JPWO2008133036A1 - Screening method for therapeutic agent for renal tubular disorder - Google Patents

Abstract

To provide a screening method for compounds capable of preventing, alleviating or treating renal tubular lesions. Tests the regulatory effect of test compounds on the expression of rBAT induced by contact of human peripheral blood mononuclear cells or human cultured cell established strains having the properties of human monocytes or human macrophages with macrophage activators A method for screening a compound for preventing, alleviating or treating renal tubular lesions of the kidney.

Description

The present invention relates to a screening method for a therapeutic agent for renal tubular disorder, and more specifically, a cultured cell established strain having properties of peripheral blood mononuclear cells or monocytes or macrophages is cultured in the presence of a test compound, and the cells A screening method for a compound for preventing, alleviating or treating renal tubular lesions, characterized by testing rBAT (related to b ^{0, +} -type amino acid transporter) About.

It has been suggested that there is a host-specific autoimmune cell response related to nerve tissue, skin wound, renal tubule injury, etc., for repair / regeneration after organ or tissue damage (Non-patent Document 1, Non-patent Document 1) Document 2, Non-Patent Document 3). A compound that induces and promotes regeneration-promoting CD11b ⁻ CD2 ⁺ macrophages and regulatory CD2 ⁻ CD4 ⁺ T lymphocytes is known to selectively repair renal tubule injury (Patent Document 1). It is also known that a compound that suppresses the induction of macrophages has an effect of improving renal tubular lesions (Patent Document 2).

  Tissue repair after organ injury requires repair at the DNA level, repair at the cellular level, and repair at the organ structure level, at the cellular level, including cell proliferation, including proliferative differentiation of progenitor cells present in the organ, Protein synthesis is required, and it has been suggested that damaged tissue is selectively repaired for structural reconstruction (Non-patent Document 3).

  In order to promote protein synthesis selectively to damaged tissue cells and lead to repair and regeneration, cells that have the action of repair and regeneration have molecules that selectively adhere to and fuse with tissue cells of the lesion, and at the same time, protein synthesis It is a good idea that a molecule that promotes is also expressed.

rBAT (related to b ^{0, +} -type amino acid transporter) constitutes a heavy chain (H chain) of a heteromeric amino acid transporter (HAT) as an amino acid transporter (Non-patent Document 4, Patent Document 5), an auxiliary activator that forms a heterodimer with BAT1 (b ^{0, +} -type amino acid transporter 1) ^, which is a light chain (L chain) of an amino acid transporter, and transport of cystine and basic amino acids It is a regulatory factor related to rBAT is localized in the apical region of the proximal tubular epithelium and functions as an amino acid transporter. RBAT is also known as a disease-causing factor of human cystinuria (Non-patent Document 4).

  However, it is not known that rBAT is expressed on human macrophages, and there is a compound that induces cells involved in repair and regeneration that expressed heteromeric amino acid transporter by regulating the expression of rBAT. There is no screening method for identifying a compound having the above-mentioned function.

International Publication No. 2004/024185 International Publication No. 01/72730 Pamphlet Renal Failure (1996); 18: 355-375 Immunology Today (2000); 21: 265-269 J Nephrology (2003); Vol.16, No.2: 186-195 Current Drug Metabolism (2001); Vol.2, No.4: 339-354 Physiology (2005); 20: 112-124

  The present invention provides a screening method for a therapeutic agent for renal tubular disorder.

The inventor of the present invention applied BAT1 (a heteromeric amino acid transporter) to a tubular lesion of an animal treated with a known compound (Patent Documents 1 and 2) having an action of selectively reducing and repairing a tubular lesion. b. The observation that mononuclear cells expressing ^{0, +} -type amino acid transporter 1) are predominantly localized in the tubule lesion site compared to the control animals, and that the tubule lesions in these animals are significantly reduced. Obtained.

  Furthermore, the present inventor used human peripheral blood mononuclear cells (PBMC) for stimulation culture with mitomycin C-treated human peripheral blood mononuclear cells, or human cultured cells having the properties of human monocytes or human macrophages. When an established strain (hereinafter sometimes simply referred to as a human cultured cell strain) was cultured with stimulation with human recombinant TNF (hereinafter also referred to as rhTNF), each of the known compounds was cultured at the same time. The present inventors have obtained knowledge that has an action of selectively regulating the expression of rBAT, which is a transporter heavy chain.

  That is, mononuclear cells express BAT1 when activated rBAT adheres and fuses with damaged cells at the time of renal tubular lesions, and rBAT / BAT1 complex is damaged in organ tissue cells. It means that the necessary amino acids are widely supplied and protein synthesis is promoted for repairing cell damage.

  The known compound selectively induces mononuclear cells expressing BAT1 to the tubule lesion site, and the known compound further converts human peripheral blood mononuclear cells (PBMC) to mitomycin C-treated human peripheral sites. Selective because it has the effect of selectively regulating rBAT expression when stimulated with blood mononuclear cells or when cultured with rhTNF on human cultured cell established strains having the properties of human monocytes or human macrophages The present inventors have obtained knowledge that can provide a screening method for easily searching and identifying a compound having an action of reducing and repairing tubular lesions.

  The present inventor has further studied based on the above findings and has completed the present invention.

