WO2016190310A1 - Tight junction mitigator, drug absorption auxiliary comprising same, and medicinal composition comprising same - Google Patents

Abstract

The present invention addresses the problem of providing a tight junction mitigator. To solve the problem, provided is a tight junction mitigator that comprises at least one of the compounds represented by formulae (33-1), (33-9), (34-1), (34-2), (34-6) and (36-1).

Description

Tight junction relaxation agent, drug absorption aid containing the relaxation agent, and pharmaceutical composition containing the relaxation agent

The present invention relates to a tight junction relaxation agent, a drug absorption aid containing the relaxation agent, and a pharmaceutical composition containing the relaxation agent.

In order to maintain homeostasis, it is important to contain moisture, sugar, ions, etc. in the body. Epithelial cells are cells that play a role in separating the outside from the inside, and cell adhesion devices that connect the cells have been developed. The tight junction is present on the outermost side among several cell adhesion devices. Tight junctions are formed by the strong interaction of the membrane protein claudin between epithelial cells, preventing the free permeation of water and ions. The function of such a tight junction is particularly important at the blood-brain barrier that prevents skin moisturization and contamination of foreign matter into the brain. Therefore, substances that control tight junction formation are attracting attention as targets for the development of cosmetics and medical products.

As a tight junction formation accelerator, a tight junction formation accelerator containing a sodium salt and a potassium salt of a compound represented by the following formula (1) is known (see Patent Document 1).

［式中、Ｌ、ＭおよびＮは、水素原子、ヒドロキシ、ハロゲン原子、低級アルキル、ヒドロキシ(低級)アルキル、低級アルカノイルオキシまたはオキソであり、ここでＬおよびＭの少なくとも１つは水素以外の基であり、５員環は少なくとも１つの二重結合を有していてもよく；Ａは、－ＣＨ_３、または－ＣＨ_２ＯＨ、－ＣＯＣＨ_２ＯＨ、－ＣＯＯＨまたはそれらの官能性誘導体であり；Ｂは、単結合、－ＣＨ_２－ＣＨ_２－、－ＣＨ＝ＣＨ－、－Ｃ≡Ｃ－、－ＣＨ_２－ＣＨ_２－ＣＨ_２－、－ＣＨ＝ＣＨ－ＣＨ_２－、－ＣＨ_２－ＣＨ＝ＣＨ－、－Ｃ≡Ｃ－ＣＨ_２－または－ＣＨ_２－Ｃ≡Ｃ－であり；Ｚは、

または単結合であり、
　ここでＲ_４およびＲ_５は、水素、ヒドロキシ、ハロゲン、低級アルキル、低級アルコキシまたはヒドロキシ(低級)アルキルであり、Ｒ_４およびＲ_５が同時にヒドロキシおよび低級アルコキシであることはなく；
　Ｒ_１は、非置換、またはハロゲン、低級アルキル、ヒドロキシ、オキソ、アリールまたは複素環基により置換された、二価の飽和または不飽和の低または中級の脂肪族炭化水素残基であり、脂肪族炭化水素中の少なくとも１つの炭素原子は所望により酸素、窒素または硫黄により置換されており；そして
　Ｒａは、非置換、またはハロゲン、オキソ、ヒドロキシ、低級アルキル、低級アルコキシ、低級アルカノイルオキシ、シクロ(低級)アルキル、シクロ(低級)アルキルオキシ、アリール、アリールオキシ、複素環基または複素環オキシ基により置換された、飽和または不飽和の低または中級の脂肪族炭化水素残基；低級アルコキシ；低級アルカノイルオキシ；シクロ(低級)アルキル；シクロ(低級)アルキルオキシ；アリール；アリールオキシ；複素環基；複素環オキシ基である］。 Moreover, the pharmaceutical composition containing the compound represented by following formula (2) is also known (refer patent document 2).

[Wherein L, M and N are a hydrogen atom, hydroxy, halogen atom, lower alkyl, hydroxy (lower) alkyl, lower alkanoyloxy or oxo, wherein at least one of L and M is a group other than hydrogen. And the five-membered ring may have at least one double bond; A is —CH ₃ , or —CH ₂ OH, —COCH ₂ OH, —COOH or functional derivatives thereof; of B, a single _{bond, -CH 2 -CH 2 -, -} CH = CH -, - C≡C -, - CH 2 -CH 2 -CH 2 -, - CH = CH-CH 2 -, - CH 2 - CH═CH—, —C≡C—CH ₂ — or —CH ₂ —C≡C—; Z is

