KR101537400B1 - Composition for treatment of prostatic hyperplasia comprising acetylcholine - Google Patents

Abstract

The present invention relates to a composition for treating hyperplasia of prostate comprising acetylcholine, and more particularly to a pharmaceutical composition for preventing or treating hypertrophy or lower urinary tract symptoms comprising acetylcholine as an active ingredient.
Since acetylcholine relaxes the contracted prostate adenomatous tissue, the pharmaceutical composition containing acetylcholine as an active ingredient according to the present invention is effective for treating the benign prostatic hyperplasia or lower urinary tract symptoms.

Description

TECHNICAL FIELD The present invention relates to a composition for treating prostatic hyperplasia comprising acetylcholine,

The present invention relates to a composition for treating hyperplasia of prostate comprising acetylcholine, and more particularly to a pharmaceutical composition for preventing or treating hypertrophy or lower urinary tract symptoms comprising acetylcholine as an active ingredient.

Prostate Stroma is not secretory mesenchyme, but it is abundant in the noradrenergic nerve distribution. Restriction of smooth muscle contraction in prostate hypertrophy is induced by α ₁ - adrenergic receptor antagonists. The mammalian prostate gland leads to nerve stimulation through the lower abdomen and pelvic nerves that regulate prostate growth and function. Although the role of adrenergic receptors in the norepinephrine function is well known, the role of cholinergic neurons in the physiological and pathophysiological aspects of the prostate is unknown.

Acetylcholinesterase-positive nerves are distributed not only in the epithelium but also in the prostate stroma (Hansen et al., Jen et al. 2000 "and" McVary KT et al., Prostate Suppl: 8: 2-13,1998 "). Activation of muscarinic receptors in the prostate gland causes glandular secretion. However, it was not concluded how muscarinic receptor activation affects smooth muscle in epileptic tissues.

Accordingly, the present inventors have studied the pathological effects of cholinergic nerves on the prostate, and discovered that acetylcholine relaxes the urethra surrounding the urethra, thus completing the present invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating hyperplasia of prostate comprising acetylcholine as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating lower urinary tract symptoms comprising acetylcholine as an active ingredient.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating enlargement of the prostate gland comprising acetylcholine as an active ingredient.

In order to accomplish another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating lower urinary tract symptoms comprising acetylcholine as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating hyperplasia of prostate comprising acetylcholine as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating lower urinary tract symptoms comprising acetylcholine as an active ingredient.

Benign prostatic hyperplasia (BPH) is the most common disease in men over the age of 50, and is associated with lower urinary tract symptoms, which can reduce quality of life. The major cause of the development of benign prostatic hyperplasia is increased age and the presence of male hormone. Since men with congenital testicular dysfunction or testicular depletion do not develop enlargement of the prostate gland, we can see that male hormones are closely involved in their induction. In addition, differences in race, environment, and dietary habits can also be the cause of benign prostatic hyperplasia.

Lower Urinary Tract Symptoms (LUTS) are caused by the obstruction of the enlarged prostate composed of epithelial cells and smooth muscle, the response of the bladder smooth muscle to it, and the strong contraction of the smooth muscle by stimulation of the alpha sympathetic nerve. The lower urinary tract symptom refers to various symptoms related to the storage and discharge process of urine. The symptoms of urinary incontinence, urge incontinence, There are weakness of urine, lack of feeling, urination. The main cause of lower urinary tract symptoms is urethral obstruction caused by enlarged prostate, and it has been thought that relieving urethral obstruction caused by enlarged prostate leads to relief of lower urinary tract symptoms.

Acetylcholine induces strong internal currents in both the sympathetic and parasympathetic nerves of the pelvic ganglia that provide autonomic nerves to the prostate gland. The presence of muscarinic receptors in the prostate epithelium suggests that acetylcholine is a major secretion promoter. Although many studies have reported that acetylcholine contracts in the prostate of various species, the contraction effect is relatively small compared to the a-adrenergic agonist (Ventura S., et al., Pharmacol Ther; 94 : 93-112, 2002).

 The relaxation effect of acetylcholine in this study is not consistent with previous studies. Previous experiments were carried out in the resting state, suggesting that no acetylcholine relaxation effect was observed. It was initially discovered by the present inventors that acetylcholine can relax the sympathetic stimulation-contracted prostate gland. When the present inventors treated acetylcholine to a prostate slice contracted with phenylephrine (a neurotransmitter causing symptoms of enlarged prostate), the acetylcholine-concentration-dependent prostate relaxed (see Experimental Result 1 and FIG. 1).

In addition, the acetylcholine relaxation effect is completely inhibited by atropine (anti-muscarinic drug), but not by hexamethonium (anti-nicotine drug). This shows that acetylcholine is secreted through activation of muscarinic receptors rather than nicotinic receptors, resulting in relaxation of the rabbit prostate smooth muscle (see Experimental Results 2 and 2).

Prostate enlargement is a disease caused by narrowing the urethra due to two mechanisms.

