JP5326101B2 - Inhibition of tachykinin NK1 receptor expression by prostanoid DP1 receptor agonist - Google Patents

Description

The present invention relates to a method for suppressing the expression of tachykinin NK1 receptor and a substance that acts on prostanoid DP1 receptor as an active ingredient and a therapeutic agent.

Prostaglandin D2 is one of the prostanoids produced by cyclooxygenase from arachidonic acid, and exhibits sleep-inducing action, bronchial smooth muscle contraction action, mucus secretion promoting action, platelet aggregation inhibitory action and the like. Two types of prostaglandin D2 receptors are known: prostanoid DP1 receptor (D prostanoid receptor 1) and CRTH2 (chemoattactant receptor homologous molecule expressed on Th2 cells). Further, studies using NC mice that spontaneously develop dermatitis have reported that prostaglandin D2 suppresses itching of NC mice via the prostanoid DP1 receptor (Non-patent Document 1).
On the other hand, tachykinin NK1 receptor is a receptor for substance P which is one of tachykinins of peptide neurotransmitters. The expression of tachykinin NK1 receptor is increased in asthmatic airways and is decreased by steroid administration (Non-patent Document 2), and tacrolimus having an immunomodulating action causes the expression of tachykinin NK1 receptor in cultured keratinocytes and vascular endothelial cells. Suppression (Non-patent Document 3) is known.
The Japanese Journal of Pharmacology, 128, 405-410 (2006) J. Mol. Endocrinol., 11, 1-7 (1993) Derma No. 101, 15-24 (2005)

Conventionally, as a treatment for nasal-airway hyperresponsiveness, antiallergic agents, antihistamines, thromboxane synthase inhibitors, thromboxane receptor antagonists, leukotriene B ₄ receptor antagonists, anticholinergics, steroids, theophylline, β-adrenergic receptor stimulants are used.
On the other hand, for the development of therapeutic agents for asthma and allergic rhinitis, exploratory research for new target molecules is being conducted. For example, the involvement of neuropeptides such as substance P in the onset of nasal cavity / respiratory hypersensitivity has been reported, but the receptor antagonist has not yet been put into practical use.

In the process of elucidating the mechanism of the occurrence of itching and developing a new antipruritic drug, the present inventors have used mice to treat itching-like reactions induced by various engulfing substances and those caused by chronic dermatitis. The effect of prostaglandin D2 was examined. As a result, it was found that the expression level of the tachykinin NK1 receptor, which is a receptor for substance P, was decreased in the skin coated with prostaglandin D2 (0.1%), although the expression level of the histamine H1 receptor was not changed.
Tachykinin NK1 receptors are present in epidermal keratinocytes, stimulation of Sabusutan P, believed to be causing itching by releasing nitric oxide is a leukotriene B ₄ and itching of enhancing agent is Okoshikayu substance. Tachykinin NK1 receptor is present not only in the epidermis but also in the respiratory tract, for example, its expression is increased in the asthmatic airway.
The present invention has been made on the basis of the above findings, a method for inhibiting the expression of tachykinin NK1 receptor by a prostanoid DP1 receptor agonist, and nasal cavity / respiratory hypersensitivity comprising a prostanoid DP1 receptor agonist as an active ingredient The purpose of this is to provide a therapeutic drug. Hereinafter, the present invention will be described in detail.

In the present invention, the prostanoid DP1 receptor agonist is not particularly limited as a natural product or a synthetic compound as long as it is an agonist or agonist-like substance that acts on the prostanoid DP1 receptor. Preferred examples include prostaglandin D2 and derivatives of prostaglandin D2.
Examples of the derivative of prostaglandin D2 include prostaglandin derivatives described in JP-A-7-233144, JP-A-2000-273082, JP-A-2001-151749, WO01 / 19790 and WO01 / 12596. It is not limited to compounds.

In order to use prostanoid DP1 receptor agonists as tachykinin NK1 receptor expression inhibitors and nasal / respiratory tract hypersensitivity agents, systemically or locally administered orally or parenterally in conventional dosage forms be able to. These can be orally administered, for example, in the form of tablets, powders, granules, powders, capsules, liquids, emulsions, suspensions and the like that can be produced by conventional methods. It can also be applied topically in the form of an aerosol. As a preparation for intravenous administration, an aqueous or non-aqueous solution, an emulsion, a suspension, a solid preparation dissolved in an injection solvent immediately before use, or the like can be used. In addition, the compound of the present invention can be formulated by forming an inclusion compound with α, β, γ-cyclodextrin, methylated cyclodextrin or the like. Furthermore, the aqueous or non-aqueous solution, emulsion, suspension and the like can be administered by injection or the like. The dose varies depending on age, weight, etc., but is 1 ng to 1 mg / day for an adult, and this may be administered once or divided into several times a day.