Or a single bond,
Wherein R ₄ and R ₅ are hydrogen, hydroxy, halogen, lower alkyl, lower alkoxy or hydroxy (lower) alkyl, and R ₄ and R ₅ are not simultaneously hydroxy and lower alkoxy;
R ₁ is a divalent saturated or unsaturated low or intermediate aliphatic hydrocarbon residue which is unsubstituted or substituted by halogen, lower alkyl, hydroxy, oxo, aryl or heterocyclic group, and is aliphatic At least one carbon atom in the hydrocarbon is optionally substituted by oxygen, nitrogen or sulfur; and Ra is unsubstituted or halogen, oxo, hydroxy, lower alkyl, lower alkoxy, lower alkanoyloxy, cyclo (lower ) Saturated, unsaturated, low or intermediate aliphatic hydrocarbon residues substituted by alkyl, cyclo (lower) alkyloxy, aryl, aryloxy, heterocyclic or heterocyclic oxy groups; lower alkoxy; lower alkanoyloxy Cyclo (lower) alkyl; cyclo (lower) alkyloxy; aryl; aryloxy; A heterocyclic group; a heterocyclic oxy group].

In addition, treatment of leaky or damaged tight junctions and enhancement of the extracellular matrix requires prevention or restriction of fluid movement, stabilization of tissues or cells, or prevention of contamination. A formulation comprising an effective amount of a self-assembling peptide that achieves them when administered at a site, wherein the self-assembling peptide is a sequence of positively charged residues within the same peptide chain And a sequence of negatively charged residues; one or more of a series of positively charged residues and one or more of a series of negatively charged residues; and a combination selected from the group consisting of The thing is also known (refer patent document 3).

On the other hand, as a preparation for alleviating tight junctions, a substance that interacts with LSR (lipolysis-stimulated receptor), that is, an LSR gene expression inhibitor or a substance that inhibits LSR function, is introduced into a tight junction such as a tricellular junction. Thus, it is known that the introduction of tricellulin is suppressed and the binding between cells is relaxed. In addition, the relaxation of the connection between cells increases the permeability of substances through tight junctions, especially the permeability of substances through tricellular junctions, and is non-invasive, non-invasive transdermally and transmucosally. It is also known that many drugs can be transferred into the body (see Patent Document 4).

Japanese Patent No. 4684906 International Publication No. 2010/137731 International Publication No. 2008/113030 Japanese Patent No. 5429736

As mentioned above, strengthening or mitigating tight junctions is known. In particular, when the tight junction is relaxed, the drug can be transdermally, transmucosally non-invasively and non-invasively transferred into the body, and therefore, application to a drug delivery system is expected.

By the way, the invention described in Patent Document 4 described above suppresses LSR by using an LSR gene expression suppressing substance such as siRNA, antisense oligonucleotide, antibody, inhibitory peptide, dominant negative mutant, or LSR function inhibiting substance. As a result, the induction of tricellulin is inhibited, and the permeability of the substance through the tricellular junction is increased. However, mass production of siRNA, antisense oligonucleotides, antibodies, inhibitory peptides, dominant negative mutants and the like has a problem of cost, and antibodies and inhibitory peptides have a problem of stability. On the other hand, low-molecular compounds are excellent in terms of cost and stability, but compounds that relieve tight junctions are not known.

The present invention has been made in order to solve the above-mentioned conventional problems. When a low molecular weight compound that acts on the cladin recognition site of LNX1, which is a factor controlling tight junction formation, was searched, (1) Although the same recognition site was targeted, surprisingly, a compound that reinforces the formation of tight junctions and a compound that relieves the formation of tight junctions can be obtained. (2) The following formulas (33-1) and (33) -9), (34-1), (34-2), (34-6) and (36-1) were newly found that the compounds can alleviate tight junction, and the present invention was completed.

That is, an object of the present invention is to provide a tight junction relaxation agent, a drug absorption aid containing the relaxation agent, and a pharmaceutical composition containing the relaxation agent.

The present invention relates to a tight junction relaxation agent, a drug absorption aid containing the relaxation agent, and a pharmaceutical composition containing the relaxation agent, as shown below.

（２）上記（１）に記載のタイトジャンクションの緩和剤を含む薬剤吸収補助剤。
（３）上記（１）に記載のタイトジャンクションの緩和剤を含む医薬組成物。 (1) Contains at least one compound represented by the following formulas (33-1), (33-9), (34-1), (34-2), (34-6) and (36-1) Tight junction relaxation agent.

