WO2007026516A1

WO2007026516A1 - Novel drug delivery system

Info

Publication number: WO2007026516A1
Application number: PCT/JP2006/315741
Authority: WO
Inventors: Junichi Ikenouchi; Sachiko Tsukita; Shoichiro Tsukita
Original assignee: Kyoto University; Sachiko Tsukita
Priority date: 2005-08-09
Filing date: 2006-08-09
Publication date: 2007-03-08
Also published as: JP2008271784A

Abstract

Disclosed is a drug delivery system or method or pharmaceutical composition which can suppress the barrier mechanism against the transfer of a substance to transfer a medicinal substance at any part of the body in a non-invasive and bloodless manner. A factor capable of suppressing the expression of a tricellulin gene or the function of a tricellulin protein; a drug delivery system or method using the factor; a pharmaceutical composition comprising the factor; and a non-human animal having a knockout tricellulin gene.

Description

Specification

New drug delivery system

Technical field

[0001] The present invention relates to a factor that suppresses the expression of a tricellulin gene or the action of a tricellulin protein.

, Drug delivery system and method using the same, pharmaceutical composition containing the factor, etc.

Background art

[0002] In various parts of the body such as the brain, retina, testis, and placenta, the transfer of substances from blood vessels is remarkably restricted. In particular, the low drug transferability to the blood-powered brain (the cerebrovascular barrier) has severely limited drug options for central nervous system diseases, and thus the means of treatment. Such poor drug transfer is due to a special barrier mechanism called tight junction. Tight junction is a mechanism that prevents the movement of substances between cells, and is composed of special adhesion molecules. As such adhesion molecules, occuludin and claudin have been studied. The present inventors conducted research on claudin among proteins constituting tight junctions (see Non-Patent Document 1), and in claudin-1 knockout mice, the permeability of the cerebrovascular barrier was investigated. It was clear that the property was increased only for compounds with a molecular weight of 800 or less (see Non-Patent Document 2).

[0003] However, the barrier mechanism against the migration of such substances functions only in blood vessels, and thus functions strictly throughout the body such as the digestive tract and skin before reaching the blood. Yes. For this reason, there has long been a demand for a method for transdermally, transmucosally noninvasively and noninvasively transferring drugs into the body. As in the case of cerebrovascular, there are 24 types of genes in claudin that forms a tight junction, a force that can be considered as one possibility to inhibit the function of claudin on the skin and mucous membranes. More generally, epithelial cells in many tissues simultaneously form multiple types of claudin force S-tight junctions. Many of them have functional redundancy, so in order to temporarily break tight junctions in each tissue and improve drug migration, Forces that need to inhibit multiple claudin functions at the same time. Such methods are currently difficult.

Non-Patent Document 1: Tsukita et al., Nature Review Molecular Cell Biology, Vol. 2, No. 4, 20 April 2001, pages 285-293

Non-Patent Document 2: Nitta et al., The Journal of Cell Biology, No. 161, No. 3, May 2003, pages 653–660

Disclosure of the invention

Problems to be solved by the invention

[0004] The problem to be solved by the present invention is to provide a drug delivery system capable of suppressing a barrier mechanism against substance transferability and transferring a drug into the body noninvasively and noninvasively throughout the body. It was to develop methods as well as pharmaceutical compositions.

Means for solving the problem

[0005] In view of the above circumstances, the present inventors have conducted extensive research and have identified a new molecule called tricellulin, which is located in a tricellular junction where three cells face each other. The inventors have found that by using a factor that suppresses the function of the tricellulin gene or tricellulin protein, the substance permeability of the living body can be improved and the drug can be efficiently taken into the body, and the present invention has been completed.

That is, the present invention provides:

(1) a factor that suppresses the expression of the tricellulin gene or the function of the tricellulin protein;

(2) The factor according to (1), selected from the group consisting of siRNA, antisense oligonucleotide, antibody, inhibitory peptide, and dominant negative mutant;

(3) The factor according to (1), which is a siRNA having the nucleotide sequence shown in SEQ ID NO: 7 or 8, or a homologous RNA having an equivalent function;

(4) The factor according to (1), which is an antisense oligonucleotide against trisenorelin DNA;

(5) The factor according to (1), which is an antibody against tricellulin protein;

(6) A drug delivery system comprising the factor according to any one of (1) to (5) (7) A pharmaceutical composition for enhancing the substance permeability of a living body, comprising the factor according to any one of (1) to (5);

