JP5923404B2 - TRPV4 activity inhibitor - Google Patents

Description

  The present invention relates to a TRPV4 activity inhibitor that suppresses activation of TRPV4 channels.

  TRPV4 is a member of the transient receptor potential (TRP) superfamily of cation channels and has been shown to be activated by hypotonic pressure, heat (24 ° C <), arachidonic acid metabolites, etc. (non- Patent Documents 1 and 2).

  TRPV4 is expressed in a wide range of tissues such as kidney, lung, bladder, heart, skin, brain and gastrointestinal tract, and is thought to play a wide variety of different physiological roles. It is particularly strongly expressed in the distal tubular epithelial cells of the kidney, and it is suggested that urine osmotic pressure and flow rate are sensed (Non-patent Document 3). It has also been suggested that it has a function of sensing low osmotic pressure in the gastrointestinal tract and enhancing sympathetic nerve activity (Non-patent Document 4).

Recently, TRPV4 channels have been reported to be involved in bladder function (Non-patent Document 5). That is, when there is TRPV4 or TRPV1 present in bladder epithelial cells inside the bladder, when urine accumulates and the bladder epithelial cells extend, calcium flows into the cells via the TRPV channel, thereby ATP is removed from the cells. Released, it has been shown that bladder swelling is transmitted to nerves. Moreover, it has been reported by the examination using TRPV4-deficient mice that the effect of TRPV4 on the urination interval is larger than that of TRPV1 (Non-patent Document 6).
Therefore, suppressing the activation of TRPV4 is considered to have a therapeutic effect on overactive bladder characterized by increased urinary intention and increased urination frequency.

On the other hand, 3,4-dimethoxycinnamic acid, which is abundant in Robusta canephora (Coffea canephora var. Robusta) and the like, has a bitterness-inhibiting action (Patent Document 1), and damages the cell membrane of bacteria to give antibiotics against the bacteria. It has been reported that the action of the agent is enhanced (Non-patent Document 7).
However, it is not known that 3,4-dimethoxycinnamic acid has a TRPV4 activity inhibitory action and is useful for bladder disorders such as overactive bladder.

Special table 2012-501634 gazette

Guler AD. J. Neurosci. 22: 6408-6414 (2002) Vriens J. Proc. Natl. Acad. Sci. USA 106: 1626-1631 (2004) Tian W. Am. J. Physiol. Renal. Physiol. 287: F17-F24 (2004) Gao F. Hypertension. 55: 1438-1443 (2010) Nilius B. Physiol. Rev. 87: 165-217 (2007) Gevaert T. J. Clin. Invest. 117: 3453-3462 (2007) S. Hemaiswarya and M. Doble, Journal of Medical Microbiology (2010), 59, 1469-1476

  The present invention relates to providing a TRPV4 activity inhibitor effective in preventing or improving overactive bladder.

  When the present inventors examined the material which suppresses the activity of TRPV4, they found out that 3,4-dimethoxycinnamic acid has the effect | action which suppresses the activation of TRPV4 effectively.

That is, the present invention relates to a TRPV4 activity inhibitor containing 3,4-dimethoxycinnamic acid or a salt thereof as an active ingredient.
The present invention also relates to an agent for preventing or improving overactive bladder comprising 3,4-dimethoxycinnamic acid or a salt thereof as an active ingredient.

  The TRPV4 activity inhibitor of this invention has the effect | action of suppressing the activation of TRPV4 effectively. Therefore, the TRPV4 activity inhibitor of the present invention is useful for improving the symptoms and diseases caused by the activation of the TRPV4 channel. Specifically, it is possible to prevent or improve overactive bladder (urinary urgency, frequent urination, urge urinary incontinence).

  The 3,4-dimethoxycinnamic acid used in the present invention can be extracted from natural products containing this, particularly plants, and can be industrially produced by chemical synthesis. Moreover, a commercial item can also be used.