(2) A drug absorption adjuvant containing the tight junction relaxation agent according to (1).
(3) A pharmaceutical composition comprising the tight junction relaxation agent according to (1) above.

The tight junction relaxation agent of the present invention can relax tight junctions. Therefore, using the tight junction relaxation agent of the present invention as a drug absorption aid, or by mixing a tight junction relaxation agent in a pharmaceutical composition containing a drug, it is impossible to transfer into the body, or A drug that has been insufficient can be transferred into the body in a large amount, quickly, noninvasively and noninvasively.

FIG. 1 is a diagram illustrating an outline of a cell adhesion device. FIG. 2 is a diagram for explaining an outline of control of tight junction formation according to the present invention. FIG. 3 shows the structure of LNX1. FIG. 4 is a drawing-substituting photograph, and the equations (33-1), (33-9), (34-1), (34-2), (34-6), (36-1), (13) and It is a photograph of the immunostaining obtained by exposing the compound represented by (14) and control (DMSO) to MDCKII.

The tight junction relaxation agent of the present invention (hereinafter sometimes simply referred to as “relaxation agent”), a drug absorption aid containing the relaxation agent, and a pharmaceutical composition containing the relaxation agent are described in detail below. To do.

FIG. 1 is a diagram for explaining an outline of a cell adhesion apparatus. Epithelial cells form a sheet that adheres to neighboring cells to separate the internal and external environments. For cell-cell adhesion, cell adhesion devices that exist on the cell membrane are important. Playing a role. In the vertebrate epithelium, there are cell adhesion devices called tight junction (TJ), adhesive junction (AJ), desmosome, and gap junction from the apical surface to the basal plane. Each cell adhesion device has a different function, and the tight junction functions so that adjacent cells in the epithelial layer are brought into close contact with each other so that molecules do not leak between the cells. AJ connects actin bundles of adjacent cells, and desmosome connects intermediate filaments of adjacent cells, both of which are involved in the binding of epithelial cells. Gap junctions form an intercellular channel and have the function of allowing inorganic ions and water-soluble small molecules to pass through.

Of these, the tight junction located on the top end surface plays an important role in partitioning the space and maintaining the homeostasis. Tight junctions have a barrier function that selectively controls the size and charge of substances that pass between cells, as well as a fence function that prevents the diffusion of proteins and lipids on the cell membrane and maintains cell polarity. . Therefore, tight junction dysfunction is thought to cause various diseases such as inflammatory bowel disease (IBD), infection, cancer, angioedema, and hematogenous metastasis. Therefore, it can be applied to medical treatment.

FIG. 2 is a diagram for explaining the control outline of tight junction formation. Tight junctions are formed by the interaction of four-transmembrane protein claudin (CLD) and ZO-1, which is a membrane lining protein. On the other hand, Ligand of Numb binding protein X-1 (LNX1), which is a protein that eliminates claudin in vivo, is also expressed. (Fig. 2 (1)) When claudin and LNX1 interact, claudin is intracellular. The tight junction disappears. In this situation, when the interaction of ZO-1 is superior to LNX1, claudin that forms a complex with ZO-1 is more complex than claudin that is taken into the cell, so that the formation of tight junctions is enhanced. (FIG. 2 (2)). Conversely, when the interaction of LNX1 is superior to ZO-1, more claudin is taken into the cell than claudin forming a complex with ZO-1, and thus the tight junction disappears (FIG. 2). (3)). In the present invention, by adding the compounds represented by formulas (33-1), (33-9), (34-1), (34-2), (34-6), (36-1) Since the tight junction relaxes (disappears), it is considered that the compound acts so that the interaction of LNX1 becomes superior to ZO-1. In addition, when using said compound as an active ingredient as a tight junction mitigating agent, a drug absorption aid containing the mitigating agent, and a pharmaceutical composition containing the mitigating agent, one selected from the above compounds is used. Or they may be used in combination.

FIG. 3 shows the structure of LNX1. LNX1 is known as a protein that interacts with the protein Numb involved in cell fate determination and endocytosis. LNX1 has two splicing variants, p70 (molecular weight about 70 kDa) and p80 (molecular weight about 80 kDa), and has a Numb binding region NPAY motif as a common structure and four PDZ domains that function as a protein-protein interaction module. . Further, only LNX1p80 has a RING finger domain on the N-terminal side and has E3 activity. In the present invention, compounds that bind to the PDZ2 domain of LNX1p80 were searched. However, since other domains of LNX1p80 and LNX1p70 are also known to bind to claudin, low molecular weight compounds that bind to other domains You may search. The amino acid sequences of LNX1p80 and LNX1p70 are known and can be obtained from UniProt (O70263) and the like.