(8) A pharmaceutical composition comprising the factor and drug according to any one of (1) to (5);

(9) The pharmaceutical composition according to (7) or (8), which is a transdermal dosage form or a transmucosal dosage form;

(10) The pharmaceutical composition according to (7) or (8), which is an eye drop;

(11) A method for increasing the substance permeability of a living body, comprising using the factor according to any one of (1) to (5);

(12) A method for delivering a drug, wherein the factor according to any one of (1) to (5) is used;

(13) The method according to (12), wherein the delivery is transdermal or transmucosal;

(14) The method according to (12), wherein the delivery is by eye drops;

(15) A method for treating and / or preventing a disease, characterized by administering the agent of the present invention before administration of a drug or simultaneously with administration of the drug;

(16) Use of the factors described in (1) to (5), or any one of them in the manufacture of a drug delivery system or pharmaceutical composition for increasing the substance permeability of a living body;

(17) Use according to (16), wherein the drug delivery system or pharmaceutical composition further comprises a drug;

(18) The use according to (16) or (17), wherein the drug delivery system or pharmaceutical composition is applied or administered transdermally or transmucosally;

(19) Use according to (16) or (17), wherein the drug delivery system or pharmaceutical composition is applied or administered by eye drops;

(20) The factor according to any one of (1) to (5), the delivery system according to (6), or any one of (7) to (: 10), wherein the tricellulin gene or tricellulin protein is derived from human. The pharmaceutical composition according to (11) to (: 15), or the use according to any of (16) to (: 19); and

(21) Non-human animals knocked out of the tricellulin gene

Is to provide.

The invention's effect [0007] According to the present invention, a drug can be efficiently taken into the body, and in particular, a large number of drugs can be transferred into the body transdermally, transmucosally and noninvasively. it can.

Brief Description of Drawings

FIG. 1 is a schematic diagram showing the structure of a tricellular junction.

[FIG. 2] FIG. 2 shows an image stained with a fluorescently labeled antibody of tricellulin (left) and an image stained with a fluorescently labeled antibody of otaldine (right).

[FIG. 3] FIG. 3 shows the results of examining the suppression of tricellulin protein production by siRNA by Western blotting. WT is a wild type Eph4 cell, KD-1 is an Eph4 cell into which an HI promoter vector is introduced so as to express the siRNA shown in SEQ ID NO: 7 (see Example 2), and KD-2 is an siRNA shown in SEQ ID NO: 8. This is an Eph4 cell (see Example 2) into which a HI promoter vector has been introduced so as to express.

[FIG. 4] FIG. 4 shows the results of examining suppression of tricellulin protein production by siRNA by the fluorescent antibody method. WT, KD-1 and KD-2 have the same meaning as in Figure 2. siRNA-introduced cells. The top row shows the results for tricellulin, and the bottom row shows the results for E-force doherin. The scale bar in the lower right panel indicates 10 μm.

FIG. 5 shows the histological observation results obtained by the fluorescent antibody method in wild-type cells and cells in which expression of tricellulin has been lost by siRNA (KD-1 and KD-2). WT, KD-1 and KD_2 have the same meaning as in Figure 2. The top row shows the results for tricellulin, and the bottom row shows the results for E-cadherin. The scale bar in the upper right panel indicates 10 μm, and the scale bar in the lower right panel indicates 3 x m.

[FIG. 6] FIG. 6 shows the results of examining tricellulin expression inhibitory effect of siRNA by quantifying the permeability in the cell gap using the TER method. WT, KD-1 and KD-2 have the same meaning as in Figure 2.

[FIG. 7] FIG. 7 shows the results of examining the effect of siRNA on the suppression of tricellulin expression by examining changes in permeability to dextran having various molecular weights labeled with FITC.

WT, KD-1 and KD-2 have the same meaning as in Figure 2.

BEST MODE FOR CARRYING OUT THE INVENTION

[0009] The target molecule of the present invention is a tricellulin gene or tricellulin protein. Therefore, In one embodiment, the present invention relates to a factor that suppresses the function of a tricellulin gene or tricellulin protein. In other words, by suppressing the function of the tricellulin gene or tricellulin protein, the permeability of substances passing through the tricellular junction and tight junction can be increased, and the drug can be efficiently taken into the body. Many drugs can be transferred into the body non-mucosally and noninvasively.

