KR20120101762A - Composition comprising ninjurin activity inhibitors for regenerating nerve cells or endothelial cells - Google Patents

Abstract

PURPOSE: A composition containing ninjurin activation inhibitors is provided to induce nerve cell and/or endothelial cell growth and to prevent and/or treat impotence. CONSTITUTION: A pharmaceutical composition for regenerating nerve cells or endothelial cells in penis contains ninjurin activation inhibitors as an active ingredient. The inhibitor is a ninjurin blocking antibody. A pharmaceutical composition for preventing or treating impotence contains the inhibitor as an active ingredient and a pharmaceutically acceptable salt thereof. A method for preventing or treating diseases caused by nerve cell or endothelial cell damage comprises a step of administering the inhibitor to an individual.

Description

Composition comprising ninjurin activity inhibitors for regenerating nerve cells or endothelial cells}

The present invention relates to a composition for regenerating neurons and / or endothelial cells using an inhibitor of ninjurin activity. Specifically, the present invention relates to a composition for preventing and / or treating erectile dysfunction using a ninjurin activity inhibitor.

Ninjurin was first reported in 1996 by Toshiyuki Araki et al., Whose expression is increased in Schwann cells after injury by transection or crushing of the distal sciatic nerve. It was discovered in the process of finding a gene.

According to the results, ninjurin is known to increase its expression after peripheral nerve injury, and ninjurin has been reported to play an important role in the proliferation of neurons (T. Araki et al., Neuron, 17: 353, 1996; T. Araki et al., J Biol Chem, 272: 21373, 1997). In addition, the administration of ninjurin-blocking peptides eliminates the effect of ninjurin's neuronal proliferation, suggesting that ninjurin plays an important role in neuronal regeneration (Araki et al., J Biol Chem, 272: 21373, 1997). . From the above, ninjurin is closely related to neuronal damage and is believed to play an important role in the survival or proliferation of neurons.

On the other hand, erectile dysfunction is a type of male sexual dysfunction, the male genitalia is not a erection or erection is not sustained is a phenomenon that can not be sexual activity. The causes of erectile dysfunction are largely divided into psychogenic causes and organic causes. Psychogenic erectile dysfunction is due to excessive secretion of noradrenaline due to excessive effects of sympathetic nerves due to psychological and psychological effects, increased cavernous smooth muscle tension, and suppression of neurotransmitter secretion. In addition, organic erectile dysfunction is classified into neurogenic, vascular, and endocrine dysfunction depending on the cause.

The neurogenic erectile dysfunction is a disorder caused by impaired secretion of releasing neurotransmitters such as acetylcholine and nitric oxide (NO) for erection at the end of the erectile nerve or fiber, spinal damage, multiple It may be caused by central nervous system diseases such as sclerosis and peripheral neurological diseases such as the abdomen-pelvis.

Recent research has focused more on organic causes, and as one of the treatment methods, sildenafil (viagra), which is an oral erectile dysfunction treatment, is used worldwide. Viagra increases the concentration of cGMP by inhibiting PDE-5 (phosphodiesterase-5) that is specifically distributed in the corpus cavernosum. However, not only side effects such as headache, hot flashes, indigestion, and heart attack have been reported, but PDE-5 family drugs such as Viagra are fundamental treatments by regulating transient protein expression and related factors at the molecular level. Can not. Furthermore, in the case of erectile dysfunction due to nerve injury after prostate cancer surgery, the effect of such a therapeutic agent is not shown well, and even if it is effective, its efficacy does not last long.

Therefore, there is a need for an erectile dysfunction treatment agent that fundamentally treats an abnormal neuro-vascular structure in an erectile dysfunction penis and whose efficacy also lasts for a long time.

The present invention is to provide a composition for treating or preventing erectile dysfunction, which has a more lasting effect by fundamentally treating the abnormalized neuro-vascular structure in the penis, and a method for treating erectile dysfunction using the same.

Specifically, the present invention is to provide a pharmaceutical composition for neuronal and / or endothelial cell regeneration comprising a ninjurin activity inhibitor as an active ingredient.

In another aspect, the present invention is to provide a pharmaceutical composition for preventing and / or treating erectile dysfunction comprising the ninjurin activity inhibitor.

In another aspect, the present invention is to provide a method for preventing and / or treating a disease caused by injury of nerve cells and / or endothelial cells by administering to a subject a composition comprising an ninjurin activity inhibitor.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention provides a pharmaceutical composition for neuronal and / or endothelial cell regeneration comprising a ninjurin activity inhibitor as an active ingredient. In one embodiment of the invention, the ninjurin activity inhibitor comprises a ninjurin blocking antibody and peptide. In another embodiment of the present invention, the nerve cells comprise penile nerve cells. In another embodiment of the present invention, the endothelial cells include penile vascular endothelial cells.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating erectile dysfunction comprising an inhibitor of ninjurin activity as an active ingredient. In one embodiment of the invention, the ninjurin activity inhibitor comprises a ninjurin blocking antibody and peptide. In another embodiment of the present invention, the composition may further comprise a pharmaceutically acceptable carrier. In another embodiment of the present invention, the composition is characterized by increasing the intracavernosal pressure. In another embodiment of the present invention, the composition is characterized by increasing the specific protein of neurons or vascular endothelial cells.