That is, the present invention
[1] The regulatory action of the test compound on the expression of rBAT induced by contact of peripheral blood mononuclear cells or cultured cell established strains having the properties of monocytes or macrophages with a macrophage activator A method for screening a compound for preventing, alleviating or treating renal tubular lesions of the kidney, characterized by comprising:
[2] A test compound is used for expression of rBAT induced by contact of a human peripheral blood mononuclear cell, or a human cultured cell established strain having the properties of human monocytes or human macrophages, and a macrophage activator. A method for screening a compound for preventing, alleviating or treating renal tubular lesions, characterized by assaying a modulatory effect,
[3] The test for the regulatory action of the test compound is carried out using a human peripheral blood mononuclear cell activated by a macrophage activator or a human cultured cell established strain having the properties of human monocyte or human macrophage. Culturing in the presence of human peripheral blood mononuclear cells or human cultured cell established strains having the properties of human monocytes or human macrophages in the presence of macrophage activator and test compound, the mononuclear cells Or the screening method according to [2], wherein the amount of rBAT expressed on the human cultured cell established strain is compared with the amount of rBAT expressed when cultured in the absence of the test compound,
[4] Human peripheral blood mononuclear cells are cultured in the presence of human AB type serum, or human cultured cell established strains having the properties of human monocytes or human macrophages are cultured in the presence of fetal bovine serum. 3] The screening method according to the above,
[5] The above [3], wherein the amount of rBAT expressed on human peripheral blood mononuclear cells, or human cultured cell established strains having the properties of human monocytes or human macrophages is calculated by FACScan analysis [4] The screening method according to the above,
[6] The macrophage activator for human peripheral blood mononuclear cells is mitomycin C-treated human peripheral blood mononuclear cells, and the macrophage activator for human cultured cell established strains having the properties of human monocytes or human macrophages is human recombinant. The screening method according to any one of the above [2] to [5], which is TNF,
[7] The screening method according to any one of [2] to [6], wherein the human cultured cell established strain having the properties of human monocytes or human macrophages is a human lymphoma cultured cell strain U937,
[8] (1) (a) culturing a mixed culture of human peripheral blood mononuclear cells and mitomycin C-treated human peripheral blood mononuclear cells in the presence of a test compound, or (b) human peripheral blood Mononuclear cells are cultured in the presence of mitomycin C-treated human peripheral blood mononuclear cells and a test compound, (2) human peripheral blood mononuclear cells are recovered from the obtained culture medium, and (3) the recovered human peripheral blood The anti-rBAT antibody is reacted with blood mononuclear cells, (4) the rBAT amount is calculated by FACScan analysis, and (5) the rBAT amount is compared with the rBAT amount in the absence of the test compound. A method for screening a compound for preventing, alleviating or treating renal tubular lesions, characterized by detecting an increase or decrease,
[9] The screening method according to [8] above, wherein the culture in the presence of the test compound is performed in human AB serum,
[10] (1) (a) A cultured human cell line having properties of human monocytes or human macrophages is cultured in the presence of human recombinant TNF and a test compound, or (b) treated with human recombinant TNF A human cultured cell established strain having the properties of human monocytes or human macrophages is cultured in the presence of a test compound, (2) the human cultured cell established strain is recovered from the obtained culture solution, and (3) the recovered An anti-rBAT antibody is reacted with a human cultured cell established strain, (4) the rBAT amount is calculated by FACScan analysis, and (5) the rBAT amount is compared with the rBAT amount in the absence of the test compound. A method for screening a compound for preventing, alleviating or treating renal tubular lesions, characterized by detecting an increase or decrease in
[11] The screening method according to [10], wherein the culture in the presence of the test compound is performed in fetal bovine serum.
[12] A compound having a regulatory action on the expression of rBAT induced by contact of human peripheral blood mononuclear cells or human cultured cell established strains having the properties of human monocytes or human macrophages with a macrophage activator And a pharmaceutical composition for preventing, alleviating or treating renal tubular lesions comprising a combination of human recombinant TNF and
[13] The pharmaceutical composition according to [11], wherein the compound is a compound selected by the screening method according to any of [2] to [11],
[14] The pharmaceutical composition according to the above [12] or [13], wherein the compound is 1-chloro-3-oxo-1,3-dihydroisobenzofuran-1-carboxylic acid benzyl ester,
[15] The pharmaceutical composition according to any one of [12] to [14] above, wherein the pharmaceutical composition for preventing, alleviating or treating renal tubular lesions is a combination drug,
[16] A pharmaceutical composition for preventing, alleviating or treating renal tubular lesions in a kidney is a contact between a human peripheral blood mononuclear cell or a human cultured cell established strain having the properties of human monocytes or human macrophages and a macrophage activator [12] The kit according to [12], wherein the kit comprises a drug comprising a compound having a regulatory action on the expression of rBAT induced by the above, and a drug comprising human recombinant TNF. And [17] a compound for preventing, alleviating or treating renal tubular lesions selected by the screening method according to any one of [2] to [11] above, and human recombinant TNF For prevention, alleviation or treatment of renal tubular lesions, characterized by administration to a patient in need of prevention, alleviation or treatment of other tubular lesions About.

  According to the screening method of the present invention, a compound capable of preventing, alleviating or treating renal tubular lesions can be easily searched and identified.

