KR101681229B1 - Use of Vorinostat as a novel TRPV4 inhibitor - Google Patents

Abstract

The present invention relates to the use of vorinostat as an inhibitor of TRPV4 activity and more particularly to a method of inhibiting the activity of transient receptor potential vanilloid 4 (TRPV4) or a pain relief comprising an effective ingredient of Vorinostat (SAHA) ≪ / RTI >
According to the present invention, by using Vorinostat, it is possible to effectively suppress the hypotensive pressure pain in TRPV4, which is very useful for the development of a pain-inhibiting agent or a pain-suppressing health food.

Description

Use of vorinostat as TRPV4 activity inhibitor [0002] Use of Vorinostat as a novel TRPV4 inhibitor [

The present invention relates to the use of vorinostat as an inhibitor of TRPV4 activity and more particularly to a method of inhibiting the activity of transient receptor potential vanilloid 4 (TRPV4) or a pain relief comprising an effective ingredient of Vorinostat (SAHA) ≪ / RTI >

Studies in the field of human physiology and genetics have found TRPV4 (transient receptor potential vanilloid 4) in the human body, and it is predicted that it will perform essential functions in survival in various tissues. In particular, TRPV4 is expressed in sensory neurons that sense external stimuli and belongs to the thermoTRP group (temperature-sensitive transient receptor potential ion channels), which is a receptor group that senses temperature and pain-induced noxious stimuli. In addition, TRPV4 is also expressed in many internal organs, especially in bile secretory tissues and has been shown to play a role in various tissues such as the regulation of vasoconstriction and relaxation regulatory bone formation. Among them, it is known that it responds to the change of mechanical osmotic pressure in the pain part and it is known as a channel to recognize when the osmotic pressure becomes low and the cell volume becomes large. On the basis of this, the role of mediator of pain due to the hypotensive pressure stimulation is attracting attention, and it is time to develop a pain relieving agent based on this.

The characteristics of the TRPV4 receptor for understanding the underlying technology used to develop the specific modulator of TRPV4 are as follows. TRPV4 is an ion channel in which cations migrate into sensory neurons and activate calcium-dependent signaling. As a technique for measuring the activation of TRPV4, there is a technique for measuring intracellular calcium concentration change, which TRPV4 is considered to move cations such as calcium ions. In addition, the TRPV4 activation measurement techniques can be implemented with the support of cell line culture technology, TRPV4 DNA management and transfection technology. That is, by administering to TRPV4-overexpressing cells a specific regulatory agent candidate for various TRPV4 activities, by measuring and comparing the inhibitory effect on 4PDD (4alpha-phorbol 12,13-didecanoate), which is a TRPV4 specific activator, And the strength thereof can be measured.

As a substance that inhibits the activity of TRPV4, ruthenium red is known, but it is a nonspecific calcium channel inhibitor and is not specific to TRPV4. It is also difficult to determine whether the effect on actual pain is for TRPV4. These problems require the development of inhibitors that can specifically inhibit TRPV4, and these inhibitors appear to be effective in reducing pain.

Vorinostat (suberoylanilide hydroxamic acid, SAHA) is known to have the effect of inhibiting tumor suppression or tumor metastasis by stopping the malignant tumor cell cycle (WO2008106524), but it is known that regulating the activity of TRPV4 and suppressing pain are known There is no way.

As a result, the present inventors have found that, in a cell line expressing TRPV4, Vorinostat inhibits TRPV4 activity and effectively suppresses low-pressure pain caused by TRPV4 activation. And the present invention was completed.

It is an object of the present invention to provide a composition for pain control comprising a substance which modulates TRPV4 activity and exhibits a pain-suppressing effect.

It is another object of the present invention to provide a method for screening for a TRPV4 activity inhibitor.

It is still another object of the present invention to provide a pain-suppressing health functional food comprising a substance that exhibits a pain-suppressing effect by modulating TRPV4 activity.

In order to achieve the above object, the present invention provides a composition for inhibiting pain comprising Vorinostat (SAHA) inhibiting the activity of transient receptor potential vanilloid 4 (TRPV4) or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a method for producing a transgenic plant, comprising: (a) preparing a transformant transformed with a gene encoding TRPV4; (b) TRPV4-specific active agent and TRPV4 activity inhibitor candidate substance (experimental group) were treated with TRPV4-specific active agent and Vorinostat or a pharmaceutically acceptable salt thereof as a control group, And TRPV4 activity of the control group, respectively; And (c) comparing the respective measurements of step (b) to select a TRPV4 activity inhibitor that exhibits a lower or similar TRPV4 activity than the control.