The present invention also provides a method for preventing and / or treating a disease caused by neuronal or endothelial cell damage, comprising administering to a subject a pharmaceutical composition comprising an ninjurin activity inhibitor as an active ingredient.

The ninjurin activity inhibitor of the present invention is effective in treating erectile dysfunction. In particular, it can effectively and fundamentally treat erectile dysfunction of various causes by showing a structural improvement of neuropathy and endothelial cell lesions of the penis simultaneously.

In addition, the erectile dysfunction treatment agent using the ninjurin activity inhibitor of the present invention does not regulate transient protein expression and related factors at the molecular level, as in the conventional PDE-5 family drugs such as Viagra, Because of the fundamental treatment of the vascular structure has the advantage that the effect lasts much longer.

Figure 1 is the result of electrophoresis to investigate the change in the expression of ninjurin protein at each stage after penile nerve injury in the mouse.
Figure 2 measures the erection power according to the electrical stimulation between the ninjurin blocking antibody administered group and the control group.
Figure 3 shows the increase in erection by the treatment of ninjurin blocking antibody through the degree of neurofilament expression, a neuronal specific protein.
Figure 4 shows the increase in erectile power by ninjurin blocking antibody treatment through the expression level of PECAM-1, a vascular endothelial cell-specific protein.

Previous studies related to the role of ninjurin indicate that ninjurin is closely related to neuronal damage and plays an important role in the survival or proliferation of neurons. However, the present inventors newly revealed that blocking of ninjurin in the penis of penile nerve-injured mice causes proliferation of vascular endothelial cells as well as nerve cells.

Accordingly, the present invention provides a composition for regenerating neurons and / or endothelial cells comprising an ninjurin activity inhibitor as an active ingredient.

The present inventors sought to develop a method for treating erectile dysfunction due to penile nerve injury that occurs frequently after prostate cancer surgery. As a result, the present inventors have found that when the ninjurin activity inhibitor is administered to a penile nerve injury animal, it shows an effect of improving, restoring, or treating the abnormal neuronal and vascular structures. In other words, it was found that the expression of ninjurin increased after penile nerve injury, and the first time that ninjurin inhibitors have the effect of improving or restoring abnormal nerves and blood vessel structures of the penis, The efficacy of treating erectile dysfunction was verified.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating erectile dysfunction comprising an ninjurin activity inhibitor as an active ingredient.

The ninjurin activity inhibitor of the present invention includes a ninjurin blocking antibody and peptide. In addition to the ninjurin activity inhibitor, the composition of the present invention may further include components such as existing therapeutically active ingredients, other adjuvants, and pharmaceutically acceptable carriers. Such pharmaceutically acceptable carriers include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and the like.

The composition of the present invention promotes the regeneration of nerve cells and endothelial cells of the penis, thereby exhibiting the activity of fundamentally treating abnormalized nerves and vascular structures in the penis without side effects. While the regeneration of nerve cells and endothelial cells is promoted by administration of the composition of the present invention, various diseases can be effectively delayed, improved, and prevented. In particular, the compositions of the present invention are effective in patients with erectile dysfunction due to erectile dysfunction due to various causes, in particular erectile nerve injury occurring after abdominal-pelvic cancer surgery such as prostate cancer surgery.

Accordingly, the present invention provides a method for preventing and / or treating a disease caused by neuronal or endothelial cell damage, the method comprising administering to a subject a pharmaceutical composition comprising a pharmaceutically effective amount of an inhibitor of ninjurin activity. Include. The composition can be used without particular limitation in the prevention and treatment of various diseases caused by neuronal or endothelial cell damage. Diseases that can be prevented and treated with the composition of the present invention include erectile dysfunction, and erectile dysfunction occurring after prostate cancer surgery may be a representative example.

As used herein, "individual" means a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses and cattle, etc. Mean mammal. Also, in the present invention, "pharmaceutically effective amount" means the type and severity of the disease to be administered, the age and sex of the patient, the sensitivity to the drug, the time of administration, the route of administration and the rate of administration, the duration of treatment, the factors including the concurrent drug and other medicines It is determined according to factors well known in the art and can be easily determined by those skilled in the art in such an amount that the maximum effect can be obtained without side effects in consideration of all the above factors.