The low molecular weight compound contained in the relaxation agent of the present invention can be obtained by creating a candidate list of compounds using an in silico screening method and confirming the usefulness of each candidate compound. Specifically, the candidate list of compounds (1) identifies the region of LNX1 that interacts with claudin, and (2) X-ray crystal structure analysis of a single three-dimensional structure of the region identified in (1) above (3) The interaction surface of the region interacting with the claudin-derived peptide is identified by a solution NMR method, and (4) the candidate low molecular weight compound that can bind to the identified interaction surface , And search using known docking software such as AutoDock, GOLD, and FlexX. In addition, the usefulness of each candidate compound may be confirmed by a known utility confirmation experiment such as NMR, in vitro, or animal experiment.

The relaxation agent of the present invention can be used for drug absorption aids, pharmaceutical compositions, quasi drugs, and the like. As the dosage form, for example, thin film, powder, pill, tablet, injection, suppository, emulsion, capsule, granule, liquid (tincture, fluid extract, alcoholic beverage, suspension, limonade) Etc.). In addition, various components used for normal quasi drugs, pharmaceuticals, etc. within a range not impairing the effects of the present invention, such as oil components, emulsifiers, moisturizers, thickeners, medicinal components, preservatives, powders, A pH adjuster, an ultraviolet absorber, an antioxidant and the like can be appropriately blended.

When using the relaxation agent of the present invention as a drug absorption aid, it may be used in combination with the drug to be absorbed. In the present invention, the “drug absorption aid” means an agent for improving the absorption efficiency of the drug. The “drug” means a substance having or expected to have a desired therapeutic effect or preventive effect when administered. Examples of the drug used together with the drug absorption aid of the present invention include known transdermal drugs, transmucosal drugs such as nasal and enteral, injection administered drugs, eye drops, inhaled drugs and the like. . The drug absorption adjuvant of the present invention may be used simultaneously with a known drug, or the drug absorption adjuvant is first applied to the skin or mucous membrane, or the eye junction or inhalation is used to alleviate tight junctions, and then the drug is added. May be used.

Further, the relaxation agent of the present invention absorbs a drug by adding it in advance to a known transdermal drug, transmucosal drug such as nasal / enteral, injection drug, eye drop drug, inhalation drug, etc. It can also be used as a pharmaceutical composition with enhanced properties.

Hereinafter, the present invention will be specifically described with reference to examples. However, these examples are provided merely for the purpose of explaining the present invention and for reference to specific embodiments thereof. These exemplifications are for explaining specific specific embodiments of the present invention, but are not intended to limit or limit the scope of the invention disclosed in the present application.

 

[Create candidate compound list]
First, a PDB file (PDB ID: 3VQF) of the PDZ2 domain of mouse LNX1p80 was obtained from Protein Data Bank (PDB). Next, on the online compound structure search site ChemCupid (registered trademark, http://www.namiki-s.co.jp/chemcupid/) of Namiki Shoji Co., Ltd., a compound having an anthranilic acid moiety was searched. The resulting compound is collated with LigandBox (http: www.ligandbox.protein.osaka-u.ac.jp/ligandbox/) to obtain the MOL2 file of the compound and build a focused library did. The molecular viewer Hermes attached to the protein-ligand docking software GOLD Suite was launched, GOLD Wizard was selected, the PDB file of the target protein was opened with the Load Protein button, and then the operation was performed according to the manual. The candidate compound list is described below. In addition, Formulas (4) to (32), Formulas (33-1) to (33-11), Formulas (34-1) to (34-14), Formulas (35-1) to (35-2), Compounds represented by the formulas (36-1) to (36-2) can be purchased from Namiki Shoji Co., Ltd.

[Cellular assay of compounds]
Compounds were picked up from candidate compounds and assayed for the effect on tight junction formation by the following procedure.

(1) Cell culture The cells used were canine kidney tubular epithelial cell line MDCKII (Madin-Darby Canine Kidney Cell Strain II; in this study, cells obtained from Professor Mikio Furuse of Kobe University. DS Pharma Biomedical Co., Ltd. Can also be purchased. The cells were cultured in D-MEM medium (Wako) containing 10% FBS (gibco) and penicillin / streptomycin (gibco).

(2) Antibody and Reagent Rabbit anti-CLD2 antibody was purchased from Sigma-Aldrich. Secondary antibodies Cy3-labeled anti-rabbit IgG antibody and FITC-labeled anti-mouse IgG antibody were purchased from Sigma-Aldrich.