  According to the method for screening a compound capable of preventing, alleviating or treating renal tubular lesions of the present invention, a peripheral cell mononuclear cell (PBMC) or a cultured cell established strain having the properties of monocytes or macrophages is contacted with a macrophage activator. The regulatory effect of the test compound on the expression of rBAT induced in this way is assayed.

  The assay of the regulatory action exhibited by the test compound comprises culturing a peripheral blood mononuclear cell activated by a macrophage activator or a cultured cell established strain having the properties of monocyte or macrophage in the presence of the test compound, The amount of rBAT expressed on mononuclear cells or the established cell line is compared with the amount of rBAT expressed when cultured in the absence of the test compound.

  As the peripheral mononuclear cells used in the present invention, human peripheral mononuclear cells are preferably mentioned. Such human peripheral mononuclear cells can be obtained from human peripheral blood by a method known per se. As a method for separating mononuclear cells from human peripheral blood, for example, an aqueous solution of 5.7% w / v Ficoll 400 and 9.0% w / v sodium diatrizoate having a specific gravity is used. And centrifuge method using Ficoll pack (Ficoll-Paque, registered trademark, Pharmacia Fine Chemicals) adjusted to 1.077 g / ml. The method includes (a) placing a predetermined amount of Ficoll pack at the bottom of the tube, (b) carefully pipetting a diluted or undiluted blood sample onto the Ficoll pack, (c) The Ficoll pack blood preparation prepared in (b) is about 10 to 40 at about 200 to 500 G so that blood components having a specific gravity greater than that of Ficoll pack travel into or pass through the Ficoll pack. Centrifuge for 1 minute, and (d) collect the mononuclear cell layer separated above the ficoll pack with a pipette.

  Preferred examples of the culture established strain having the properties of monocytes or macrophages used in the present invention include human culture established strains having the properties of human monocytes or human macrophages. The human culture established strain refers to a strain exhibiting a stimulus response by a macrophage activator similar to normal human monocytes or macrophages. Examples of such a human culture established strain include a human lymphoma cultured cell line, and specifically a human lymphoma cultured cell line U937. The U937 stock can be purchased and used from the Human Science Foundation, Health Science Research Resources Bank, (Sennan City, Osaka). U937 is a cultured cell line having macrophage-like cell activity established from a patient with human-derived diffuse histiocytic lymphoma, and is a non-adherent floating cell having many monocyte-like properties.

  The macrophage activator used in the present invention is not particularly limited as long as it is a substance that expresses rBAT in peripheral mononuclear cells or the cultured cell established strain, but rBAT is expressed in human peripheral mononuclear cells or the human cultured cell established strain. Substances to be expressed are preferred. For example, mitomycin C-treated human peripheral blood mononuclear cells are preferred for human peripheral blood mononuclear cells, and human recombinant TNF is preferred for the human cultured cell established strain. The human recombinant TNF may be human recombinant TNF-α or human recombinant TNF-β, but human recombinant TNF-β is preferred. The mitomycin C-treated human peripheral blood mononuclear cells are preferably those treated with mitomycin C using human allogeneic peripheral blood mononuclear cells.

The activation of peripheral blood mononuclear cells or the cultured cell established strain by a macrophage activator will be described below by taking as an example the case of using human peripheral blood mononuclear cells or the cultured human cell established strain.
Activation of human peripheral blood mononuclear cells or the aforementioned human cultured cell established strain with a macrophage activator can be performed as follows.
That is, activation of human peripheral blood mononuclear cells by a macrophage activator is performed by treating human peripheral blood mononuclear cells (responding PBMC) and human peripheral blood mononuclear cells of the same type as the mononuclear cells with mitomycin C. It can be performed by co-culturing the resulting mononuclear cells (stimulated PBMC).
In the mitomycin C treatment, mitomycin C is added to human peripheral blood mononuclear cells to a final concentration of about 3 to 300 μM, preferably about 120 μM, and is usually treated at 37 ° C. for about 20 to 40 minutes, preferably about 30 minutes. Can be done. In the mixed culture, stimulated PBMC and responding PBMC are suspended in a medium containing human AB serum (for example, RPMI medium), and the cells are added in the same ratio into a well of a microplate, for example, Falcon 3046 (Becton Dickinson), 5% CO2. _2. It can be suitably carried out by culturing usually in a carbon dioxide gas incubator at 37 ° C. for 2 to 14 days, preferably 6 to 7 days.
In addition, activation of human peripheral blood mononuclear cells by mitomycin C-treated human peripheral blood mononuclear cells is performed in the presence of a test compound in addition to the test compound in addition to mitomycin C-treated human peripheral blood mononuclear cells. It can be performed by culturing in the presence of (stimulated PBMC).

  On the other hand, activation of human cultured cell established strains having the properties of human monocytes or human macrophages with human recombinant TNF is carried out by contacting the human cultured cell established strain with human recombinant TNF or the presence of a test compound. When cultivating below, it can be carried out by culturing in the presence of human recombinant TNF in addition to the test compound.

  As human recombinant TNF (rhTNF), for example, rhTNF-β, a commercially available product (for example, catalog number: PHP052, Serotec) can be used. When contacting the human cultured cell line with human recombinant TNF, rhTNF can be used by dissolving in an appropriate solvent (preferably RPMI1640). The concentration at that time is usually about 1 to 40 ng / ml, preferably about 1 to 40 ng / ml. Is about 5 to 20 ng / ml, more preferably about 10 ng / ml. The culture medium used for dilution is RPMI 1640, which contains 10% fetal calf serum or no serum.