[0010] As described above, the tight junction is formed by a pair of adhesion molecules (cludins) between adjacent cells. However, the structure that seals such cells has three cells. Are weakened in the tricellular junction structure facing each other. Figure 1 shows a schematic diagram of the tricellular junction structure. We have identified for the first time the adhesion molecule tricellulin localized at the tricellular junction. The Tricellular Large Cyan has only been described morphologically so far, and its molecular construction was completely unknown. The present inventors have identified the adhesion protein tricellulin localized in the tricellular junction, and revealed the nucleotide sequence of the DNA and the amino acid sequence encoded thereby.

[0011] Tricellular junction structures and tricellulin are widely distributed in mammals.

As a typical example, the nucleotide sequence (Genebank Accession number AB219936) of DNA encoding human 'Tricellulin is shown in SEQ ID NO: 1, and the amino acid sequence of human' Tricellulin is shown in SEQ ID NO: 2. The nucleotide sequence of the DNA encoding another form of human 'tricellulin (Genebane Accession number AB219937) is shown in SEQ ID NO: 3, and the amino acid sequence of another form of human' tricerulin is shown in SEQ ID NO: 4. Further, the nucleotide sequence (Genebank Accession number AB219935) of the DNA encoding mouse 'Tricellulin is shown in SEQ ID NO: 5, and the amino acid sequence of mouse' Tricellulin is shown in SEQ ID NO: 6. The target molecules of the present invention are a tricellulin gene and a tricellulin protein, and preferred target molecules are a human-derived tricellulin gene and a tricellulin protein.

[0012] In the present specification, the term "tricellulin gene" is included, and in this case, "tricellulin DNA" is included. Therefore, “expression of tricellulin gene” includes “expression of tricellulin DNA”. In addition, “gene” or “oligonucleotide” refers to DNA. And both RNA. In the present specification, the nucleotide sequence is indicated with 5 'force and 3' direction to the left and right.

In the present specification, unless otherwise specified, “suppressing the expression” of a gene includes reducing the expression of the gene from a normal level and completely eliminating it. Specifically, “inhibiting expression” of the tricellulin gene includes inactivating or eliminating the synthesis of the tricellulin gene and shifting the control / regulatory mechanism of the tricellulin gene in the direction of expression suppression. To do. For example, the tricellulin gene of a cell may be knocked down, knocked out, or deleted by known methods. For example, in the case where there are a plurality of genes encoding the same type of function, for example, isozymes, the expression of one or more or all of these genes may be suppressed.

[0014] Examples of factors that suppress the expression of the tricellulin gene include, but are not limited to, factors and drugs such as siRNA, antisense oligonucleotides, antibodies, inhibitory peptides, and dominant negative mutants. By treating cells at the desired site for drug delivery with these factors, for example, by contacting the cells with these factors or introducing these factors into the cells, trycellulin in the cells at the desired site. Gene expression can be suppressed. Treat cells with genes that encode these factors. Methods for treating cells with these factors and genes, for example, methods for bringing these factors or genes encoding these factors into contact with cells or introducing them into cells are known in the art. For example, a method using a vector (for example, a method using an adenovirus vector, etc.), cell fusion, electoporation, gene gun, lipofection, a direct introduction method, etc. can be used to introduce a gene into a cell. . Those skilled in the art can appropriately select and use these methods according to the type of cells at the desired site, the type of factors used, and the like. In addition, expression of the trisenorelin gene can also be suppressed by methods other than those described above known in the art.

[0015] Examples of preferable factors that suppress the expression of the tricellulin gene used in the present invention include tricellulin siRNA, antisense oligonucleotides to the tricellulin gene, and the like. Anti-sense oligo for tricellulin siRNA and tricellulin gene Nucleotides have the advantage of high knock-down specificity for the tricellulin gene, high knock-out efficiency, and low risk of side effects. Methods for producing antisense oligonucleotides and siRNA, preferred sequences and sequences are also known to those skilled in the art, and can be easily obtained. Methods for introducing antisense oligonucleotides and siRNA into cells (for example, the transfection method when introducing antisense DNA) have also been established, which has the advantage of being easy to introduce and use. As a method for introducing the siRNA of the present invention into the body, a known method such as a direct introduction method, a method using an adenovirus vector, a transdermal lipofusion, or an electoral position can be used. A preferred method for introducing the siRNA of the present invention into the body is a direct introduction method. By adopting the direct introduction method, it is possible to simplify the configuration and use of the drug delivery system and the pharmaceutical composition described below. Conditions and factors for direct introduction of siRNA can be appropriately selected by those skilled in the art.