The salt of 3,4-dimethoxycinnamic acid may be a pharmaceutically acceptable salt. Examples of the basic substance for salt formation include alkalis such as lithium hydroxide, sodium hydroxide, and potassium hydroxide. Metal hydroxides; alkaline earth metal hydroxides such as magnesium hydroxide and calcium hydroxide; ammonia; basic amino acids such as arginine, lysine, histidine, ornithine; monoethanolamine, diethanolamine, triethanolamine, etc. Examples thereof include organic bases, and alkali metal or alkaline earth metal hydroxides are particularly preferable.
In addition, 3,4-dimethoxycinnamic acid salt may be added to a pharmaceutical or food after preparing the above salt, or 3,4-dimethoxycinnamic acid and a salt-forming component may be added. It may be added separately to a pharmaceutical or food to form a salt in the formulation system.

  3,4-dimethoxycinnamic acid, as shown in the Examples below, is brought into contact with TRPV4 stimulating substances (agonists) and cells transduced with TRPV4 (TRPV4 expressing cells). It has a TRPV4 activity inhibitory action of suppressing the inflow of the amount of cation (Example).

  Here, “inhibition of TRPV4 activity” means that the activity of the receptor TRPV4 is suppressed, specifically, for example, the activity expressed by binding of TRPV4 stimulating substance to TRPV4, for example, the ability to regulate ion flux. It refers to suppressing or inhibiting (for example, the ability to transport cations such as calcium ions and sodium ions from the outside of the cell into the cell) and the ability to regulate membrane potential (for example, the ability to generate current).

  TRPV4 is present in bladder epithelial cells inside the bladder, and when urine accumulates and the bladder epithelial cells extend, calcium is taken into the cells through the TRPV channel, whereby ATP is released from the cell surface, It has been clarified that the bulge of the worm is transmitted to the nerve (Non-Patent Document 5). Therefore, suppressing the activity of TRPV4 is considered to bring about a therapeutic effect on overactive bladder characterized by increased urinary intention and increased urination frequency.

  Therefore, 3,4-dimethoxycinnamic acid or a salt thereof can be used for suppressing TRPV4 activity or for preventing or improving overactive bladder. The use can be in humans or non-human animals, or specimens derived therefrom, and can be therapeutic or non-therapeutic. Here, “non-therapeutic” is a concept that does not include a medical act, that is, a treatment act on the human body by treatment.

  Moreover, 3,4-dimethoxycinnamic acid or a salt thereof can be used as a TRPV4 activity inhibitor, a preventive or ameliorating agent for overactive bladder (hereinafter referred to as a TRPV4 activity inhibitor, etc.), and further, these agents are produced. Can be used to

The TRPV4 activity inhibitor or the like may itself be a drug for humans or animals, a quasi-drug, or a food that exhibits an effect of inhibiting or improving TRPV4 activity, overactive bladder, or the drug, It may be a material or a preparation used by blending with a quasi drug. At this time, the TRPV4 activity inhibitor or the like is allowed in the subject to be blended as described below, alone or in addition to the 3,4-dimethoxycinnamic acid or a salt thereof of the present invention. A carrier or the like may be blended. In addition, the said formulation can be manufactured by a conventional method according to the target object which should be mix | blended.
In addition, as food, the concept is physiological functions such as TRPV4 activity suppression, prevention or improvement of overactive bladder, and food / beverage products, functional food / beverage products, food / beverage products for the sick, specified health Includes food products.

  In the present invention, “overactive bladder” is a symptom syndrome in which urinary urgency is essential, and is usually accompanied by frequent urination and nocturia.

The above pharmaceutical products (including quasi-drugs) include tablets, capsules, granules, powders, syrups, intravenous injections, intramuscular injections, suppositories, inhalants, transdermal absorption agents, eye drops, It may be any of nasal drops, poultices, poultices, ointments, lotions, creams, oral preparations, etc. The administration form may be either oral (internal) or parenteral (external, injection). Good.
Such pharmaceutical preparations of various dosage forms include 3,4-dimethoxycinnamic acid or a salt thereof alone or other pharmaceutically acceptable excipients, binders, extenders, disintegrants, interfaces. It can be prepared by appropriately combining activators, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, diluents and the like.