Culture of activated human peripheral blood mononuclear cells or human cultured cell established strains having the properties of human monocytes or human macrophages in the presence of a test compound, or human peripheral blood mononuclear cells, human monocytes or human macrophages Culturing of a human cultured cell established strain having the above properties in the presence of a macrophage activator and a test compound can be suitably performed in an appropriate medium in the presence of serum. As the medium, RPMI medium (Goding, JW (1980) J. Immunol. Methods 39, 285, JAMA 199 (1957) 519) can be preferably used. As the serum, human AB serum is preferable in the case of human peripheral blood mononuclear cells, and fetal bovine serum is preferable in the case of a human cultured cell established strain having the properties of human monocytes or human macrophages. The culture time is usually 2 to 14 days, preferably 6 to 10 days. The culture temperature is preferably about 37 ° C. The main culture is preferably performed under conditions of about 5% CO ₂ .

  After the culture, a human cultured cell established strain having the properties of human peripheral blood mononuclear cells or human monocytes or human macrophages is collected. The recovered mononuclear cells or rBAT expressed on the human cultured cell established strain is reacted with an anti-rBAT antibody, and the human peripheral blood mononuclear cells bound to the antibody or the human cultured cell established strain are treated with a flow cytometer, for example, By analyzing with FACScan (Becton Dickinson), the amount of rBAT expressed on the mononuclear cells or the established cultured cell line is calculated.

An anti-rBAT antibody used in the present invention, for example, an anti-human rBAT polyclonal antibody, can be prepared by the method of Bauch and Very et al. (Am J Physiol Renal Physiol (2002); 283: 181). KLH (keyhole-limpet hemocyanin) which is a synthetic peptide consisting of amino r-terminus of human rBAT (MAEDKSKRDSIEMSMKGC (SEQ ID NO: 1)) and carboxyl end of mouse b ^{0, +} AT (BAT1) (CHLQMLEVVPEKDPE (SEQ ID NO: 2)) ) To immunize rabbits to obtain antisera. A purified antibody is obtained on an affinity column using the peptide used for immunization as an antigen. When the obtained purified antibody of rabbit anti-human rBAT polyclonal antibody is used as a primary antibody, the final concentration can be about 0.001 to 0.1 mg / ml.

In the case of analyzing with a flow cytometer, for example, the case where FACScan analysis is performed will be described below as an example. First, the cells are fixed. That is, about 4% formaldehyde solution is added to human peripheral blood mononuclear cells or human cultured cell lines collected as described above and allowed to stand at about 37 ° C. for about 10 minutes for immobilization (Cytometry Part A (2003); 55A: 61-70). For cell fixation, for example, a commercially available cell fixation / cell membrane permeabilization kit (trade name: BD Cytofix / Cytoperm ^™ Kit, catalog No. 554714, BD Bioscience) BD Cytofix / Cytoperm ^™ solution can be used. Next, a membrane permeation treatment is performed so that the antibody can pass through the cell membrane. That is, about 0.1% saponin solution is added to human peripheral blood mononuclear cells or human cultured cell lines after immobilization, and left at about 4 ° C. for about 30 minutes. For the membrane permeabilization treatment, for example, a commercially available BD Perm / Wash ^™ staining buffer of the cell fixation / cell membrane permeabilization kit containing 0.1% saponin can be used. Next, immunostaining is performed. That is, a human anti-human rBAT polyclonal antibody, which is a primary antibody, is reacted with human peripheral blood mononuclear cells or human cultured cell lines after membrane permeabilization. Subsequently, a fluorescent labeled antibody (for example, goat FITC-anti-rabbit immunoglobulin) is reacted as a secondary antibody. Human peripheral blood mononuclear cells or human cell lines stained with the fluorescent-labeled antibody are flowed in a liquid flow, passed through the focal point of a laser beam, and the fluorescence emitted by individual cells is measured. The amount of rBAT expressed in mononuclear cells or human cell lines is measured.

  By comparing the amount of rBAT measured above with the amount of rBAT in the absence of the test compound, an increase or decrease in the amount of rBAT is detected, and thereby the modulating effect of the test compound on the expression of rBAT. Can be tested. Here, prevention and alleviation of renal tubular lesions by identifying and selecting compounds that increase the amount of rBAT decreased in renal tubular lesions and decrease the amount of rBAT increased in renal tubular lesions. Alternatively, compounds that can be treated can be screened.

（式中、記号は国際公開第０１／７２７３０号パンフレットに記載されたものと同一意味を有する。）
で示される化合物が挙げられる。具体的には、１−クロロ−３−オキソ−１，３−ジヒドロイソベンゾフラン−１−カルボン酸ベンジルエステルなどが挙げられる。また、前記有効成分化合物として下記式（４−２）、（９−１）で示される化合物も挙げられる。

Another aspect of the present invention relates to the expression of rBAT induced by contact of human peripheral blood mononuclear cells or human cultured cell established strains having the properties of human monocytes or human macrophages with a macrophage activator. A pharmaceutical composition for preventing, alleviating or treating renal tubular lesions comprising a combination of a compound having a regulatory action (hereinafter also referred to as an active ingredient compound) and human recombinant TNF. By using the active ingredient compound and human recombinant TNF in combination, the effect of preventing, alleviating or treating renal tubular lesions in the kidney can be enhanced compared to the use of the compound alone.
Examples of the active ingredient compounds include compounds that prevent, alleviate or treat renal tubular lesions selected by the screening method. Examples of such a compound include those described in WO 01/72730, for example, the following formula (7):

(In the formula, the symbols have the same meaning as those described in WO 01/72730.)
The compound shown by these is mentioned. Specific examples include 1-chloro-3-oxo-1,3-dihydroisobenzofuran-1-carboxylic acid benzyl ester. Moreover, the compound shown by following formula (4-2) and (9-1) as said active ingredient compound is also mentioned.

