KR20170065447A - Pharmaceutical compositions for contraception containing Conoidin A as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising a compound containing Conoidin A or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition according to the present invention comprises Conoidin A as an active ingredient, It was confirmed that sperm motility, acquisition of fertilization ability, acrosome reaction, expression of phospho-PKA substrates, and expression of tyrosine phosphorylated protein were reduced by the treatment, and thus it is expected to be useful as a pharmaceutical composition for parenteral administration.

Description

[0001] The present invention relates to a pharmaceutical composition comprising Conoidin A as an active ingredient,

The present invention relates to a pharmaceutical composition for parenteral administration containing Conoidin A as an active ingredient.

Studies have been carried out on anti-wrinkle agents to prevent unwanted pregnancies through methods such as controlling hormones in women. Since the composition for oral use consisting of progestogen and an estrogen component was first marketed in the early 1960s, a composition for parenteral administration containing ethinylestradiol or drospirenone or the like was commercially available. Recently, However, such a parenteral composition has side effects such as arterial and venous thrombosis, and has a problem that it has a large effect on carbohydrate and lipid metabolism. Therefore, there is a need to develop a new composition for parenteral administration which has few side effects.

In addition, Conoidin A, a cell permeable compound, is known to inhibit the function of peroxiredoxins (PRDXs) in human epithelial cells by binding to peroxidative cysteine. The inactivation of PRDXs increased the production of reactive oxygen species (ROS) in the sperm and the sperm production of ROS was significantly correlated with the cytotoxicity due to the increase of lactate dehydrogenase (LDH). Thus, extensive studies have been conducted on Conoidin A, which inhibits the function of PRDXs, but no study has yet been conducted on the direct role of Conoidin A in spermatozoa.

Under these circumstances, research on a new pharmaceutical composition for parenteral administration has been under way (Korean Patent No. 10-1501209) to solve the problem of conventional parenteral compositions.

On the other hand, in order for the sperm to undergo fertilization, acupuncture ability acquisition and acrosome reaction must take place. The intracellular calcium concentration of the sperm increases, causing the activity of cAMP (cyclic AMP), followed by the sequential activation of protein kinase A (PKA), resulting in tyrosine phosphorylation. Through this process, the sperm is able to acquire corrective ability and produce acrosome reaction afterwards.

Thus, when spermatozoa treated with Conoidin A, which is a cell permeable compound and known as an inhibitor of PRDXs, did not show cytotoxicity changes due to ROS production and LDH increase, sperm motility, viability , The ability to obtain fertilization ability, the expression of phospho-PKA-substrates and the expression of tyrosine phosphorylated proteins, and to overcome the problems of conventional pumping compositions, the present invention has been completed.

It is an object of the present invention to provide a pharmaceutical composition for parenteral administration, which comprises a compound containing Conoidin A or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to accomplish the object of the present invention, the present invention provides a pharmaceutical composition for parenteral administration, which contains, as an active ingredient, a Conoidin A compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

In one embodiment of the present invention, the composition may reduce sperm motility, viability, or intracellular ATP concentration in the sperm.

In another embodiment of the present invention, the composition may reduce sperm's ability to acquire fertility or reduce acrosome response.

In another embodiment of the present invention, the composition can reduce the expression of phospho-PKA substrates or tyrosine phosphorylated proteins.

In another embodiment of the present invention, the Conoidin A compound is contained at a concentration of 1 μM to 150 μM.

The present invention provides a method of contraception comprising the step of administering the pharmaceutical composition to a subject.

The present invention provides a contraceptive use of a composition comprising Conoidin A.

The composition according to the present invention contains Conoidin A as an active ingredient and shows the effect of sperm motility, viability, fertilization ability, acrosome reaction, phospho-PKA substrates expression and tyrosine phosphorylated protein expression by the treatment of the composition , Which is expected to be useful as a pharmaceutical composition for parenteral administration.

