder afferent and a novel overactive bladder therapeutic agent.

  As a result of intensive studies, the present inventors have found that epithelial sodium channel protein in the urinary bladder is involved in transmission of mechanosensitive information in the afferent nerve tract of the urinary bladder. And discovered that the epithelial sodium channel protein can be used as a target substance in the development of new overactive bladder therapeutics that act on the bladder afferents. The invention has been completed.

That is, the present invention provides an agent for increasing bladder capacity , comprising amiloride as an active ingredient and an inhibitor of epithelial sodium channels expressed in the bladder, wherein the bladder capacity increases.

  The present invention can provide a novel target substance that can be used in the development of a new overactive bladder therapeutic agent that acts on the bladder afferent, and a novel overactive bladder therapeutic agent.

  The human epithelial sodium channel protein (Epithelial Sodium Channel: sometimes referred to as ENaC) according to the present invention is a method described in Wicker L, Steinkruger et al. (Biochemical and Biophysical Research Communications Vol 295, p330-335, 2002). The cDNA is obtained from the above, and is represented by SEQ ID NO: 5 or 6, 7 and 8 among the nucleotide sequences of SEQ ID NO: 5, 6, 7 and 8 corresponding to human αENaC, human δENaC, human βENaC and human γENaC, respectively. The full-length cDNA having the base sequence can be obtained by expressing it in E. coli using an E. coli expression vector and purifying it by a known method. The recombinant protein, and ENaC and a fragment of the recombinant protein are obtained by genetic recombination of the cDNA having the base sequence represented by SEQ ID NO: 5, 6, 7, or 8. Furthermore, analogs include rat epithelial sodium channel protein subunit α (hereinafter also referred to as rat αENaC), rat epithelial sodium channel protein subunit β (hereinafter also referred to as rat βENaC) and rat epithelial sodium channel protein, respectively. Rat epithelium having amino acid sequences of SEQ ID NOs: 9, 10, and 11 obtained by expressing a gene having the nucleotide sequence represented by SEQ ID NOs: 12, 13, and 14 corresponding to subunit γ (hereinafter also referred to as rat γENaC) Type sodium channel protein, rabbit epithelial type sodium channel protein subunit α (hereinafter also referred to as rabbit αENaC), rabbit epithelial type sodium channel protein subunit β (hereinafter also referred to as rabbit βENaC) and rabbit epithelial type sodium cha Examples of epithelial sodium channel proteins obtained from mammals such as rabbit epithelial sodium channel proteins having the amino acid sequences of SEQ ID NOs: 15, 16, and 17 corresponding to flannel protein subunit γ (hereinafter also referred to as rabbit γENaC) be able to. Among the nucleotide sequences for the amino acid sequences of SEQ ID NOs: 15, 16, and 17, the sequences of known portions are shown as SEQ ID NOs: 18, 19, and 20. Rat epithelial sodium channel protein subunit α and rabbit epithelial sodium channel protein subunit α are both subunits corresponding to human ENaCα and human ENaCδ. Here, one of the biological functions of the epithelial sodium channel protein is any one of a mechanosensitive sodium ion active transport function, a voltage-dependent sodium active transport function, or a receptor-related sodium active transport function. It is.

  Mammalian epithelial sodium channel proteins are involved in the mechanosensitive active transport of sodium ions performed by baroreceptors, skin sensory structures, and the like. Mammalian epithelial sodium channel protein is also expressed in the urothelium (renal pelvis, ureter, bladder), for example, rabbit bladder epithelial sodium channel protein responds to hydrostatic pressure changes Thus, the sodium ion active transport property is changed to mechanical sensitivity. Furthermore, it was shown that the epithelial sodium channel protein of rat renal pelvic epithelium is involved in the activation of renal afferent nerve with increasing intrarenal pressure. Therefore, epithelial sodium channel protein in the bladder is involved in the transmission of mechanosensitive information in the afferent nerve tract of the bladder and may cause dysfunction of the detrusor associated with lower urinary tract obstruction. By suppressing the bladder sensory nerve activity using epithelial sodium channel protein as a target substance, it is considered that an abnormality in detrusor function associated with dysuria can be prevented and overactive bladder can be treated. In addition, there is an advantage that a drug acting on the bladder afferent does not affect the urinary excretion.

  Therefore, the sodium channel protein according to the present invention is used for screening a therapeutic agent for overactive bladder syndrome, for example, by screening a drug that inhibits the mechanosensitive sodium ion active transport function and voltage-dependent sodium active transport function of the protein. It can be used as a target substance. Furthermore, it can be preferably used as a target substance for screening for a therapeutic agent for overactive bladder syndrome associated with prostatic hypertrophy. Further, amiloride, benzamyl, phenamyl, CDPC and the like relating to the therapeutic agent for overactive bladder syndrome of the present invention can be obtained commercially.