The human recombinant TNF may be human recombinant TNF-α or human recombinant TNF-β, but human recombinant TNF-β is preferred.
The pharmaceutical composition of the present invention may be a combination of the above-mentioned active ingredient compound and human recombinant TNF, or may be a kit comprising a drug containing each component. In the case of a kit, the drug containing the active ingredient compound and the drug containing human recombinant TNF may be administered separately or simultaneously. The molar ratio of the active ingredient compound and human recombinant TNF is usually 1: 0.001 to 1: 0.5, preferably 1: 0.02 to 1: 0.1.

（尿細管間質病変を選択的に抑制する化合物）

Hereinafter, although an example of an experiment and a test example are given and the present invention is explained concretely, the present invention is not restricted to these. The test compound used in this experimental example is known to selectively inhibit renal glomerular lesions and compound [I], which selectively inhibits renal glomerular lesions. Compound [II], which has the following structural formula (see Patent Documents 1 and 2).
(Compound that selectively suppresses glomerular lesions)

(Compound that selectively inhibits tubulointerstitial lesions)

[Experimental Example 1]
(1) Separation of human peripheral blood mononuclear cells (PBMC) About 30 ml of normal human peripheral blood was collected using a syringe to which a small amount of heparin was added, and immediately equal volume of 0.9% physiological saline (added with 1 mM EDTA). Mixed with. A 50 ml tube was overlaid with 12 ml histopack (HISTOPAQUE-1119, catalog No. 1119-1, Sigma-Aldrich) and 10 ml Ficoll pack (Ficoll-Paque Plus, Amersham Bioscience), and then the blood was gently overlaid. Centrifugation at room temperature (400 G, 20 minutes), about 8 ml of plasma components were collected and passed through a 0.2 μm Millipore filter. Mononuclear cell fractions containing lymphocytes were collected, a sufficient amount of cooled PBS (Ca ⁺⁺ ) was added and mixed, and the mixture was centrifuged at 250C (250 G, 10 minutes). The supernatant was discarded and the same operation was performed twice. About 8 ml of the previously collected plasma was added to the pellet, mixed, and allowed to stand at 37 ° C. for 10 minutes. Centrifugation (250 G, 10 minutes) at 4 ° C., serum-free RPMI 1640 (2 mM L-glutamine, 5 μg / ml gentamicin, Sigma, hereinafter referred to as culture medium RPMI) was added to the pellet, and centrifugation was performed in the same manner. It was. The final cell count of human peripheral blood mononuclear cells (PBMC) was adjusted to 2 × 10 ⁶ cells / ml.

(2) Dilution of the test compound After dissolving the test compound in a sterilized DMSO stock solution, the test compound was diluted with the culture solution RPMI so that the final DMSO concentration was 1 × 10 ⁻⁶ (v / v). A test compound was prepared by adjusting the dilution of the compound. The activity of the test compound was examined at a concentration of 1000 nM or less.

(3) Culture and recovery Human PBMC were treated with mitomycin C (final concentration 120 μM) at 37 ° C. for 30 minutes to obtain stimulated PBMC. In each well of a microplate (Falcon 3046, Becton Dickinson), 1000 μl of response PBMC (untreated allogeneic human PBMC) solution (2 × 10 ⁶ cells / ml), the same amount of the stimulated PBMC (2 × 10 ⁶ cells / ml). ml), 250 μl of the test compound solution and 250 μl of human AB serum (10% final concentration) were added and cultured at 37 ° C. for 5 days at 5% CO ₂ . After completion of the culture, human PBMC including the adherent cells was collected with a rubber. The cells were washed twice with PBS (+), and 0.1-1 × 10 ⁶ cells / ml were collected.