FIG. 1A shows a double staining pattern of sperm following Conoidin A treatment.
Fig. 1B shows the inhibitory effect of acoid reaction (AR pattern) spermatozoa on conoidin A treatment.
FIG. 1C shows the effect of inhibiting the acquisition of fertilization ability (B pattern) sperm by the Conoidin A treatment.
FIG. 1D shows the degree of acquisition of correction ability according to Conoidin A treatment of non-fertilization ability acquisition (F pattern) sperm.
Fig. 2 shows the effect of spermatozoa survival inhibition by Conoidin A treatment.
Fig. 3 shows the effect of spermatozoa ATP level reduction by Conoidin A treatment.
FIG. 4a shows the change in sperm peroxiredoxins (PRDXs) function by Conoidin A treatment by measuring the intracellular reactive oxygen species (ROS).
FIG. 4B shows the change in sperm peroxiredoxins (PRDXs) function by Conoidin A treatment through intracellular lactate dehydrogenase (LDH) measurement.
FIG. 5A is a Western blot graph showing the inhibitory effect of Conoidin A treatment on the expression of phospho-PKA-substrates.
FIG. 5b shows the results of western blotting on the inhibition of phospho-PKA substrates expression by Conoidin A treatment through the ratio of phospho-PKA substrates to α-tubulin.
FIG. 6A shows Western blotting effect of inhibition of tyrosine phosphorylated protein expression by Conoidin A treatment.
FIG. 6B shows the effect of inhibition of tyrosine phosphorylated protein expression by Conoidin A treatment by Western blotting through the ratio of tyrosine phosphorylated protein to? -Tubulin.
Figure 7a shows the cleavage and blastocyst after in vitro fertilization (IVF) following conoidin A treatment.
FIG. 7B shows the modification rate according to the Conoidin A treatment as a result of the IVF technique.
FIG. 7C shows the initial embryo development rate according to the Conoidin A treatment as a result of the IVF technique.

The inventors of the present invention confirmed that the sperm motility, viability, corrective ability acquisition, acrosome reaction, phospho-PKA substrates and expression of tyrosine phosphorylated proteins were reduced when the Conoidin A compound, which is a cell permeable compound, The present invention has been completed.

Accordingly, the present invention provides a pharmaceutical composition for parenteral administration, which contains, as an active ingredient, a conoidin A (2,3-bis (bromomethyl) quinoxaline 1,4-dioxide) compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof .

[Chemical Formula 1]

The term " contraceptive " used in the present invention means to inhibit the phenomenon that reproductive cells of male and female are combined into a single zygote. In the present invention, the sperm motility, viability, .

The compound of the present invention may be contained at a concentration of 1 [mu] M to 150 [mu] M.

The compound of the present invention can be used in the form of a pharmaceutically acceptable salt. As the salt, acid addition salt formed by a pharmaceutically acceptable free acid is useful.

As used herein, the term " salt " is useful as an acid addition salt formed by a pharmaceutically acceptable free acid. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Sulfonate, xylene sulfonate, phenylacetate, phenyl propionate, phenyl butyrate, citrate, lactate, β - hydroxybutyrate, glycolate, maleate, tart rate, methanesulfonate, propanesulfonate, naphthalene-1 Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the above compound in an excess amount of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile . It may also be prepared by evaporating a solvent or excess acid in this mixture and then drying or by suction filtration of the precipitated salt.

In addition, the base may be used to make a pharmaceutically acceptable metal salt. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

In addition, the compounds of the present invention include not only pharmaceutically acceptable salts, but also all salts, isomers, hydrates and solvates which can be prepared by conventional methods.

In one embodiment of the present invention, a culture containing Conoidin A was prepared and rat sperm were prepared (see Example 1). To determine the effect of conoidin A on the motility and dynamic parameters of sperm, (Computer-assisted sperm analysis (CASA)), it was confirmed that sperm motility was significantly reduced in the group treated with Conoidin A stock solution (see Example 2).

In addition, Hoechst 33258 / chlortetracycline fluorescence evaluation (H33258 / CTC) was performed to confirm the effect of conoidin A on the sperm fertilization ability. As a result, Confirming that the sperm fertilization ability and acrosome response were significantly reduced in the group (see Example 3).

In addition, to confirm the effect of conoidin A on the viability and membrane integrity of spermatozoa, we performed a Hypo-Osmotic Swelling Test (HOST) and found a significant decrease in sperm viability See Example 4).