The present invention also provides a health food for improving pain comprising Vorinostat or a pharmaceutically acceptable salt thereof as an active ingredient.

According to the present invention, by using Vorinostat, it is possible to effectively suppress the hypotensive pressure pain in TRPV4, which is very useful for the development of a pain-inhibiting agent or a pain-suppressing health food.

FIG. 1 is a graph showing that the 4PDD-induced TRPV4 activity is inhibited by Vorinostat in the HEK293 cell line by calcium imaging method (PDD: 4αPDD, Vorinostat).
FIG. 2 shows that the Vorinostat inhibits TRPV4 in a concentration-dependent manner by Hill-plot through calcium imaging.
FIG. 3 shows that the 4PDD-induced TRPV4 activity in dorsal root ganglia (DRG) sensory neurons is inhibited by Vorinostat (PDD: 4αPDD, Vorinostat).
FIG. 4 shows that the number of flinches decreased in the simultaneous injection of Vorinostat, while the number of flinches increased in the injection of DMAPP, which is a TRPV4 activator and pain inducer, in the acute foot flinch test.
FIG. 5 shows that the number of flinches decreased in the simultaneous infusion group of Vorinostat, while the number of flinches increased in the infusion of PGE2, an inflammatory mediator, in the acute foot flinching behavior test.
FIG. 6 shows the results of Von frey, Randall Sellito, and Hargreaves. As a result, 4A PDD and CFA, an inflammation inducing substance, increased Von frey pain, Randall Sellito pressure intensity and Hargreaves heat intensity In vorinostat simultaneous infusion group, Von frey pain, Randall Sellito pressure intensity, and Hargreaves heat intensity intensity decreased.

In one aspect, the present invention relates to a composition for inhibiting pain comprising Vorinostat (SAHA) inhibiting the activity of transient receptor potential vanilloid 4 (TRPV4) or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, the pain may be characterized as hypotonic pain caused by TRPV4 activation.

Vorinostat used in the present invention is also known as SAHA (eroylanilide hydroxamic acid), and is known as histone deacetylase inhibitors (HDI) which inhibits histone deacetylases (HDAC). .

Vorinostat is marketed under the trade name Zolinza and is a CTCL (cutaneous T cell lymphoma) drug approved by the US FDA.

In the present invention, the use of vorinostat as an inhibitor of pain was confirmed.

In a specific example of the present invention, 4αPDD (4alpha-phorbol 12,13-didecanoate (TRPV4)), known as a TRPV4-specific activator, is expressed in transgenic cells overexpressing TRPV4 and in dorsal root ganglia (DRG) sensory neurons expressing TRPV4 ) By Vorinostat was confirmed by calcium imaging, a kind of a technique for measuring intracellular calcium concentration change (FIGS. 1 and 3). In addition, the Vorinostat of the present invention showed TRPV4-inhibiting effect in a concentration-dependent manner within the range of nanomole (nM) concentration. The EC ₅₀ (effective concentration 50%; 50% effect concentration) for TRPV4 was approximately 28 nM, Was about 2.88 (see Fig. 2). In addition, it was confirmed that Vorinostat suppresses pain sensitivity to hypotonic mechanical stimulation based on TRPV4 activity in animal models (Fig. 5). Accordingly, the Vorinostat or a pharmaceutically acceptable salt thereof may be usefully used as a composition for inhibiting pain.

The hypotensive pressure pain indicates that it is based on TRPV4 activity.

The Vorinostat can be used in the form of a pharmaceutically acceptable salt, and includes all salts, hydrates and solvates produced by conventional methods. Such pharmaceutically acceptable salts are useful as addition salts formed by free acids. Suitable inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid. Examples of the organic acids include citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, There may be mentioned fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid or aspartic acid Etc. may be used. Furthermore, vorinostat according to the present invention may include not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates which can be prepared by conventional methods.

The pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions and syrups, external preparations, suppositories and sterilized injection solutions according to a conventional method.

Solid form preparations for oral administration include powders, granules, tablets, capsules, soft capsules, and the like. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . As the agent for parenteral administration, the external preparation such as powders, granules, tablets, capsules, sterilized aqueous solutions, liquid preparations, non-aqueous solutions, suspensions, emulsions, syrups, suppositories and aerosols, The composition may be formulated in the form of a cream, a gel, a patch, a spray, an ointment, a warning agent, a lotion, a liniment, a pasta or a cataplasma, , But is not limited thereto. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The pharmaceutical composition according to the present invention may further comprise a commonly used carrier, excipient, disintegrant, sweetening agent, lubricant, flavoring agent and diluent. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Examples of the disintegrant include sodium starch glycolate, crospovidone, croscarmellose sodium, alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium, chitosan, guar gum, low substituted hydroxypropylcellulose, magnesium aluminum silicate, Potassium and the like.