(4) Analysis by FACScan 0.5 ml of the collected human PBMC solution is put into a tube for FACScan measurement, and an equal amount of BD Cytofix / Cytoperm ^TM solution (catalog No. 554714, BD Bioscience) containing about 4% formaldehyde solution is previously prepared. The cells were fixed by heating and treating at 37 ° C. for 10 minutes. After centrifugation (300 G, 5 minutes), the supernatant was discarded, and BD Perm / Wash ^™ staining buffer (catalog No. 554714, BD Bioscience) containing 0.1% saponin in the cell pellet was used as a membrane permeabilization treatment. 5 ml was added and allowed to stand at 4 ° C. for 30 minutes. Centrifugation (300 G, 5 minutes) with Perm / Wash ^™ staining buffer. After centrifugation, 100 μl of a primary antibody (rabbit anti-human rBAT polyclonal antibody) diluted to a final concentration of 1.976 × 10 ⁻² mg / ml with BD Perm / Wash ^™ staining buffer was added to the tube, and 10 minutes at 37 ° C. The reaction was carried out at 4 ° C. for 20 minutes. After completion of the reaction, 100 μl of goat FITC-labeled anti-rabbit immunoglobulin diluted to the appropriate concentration with Perm / Wash ^™ staining buffer was added as a secondary antibody, reacted at 4 ° C. for 30 minutes, and then 300 μl of 1% formalin-PBS solution. Add ~ 500 μl to float. The peak value of HAT (Heterodimeric Amino acid Transporter: HAT) expressed in a larger fraction than monocyte macrophages in human PBMC was measured for 10,000 cells by FACScan.
In addition, using a rabbit anti-human 4F2hc (4F2 heavy chain) polyclonal antibody (see Biol Pharm Bull 30: 415-422, 2007) as a primary antibody, a fraction larger than monocyte macrophages in human PBMC (see FIG. The peak value of HAT expressed in large-macropharge) was measured.
In addition, using a test compound to which no test compound was added as a control, a rabbit anti-human 4F2hc polyclonal antibody was synthesized by synthesizing a peptide corresponding to amino acid residues 164 to 175 of human 4F2hc (HKNQKDDVAQTD (SEQ ID NO: 3)). A cysteine residue was introduced at the terminal, KLH was coupled, and the rabbit was immunized.

The results are shown in Table 1.

  From Table 1 above, it can be seen that Compound [II] significantly activated the expression of rBAT, but did not activate the expression of 4F2hc, which is another heavy chain of HAT. In addition, Table 1 shows that Compound [I] did not increase the expression of either rBAT or 4F2hc. From these results, it can be seen that compound [II] acts specifically on the expression of rBAT.

[Experiment 2]
(1) Culture of human-derived cultured cell line U937 In a culture liquid (10% FBS-RPMI) in which fetal bovine serum is added to the culture liquid RPMI to a final concentration of 10%, the human-derived cultured cell line U937 is treated with 5% CO2. _2. Cultured at 37 ° C. and maintained under the same conditions. At the time of use, after centrifugation (300 G, 5 minutes) once, the supernatant was discarded and 10% FBS-RPMI was added to adjust the final cell number to 1 × 10 ⁶ cells / ml.

(2) Preparation of macrophage activating substance Human recombinant TNF-β (catalog number: PHP052, manufactured by Serotec) (Blood, (2006); 107: 1996-2003) was dissolved in 10% FBS-RPMI1640, and rhTNF-β A solution was prepared.

(3) Dilution of test compound After the test compound is dissolved in a sterilized DMSO stock solution, the test compound is diluted with a culture solution RPMI to obtain a final DMSO concentration of 1 × 10 ⁻⁶ (v / v). The compound was diluted and the activity was examined at a test compound concentration of 1000 nM or less.

(4) Culture and collection In each well of the microplate (Falcon 3046), 1000 μl of cultured cells U937 (1 × 10 ⁶ ), 1000 μl of rhTNF-β solution (final concentration 10 ng / ml), 250 μl of the test compound solution, 250 μl Of 10% FBS-RPMI was added and cultured at 5% CO ₂ and 37 ° C. for 10 days. After the culture was completed, U937 was collected with rubber, including adherent cells. The cells were washed twice with PBS (+), and a cell count of 0.1 to 1 × 10 ⁶ cells / ml was collected.

(5) Analysis by FACScan 0.5 ml of the collected U937 solution was put into a tube for FACScan measurement, about 4% formaldehyde solution and an equal amount of BD Cytofix / Cytoperm ^TM solution preheated were added, and treated at 37 ° C. for 10 minutes. Cells were fixed. After centrifugation (300G, 5 minutes), the supernatant is discarded, and as a membrane permeabilization treatment, 0.5 ml of BD Perm / Wash ^™ staining buffer with 0.1% saponin concentration is added to the cell pellet and allowed to stand at 4 ° C. for 30 minutes. I put it. Centrifugation (300 G, 5 minutes) with Perm / Wash ^™ staining buffer. After centrifugation, 100 μl of primary antibody and rabbit anti-human rBAT polyclonal antibody diluted to a final concentration of 1.976 × 10 ⁻² mg / ml with BD Perm / Wash ^™ staining buffer were added to the tube, and the mixture was added at 37 ° C. for 10 minutes. The reaction was carried out at 20 ° C. for 20 minutes. After completion of the reaction, 100 μl of goat FITC-labeled anti-rabbit immunoglobulin was added as a secondary antibody, reacted at 4 ° C. for 30 minutes, 300 μl to 500 μl of 1% formalin-PBS solution was added and suspended. The peak value of HAT expressed in a larger fraction (Large-cell) was determined for 10,000 cells on FACScan compared to those in U937 that were not stimulated with human recombinant TNF-β. .
In addition, using a rabbit anti-human BAT1 polyclonal antibody as a primary antibody, HAT expressed in a larger fraction (Large-cell) as compared with those not stimulated by human recombinant TNF-β in the same manner. The peak value was determined.
In addition, the thing which does not add a test compound was made into the control | contrast.

The results are shown in Table 2.

  From Table 1 above, it can be seen that Compound [II] significantly activated the expression of rBAT, the heavy chain of HAT, but did not activate the expression of BAT1, the light chain. Table 1 also shows that Compound [I] hardly activated the expression of either heavy chain rBAT or light chain BAT1 of HAT. From these results, it can be seen that compound [II] acts specifically on the expression of rBAT.