[0016] The siRNA homologue of the siRNA having the nucleotide sequence shown in SEQ ID NO: 7 or 8 means all the tricellulin gene expression inhibitory effects equivalent to the siRNA having the nucleotide sequence shown in SEQ ID NO: 7 or 8. Includes RNA. Preferably, siRNA homologue RNA of the present invention has one to several nucleotides deleted, substituted and / or added (set of them) in the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8. (Which may be combined). The term “several” means usually 5, preferably 3, more preferably 2, and most preferably 1.

In the present specification, unless otherwise specified, “suppressing the function” of a protein includes reducing the function of the protein below a normal level and completely eliminating the function. When there are a plurality of proteins of the same kind (for example, isozymes), one or more of them may be suppressed, or all functions may be suppressed.

[0018] Examples of factors that suppress the function of tricellulin include, but are not limited to, a tricellulin antibody, a tricellulin-inhibiting peptide, and a dominant-negative mutant of tricellulin. An example of a preferable factor that suppresses the function of tricellulin used in the present invention is an antibody against trisenorelin protein. Various antibodies are known, such as polyclonal antibodies. It may be an internal antibody or a monoclonal antibody. Chimeric antibodies and humanized antibodies may be prepared and used. Methods for producing antibodies are known to those skilled in the art, and can be appropriately selected depending on the type and use of the antibody to be prepared. By treating cells at the desired site to be delivered with these factors, for example, by contacting the cells with these factors or introducing these factors into the cells, trycellulin in the cells at the desired site. Can be suppressed. Alternatively, the cells may be treated with a gene encoding a factor that suppresses the function of these tricellulins. Methods for treating cells with these factors and genes, for example, methods for contacting or introducing these factors or genes encoding these factors into cells are known in the art, and those skilled in the art can These methods can be appropriately selected and used according to the type, the type of factor, and the like. For the introduction of the gene into the cell, for example, the means and method as described above can be used. In another embodiment, the present invention is a factor that suppresses the expression of the tricellulin gene or the function of the tricellulin protein (hereinafter, “ The present invention relates to a drug delivery system characterized by using the “factor of the present invention”. By applying the factor of the present invention to a living body and increasing the permeability of the applied portion, the drug can be transferred into the body in a large amount, rapidly, noninvasively and noninvasively. When using the drug delivery system of the present invention, the agent of the present invention may be applied to the drug application site in advance, and the permeability of the drug application site may be increased to administer the drug with force. In addition, when using the drug delivery system of the present invention, the agent of the present invention may be administered to the application site together with the drug. In the drug delivery system of the present invention, other components and means other than those described above may be used. For example, a drug delivery device (for example, an application brush, a patch, an eye drop container, etc.) may be used. Alternatively, a technique that increases penetration into the skin by mixing a drug to be permeated into a fat-soluble cream or the like by using a tight seal may be applied.

By using the drug delivery system of the present invention, it is possible to suppress the expression of the tricellulin gene or the function of the tricellulin protein, increase the permeability of the drug to the cell layer, and deliver a large amount of drug to the body quickly and forcefully. Can do. In other words, drugs that could not be delivered to the body by administration to these sites or could not be delivered to the body. By applying the drug delivery system of the invention to these sites, the amount of force can be transferred into the body in a noninvasive and noninvasive manner. This also reduces the burden on the target.

There are no particular limitations on the drug delivered by the drug delivery system of the present invention. The drug to be delivered may be one type or multiple types. The application site of the drug delivery system of the present invention is not particularly limited, and may be applied to 2 or more sites. Preferred delivery forms include transdermal, transmucosal, or ophthalmic. Preferable application sites include skin, mucous membrane, eyeball, especially cornea. The amount of the factor of the present invention is not particularly limited and can be set according to the purpose. For example, the amount and number of uses of the factor of the present invention can be changed according to the required degree of permeability, the drug to be delivered, the type of disease, the state of the subject, the application site, and the like. As used herein, “drug” refers to a substance that has or is expected to have a desired therapeutic or prophylactic effect when administered.