  The therapeutic agent for overactive bladder syndrome of the present invention may contain an adjuvant. Adjuvants are roughly divided into several groups based on their composition.

These groups include, for example, oil adjuvants such as Freund's complete and incomplete adjuvants, inorganic salts (eg, AlK (SO ₄ ) ₂ , AlNH ₄ (SO ₄ ), silica, alum, Al (OH) ₃ , Ca ₃ (PO ₄ ) ₂ , kaolin and carbon), for example polynucleotides such as poly IC and poly AU acids, and, for example, wax D from Mycobacterium tuberculosis and members of the Corynebacterium parvum, Bordetella pertussis and Brucella families Certain natural substances such as those found in The therapeutic agent for overactive bladder syndrome of the present invention can be used not only for humans but also for animals such as domestic animals and pets.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, this is merely an example and does not limit the present invention.

<Bladder mucosa specimens collected from patients>
Normal bladder mucosa specimens were collected from 9 patients suffering from bladder cancer or prostate cancer without prostate enlargement. Mucosal specimens of obstructed bladder were collected from 9 patients with prostatic hypertrophy (hereinafter also referred to as BPH) who had undergone endoscopic surgical treatment. The criteria for dysuria due to obstructed bladder are a prostate volume of 30 cm ³ or more, an international prostate symptom score total score (hereinafter also referred to as IPSS) of 12 points or more, and a maximum urine flow rate of 10 ml / sec or less. Adopted the standard. Pressure flow measurements were performed on 7 of 9 patients with BPH to confirm bladder obstruction. Involuntary detrusor contraction (detrusor overactivity) was observed in 7 out of 8 patients with obstructed bladder who underwent intravesical pressure measurements. A 6 Fr double lumen catheter was inserted through the urethra into the bladder. The bladder was filled with physiological saline, which is an isotonic solution, at an injection rate of 50 ml / min, and the intravesical pressure was measured. Abdominal pressure was measured in the rectum using a balloon catheter. The detrusor pressure was determined by subtracting the abdominal pressure from the intravesical pressure. Although nine patients without prostate enlargement did not undergo a micturition test, the six patients who calculated the total score for the international prostate symptom score question had a total score of 12 or less. there were. The study protocol was approved by the University of Yamanashi Ethics Committee, and all specimens were collected with sufficient informed consent from the patient. Table 1 shows the prostate volume and urine flow rate measurement results for each patient, the presence or absence of involuntary contraction, and IPSS.

注）表１中、患者番号におけるNは正常膀胱を示し、Oは閉塞膀胱を示し、Fは女性を示し、NSは直腸指診及びコンピュータによる断層X線写真法において正常な大きさの前立腺であったことを示し、Qmaxは尿流量測定におけるピーク流速を示し、IPSSは、国際前立腺症状スコアのスコア合計を示し、蓄積は、蓄尿症状スコアを示す。

Note) In Table 1, N in patient number indicates normal bladder, O indicates obstructed bladder, F indicates female, NS indicates prostate of normal size in digital rectal examination and computed tomography Qmax indicates the peak flow rate in the urine flow measurement, IPSS indicates the total score of the international prostate symptom score, and accumulation indicates the urine accumulation symptom score.

<Staining by immunofluorescence>
Tissue frozen by embedding in OCT compound (manufactured by Tissue-Tek) (bladder epithelial mucosa provided by 9 patients with normal bladder and 9 patients with obstructed bladder) was sliced to 8 μm thickness. . Each slice on the glass slide was fixed in acetone for a sufficient time at 4 ° C. and washed with Tris-buffered saline. The obtained slide was treated with a hydrogen peroxide solution having a concentration of 3% to block peroxidase, and then treated with a blocking reagent (manufactured by DakoCytomation) to block nonspecific protein binding. Rabbit polyclonal antibodies (Chemicon) against rat αENaC, βENaC, and γENaC subunits diluted 1: 600 were reacted as primary antibodies for 60 minutes at room temperature. When the subunits of sodium channel proteins in human and rat are compared with each other, the homology is 85% or more and the homology is very high. Therefore, the polyclonal antibody is considered to function in human tissues. The secondary antibody reaction was performed by diluting an anti-rabbit immunoglobulin derived from pig conjugated with tetramethyl isocyanate (DakoCytomation) 1:50 and observed with a fluorescence microscope BX50WI (Olympus). In the fluorescence microscope, the excitation wavelength and the emission wavelength were 520 nm and 550 nm, respectively. The results of observing the tissues of a patient having normal bladder and a patient having obstructed bladder with a fluorescence microscope are shown in FIGS. In FIG. 1, (A), (B), and (C) show the measurement results by immunofluorescence of human αENaC, human βENaC, and human γENaC in the tissues of patients with normal bladder, respectively (D), (E) and (F) show the measurement results of human αENaC, human βENaC, and human γENaC by immunofluorescence in the tissues of patients with obstructed bladder, respectively.