[Test Example 1]
(1) Creation of rat one-side ureteral obstruction release model An experimental model was created using 8 to 9 week old, approximately 280 g SD rat males (Michio Ishibashi et al .: Journal of the Nephrological Society of Japan 42: 248, 2000). That is, the rat was laparotomized under ether anesthesia, the ureter was ligated and closed with 7-0 nylon at the level of the left lower pole, the obstruction was released on the 14th day, and the urinary tract was reconstructed using a cuff. Specifically, the occluded ureter ligated 14 days later is partially excised, and a 25 gauge polyethylene tube (manufactured by Sherwood Japan) is inserted into the lumen from the lower normal ureter stump, and then the upper Cuffs were also placed in the expanded ureters, and each ligature was fixed with 7-0 nylon to reconstruct the urinary tract. At the same time, the contralateral right kidney was removed. Seven days after the release of the occlusion, the patient was sacrificed under anesthesia, and the left occlusion-released kidney was removed. Pathomorphological examination was performed on the removed kidney. In this model, when no test compound was administered, renal structure was destroyed during the occlusion period and after the occlusion, and the renal structure was destroyed, glomerular Bowman sac wall thickening, mesangial cell hyperplasia, thread Spherical sclerosis, tubular atrophy, dilatation, interstitial cell infiltration, and fibrosis were exhibited.

(2) Administration of test compound The in vivo biological effect of the test compound was examined using the model obtained in (1) above. The test compound was prepared by dissolving the raw powder of the test compound together with gum arabic in sterile physiological saline, and adjusting the gum arabic to 5% and the test compound to 30 mg / ml. The test compound was injected subcutaneously with gum arabic every day at 30 mg / kg / day. The administration was continued every day for 21 days, with a 14-day occlusion period and a 7-day occlusion release. As a control, only 5% gum arabic as a solvent was administered.

(3) Observation of glomerular lesions On the 22nd day from the start of administration of the test compound, the mice were sacrificed under anesthesia, and the left occlusion-released kidney was removed and fixed with neutral formalin. The fixed paraffin-embedded kidney tissue was sliced at a thickness of 4 microns and examined.
Microscopic observation of the tissue section shows the number of glomeruli that fall within the field of view and the number of glomeruli with lesions (expansion with enlargement of Bowman's sac epithelial cells at the tubule pole or thickening of the Bowman's sac basement membrane) Counting was performed, and the number of glomeruli (referred to as lesion glomeruli) in which lesions were observed per 50 glomeruli was calculated.

(4) Observation of tubulointerstitial lesions Tissue sections obtained in the same manner as in (3) above were observed with a microscope, tubule atrophy accompanying tubule dilation, tubule basement membrane thickening, and tubule stroma. Observed fibrosis, ± (very mild change was observed) 0.5 point, + (mild change was recognized) 1 point, ++ (moderate change was observed) Two points and ++++ (altitude change was recognized) were taken as three points, and the total was calculated.

(5) Examination of the number of BAT1-positive cells For the production of a rabbit anti-human BAT1 polyclonal antibody, a peptide (J Biol Chem 274: 28845) corresponding to the amino acid residue of 474-487 of rat BAT1 (QMLMEVVPPEKDPEC (SEQ ID NO: 4)) -28848, 1999) by synthesizing an oligopeptide in which 484 (K) is changed to glutamic acid (E) (Kidney Int, (2001); 59: 1821-1833), and the cysteine residue at the C-terminal is the key as an adjuvant. Rabbits were immunized by binding keyhole limpet hemocyanine. Serum was collected on days 7 and 14 after immunization. Serum was concentrated by an adsorption dissociation operation using an affinity column bound with an oligopeptide corresponding to BAT1 (474-487) to obtain an antigen-affinity purified anti-human BAT1 polyclonal antibody (rabbit) with a protein amount of 0.5 mg / ml. It was. In the immunopathological examination, paraffin rat kidney tissue was diluted with PBS and used at an optimum concentration.
Immunohistochemical staining was performed based on the protocol of Ventana HX System Benchmark (Ventana Japan) including an automatic immunostaining device manufactured by Ventana. The tissue section was heat-treated at 100 ° C. for 1 hour after deparaffinization. As blocking, 10% goat serum (Histofine SAB-PO (R) kit, Nichirei Bioscience Co., Ltd.) was used, and an affinity purified anti-human BAT1 polyclonal antibody (rabbit) was reacted at 4 ° C. for 18 hours as a primary antibody. As a secondary antibody, a biotin-labeled anti-rabbit IgG antibody (goat) (histofine SAB-PO (R) kit, Nichirei Bioscience Co., Ltd.) was reacted at room temperature for 30 minutes. Subsequently, color was developed using a simple stain DAB solution (Histfine SAB-PO (R) kit, Nichirei Bioscience Co., Ltd.).
The stained cells are observed under a microscope, the number of BAT1 ⁺ cells in the entire visual field present in the tubulointerstitial lesion is counted, and the number of BAT1 ⁺ cells in the glomerular lesion present per 50 glomeruli is determined. Calculated.

(6) Results The results are shown in Table 3 (Compound [II]) and Table 4 (Compound [I]).