First, the prostate tissue around the urethra is proliferating and hypertrophic, and the urethra is mechanically depressed. Second, the prostate tissue is not easily relaxed or tensioned, so the urethra does not develop well. Most of the prostate hypertrophy has two mechanisms Occurs.

The nerve involved in the tension of the prostate is the alpha receptor, especially alpha-1, which is a drug that increases the degree of tension in the prostate with alpha adrenergics. Therefore, suppression of prostate cancer will be helpful in the treatment of prostate. In addition, from the bladder side, one of the mechanisms that the bladder responds to the external nociceptive stimuli is sympathetic nerve, and it is known that the sensitive bladder has many subtypes of aplpha 1d.

The main point of the result of the present invention is that the prostate tissue is relaxed by the cholinergic receptor and the relaxation effect can be further strengthened by NOS, so that the combination of the two agents can be clinically effective and can be developed as a combination of prostate hypertrophy It is.

In particular, cholinergic nerve (muscarinic receptor) is important for bladder contraction. Therefore, it is considered that bladder contraction is most effective when bladder contraction is absent and concomitant with benign prostatic hyperplasia.

The composition for preventing or treating benign prostatic hyperplasia or lower urinary tract symptoms comprising acetylcholine as an active ingredient of the present invention may further comprise a nitrogen monoxide donor.

The nitrogen monoxide donor may be selected from the group consisting of L-arginine, Nitroglycerine, Isosorbide Dinitrate, Isosorbide Mononitrate, have.

In the experimental results of the present invention, the acetylcholine-induced relaxation effect was completely blocked by L-NAME (non-specific NOS inhibitor) treatment, but the degree of relaxation increased when treated with L-arginine. These results suggest that nitrate - mediated nerves are present in the prostate gland. N aminoguanidine (iNOS inhibitor) did not affect the relaxation effect of acetylcholine, but the relaxation effect of acetylcholine was greatly inhibited when 7-NI (mainly inhibiting nNOS and weakly inhibiting eNOS) was treated. These results show that the relaxation effect of acetylcholine in the prostate is mediated by the release of NO via nNOS and eNOS.

L-arginine produced nitrogen monoxide, and the addition of L-arginine further increased the relaxation effect (see FIG. 4).

The pharmaceutical composition for preventing or treating benign prostatic hyperplasia or lower urinary tract symptoms comprising acetylcholine as an active ingredient according to the present invention can be used in the form of a general pharmaceutical preparation. That is, the pharmaceutical composition of the present invention can be administered in various formulations, oral or parenteral, at the time of actual clinical administration, and oral administration is preferred in the present invention. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid formulations for oral administration may include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium Calcium carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used.

The liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like May be included. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

In addition, the dosage or dose of the pharmaceutical composition according to the present invention may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, It is preferable to take 20 to 200 mg per day for one to several times on an adult basis.

Since acetylcholine relaxes the contracted prostate adenomatous tissue, the pharmaceutical composition containing acetylcholine as an active ingredient according to the present invention is effective for treating the benign prostatic hyperplasia or lower urinary tract symptoms.

Fig. 1 shows the degree of shrinkage of the prostate according to the treatment concentration when acetylcholine was treated with the phenylpurine-shrunk prostate gland.
FIG. 2 shows the results of treatment with 10 μM of hexamethonium and atropine, respectively, on the prostate shrunk with phenylphrine, giving the degree of shrinkage of the prostate. (Control: Controlled prostate-shrinked prostate with acetylcholine )
Figure 3 gives the degree of shrinkage of the prostate when treated with 10 ^-6 M of the M ₂ , M ₃ receptor antagonist, respectively, on the prostate shrunk with phenylphrine. (Control: Acetyl Choline-treated control group)
Figure 4 shows the degree of shrinkage of the prostate gland when treated with 10 mM L-arginine and 10 μM L-NAME, respectively, on the prostate treated with phenylphrine. (Control: Treatment of the prostate with shrinking phenylephrine with acetylcholine Control group)
FIG. 5 shows the degree of shrinkage of the prostate gland when treating aminoguanidine, 7-nitroindazole, at a concentration of 10 μM, to the prostate shrunk with phenylphrine (Control: treated with acetylcholine in a phenylpurine-shrunk prostate Control group)

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

<Experimental Method>

1. Tissue preparation and shrinkage measurement

The experiment was conducted in accordance with the guidelines of the Human and Animal Care Committee of the Medical Science Center of Chung - Ang University, Korea.

30 New Zealand white rabbits (3-4 kg) were prepared and anesthetized with pentobarbital (60 mg / kg, intraperitoneal injection), and the carotid artery was incised and sacrificed. Prostate tissue was cut and transferred to HEPES buffered with 100% oxygen-saturated PSS (physiological salt solution).