In addition, the intracellular ATP levels of spermatozoa treated with Conoidin A treatment were evaluated. As a result, it was confirmed that ATP levels of sperm were significantly decreased in all groups treated with Conoidin A (see Example 5).

In addition, after sperm treatment of the Conoidin A stock solution, which is known as an inhibitor of PRDXs, it was confirmed that there was no change in the production of reactive oxygen species (ROS) and cytotoxicity due to lactate dehydrogenase (LDH) Thus, it was confirmed that the composition comprising the Conoidin A compound did not affect the ROS metabolism through inhibition of PRDXs in the sperm (see Example 6).

Western blot analysis of sperm proteins to confirm the effect of Conoidin A on the expression of phospho-PKA substrates and tyrosine phosphorylated proteins revealed that phospho-PKA substrates and tyrosine phosphorylation Protein expression was decreased (see Example 7).

In addition, IVF (in vitro fertilization) method was applied to evaluate sperm fertilization ability by Conoidin A treatment. As a result, both fertilization rate and early embryonic development were significantly decreased by treatment with Conoidin A, Fertilization ability and early embryonic development (see Example 8).

Therefore, in the present invention, the spermatozoa treated with Conoidin A, which is known as an inhibitor of PRDX, showed a significant decrease in ATP concentration and an increase in ROS and a significant decrease in sperm viability after ATP reduction. However, there was no significant change in LDH concentration. Based on the above results, Conoidin A is considered to be non-toxic to sperm.

Conoidin A compounds inhibit PKA-substrate and tyrosine phosphorylation, resulting in decreased ability to acquire fertility and acrosome responses. These conoidin A-induced mobility, fertility, and acrosome responses reduce subsequent fertilization and embryo development Therefore, Conoidin A can be used to develop a contraceptive.

The pharmaceutical composition according to the present invention may contain a pharmaceutically acceptable carrier in addition to the active ingredient. The pharmacologically acceptable carriers are those conventionally used at the time of formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like may be further included.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may vary depending on the condition and the weight of the patient, The mode of administration, the route of administration, and the time, but may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, body weight, the degree of absorption of the active ingredient in the body, the rate of inactivation and excretion, the type of disease, 100 to 500 mg per kg of body weight may be administered daily or every other day, or one to three times a day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

In another aspect of the present invention, the present invention provides a method of contraception comprising the step of administering the pharmaceutical composition to a subject. As used herein, the term "individual" refers to a subject in need of the effect of the composition, and more specifically refers to a mammal such as a human or non-human primate, mouse, dog, cat, horse, do.

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 1. Preparation of experiment

1-1. Cultures and Chemicals

All reagents were purchased from Sigma-Aldrich (St Louis, MO, USA). 0.75 mM MgCl ₂ .H ₂ O, 0.36 mM NaH ₂ PO ₄ .H ₂ O, 5.56 mM D-glucose, 25 mM NaHCO ₃ , 1.78 mM CaCl ₂ .H ₂ O, 24.9 mM The transformed tie-rod culture with the composition of Na-lactate, 0.47 mM Na-pyruvate and 50 μg / ml gentamycin was pre-cultured the day before the experiment and used as a basic culture. In addition, bovine serum albumin (4 mg / ml BSA) was added to the medium to obtain sperm fertilization ability. Conoidin A was diluted with dimethyl sulfoxide and stored at -20 ° C. The Conoidin A stock solution was diluted in the basic culture medium so that the final molar concentrations were 1 μM, 5 μM, and 10 μM.

1-2. Preparation and processing of sperm

Spermatozoa from 8- to 12-week-old male ICR rats were collected to prepare mouse sperm suspension. 2 ml of the basic culture solution was placed in a 35 mm aseptic dish and the cauda epididymides of the rats were placed and the sperm were released by incision. The Conoidin A stock solutions at concentrations of 1 μM, 5 μM, and 10 μM, respectively, were added and incubated at 37 ° C. in 5% CO ₂ (CO ₂ ) for 90 minutes. The spermatozoa concentration was 10 × 10 ⁶ cells / ㎖, and the control group was treated with medium alone without addition of Conoidin A stock solution.