<Preparation of cDNA>
Total RNA was isolated from the bladder mucosa using RNeasy mini kit and RNase-free DNase set (Qiagen) and quantified using a spectrophotometer. Using the first strand cDNA synthesis kit (Roche) containing the reverse transcriptase of avian myeloid leukemia virus, the extracted RNA was reverse transcribed using a temperature program heated at 42 ° C for 60 minutes and then at 99 ° C for 5 minutes. CDNA was prepared.

<Quantitative real-time PCR>
Using SYBR green I (Molecular Probes) as a fluorescent dye, quantitative real-time PCR assay was performed using Smart Cycler System (Cephied). A pair of gene-specific primers designed by RT-PCR product cDNA using Ex taq R-PCR version (Takara) and Primer3, an online program described in Table 2 below. 40 PCR cycles of 94 ° C. for 10 seconds and 67 ° C. for 25 seconds with a measurement point of 6 seconds were performed in a 25 μl mixture containing 2 μM. The base sequences corresponding to the primer SEQ ID NOs: 21 to 30 are shown in Table 2. In order to reduce non-specific DNA products, the temperature of the measurement point was 2-3 ° C. lower than the specific melting peak point of the target gene. The expression level for each sodium channel protein subunit was determined for each patient by standardizing the ratio to the expression level of GAPDH by 100. Further, the average value of 9 normal bladder patients and 9 abnormalities were obtained. The average value of patients with bladder was determined. The amplified PCR product was electrophoresed on a 2% agarose gel visualized with ethidium bromide. The results are shown in FIG. FIG. 2A shows the results of electrophoresis of PCR products of human αENaC cDNA, human βENaC cDNA, human γENaC cDNA, human δENaC cDNA, and GAPDH cDNA. FIG. 2B is a diagram showing the standardized expression levels of human αENaC, human βENaC, human γENaC, and human δENaC. Moreover, the bar | burr part in FIG.2 (B) shows the standard error of the average value obtained from the sample of nine patients, and the number in a parenthesis shows the number of the samples by which the target gene was expressed. A portion of the PCR product was purified and sequenced using an automated sequencer to confirm that it was the target gene. Moreover, the numerical value of the expression level of αENaC, βENaC, γENaC, and δENaC for each patient is described in Table 1 described above and used in the statistical analysis described below.

<Statistical analysis>
A two-tailed t-test was used to statistically analyze the expression level of sodium channel protein subunits. In order to statistically analyze the correlation between the expression level of the sodium channel protein subunit and the IPSS and the urine accumulation symptom score, a linear regression analysis method was used. If the p value is less than 0.05, a statistically significant correlation will be recognized. In FIG. 2B, the asterisk indicates that the p-value is less than 0.01. In this case, the expression level of sodium channel protein subunit in normal bladder and the sodium channel protein subunit in abnormal (obstructed) bladder There is a statistically significant difference between the expression levels.

<Expression of ENaC mRNA in rat bladder epithelium>
FIG. 3 shows the results of examining the expression of αENaC, βENaC, and γENaC in the bladder epithelium collected from the rat normal bladder and obstruction model (urethal partial occlusion) bladder according to <cDNA preparation> and <quantitative real-time PCR> described above. Shown in

<Measurement of intravesical pressure in rats>
In female rats under urethane anesthesia, the micturition reflex induced by continuous infusion of saline from the bladder fistula at a rate of 4 ml / h was observed before refluxing 1 mM amiloride into the bladder, after refluxing amiloride, And amiloride was observed after shed. The results for female rats with normal bladder are shown in FIG.

  From the results of FIGS. 1 (A), (B), and (C), αENaC and γENaC were not detected by staining in human normal bladder, but in FIGS. 1 (D), (E), and (F) From the results, all of αENaC, βENaC, and γENaC were detected by staining in human obstructed bladder. From these results, it was found that the expression of αENaC and γENaC was more remarkable in the obstructed bladder than in the normal bladder. Furthermore, from the results shown in FIGS. 1A to 1F, the site of immunostaining of bladder epithelial cells covering the bladder lumen was present at the cell apex (bladder cavity side).