In another aspect, the present invention relates to a pharmaceutical composition for increasing the substance permeability of a living body, comprising the factor of the present invention. The pharmaceutical composition of the present invention suppresses the expression of the tricellulin gene or the function of the tricellulin protein by the action of the factor of the present invention, thereby increasing the permeability of the substance to the cell layer and thus increasing the substance permeability of the living body. Can be increased. Therefore, by administering the pharmaceutical composition of the present invention, it is possible to promote the transfer of a drug administered at the same time or later into the body. Therefore, a large amount of drugs can be quickly transferred into the body. For example, the pharmaceutical composition of the present invention may be administered to a drug administration site to increase the substance permeability at that site, and then the drug may be administered to the site. Further, for example, the pharmaceutical composition of the present invention and the drug may be simultaneously administered to a desired site in the living body.

In this respect, the present invention relates to a pharmaceutical composition comprising the agent according to any one of claims:! To 5 and a drug.

By using the pharmaceutical composition of the present invention as described above, a large amount and a high speed of a drug that could not be transferred into the body by the conventional pharmaceutical composition or could only be insufficient. The force can be transferred into the body noninvasively and noninvasively. This also reduces the burden on the subject. The amount of the factor of the present invention, drug, other components, application site, etc. in the pharmaceutical composition of the present invention are as described in the drug delivery system of the present invention. The dosage form of the pharmaceutical composition of the present invention is not particularly limited, but is preferably a transdermal dosage form (such as lotion, cream, ointment, clothing agent) or transmucosal dosage form. Or eye drops. The manufacturing method of these dosage forms is well-known, and those skilled in the art can select suitably.

[0021] In another aspect, the present invention relates to a method for enhancing substance permeability of a living body, characterized by using the factor of the present invention. In yet another embodiment, the present invention relates to a method for delivering a drug, characterized in that the agent of the present invention is used. In the drug delivery method of the present invention, the factor of the present invention may be applied before the drug administration, or the factor of the present invention may be applied simultaneously with the drug administration. In the method of the present invention, the drug delivery is preferably a force transcutaneous or transmucosal that can be performed by any means or method. Delivery by eye drops is also preferred. The drug delivery is as described above.

In yet another embodiment, the present invention also relates to a method for treating and / or preventing a disease, characterized in that the agent of the present invention is administered before or simultaneously with the administration of the drug. According to the method of the present invention, a large amount of a drug that could not be transferred into the body by the conventional method or was insufficiently transferred to the body in a non-invasive and non-invasive manner. Therefore, excellent treatment and / or prevention effects can be obtained, and the burden on the subject can be reduced.

[0022] In yet another embodiment, the present invention relates to the use of the agent of the present invention for producing the drug delivery system or pharmaceutical composition of the present invention described above. The drug delivery system or pharmaceutical composition produced using the factor of the present invention is a drug delivery system or pharmaceutical composition that cannot be transferred to the body by the conventional drug delivery system or pharmaceutical composition. It can be transferred to the body in a large amount, promptly, and noninvasively and noninvasively, and the burden on the subject can be reduced. The above description applies to the delivery system or pharmaceutical composition produced. The delivery system or pharmaceutical composition produced by the above use may not contain a drug. In such cases, the delivery system or pharmaceutical composition is applied or administered prior to administration of the drug, or at the same time as administration of the drug. In addition, the manufactured delivery system or pharmaceutical composition may contain a drug. Application or administration of the delivery system or pharmaceutical composition to the living body may be by any means, but those applied or administered transdermally or transmucosally are preferred. Also preferred are those that are applied or administered by eye drops.

[0023] In yet another embodiment, the present invention relates to a non-human animal in which the tricellulin gene is knocked out. Methods for producing knockout animals are known to those skilled in the art and can be produced from any animal. Preferred animal species are mammals, such as monkeys, dogs, cats, horses, horses, hidges, mice, rats and the like. The tricellulin gene knockout animal of the present invention can be used for drug research and development as a model animal for drug delivery since substance transfer through the skin and mucous membrane is enhanced. In addition, conditional knockout animals (tricellin gene knocked out only in a specific organ) are prepared by a known method, and those animals are determined according to the nature of the barrier mechanism of each organ and its breakdown. Use as a model animal for pathological analysis.

[0024] The present invention will be described in more detail and specifically with reference to the following examples. However, the examples should not be construed as limiting the present invention.