  Among these dosage forms, the preferred dosage form is oral administration. In this case, the content of 3,4-dimethoxycinnamic acid or a salt thereof in the preparation is usually 0.01% by mass or more, preferably 0. 1% by mass or more, more preferably 0.5% by mass or more, and 20% by mass or less, preferably 10% by mass or less, more preferably 5% by mass or less. For example, 0.01-20 mass%, Preferably it is 0.1-10 mass%, More preferably, 0.5-5 mass% is mentioned.

The form of the food may be solid, semi-solid or liquid, for example, various foods such as breads, cakes, noodles, confectionery, jelly, frozen foods, ice creams, dairy products, beverages, Examples include the same forms (tablets, capsules, syrups, etc.) as the above-mentioned oral administration preparations.
Various forms of food include 3,4-dimethoxycinnamic acid or its salt alone, or other food ingredients, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, It can be prepared by appropriately combining an antioxidant, a humectant, a thickener and the like. The content of 3,4-dimethoxycinnamic acid or a salt thereof in the food (in terms of a dried product of the extract) is usually 0.001% by mass or more, preferably 0.005% by mass or more, more preferably 0.8%. It is 01 mass% or more, and is 10 mass% or less, preferably 5 mass% or less, more preferably 1 mass% or less. For example, 0.001-10 mass%, Preferably it is 0.005-5 mass%, More preferably, 0.01-1 mass% is mentioned.

  The dose or intake of the TRPV4 activity inhibitor or the like of the present invention may vary according to the subject's condition, body weight, sex, age or other factors, but in the case of oral administration or intake, 3,4- Dimethoxycinnamic acid or a salt thereof is usually 0.1 mg or more per day, preferably 1 mg or more, more preferably 5 mg or more, and 1000 mg or less, preferably 300 mg or less, more preferably 5 mg or less. For example, 0.1 mg-1000 mg, Preferably 1 mg-300 mg, More preferably, 5 mg-100 mg is mentioned. Moreover, although the said formulation can be administered or ingested according to arbitrary administration schedules, it is preferable to administer or ingest it once to several times a day.

  The subject of administration or ingestion of the TRPV4 activity inhibitor or the like of the present invention is not particularly limited as long as it is a person who needs it, but in particular, since TRPV4 activity can be suppressed and overactive bladder can be prevented or improved. In addition, administration or ingestion to humans with urgency, frequent urination or urinary leakage is effective.

With respect to the above-described embodiment, the following aspects are disclosed in the present invention.
<1> A TRPV4 activity inhibitor comprising 3,4-dimethoxycinnamic acid or a salt thereof as an active ingredient.
<2> A preventive or ameliorating agent for overactive bladder comprising 3,4-dimethoxycinnamic acid or a salt thereof as an active ingredient.

<3> Use of 3,4-dimethoxycinnamic acid or a salt thereof for producing a TRPV4 activity inhibitor.
<4> Use of 3,4-dimethoxycinnamic acid or a salt thereof for producing an agent for preventing or improving overactive bladder.
<5> 3,4-dimethoxycinnamic acid or a salt thereof for use in inhibiting TRPV4 activity.
<6> 3,4-dimethoxycinnamic acid or a salt thereof for use in preventing or improving overactive bladder.
<7> A method for inhibiting TRPV4 activity, comprising administering or ingesting 3,4-dimethoxycinnamic acid or a salt thereof to a subject in need thereof.
<8> A method for preventing or improving overactive bladder, comprising administering or ingesting 3,4-dimethoxycinnamic acid or a salt thereof to a subject in need thereof.
<9> The method <7> to <8>, which is a non-therapeutic method.

Reference Example 1 Production of human TRPV4 gene expression vector Published using reverse transcribed totalRNA extracted from human duodenum-derived cell line Hutu-80 cells (purchased from American Type Culture Collection) as a template Polymerase chain reaction (PCR) was performed under the following conditions using a primer set composed of oligonucleotides represented by the following base sequences synthesized with reference to the human TRPV4 gene sequence.