1-3. Statistical analysis

Data statistical analysis of all the embodiments was analyzed using the one-way ANOVA (v. 12.0; IL, USA) of the SPSS program and Tukey's test was used for post-analysis. P <0.05 was considered statistically significant, and data were expressed as mean ± standard error.

Example  2. Conoidin  Evaluation of Sperm Motility Suppression Effect by Treatment of A

Sperm motility and exercise were measured using computer-assisted sperm analysis (SAIS Plus version 10.1, Medical Surfle, Seoul, Korea) for the sperm motility inhibition by Conoidin A treatment The dynamic parameters were analyzed. 10 [mu] l of sample was placed in a Makler chamber (Makler, Haifa, Israel) preheated to 37 &lt; 0 &gt; C. Images were transmitted and digitized by SAIS using 10 X objective in phase contrast mode. At least 250 sperm cell movements per sample were found from an arbitrary field (> 5). The custom settings are as follows. Frame acquisition, 20; Frame rate, 30 Hz; Minimum contrast, 4; Minimum size, 5; Small / large size gates, 0.4-1.5; Low / high saturation gates, 0.4-1.5; Non-magnetic head size, 16; Non-dynamic brightness, 14. Based on this, we analyzed sperm motility and dynamic parameters by Conoidin A treatment using CASA system as follows. Sperm motility (MOT), hyper-activated motile (HYP), and curvilinear velocity (SEM) were measured after sperm cultivation for 90 minutes in Conoidin A stock solution at 0 μM (control), 1 μM, 10 μM and 100 μM VCL), straight-line velocity (VSL), mean path velocity (VAP), mean amplitude of head lateral displacement (ALH), linearity (LIN) and wobble (WOB).

As a result, as shown in Table 1, sperm motility was significantly reduced in the group treated with 10 μM and 100 μM Conoidin A ( p <0.05). At the same time, the kinetic parameters were also significantly reduced in the group treated with 100 μM Conoidin A ( p <0.05).

Example 3 Evaluation of Inhibitory Effect of Conoidin A on Acquisition of Correcting Ability of Spermatozoa

Perez's double staining method using Hoechst 33258 / chlortetracycline fluorescence evaluation (H33258 / CTC) was used to confirm the inhibitory effect of conoidin A treatment on sperm retrieval ability. To 135 의 of mouse sperm samples, 15 H of H33258 (10 H H33258 / ㎖ in Dulbecco's Phosphate-Buffered Saline (DBPS)) was added and incubated at room temperature for 2 minutes. The excess staining solution was layered on 250 μl of 2% (w / v) polyvinyl-pyrrolidone in DBPS. The supernatant was removed and the pellet was resuspended in 100 μl of DPBS and 100 μl of chlortetracycline fluorescence (CTC) solution (750 CTC in 5 μl buffer: 20 mM Tris, 130 mM NaCl, 5 mM cysteine). H33258 and CTC were obtained under epifluorescence illumination using the Nikon Microphot-ly fluorescent microscope using UV BP 340-380 / LP 425 and BP 450-490 / LP 515 stimulation / emission filters, respectively. And observed with an FXA microscope. Two slides per sample, at least 400 sperm per slide, were obtained.

The spermatozoa were classified as shown in Figure 1a and below.

- AR pattern: A live acrosome (spermatozoa) that does not show green fluorescence in the mottled pattern on the sperm head, green fluorescence only in the posterior acrosome, or fluorescence in the sperm head, reactive sperm).

- B pattern: live capacitated sperm, which shows green fluorescence in the acrosome of the sperm and dark in the acrosome rear, where acupuncture acquisition occurs but acrosome reaction does not occur.

- F pattern: shows uniformly bright green fluorescence in the head of the sperm and no stronger fluorescence band in the equatorial segment of the sperm, Live non-capacitated sperm, which have not acquired both fertility and acrosome responses.

- D pattern: Spermatozoa dyed in blue.

As a result, as shown in Figs. 1B and 1C, the spermatozoa (AR pattern) in which the acrosome reaction occurred and the sperm obtained with the correction ability (B pattern) were significantly decreased in all groups treated with Conoidin A ( p <0.05 ). In addition, as shown in Fig. 1D, sperm (F pattern) in which neither acupuncture nor acrosome reaction occurred was significantly increased in all treatment groups ( p <0.05).