By using a prostanoid DP1 receptor agonist and suppressing the expression of tachykinin NK1 receptor, various brain diseases including pain, anxiety, panic, depression, schizophrenia, neuralgia and drug dependence; arthritis, asthma and Inflammatory diseases such as psoriasis; gastrointestinal diseases such as colitis, Crohn's disease and irritable bowel syndrome; allergic diseases such as rhinitis; vascular diseases such as angina and migraine; Parkinson's disease, multiple sclerosis and Alzheimer's disease Treatment or prevention of ocular diseases. More specifically, the prostanoid DP1 receptor agonist is useful as a therapeutic agent for nasal cavity / respiratory hypersensitivity, for example.

The present invention clarifies a new target molecule for the development of therapeutic agents for asthma and allergic rhinitis whose Rakan rate is increasing year by year, and a drug that uses this new target agonist as an active ingredient makes it a conventional therapeutic method. Higher effects can be expected.

Example 1
[Effect of prostaglandin D2 (PGD ₂ ) on the expression level of histamine H1 receptor (H1R) and tachykinin NK1 receptor (NK1R) in the skin of ICR mice]
PGD ₂ was dissolved in 100% ethanol (0.1%, V / W), and 200 μL was applied to the rostral back of ICR mice. As a control, only 100% ethanol was applied. Thirty minutes later, ICR mice treated with solvent and PGD ₂ were exsanguinated using physiological saline, and the rostral dorsal skin was collected. The collected skin was frozen with liquid nitrogen and stored at −80 ° C. until used for experiments. 1 mL of lysis buffer (137 mM NaCl, 20 mM Tris-HCl; pH 7.5, 1% NP-40, 10% glycerol, 1 mM PMSF, 10 μg / mL aprotinin, 1 μg / mL leupeptin) was added to the collected skin and homogenized. Thereafter, the mixture was centrifuged at 1500 rpm for 5 minutes, and the supernatant was collected. Dilute with sterilized water so that the protein in the supernatant is 2.5 μg / μL, and add 0.3125 M Tris-HCl buffer (pH 6.8; 8.3% SDS, 25% glycerol, 25% mercaptoethanol, 0.01% bromophenol blue) to the sample. 1/5 volume was added, and after heating (100 ° C., 5 minutes), it was quenched in an ice bath. The prepared sample was subjected to protein separation by SDS-PAGE (200V). After completion of SDS-PAGE, the gel was transferred to a PVDF (Polyvinylidene difluoride) membrane (200 mA, 70 minutes). After completion of the transfer, blocking treatment was performed for 60 minutes with a TBS-T (0.1% Tween 20, 0.1M NaCl, 0.01M Tris-HCl) solution containing 5% skim milk. After that, the primary antibody [rabbit anti-histamine H1 receptor polyclonal IgG (1: 200; Santa Cruz Biotechnology, goat anti-NK1R polyclonal IgG (1: 1000; Santa Cruz Biotechnology, mouse anti-β-actin IgG (1: 5000; Sigma)] (4 ° C., overnight) After completion of the primary antibody reaction, the PVDF membrane was washed with a TBS-T solution for 10 minutes, and the secondary antibody [for H1R; ECL ^™ anti-rabbit IgG, Horseradish] Peroxidase (HRP) linked whole antibody from donkey (1: 2500; GE Healthcare UK), for NK1R; donkey anti-goat IgG-HRP (1: 20000; Santa Cruz Biotechnology), for β-actin; ECL ^TM anti-mouse IgG , HRP linked whole antibody from sheep (1: 20000; GE Healthcare UK)] was allowed to react at room temperature for 60 minutes, then washed with TBS-T solution (15 minutes × 3 times), and detection reagent (ECL solution, Amersham) Was reacted for 1 minute to induce chemiluminescence with peroxidase, and the luminescence was exposed to X-ray film, so The β-actin band was detected, and the band intensity was quantified by the image analysis software Scion Image.
The results of Western blot analysis of the expression levels of H1R and NK1R are shown in FIG.
In the group treated with PGD ₂ , there was almost no change in the expression level of histamine H1 receptor compared with the solvent application group (FIG. 1A), but the expression level of tachykinin NK1 receptor was significantly decreased (FIG. 1B). ).

The method for suppressing the expression of tachykinin NK1 receptor by a substance that acts on the prostanoid DP1 receptor of the present invention is a method useful for developing therapeutic agents for asthma and allergic rhinitis whose rakan rate is increasing year by year. A substance that acts on the noid DP1 receptor is useful as a therapeutic agent for nasal cavity / respiratory hypersensitivity.

It is a figure of the Western blot analysis regarding histamine H1 receptor (H1R) and tachykinin NK1 receptor (NK1R). PGD ₂ in the figure indicates prostaglandin D2, and VH indicates a solvent (100% ethanol). * Indicates that tachykinin NK1 receptor (NK1R) expression was significantly suppressed by PGD2 application compared to VH application.

Claims (1)

A skin tachykinin NK1 receptor expression inhibitor comprising prostaglandin D2 as an active ingredient.

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