<Primer set>
Forward primer; 5'-CACCATGGCGGATTCCAGCGAAGGCCC-3 ': SEQ ID NO: 1
Reverse primer; 5'-CTAGAGCGGGGCGTCATCAGTCC-3 ': SEQ ID NO: 2

<PCR conditions>
a) PCR solution composition
cDNA (Template) 15μl
5x PrimeStar GXL Buffer 10μl
dNTPs mixture (2.5mM) 4μl
PrimeStar GXL DNA Polymerase (Takara Bio) 1μl
Forward Primer (10μM) 1μl
Reverse Primer (10μM) 1μl
Water 18μl
b) Temperature and cycle conditions
95 ℃ 2min
↓
98 ℃ 10sec 33 cycles
70 ℃ 2min

  The obtained PCR product was purified using High Pure PCR Product Purification Kit (Roche). An expression vector was prepared using the purified PCR product and pcDNA3.1 Directional TOPO Expression Kit (Invitrogen).

Example 1 Production of human TRPV4-expressing cells and measurement of intracellular Ca ²⁺ influx activity HEK293 cells (purchased from American Type Culture Collection) were cultured using DMEM / F12 medium (Invitrogen) containing 10% fetal bovine serum. It was. HEK293 cells were seeded at 5 × 10 ⁵ cells / Flash in a T-75 cell culture flask. After 3 days of culture, the human TRPV4 expression vector (8 μg) prepared in Reference Example 1 was transfected into cells using TransIT-293 (Mirus) and cultured for 1 day. The cells were peeled off with Detachin (Genlantis), seeded in DMEM / F12 medium containing 10% fetal bovine serum in 96-well Optical bottom plate (Nunc) at a cell density of 1.5 × 10 ⁴ cells / 90 μl / well, and further cultured for 1 day. .

Intracellular Ca ²⁺ influx activity was measured using Calcium Kit II-fluo 4 (DOJINDO). 90 μl / well of loading buffer containing fluo4-AM was added to the above cells, incubated at 37 ° C. for 1 hour, and then fluorescence intensity (excitation wavelength; 488 nm, fluorescence wavelength) at 37 ° C. using a fluorescence plate reader FDSS3000 (Hamamatsu Photonics). 524 nm) every 2 seconds. 30 seconds after the start of measurement, 20 μl / well of GSK1016790a (Sigma), a TRPV4 agonist, and a solution containing the following specimens (GSK1016790a; 50 nM (final concentration 5 nM), specimen; 10 times the final concentration) were added. The change in fluorescence intensity was measured every 2 seconds up to 300 seconds.

<Sample>

  The specimens used were 3,4-dimethoxycinnamic acid (Tokyo Kasei) and 3-methoxycinnamic acid (Tokyo Kasei) dissolved in dimethyl sulfoxide (DMSO) (Nacalai Tesque).

Further, the inhibition rate of TRPV4 activity of the specimen was calculated by the following formula.
(Equation 1)
Inhibition rate (%) = [(F300C1 / F0C1-F300 / F0) / (F300C1 / F0C1-F300C2 / F0C2)] x 100
F300: Fluorescence intensity of well added with GSK1016790a and specimen 300 seconds after the start of measurement
F300C1; Fluorescence intensity of wells added with GSK1016790a and DMSO 300 seconds after the start of measurement
F300C2; fluorescence intensity of wells added with DMSO only 300 seconds after the start of measurement
F0: GSK1016790a immediately after the start of measurement and fluorescence intensity of wells to which the sample was added
F0C1; Fluorescence intensity of wells with GSK1016790a and DMSO added immediately after the start of measurement
F0C2; Fluorescence intensity of well added with DMSO only immediately after the start of measurement

<Testing method and results>
When the inhibition rate of DMSO as a control was 0%, the inhibition rate of the specimen relative to the control was assayed by Dunnet. The results are shown in Table 1.
From Table 1, the 3,4-dimethoxycinnamic acid of the present invention significantly inhibited the TRPV4 activity at 300 μM compared to the control. In contrast, no significant inhibition was observed in Comparative Compound 1, which is a related compound of 3,4-dimethoxycinnamic acid.

Claims (2)

  A TRPV4 activity inhibitor comprising 3,4-dimethoxycinnamic acid or a salt thereof as an active ingredient.   A prophylactic or ameliorating agent for overactive bladder comprising 3,4-dimethoxycinnamic acid or a salt thereof as an active ingredient.