Example 4. Evaluation of survival and membrane integrity of spermatozoa treated with Conoidin A

Hypo-Osmotic Swelling Test (HOST) was performed to evaluate the survival and membrane integrity of sperm. 900 μl of a hypo-osmotic solution (distilled water: 0.9% NaCl [1: 1], 150 mOsm / kg) was added to 100 μl of the rat sperm suspension prepared in Example 1-2 and cultured at 37 ° C for 30 minutes. After culturing, 10 占 퐇 was plated on a slide, followed by natural drying. The dried slides were read at 20x magnification using a Microphot-FXA microscope (Nikon, Osaka, Japan). Spermatozoa were classified into live sperm and dead sperm according to WHO 2010 criteria.

As a result, as shown in Fig. 2, the viability of the sperm was significantly decreased at 10 μM and 100 μM ( p <0.05).

Example  5. Conoidin  ATP levels in the sperm after treatment of A

ATP concentration was measured using ATP Bioluminescence Assay Kit HS II (Roche Molecular Biochemicals, Mannheim, Germany) to evaluate intracellular ATP concentration in sperm. 10 ⁵ to 25 μl of sperm diluted to 10 ⁸ cells / μl was placed on a 96-well plate, treated with the same amount of cell lysis reagent, and incubated at room temperature for 5 minutes. 50 μl of luciferase reagent was added to each well and ATP bioluminescence intensity (RLU) was measured using a Microplate Multimode Reader (Promega, Madison, WI, USA).

As a result, as shown in FIG. 3, sperm ATP levels were significantly decreased in all groups treated with Conoidin A ( p <0.05).

Example 6. Functional evaluation of sperm peroxiredoxins (PRDXs) by treatment with Conoidin A

6-1. Analysis of Functional Changes of PRDXs by Measuring Intracellular Reactive Oxygen Species (ROS) by Conoidin A Treatment

For intracellular ROS measurement, quantitative determination was performed using DCFDA (Abcam, Cambridge, UK), which is sensitive to oxidative action. The fluorescence activity of the control and treatment groups was measured using a Microplate fluorescence spectrometer (Gemini Em, Molecular Devices, Sunnyvale, Calif., USA) and quantified using SoftMax Pro 5 (Molecular Devices). Fluorescence of the control and all treatment groups was calculated by the fluorescence ratio (485/535) and the fluorescence was measured by the fluorescence ratio of the control group treated with Conoidin A.

As a result, as shown in FIG. 4A, intracellular ROS was significantly increased in the group treated with Conoidin A at the highest concentration of 100 μM.

6-2. Analysis of the function of PRDXs by measuring intracellular lactate dehydrogenase (LDH) by Conoidin A treatment

Cytotoxicity was measured using the CytoTox 96 Nonradioactive Cytotoxicity Assay Kit (Promega, Fitchburg, WI, USA) for the measurement of intracellular LDH. Sperm were placed in lysis buffer and incubated for 1 hour at 37 ° C in 5% CO ₂ . After incubation, the supernatant (50 μl / well) and the substrate (50 μl / well) were placed in a 96-well plate and incubated in a dark room at room temperature for 30 minutes. 50 μl of stop solution was added to each well, and the activity of LDH was measured by absorbance (OD) using SoftMax Pro 5 software. LDH activity was measured by absorbance (OD) as the fluorescence ratio of the sample treated with Conoidin A in the control group.

As a result, no significant change in intracellular LDH was observed in sperm treated with Conoidin A, as shown in Fig. 4B.