Example 1

[0025] Production of antibodies against tricellulin and confirmation of the presence of tricellulin in tricellular junctions

The N-terminal and C-terminal cytoplasmic regions of mouse tricellulin were expressed in E. coli in the form of GST fusion proteins and purified by conventional methods. Purified protein mixed well with Mycobacterium tuberculosis adjuvant was injected subcutaneously into rabbits and rats. Polyclonal antibodies were made from rabbits and monoclonal antibodies were made from rats. Secondary antibodies include commercially available donkey anti-rabbit IgG with FITC and Cy3 fluorescent dyes, or donkey anti-rat IgG with FITC and Cy3 fluorescent dyes. Using. When cultured epithelial cells (Eph4) derived from the mammary gland of mice were stained with the fluorescent antibody method, as shown in Fig. 2 (left), a signal for tricellulin was observed only in the tricellular large junction formed by three cells. . Figure 2 (right) is an antibody staining image of otaldine that is normally found between cells.

Example 2

[0026] Effect of siRNA on tricellulin

Nucleotides 1590-1608 of the protein coding region of mouse 'Tricellulin cDNA (GAAC

AAACTCTCTCACATA) was incorporated into the HI promoter vector. Similarly, nucleotides 1542 to 1560 (GAATGA of the protein coding region of mouse 'Tricellulin cDNA

CCCTTCGTTTCTG) was incorporated into the HI promoter vector. The neomycin resistance gene was integrated into one HI promoter vector. In preparing these vectors, the description of T. R. Brummelkamp, R. Bernards, R. Agami, Science 296, 550 (2002) was referred to.

[0027] Each of the prepared vectors was introduced into cultured mammary gland-derived epithelial cells (Eph4) using lipofectamin plus from Invitrogen and cultured in DMEM medium for 48 hours. Thereafter, the cells were cultured for about 2 weeks in a DMEM medium containing neomycin (400 ug / ml) to obtain cells having acquired neomycin resistance (that is, having stably acquired the HI promoter vector). By the above operation, cells that stably express 19mer siRNA: GAACAAACUCUCUCACAUA (SEQ ID NO: 7) (KD _ 1) and cells that stably express 19mer siRNA: GAAUGA CCCUUCGUUUCUG (SEQ ID NO: 8) (KD -2) got.

[0028] The cells thus obtained were subjected to histological observation using Western blotting of the cell extract, staining by the fluorescent antibody method, and staining by the fluorescent antibody method. It was confirmed by Western blotting that RNAi against trisenorelin lost about 95% of the tricellulin protein (Fig. 3). Also by the fluorescent antibody method, it was confirmed that RNAi for tricellulin has lost the protein of tricellulin. That is, compared to wild-type (WT) cells, the fluorescence in the tricellular junction disappeared in the cells (KD-1 and KD-2) in which the expression of tricellulin disappeared (FIG. 4, upper panel). On the other hand, E—cadherin (adherens junction adhesion molecule) does not change. (Figure 4 bottom). As shown in Fig. 5, in cells where the expression of tricellulin was lost in cultured epithelial cells (Fig. 5, upper panel, middle and right panels), the ability to construct tight junctions (1) Compared to the normal formation of the crossing point (tricellular center) in the wild type (WT) (Fig. 5, upper panel, left panel), cells in which the expression of tricellulin disappeared (KD_1 and KD-2) was confirmed to be open, and (2) as shown by the arrowheads, KD-1 and KD-2 were found to be vulnerable to tight junction formation between the two cells. I was strong. These results were judged by comparison with staining for otaldin (a membrane protein localized at the tit junction) (bottom of Fig. 5).

Example 3

[0029] Quantitative measurement of permeability change in cell gap due to suppression of tricellulin expression by siRNA

Permeability in the cell gap was quantified using the TER (trans-mark ithelial electrical resistance) method, which is one of the functional quantification methods of tight junctions. In the TER method, epithelial cells are cultured in a two-chamber culture dish, and the electrical resistance values (TER) generated above and below the epithelial cell sheet are measured to facilitate the permeation of solutes between epithelial cells. It is a means to measure. In this experiment, cells in which expression of tricellulin disappeared (KD-1 and KD-2) were cultured in a DMEM medium using a two-chamber culture dish. The results are shown in Fig. 6. Compared to normal wild type cells (WT), TER was greatly reduced in cells in which expression of tricellulin was lost (KD-1 and KD-2). This result indicates that the function of the tight junction due to the disappearance of trisenorelin is reduced, and the permeability of ions in the cell gap is increased.