(3) Cell immunostaining A cover glass was placed in each well of a 6-well dish, 30 × 10 ⁴ MDCKII cells were seeded therein, and incubated at 37 ° C. in a 5% CO ₂ environment for 24 hours. After that, the formulas (33-1), (33-9), (34-1), (34-2), (34-6), 100 μM in the D-MEM medium and DMSO concentration of 0.1%, The compounds represented by (36-1), (13) and (14) were mixed, added to the cells, and then incubated at 37 ° C. in a 5% CO ₂ environment for 48 hours.
After the incubation, the cells were fixed to the cover glass by washing twice with D-MEM medium not containing FBS or antibiotics, applying cold methanol at −20 ° C., and allowing to stand at −20 ° C. for 20 minutes. Thereafter, after washing with TBS-T (100 mM Tris-HCl, 150 mM NaCl, pH 7.5, 0.1% Tween 20), a blocking solution (5% skim milk in TBS-T) was added and incubated at room temperature for 1 h. Then, the primary antibody diluted solution diluted with the blocking solution was placed on the parafilm, covered with a cover glass so that the cell fixing surface faced, and incubated at 4 ° C. in a humid environment for 23 hours. Thereafter, the cover glass was recovered and washed twice with TBS-T. Then, the secondary antibody diluted solution diluted with the blocking solution was placed on the parafilm, covered with a cover glass so that the cell fixing surface faced, and incubated at room temperature in a light-shielded environment for 1 hour. Thereafter, the cover glass was recovered, washed twice with TBS-T, and then diluted with TBS-T to obtain a DAPI (4 ′, 6-diamidino-2-phenylindole dihydrochloride) solution (DOJINDO, (1: 1000)). And incubated for 5 min at room temperature under light-shielding conditions. Thereafter, after washing with TBS-T three times, a cover glass was sealed with a mounting agent. These samples were observed with a microscope using OLYMPUS microscope IX71. As a control, cells exposed to 0.1% dimethyl sulfoxide (DMSO) were used.

FIG. 4 shows the above formulas (33-1), (33-9), (34-1), (34-2), (34-6), (36-1), (13), and (14). It is the photograph of the immuno-staining obtained by exposing the compound represented and control (DMSO) to MDCKII. From the photograph of FIG. 4, the compounds represented by the formulas (33-1), (33-9), (34-1), (34-2), (34-6) and (36-1) are added to the cells. When exposed, the white part between the cells was clearly thinner than the control, and relaxation (disappearance) of the tight junction was confirmed. On the other hand, when the compounds represented by the formulas (13) and (14) were exposed to cells, white portions between the cells were clearly darkened, and accumulation of claudin to tight junctions was confirmed. .

From the above results, the compounds represented by the formulas (33-1), (33-9), (34-1), (34-2), (34-6) and (36-1) show tight junctions. It became clear to ease.

In addition, the amino acid sequence located on the surface of the human, mouse, and dog LNX1PDZ2 domain claudin interacting was examined. The amino acid sequences examined are all the following parts obtained from UniProt.
<Human> ID: Q8TBB1 amino acid number 377-463 (87 sequences)
<Mouse> ID: O70263 amino acid number 381-467 (87 sequences)
<Canine> ID: E2RBE8 amino acid number 381-467 (87 sequences)
The homology of the amino acid sequences of the human, mouse and dog LNX1PDZ2 domains (above 87 sequence) was as high as 95% for humans and mice, 94% for humans and dogs, and 94% for mice and dogs. In addition, the sequences of amino acids located on the surface where claudin interacts (the 18th glycine to the 23rd arginine of the 87 sequence and the 66th proline to the 74th glutamine) are completely present in humans, mice and dogs. Was in agreement. Therefore, the compounds represented by the formulas (33-1), (33-9), (34-1), (34-2), (34-6) and (36-1) are human tight junctions. It can also be used for relaxation.

The compounds represented by the formulas (33-1), (33-9), (34-1), (34-2), (34-6) and (36-1) of the present invention alleviate tight junctions. can do. Therefore, it can be applied to drug absorption aids, pharmaceutical compositions and the like.

Claims (3)

A tight junction comprising at least one compound represented by the following formulas (33-1), (33-9), (34-1), (34-2), (34-6) and (36-1) Relaxation agent.

A drug absorption adjuvant containing the tight junction relaxation agent according to claim 1. A pharmaceutical composition comprising the tight junction mitigating agent according to claim 1.

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