In addition, the pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, Corn starch, powdered cellulose, hydroxypropyl cellulose, opaques, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, calcium stearate, calcium stearate, calcium phosphate, calcium hydrogen phosphate, lactose, mannitol, Aluminum stearate, calcium stearate, white sugar, dextrose, sorbitol, talc, and the like can be used. The pharmaceutically acceptable additives according to the present invention are preferably contained in an amount of 0.1 to 90 parts by weight based on the pharmaceutical composition.

The pharmaceutical composition of the present invention may further contain one or more active ingredients which exhibit the same or similar functions in addition to the above components. The composition of the present invention comprises 0.0001 to 10% by weight, preferably 0.001 to 1% by weight, of the above-mentioned Vorinostat or a pharmaceutically acceptable salt thereof, based on the total weight of the composition.

In addition, the pharmaceutical composition of the present invention can be administered orally or parenterally, and parenteral administration can be performed by external or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, .

In another aspect, the present invention relates to a method of inhibiting the activity of TRPV4 comprising the step of treating the isolated sensory neuron cells expressing TRPV4 with the Vorinostat or a pharmaceutically acceptable salt thereof.

In the specific examples of the present invention, it was confirmed that the increased activity by 4αPDD known as TRPV4 activator was inhibited by the Vorinostat in a concentration-dependent manner (FIGS. 1 to 3). Therefore, Vorinostat may be useful for inhibiting the activity of TRPV4.

Also provided is a method of inhibiting hypotensive pressure pain comprising administering to a subject a pharmaceutically effective amount of Vorinostat or a pharmaceutically acceptable salt thereof.

In the specific examples of the present invention, it was confirmed that the increased activity by 4αPDD known as TRPV4 activator was inhibited by the Vorinostat in a concentration-dependent manner (FIGS. 1 to 3). In addition, it has been shown that Vorinostat inhibits pain sensitivity to hypo-osmotic mechanical stimulation based on TRPV4 activity in animal models (Figure 5). Therefore, Vorinostat may be useful for pain suppression.

The hypotensive pressure pain indicates that it is based on TRPV4 activity.

The subject is a vertebrate animal, preferably a mammal, more preferably an experimental animal such as a mouse, a rabbit, a guinea pig, a hamster, a dog or a cat, and most preferably an ape-like animal such as a chimpanzee or a gorilla. In addition, the administration method can be oral or parenteral administration and can be administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, intramuscular injection, intramural injection, intracerebroventricular injection, or chest May be administered by intramuscular injection. In addition, the Vorinostat or a pharmaceutically acceptable salt thereof may be used alone or in combination with methods using surgery, hormone therapy, drug therapy and biological response modifiers for pain suppression.

The pharmaceutically effective amount of the present invention will vary depending on the condition and the weight of the patient, the degree of the disease, the type of the drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, it is preferable to administer the Vorinostat of the present invention or a pharmaceutically acceptable salt thereof at a dose of 400 mg per day. The administration can be administered once a day.

In another aspect, the present invention provides a method for producing a transgenic plant, comprising: (a) preparing a transformant transformed with a gene encoding TRPV4; (b) TRPV4-specific active agent and TRPV4 activity inhibitor candidate substance (experimental group) were treated with TRPV4-specific active agent and Vorinostat or a pharmaceutically acceptable salt thereof as a control group, And TRPV4 activity of the control group, respectively; And (c) comparing the respective measurements of step (b) to select a TRPV4 activity inhibitor that exhibits a TRPV4 activity that is lower or similar than the control group.

In the specific examples of the present invention, it was confirmed that the activity increased by 4αPDD known as TRPV4 activator was inhibited by Vorinostat in a concentration-dependent manner (see FIGS. 1 to 3). Thus, Vorinostat may be useful for screening for the activity inhibitor of TRPV4.

In the present invention, the TRPV4-specific activator of step (b) may be characterized as 4αPDD (4alpha-phorbol 12,13-didecanoate), and the TRPV4 activity of the step (b) Technology or by calcium imaging.

In the present invention, it is preferable to treat the Vorinostat or a pharmaceutically acceptable salt thereof at a concentration of 0.1 to 1000 μM, more preferably at a concentration of 1 to 100 μM, and most preferably 1 μM to 30 μM mu] M.