  From the results of FIG. 2 (B), in 9 patients with normal bladder, human αENaC, human βENaC, human γENaC, and human δENaC mRNAs are 1, 6, 4, and 1 respectively. Expression was confirmed. Therefore, human αENaC and human δENaC are hardly expressed in human normal bladder. On the other hand, in 9 patients with obstructed bladder, human αENaC, human βENaC, and human γENaC mRNA were expressed in all patients, but human δENaC mRNA was not substantially expressed. In addition, from the statistical analysis results of the results of quantitative real-time PCR, it was found that the expression levels of all human ENaC subunits except human δENaC were significantly higher in the obstructed bladder than in the normal bladder. (The p-value is 0.00002 for αENaC, 0.007 for βENaC, and 0.0005 for γENaC.) In particular, the expression level of αENaC was found to be significantly increased in obstructed bladders compared to normal bladders. The occurrence of detrusor overactivity (overactive bladder) in lower urinary tract obstruction diseases such as prostatic hypertrophy is considered to be associated with increased activity of bladder afferent nerves. On the other hand, epithelial sodium channel protein has been suggested to be a promising mechanosensitive molecule in various afferents (sensory nerve tracts) and is also expressed in the urothelium of mammals such as rats . Thus, it is believed that epithelial sodium channels may be involved in the development of overactive bladder in obstructed bladder through increased bladder afferent nerve activity. From the result of FIG. 2 (B) above, it is considered that the increase in ENaC expression in the bladder mucosa is involved in the occurrence of detrusor overactivity in the obstructed bladder, causing overactive bladder symptoms.

  From the results of FIG. 3, unlike the normal human bladder, in the rat normal bladder (5 animals), all proteins of rat αENaC, rat βENaC, and rat γENaC were expressed in the bladder epithelium.

  As a result of statistical analysis of the expression level data in FIG. 2 (B) and the correlation with the total score of IPSS and the urine accumulation symptom score, the expression level of human αENaC, human βENaC, and human γENaC is It was found that there was a high correlation with the urine accumulation symptom score. In the statistical analysis result of the expression level of human αENaC and the urine storage symptom score, r = 0.58, p = 0.02, and in the statistical analysis result of the expression level of human βENaC and the urine storage symptom score, r = 0.68, p = In the statistical analysis results of the expression level of human γENaC and the urine accumulation symptom score, r = 0.81, p = 0.0003, and in the statistical analysis result of the expression level of human γENaC and IPSS, r = It was 0.59 and p = 0.02. The expression level of human αENaC was also weakly correlated with age, r = 0.50, p = 0.03.

From the results of FIG. 4, the administration of amiloride to the rat bladder extended the urination interval,
It can be seen that there was a significant increase in bladder capacity. This change is, after you poured off the Amirora Lee de,
It recovered to the original. Since there was no change in bladder contraction force, it is considered that amiloride suppressed bladder afferent activity. Similar results were obtained in rats of the occlusion model (urethral partial occlusion).

  From the results of FIG. 4 above, it was found that an inhibitor of epithelial sodium channel can be used as a therapeutic agent for dysuria such as overactive bladder.

  As described above, the involvement of epithelial sodium channels in bladder afferent nerve transmission and the possibility of involvement in the development of overactive bladder revealed in the Examples is the development of a new overactive bladder therapeutic agent that acts on bladder afferents. It is thought to give a perspective. At present, drugs that act on epithelial sodium channels are limited to amiloride, etc. and have low specificity. However, there is a possibility of clinical application (development of therapeutic drugs) through joint research and development with appropriate pharmaceutical company laboratories. high.

The measurement results by immunofluorescence against human αENaC in tissue of patients with normal bladder. The measurement results by immunofluorescence against human βENaC in tissue of patients with normal bladder. The measurement results by immunofluorescence against human γENaC in tissue of patients with normal bladder. Against human αENaC in tissue of patients with obstructive bladder showing the result measured by immunofluorescence. Against human βENaC in tissue of patients with obstructive bladder showing the result measured by immunofluorescence. Against human γENaC in tissue of patients with obstructive bladder showing the result measured by immunofluorescence. Diagram showing the results of electrophoresis of human αENaC cDNA, human βENaC cDNA, human γENaC cDNA, human δENaC cDNA, and GAPDH PCR products obtained from the bladder mucosa of patients with normal bladder and patients with obstructed bladder It is. It is a figure which shows the expression level in normal bladder and obstruction | occlusion bladder of each of (alpha) ENaC, (beta) ENaC, (gamma) ENaC, (delta) ENaC normalized by the GAPDH expression level measured by real-time PCR method. It is the agarose gel electrophoresis image of the product by RT-PCR method for rat αENaC, rat βENaC and rat γENaC of bladder epithelium collected from rat normal bladder and obstructed bladder. It is a figure which shows the result of the bladder pressure measurement in the rat under urethane anesthesia. Intravesical reflux of 1 mM amiloride solution prolonged the urination interval and increased bladder capacity. This effect was restored by washing off amiloride. There was no change in bladder contraction force.

Claims (2)

An agent for increasing bladder capacity, comprising amiloride as an active ingredient and an inhibitor of epithelial sodium channels expressed in the bladder, wherein the bladder capacity increases . 2. The bladder capacity increasing drug according to claim 1, which acts on the bladder .

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