The tissue around the prostate gland was carefully removed, and a piece of 1 x 1 x 10 mm was cut in the longitudinal direction parallel to the urethra. I obtained four sections per section, and I used two prostate tissue sections surrounding the urethra. Each slice is suspended in a bath containing 30 ml of PSS solution [114 mM NaCl, 26 mM NaHCO ₃ , 4.7 mM KCl, 2.5 mM CaCl ₂ , 1.2 mM NaH ₂ PO ₄ , and 11 mM D-glucose]. During the experiment, the bath was continuously fed with 95% oxygen and 5% carbon dioxide gas at 37 DEG C, maintaining the pH between 7.3 and 7.4. The shrinkage of each prostate tissue section was measured using a force transducer (52-9545, Harvard Apparatus, UK) and the analog signal from the machine was converted to a digital signal using a MacLab 4e recording system (AD Instruments, Australia). Two grams of the slices were kept at resting tension and homogenized using PSS solution at intervals of 30 minutes.

2. Reagents

The following reagents were used in the present invention.

1-piperdinyl] acetyl) - (2 - [(diethylamino) methyl]) - phenyl- Dihydro-6H-pyrido [2,3- b] [1,4] benzodiazepine-6-one, 4-DAPH (4-diphenylacetoxy-N-methyl-piperidine), tropicamide, L-NAME, 7-NI (3-bromo-7-nitroindazole), aminoguanidine chloride and L-arginine were used. All of the reagents were purchased from Sigma Chemical Company (St. Louis, Mo., USA).

3. Phenylpyridine  Measurement of the response of acetylcholine to induced shrinkage

In the steady state, we observed how the prostate slice reacted with acetylcholine. Tensile conditions were induced by contacting the sections with 10 ^-5 M pentylphrine for 5 minutes and treated with 10 ^-9 to 10 ^-4 M acetylcholine for 3 minutes each.

4. Effects of acetylcholine relaxation and Choline  Observation of relationship with receptor

To determine whether acetylcholine prostate relaxation was associated with cholinergic receptors, cholinergic receptor antagonists were treated for 10 minutes each, and then the prostate relaxation effect of acetylcholine was observed.

The antagonist used was 10 ^-5 M hexamethonium, 10 ^-5 M atropine, selective muscarinic receptor (M ₁ , M ₂ , M ₃ , M ₄ ) antagonist (10 ^-6 M pyrene ^{benzodiazepine, 10 -6 M AF-DX116,} 10 -6 M 4-DAMP, 10 -6 M tropicamide each treatment)

5. Effect of acetylcholine relaxation and Nitric oxide synthase NOS )

10 μM L-NAME (non-specific NOS inhibitor), 10 μM 7-NI (predominantly nNOS, partially eNOS inhibited), 10 μM aminoguanidine (irreversible iNOS inhibitor), 10 mM L -arginine (NO donor). Thereafter, acetylcholine prostate relaxation effect was observed.

6. Results Analysis

The results of the experiments were expressed as mean ± standard deviation (SEM). The maximum degree of shrinkage induced by phenylphrine was regarded as 100% shrinkage, and the relaxation effect of acetylcholine was regarded as the rate of reduction of the maximum shrinkage. IC ₅₀ and EC ₅₀ values were obtained by regression analysis. The regression results were plotted using Logistic sigmidal fitting model (MicroCal Origin, version 7.5) using 4 to 5 values.

In all experiments, "n" means number of intercepts, t-test and ANOVA were used for analysis, and significance level was set at p <0.05.

<Experimental Results>

1. Reaction of prostate slice to acetylcholine

In the stable prostate slice, there was no change in treatment with acetylcholine (10 ^-9 M to 10 ^-4 M) (results not shown). However, the prostate slice induced contraction by phenyl-prine showed a concentration-dependent relaxation effect (see FIG. 1). Acetylcholine IC ₅₀ values and the maximum relaxation value was turned out to 45.44 ± 40.66 μM and 39.21 ± 2.56%.

2. Relaxation effect of acetylcholine Choline  Receptor relationship

The relaxation effect of acetylcholine was inhibited when treated with atropine (a non-selective muscarinic receptor antagonist), but not when treated with hexamethonium (nicotinic receptor antagonist) (see FIG. 2).

M ₂ (10 ^-6 M) and M ₃ (10 ^-6 M) receptor antagonists almost completely blocked the acetylcholinergic effect (see FIG. 3). On the other hand, M ₁ (10 ^-6 M) and M ₄ (10 ^-6 M) receptor antagonists did not affect the relaxation effect of acetylcholine (not shown).

3. Relaxation effect of acetylcholine NOS Relationship

The experimental group treated with L-NAME (10 μM) and 7-NI (10 μM) inhibited the acetylcholine relaxation effect, whereas the experimental group treated with aminoguanidine (10 μM) did not affect the acetylcholine relaxation effect, but L-arginine 10 mM) increases the acetylcholinesterase effect (see FIGS. 4 and 5).

Since acetylcholine relaxes the contracted prostate adenomatous tissue, a pharmaceutical composition capable of treating the benign prostatic hyperplasia or lower urinary tract symptoms using acetylcholine can be prepared, which is highly industrially applicable.

Claims (4)

A pharmaceutical composition for the treatment of benign prostatic hyperplasia comprising acetylcholine as an active ingredient.


delete 2. The pharmaceutical composition according to claim 1, wherein the composition further comprises L-arginine.

delete

Images