The host cell may be, but is not limited to, a cell line that can be used for studies of calcium channel activity and for searching for high-efficiency inhibitors such as HEK cell line, CHO cell line, HeLa cell line, RBL-2H3 cell line, HaCat cell line and the like.

The RPV4 activity inhibitor candidate is preferably a metabolite of a natural compound, a synthetic compound, RNA, DNA, a polypeptide, a ligand, an aptamer, an enzyme, a protein, an antibody, an antigen, a bacterium or a fungus, But is not limited thereto.

In another aspect, the present invention relates to a health food for the reduction of pressure pain comprising Vorinostat or a pharmaceutically acceptable salt thereof as an active ingredient.

The Vorinostat of the present invention can be added directly to food or can be used together with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose (for prevention or improvement). Generally, when preparing foods or beverages, Vorinostat of the present invention is added in an amount of 0.2 to 20% by weight, preferably 0.24 to 10% by weight based on the raw material. However, in the case of long-term consumption intended for health or hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health food of the present invention may contain various flavors or natural carbohydrates as an additional ingredient. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is preferably 0.01 to 0.04 part by weight, more preferably 0.02 to 0.03 part by weight per 100 parts by weight of the health food of the present invention.

There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products including drinks, sausages, chocolates, candies, snacks, confectionery, gums and ice cream, drinks, alcoholic beverages and vitamin complexes. .

In addition to the above, the health food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, , A carbonating agent used in carbonated drinks, and the like. In addition, the health food of the present invention may contain flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

< Example  1> TRPV  Production of transformed cell line

The HEK293 cell line (ATCC CRL-11268) was transiently transformed with plasmid DNA containing the polynucleotide of mTRPV4 (SEQ ID NO: 1).

Specifically, the HEK293T cell line was treated with Fugene HD (Roche Diagnostics, Inc., USA) with 0.25 쨉 g of polynucleotide containing 0.55 쨉 g of mTRPV4 per 24 well plate and 0.2 ㎍ / well of pCDNA3 (Invitrogen Corp., USA; USA) according to the manufacturer's protocol. The transformed cells were cultured in a DMEM medium containing 10% FBS, 1% penicillin / streptomycin in a CO ₂ incubator for 24 hours, plated on a poly-L-orthotin coated glass cover slip, Lt; / RTI &gt; for 24 hours.

< Example  2> Spinal cord posterior ganglion ( dorsal root ganglia : DRG ) Sensory neuron preparation

The DRG (dorsal root ganglia) of adult ICR mice (6-week-old male, Hallym University, Korea) was isolated from cold PBS and subdivided into 3 mg / ml collagenase type 2 (Invirogen, USA ) Were incubated at 37 &lt; 0 &gt; C for 45 minutes and then completely digested by a mechanical method. The DRG sensory neurons prepared by the above method were cultured in DMEM / F12 medium containing 10% FBS, 1% penicillin / streptomycin and 10 ng / ml 2.5S NGF (calbiochemn, USA) After coating on the cover slip, it was incubated in a CO ₂ incubator for 48 to 72 hours.

< Example  3> Using calcium imaging Intracellular  Measurement of calcium level change

The mTRPV4 transformed cell line prepared in Example 1 was treated with 100 nM Vorinostat (Sigma-Aldrich, USA) and then treated with 4αPDD (Sigma-Aldrich, USA) for some periods during the drug treatment period. Calcium imaging was performed on the transformed cells treated in this manner.

Specifically, a solution containing 0.02% pluronic acid (Invitrogen, USA) (bath solution, 140 mM NaCl, 5 mM KCl, 2 mM CaCl ₂ , 2 mM MgCl ₂ , 10 mM HEPES, (5 μM; Invitrogen, USA) was injected at 37 ° C for 1 hour to the transformed cells treated in the above manner at a pH of 7.4 with the use of a microplate reader. Later, calcium imaging was performed using a fura2 calcium imaging system olympus fluorescence microscope (Olympus, Japan) and time-lapse images (340 nm / 380 nm) were measured using Meta flow (Morecular device, USA) Nm) was measured every 3 seconds and expressed as the ratio.

As a result, as shown in Fig. 1, it was confirmed that Vorinostat inhibited TRPV4 activation.

< Example  4> TRPV4 of Vorinostat  Concentration-dependent response study

The TRPV4 transformed cell line prepared by the method of Example 1 was treated with 0.1 to 100000 nM of Vorinostat, and then subjected to calcium imaging by the method of Example 3. The measured values were then plotted with density-response curves using Hill-plot.