[0030] Further, as shown in Fig. 7, changes in permeability to dextran having various molecular weights labeled with FITC were examined. In this experiment, cells in which expression of tricellulin disappeared (KD-1 and KD-2) were cultured in DMEM medium using a two-chamber culture dish. In cells where the expression of tricellulin disappeared (KD-1 and KD-2), it was found that the permeability to substances below 4 kD was greatly enhanced. Considering that the molecular weight of many drugs and growth factors such as bioactive substances is at most several kD, this It is of great significance to selectively penetrate those with the same molecular weight. The data obtained in the above examples show that the function of tricellulin is temporarily and locally inhibited, and the barrier mechanism of the relevant part is temporarily broken, so that the drug and physiologically active substance can be freely diffused. This indicates that it can be transferred to a desired site.

Industrial applicability

[0031] The present invention can be used in fields such as pharmaceutical development and manufacture, and medicine, physiology, and pharmaceutical research.

[0032] Sequence Listing Free Text

SEQ ID NO: 1 shows the nucleotide sequence of DNA encoding human tricellulin.

SEQ ID NO: 2 shows the amino acid sequence of human tricellulin.

SEQ ID NO: 3 shows the nucleotide sequence of DNA encoding another form of human 'Tricellulin.

SEQ ID NO: 4 shows the amino acid sequence of another form of human tricellulin.

SEQ ID NO: 5 shows the nucleotide sequence of DNA encoding mouse 'Tricellulin. SEQ ID NO: 6 shows the amino acid sequence of mouse 'Tricellulin.

SEQ ID NO: 7 shows the nucleotide sequence of the siRNA of the present invention that suppresses the expression of the tricellulin gene.

SEQ ID NO: 8 shows the nucleotide sequence of the siRNA of the present invention that suppresses the expression of the tricellulin gene.

Claims

The scope of the claims

[I] A factor that suppresses the expression of the tricellulin gene or the function of the tricellulin protein.

[2] The factor according to claim 1, wherein the factor is selected from the group consisting of siRNA, antisense oligonucleotide, antibody, inhibitory peptide, and dominant negative mutant.

[3] The factor according to claim 1, which is a siRNA having the nucleotide sequence shown in SEQ ID NO: 7 or 8, or a homologous RNA having a function equivalent thereto.

[4] The factor according to claim 1, which is an antisense oligonucleotide against tricellulin DNA.

5. The factor according to claim 1, which is an antibody against a tricellulin protein.

[6] Claim: A drug delivery system comprising the factor according to any one of! To 5.

[7] A pharmaceutical composition for enhancing substance permeability of a living body, comprising the factor according to any one of claims 1 to 5.

[8] Claims: A pharmaceutical composition comprising the factor according to any one of! To 5 and a drug.

9. The pharmaceutical composition according to claim 7 or 8, which is a transdermal dosage form or a transmucosal dosage form.

[10] The pharmaceutical composition according to claim 7 or 8, which is an eye drop.

[II] Claim: Use of the factor according to any one of! To 5 to increase the material permeability of a living body.

[12] Claim: A method for delivering a drug, characterized by using the factor according to any one of! To 5.

13. The method according to claim 12, wherein the delivery is transdermal or transmucosal.

14. The method according to claim 12, wherein the delivery is by eye drops.

[15] A method for treating and / or preventing a disease, which comprises administering the agent of the present invention before administration of a drug or simultaneously with administration of the drug.

[16] Use of the factor according to any one of claims 1 to 5 in the manufacture of a drug delivery system or a pharmaceutical composition for increasing the substance permeability of a living body.

[17] The use according to claim 16, wherein the drug delivery system or the pharmaceutical composition further comprises a drug.

[18] The drug delivery system or pharmaceutical composition is applied or administered transdermally or transmucosally 18. Use according to claim 16 or 17, wherein:

[19] A drug delivery system or pharmaceutical composition is applied or administered by eye drops

Use according to claim 16 or 17.

[20] The tricellulin gene or tricellulin protein is derived from a human, claim: any one of! To 5, the factor according to 1, the delivery system according to claim 6, and any one of claims 7 to 10 A pharmaceutical composition according to claim 11, claims 11 to 15, or a method according to claim 1, or claims 16 to 1.

Use of 9 items, deviation or 1 item.

[21] A non-human animal knocked out of the tricellulin gene.