The composition of the present invention is not limited in the method of administration as long as it can reach the target tissue. Examples include oral administration, arterial injection, intravenous injection, transdermal injection, intranasal administration, coronary administration or intramuscular administration. The daily dosage is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, preferably administered once to several times a day.

In an embodiment of the present invention, to determine the relationship between penile nerve damage and ninjurin expression, the expression level of ninjurin protein was confirmed in the damaged penile tissues of 12-week-old mice. As a result, it was confirmed that ninjurin protein expression was significantly increased in penile erectile tissue after induction of penile nerve damage (see FIG. 1).

In another embodiment of the present invention, in order to determine the effect of the ninjurin blocking antibody erectile dysfunction, by dividing the damaged penis nerves of 12-week-old mice into various groups to measure the erection, nerve cells or vascular endothelial cell-specific protein The expression changes of neurons or vascular endothelial cells were confirmed. As a result, it showed an improvement in erectile power in the group to which the ninjurin blocking antibody was administered (see FIG. 2), and the expression of neuronal and vascular endothelial specific proteins was increased in the group to which the ninjurin blocking antibody was administered. (See FIGS. 3 and 4). The results suggest that the ninjurin blocking antibody improves or repairs abnormal nerves and blood vessel structures of the penis, thereby treating erectile dysfunction due to penile nerve damage.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  1. In the penile tissue of penile nerve injury model ninjurin  Changes in protein expression

12-week-old mice (C57BL / 6J) were used and anesthesia was performed by intramuscular injection of ketamine (100 mg / kg) and xylazine (5 mg / kg). After the medial incision of the lower abdomen, the prostate gland was approached, and after carefully detaching the prostate gland, the penile nerves located on both sides of the prostate gland were reached. Both penile nerves were excised to a length of 1 mm and the nerve injured, and then the skin incision site was closed. Penile tissues were collected at 1, 3, 7, 14 and 28 days after nerve injury. Electrophoresis was performed after protein separation from the penile tissues, followed by electrophoresis. The ninjurin blocking antibody was used to evaluate the expression level of ninjurin protein in each group, and the results are shown in FIG. 1.

As shown in FIG. 1, the expression of ninjurin in the penile erectile tissues was significantly increased in the penile erectile tissues on the 1st, 3rd and 7th day after the induction of penile nerve injury, and the 14th and 28th day after the penile nerve injury. There was a return to normal levels.

Example  2. Ninjurin  Effect of Blocking Antibodies on Erectile Dysfunction

12-week-old mice (C57BL / 6J) were used and anesthesia was performed by intramuscular injection of ketamine (100 mg / kg) and xylazine (5 mg / kg). After the medial incision of the lower abdomen, the prostate gland was approached, and after carefully detaching the prostate gland, the penile nerves located on both sides of the prostate gland were reached. Bilateral penile nerves were crushed with vascular forceps (Karl Stortz, Germany) for about 1 minute and then the skin incision site was closed. The experiment was divided into four groups (N = 4 / group, group 1-Sham surgery group; group 2-penis nerve injury + PBS (Phosphated buffered saline) group; group 3-penis nerve injury + ninjurin blocking antibody administration group) [1 μg / 20 μl]; Group 4-Penile nerve injury plus ninjurin blocking antibody group [2.5 μg / 20 μl]). PBS and ninjurin blocking antibodies were administered into the corpus cavernosum immediately after penile nerve injury, and erection was measured after corpus cavernous nerve stimulation at 1 week. Immunohistochemical staining of neurofilament and platelet / endothelial cell adhesion molucule-1 (PECAM-1) in the corpus cavernosum tissue changes expression of neurons and vascular endothelial cells Was investigated.

As a result, it showed the highest improvement of erection at 2.5 μg concentration among the ninjurin-blocking antibody-administered group, and the erection was restored to about 80-85% of the normal control group. After penile nerve injury, the expression of neurons and vascular endothelial cells in the penis was significantly decreased compared to the Sham surgical group. After administration of ninjurin blocking antibody, the amount of neurons and vascular endothelial cells was remarkably restored to normal levels.

It will be described in more detail below.

(1) Analysis of erection power according to electrical stimulation

In the experimental method for erectile power measurement, the left epidermis was opened in the lower abdomen of the prepared mouse, and the cavernous corpus cavernoscopy (penis nerve) located in the posterior and outer sides of the prostate gland was prepared. For electrical stimulation of the penis nerve, a platinum electrode was placed on the penis nerve, and then electrostimulated for about 1 minute at a 5 volt 12 hertz intensity. When the electrical stimulation is applied, the penis is erection, the pressure inside the penis (penal cavernosal pressure) during the erection through the catheter inserted into the penis was measured by a pressure measuring device, the results are shown in Figure 2 . The measured pressure values will reflect penile erection.