Example 7. Evaluation of inhibitory effect of phospho-PKA substrates and tyrosine phosphorylated protein expression by treatment with Conoidin A

Rat sperm were treated with Conoidin A and subjected to Western blotting to determine the expression level of phospho-PKA substrates and tyrosine phosphorylated proteins. Phospho-PKA substrates were incubated with a rabbit monoclonal anti-phospho-PKA substrate antibody diluted 1: 10,000 in blocking solution (Cell Signaling Technology, MA, USA) Overnight. The HRP-conjugated mouse monoclonal anti-phosphotyrosine antibody (PY20, Abcam) diluted at a ratio of 1: 2,500 to the blocking solution was incubated overnight at 4 ° C Respectively. α -tubulin is at room temperature for 2 hours in blocking solution 1 was detected by incubation with a monoclonal anti-diluted at a rate of 10,000 day α -tubulin mouse antibody (monoclonal anti- α -tubulin mouse antibody, Abcam). The concentration of the band was quantified according to the ratio of α -tubulin (phospho-PKA substrates / α -tubulin or tyrosine phosphorylated protein / α -tubulin) and repeated three times.

As a result, as shown in FIG. 5A, spermatozoa were cultured while increasing concentrations of Conoidin A by a Western blot analysis to the control group, 1 μM, 10 μM and 100 μM, and four kinds of phospho-PKA substrates ~ 55, 35, 30 and 21 kDa). Phospho-PKA substrates were standardized based on α -tubulin expression.

In addition, as shown in FIG. 5B, phospho-PKA substrates of 21 kDa were significantly decreased in the treatment group treated with 10 μM and 100 μM Conoidin A ( p <0.05).

As shown in FIG. 6A, spermatozoa were cultured while increasing concentration of Conoidin A in the control group, 1 μM, 10 μM and 100 μM by Western blot analysis, and two different types of tyrosine phosphorylation-related proteins (~ 70 and 55 kDa). Tyrosine phosphorylation-related proteins were standardized based on the expression of alpha -tubulin.

In addition, as shown in Fig. 6 (b), 70 kDa and 55 kDa tyrosine phosphorylation-related proteins were significantly decreased in all the treatment groups treated with 1 μM, 10 μM and 100 μM of Coloidin A ( p <0.05).

Example 8. Evaluation of sperm fertilization ability by treatment with Conoidin A

In vitro fertilization (IVF) was performed to evaluate the fertility of sperm following conoidin A treatment. A female hybrid B6D2F1 / CrljOri (8-12 weeks old) purchased from Nara Biotech (Seoul, Korea) was used. Five IU peritoneal injection of pregnant mare serum gonadotropin (PMSG) hCG) were intraperitoneally injected at 5 IU. After 15 hours, cumulus-oocyte complexes (COCs) were collected from the bulge. The collected COCs were incubated in mineral oil at 50% of BM containing 10% Fetal bovine serum (FBS) for 1 hour at 37 ° C and 5% CO ₂ . A culture medium containing 0.4% BSA was used to obtain fertility of the sperm. After culturing, COCs were fixed in 1 × 10 ⁶ / μl of sperm suspension prepared in Example 1-2, and then cultured in an environment of 37 ° C. and 5% CO ₂ for 6 hours. The fertilized embryos were cultured in 50 μl of culture medium containing 0.4% BSA at 37 ° C and 5% CO ₂ , and after 18 hours, the number of 2-cell embryos was counted to calculate the fertility. The identified 2-cell embryos were re-cultured in 50 μl of culture medium containing 0.4% BSA at 37 ° C and 5% CO ₂ . Five days after fertilization, blastocyst development was confirmed in each treatment group.

In order to determine whether conoidin A directly affects fertility and early embryo development, the IVF technique was used to treat both fertility and early embryonic development, as shown in Figures 7a, 7b and 7c, ( P < 0.05). &Lt; / RTI &gt;

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics 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 (5)

A concomitant pharmaceutical composition comprising, as an active ingredient, a conoidin A compound represented by the following formula (1), or a pharmaceutically acceptable salt thereof.
[Chemical Formula 1]

2. The composition of claim 1, wherein the compound reduces sperm motility, viability or intracellular ATP concentration in the sperm.
2. The composition of claim 1, wherein the compound reduces the ability of the sperm to obtain fertility or reduces acrosome reaction.
2. The composition of claim 1, wherein the compound reduces the expression of phospho-PKA substrates or the expression of tyrosine phosphorylated proteins.
2. The composition of claim 1, wherein the compound is included at a concentration of 1 [mu] M to 150 [mu] M.

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