As a result, the EC ₅₀ (effective concentration 50%; 50% effect concentration) of Vorinostat against TRPV4 was approximately 28 nM and the N value was approximately 2.88. This indicates that Vorinostat exhibits an activity effect on TRPV4 in a range of nanomolar concentrations.

< Example  5> Sensory neurons Vorinostat  Specific response investigation

The sensory neurons isolated as in Example 2 were treated with 100 nM Vorinostat followed by 4 alpha PDD for some periods during the drug treatment period. Calcium imaging was performed on the transformed cells treated in this manner.

As a result, as shown in FIG. 3, Vorinostat effectively inhibited TRPV4 activation in DRG sensory neurons.

< Example 7> Vorinostat pain suppression test through animal pain behavior test

<7-1> Inflammatory Sensitization  cause

In order to observe the inflammatory sensitization by Vorinostat, 100 ng PGE2 (carrageenan; Sigma aldrich, USA), an inflammation mediator, was injected into the soles of the right hind paw 3 hours before the injection of the Vorinostat. Prior to the experiment, the rats were allowed to adapt to the experimental environment for 1 hour, and 25 ml of each of the following experimental groups was injected intradermally into the right hind paw of the rats.

Control: untreated;

Experimental group 1: 100 uM of Vorinostat;

Experimental group 2: DDW + 100 ng PGE2;

Experimental group 3: 100 uM Vorinostat + DDW + 100 ng PGE2.

<7-2> Acute Invasiveness  Behavior (foot Flinch  Behavior) analysis

The number of hindpaw flinching behaviors was measured for 10 minutes.

As a result, as shown in FIG. 5, in the case of DDW + 100 ng PGE2, the number of times that mice did not reach to the floor due to foot elevation was increased, whereas the number of times of administration of Vorinostat was significantly reduced in rats.

<7-3> Von frey , Randall Sellito , Hargreaves Behavioral testing through

In the examples of the present invention, each data was expressed as mean ± standard error from each culture. T-test: Fluo-3 Ca2 + imaging experiment response, licking / flicking time, flinch count, Von frey time, Randall Sellito pressure measurement and Hargreaves heat intensity measurement were analyzed using two groups of equally distributed assumptions. *** P <0.001, ** P <0.01 and * P <0.05.

Von frey, Randall Sellito, and Hargreaves. As shown in FIGS. 6A and 6B, Von frey pain, Randall Sellito pressure intensity, and Hargreaves heat during injection of 4αPDD and CFA, an inflammation inducing substance, In contrast, the simultaneous infusion of Vorinostat resulted in a decrease in Von frey pain, Randall Sellito pressure, and Hargreaves heat intensity.

< Manufacturing example  1> Preparation of pharmaceutical preparations

1. Manufacturing of powder

vorinostat 2 g

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

2. Preparation of tablets

vorinostat 100 mg

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of capsules

vorinostat 100 mg

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

4. Manufacture of rings

vorinostat 1 g

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

5. Manufacture of granules

vorinostat 150 mg

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 캜 to form granules, which were then filled in a capsule.

&Lt; Preparation Example 2 > Production of dairy product

5 to 10 parts by weight of the Vorinostat of the present invention and its analogue were added to milk, and the milk was used to make various dairy products such as butter and ice cream.

< Manufacturing example  3> Manufacturing of beverage

1. Manufacture of health drinks

vorinostat 1000 mg

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 ° C for about 1 hour. The resulting solution was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, It is used in the production of the health beverage composition of the invention. Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

delete delete A TRPV4 activity inhibitor screening method comprising the steps of:
(a) preparing transformants (except for humans) transformed with a gene encoding TRPV4;
(b) TRPV4-specific active agent and a TRPV4 activity inhibitor candidate substance were treated with the TRPV4-specific active agent and a pharmaceutically acceptable salt thereof as a control group, 0.03 to 10 [mu] M, and measuring the TRPV4 activity of the experimental group and the control group, respectively; And
(c) comparing the respective measurements of step (b) above to select a TRPV4 activity inhibitor that exhibits a lower or similar TRPV4 activity than the control.
4. The screening method according to claim 3, wherein the TRPV4-specific activator of step (b) is 4 alpha PDD (4alpha-phorbol 12,13-didecanoate).
4. The screening method according to claim 3, wherein the measurement of TRPV4 activity of step (b) is performed by whole-cell voltage clamp technique or calcium imaging.
delete delete

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