In FIG. 2, ICP (Intracavernous Pressure) refers to the pressure inside the penis (intracavernosal cavernosal pressure) during erection, and is generally an indicator of erection power. Mean Systolic Blood Pressure (MSBP) also represents mean systolic blood pressure. In general, the erection power is expressed by dividing the maximum ICP or the area of the cavernosal pressure curve by the mean systolic blood pressure (MSBP). This may affect the internal pressure.

As shown in Figure 2, ninjurin blocking antibody showed a much higher erection than PBS injection group at 1 μg, 2.5 μg concentration, and showed a better erection improvement effect compared to 1 μg at 2.5 μg concentration. Two variables for penile erection, namely the peak corpus cavernosum and the area of the penile pressure curve, were markedly increased after ninjurin blocking antibody.

(2) neuronal and vascular endothelial specific proteins Neurofilament , PECAM -1 expression analysis

Penile erection is a type of neuro-vascular response. Therefore, the normal activity of the penis nerves and blood vessels is essential for normal erection. Abnormal structure or activity of nerves and blood vessels can cause erections to decrease. In order to investigate structural changes in nerves and blood vessels in penile erectile tissues after penile nerve injury, the present inventors have examined neurofilament (Neurofilament), a protein specific to neurons, and PECAM-1 (platelet / endothelial cell, a protein specific for vascular endothelial cells). The expression of adhesion molucule-1) was confirmed.

Tissues on slides prepared for neurofilament, PECAM-1 expression analysis were fixed in 3.7% paraformaldehyde for about 5 minutes. The fixed penile tissue sections were washed three times in wash buffer (2% FBS + 0.1% Sodium Azid in PBS), and the first antibody (anti-Neurofilament, anti-PECAM-1) was added at a ratio of 1: 100 at 4 ° C at 16 ° C. The reaction was carried out for a time. The second antibody (FITC-labeled anti-mouse) was then washed three times with a washing buffer to remove the remaining antibody, and then fluorescently identified the antibody specifically reacting with the neurofilament, PECAM-1. or TRITC-labeled anti-hamster antibody) at 1: 100 ratio for 1 hour. After the reaction was completed, washed again three times with a wash buffer to remove the remaining antibody and then analyzed the expression level by a fluorescence microscope or a confocal microscope that can identify the fluorescent substance, the results are shown in Figures 3 and 4 It was. At this time, DAPI (4 ', 6-diamidino-2-phenylindole) was used to stain the nucleus of cells, which is to more objectively show the regeneration of penile neurons and vascular endothelial cells.

As shown in Figure 3, it can be seen that the number of nerve cells in the penis of penile nerve injury mouse is significantly reduced compared to the Sham surgical mouse. It can be seen that the expression of neuronal specific protein was increased in the group administered the ninjurin blocking antibody, which proves that the ninjurin blocking antibody restores erection by inducing penile neuronal regeneration.

In addition, as shown in Figure 4, it can be seen that the number of vascular endothelial cells in the penis of the penile nerve injury mouse is significantly reduced compared to the Sham surgical mouse, which is not only nerve cells but also penile vascular endothelial cell damage It is the result that proves. That is, the expression of vascular endothelial cell-specific protein was increased in the group to which the ninjurin blocking antibody was administered, which means that the reduction of vascular endothelial cells was restored to normal level by the injection of the ninjurin blocking antibody.

The results suggest that erectile dysfunction due to penile nerve injury after prostate cancer surgery is closely related to penile nerve and vascular endothelial cell damage.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (14)

A pharmaceutical composition for regenerating neurons or endothelial cells comprising a Ninjurin activity inhibitor as an active ingredient. The method of claim 1,
Wherein said ninjurin activity inhibitor is a ninjurin blocking antibody. The method of claim 1,
The nerve cell is a penis nerve cell, composition. The method of claim 1,
Wherein the endothelial cells are penile vascular endothelial cells. Pharmaceutical composition for preventing or treating erectile dysfunction comprising a ninjurin activity inhibitor as an active ingredient. The method of claim 5, wherein
Wherein said ninjurin activity inhibitor is a ninjurin blocking antibody. The method of claim 5, wherein
The composition further comprises a pharmaceutically acceptable carrier. The method of claim 5, wherein
The composition is characterized in that to increase the intracavernosal pressure. The method of claim 5, wherein
The composition is characterized in that for increasing specific proteins of neurons or vascular endothelial cells. The method of claim 9,
Wherein said neuronal specific protein is a neurofilament. The method of claim 9,
The specific protein of the vascular endothelial cell is platelet-endothelial cell adhesion substance-1 (PECAM-1). A method of preventing or treating a disease caused by neuronal or endothelial cell damage, comprising administering to a subject a pharmaceutical composition comprising a pharmaceutically effective amount of an inhibitor of ninjurin activity as an active ingredient. The method of claim 12,
The disease is erectile dysfunction. The method of claim 13,
The erectile dysfunction is due to an erectile nerve injury occurring after prostate cancer surgery.

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