From Table 3 and Table 4, regarding the tubulointerstitial lesion, the lesion was significantly improved in the group administered with Compound [II] (significant difference: p = 0.01, score-decrease rate with respect to control: 60). %) And the number of BAT1-positive cells present in the same lesion was significantly increased (average increase over control: 16.5 cells), but in the group administered with Compound [I], BAT1-positive cells present in the same lesion It can be seen that there was no significant increase in the number of tubulointerstitial lesions (significant difference: none, score-decrease rate relative to control: 18%).
On the other hand, with respect to glomerular lesions, the group administered with Compound [II] did not improve the lesions much (reduction rate of lesioned glomeruli with respect to the control: 44%), and the number of BAT1-positive cells present in the lesions was also small. Although it did not increase (increase with respect to the control: 0.3), it was found that the glomerular lesion was remarkably improved in the compound [I] administration group (reduction rate of lesioned glomeruli with respect to the control: 67%).
From the above, it can be seen that compound [II] is a drug that selectively suppresses tubulointerstitial lesions, whereas compound [I] is a drug that selectively suppresses glomerular lesions.

  From the results of the experimental examples 1 and 2 and the results of the test examples, human peripheral blood mononuclear cells or human cultured cell established strains having the properties of human monocytes or human macrophages and a macrophage activator are brought into contact with each other. It was confirmed that preventive, alleviating and therapeutic agents for renal tubular lesions can be screened using fluctuations in the expression of rBAT as an index.

[Test Example 2]
(1) Creation of rat one-side ureteral obstruction release model An experimental model was created using 9-week-old, approximately 300-320 g SD rat males (Michio Ishibashi et al .: Journal of the Nephrological Society of Japan 42: 248, 2000). That is, the rat was laparotomized under ether anesthesia, the ureter was ligated with 7-0 nylon at the level of the left lower pole, and the obstruction was excised on the 14th day after the obstruction was created, and the cuff was used. The urinary tract was reconstructed by left uretero-ureteral anastomosis. Specifically, the obstructed ureter ligated 14 days later was partially excised, and a 25 gauge polyethylene tube (manufactured by Sherwood Japan) was cuffed to a length of about 3 to 5 mm. Insert and place in the end lumen, fix the cuff with 7-0 nylon, then insert the tube placed in the lower ureter into the upper expanded ureter, and expand the ureter with 7-0 nylon The cuff was ligated and fixed, the left uretero-ureteral anastomosis was completed, and the urinary tract was reconstructed. At the same time, the contralateral right kidney was removed.
Seven days after the release of the occlusion, the rat was sacrificed under anesthesia, and the left occlusion-released kidney was removed. Pathomorphological examination was performed on the removed kidney. In the control group to which no test compound was administered in this model, the renal structure was destroyed during the occlusion period and after the release of the occlusion, resulting in destruction of the glomerular Bowman sac wall, glomerular urine. The urinary space showed a tubule pole opening enlargement, tubule atrophy, dilation, interstitial cell infiltration, and fibrosis. In terms of renal function, the severe renal failure state recovered to a normal 30% renal function level and a plasma creatinine level of 2.0 mg / dl on the 6th to 7th day after the release of the obstruction.

(2) Administration of test compound and combined administration with TNF As test compound, compound [II] was used in the same manner as in Test Example 1, and the raw powder of the test compound was dissolved in sterile physiological saline together with gum arabic. The rubber was adjusted to 5% and the test compound was adjusted to 30 mg / ml. The test compound was injected subcutaneously daily with gum arabic at a dose of 30 mg / kg / day.
Other than humans, TNF-β (Tumor Necrosis Factor-beta) has not been isolated and identified (Aggarwal BB. Comparative analysis of the structure and function of TNF-α and TNF-β. In Aggarwal BB, Vilcek J (eds .): Tumor necrosis factors: Structure, Function and Mechanism of action. See New York, NY, Dekker, 1992, p61), because the experiment using rat recombinant TNF-β is not possible. Rat recombinant TNF-α (R & D System, Cat Number: 510-RT) was used to predict that rat recombinant TNF-β activity was also included in rat recombinant TNF-α. As the dosage, non-patent literature (Reynolds JL et al. J Pharmacol Exp Ther 2004; 310: 1216-1225) was administered 1 μg of rat recombinant TNF-α (R & D Systems) by continuous daily intravenous injection. Therefore, the intraperitoneal dose once a day was 0.45 μg. A solution diluted with physiological saline was administered intraperitoneally into 0.45 ml (including 0.45 μg) every other day.
In the experimental group, Compound [II] alone and the compound [II] alone administration group in which 0.45 ml of physiological saline was administered intraperitoneally instead of administration of Compound [II] and rat recombinant TNF-α were group 1; The combined administration group of recombinant TNF-α was group 2.
The administration period started from 5 days before the left ureteral obstruction was created, and lasted up to a 14-day obstruction period and an additional 7-day obstruction release period.
Using the model obtained in (1) above, it was examined that the combined regeneration of compound [II] and TNF promotes repair and regeneration after renal tubular injury than compound [II] alone. That is, blood was collected on the 3rd and 6th days after the release of the occlusion, and the plasma creatine level was measured. Since the right kidney is removed when the left kidney is deoccluded, the measured plasma creatine value represents the renal function of the left kidney that has been deoccluded. Renal function was expressed as 1 / plasma creatine mg ⁻¹ · dl.

注）化合物［ＩＩ］およびＴＮＦ−α共に投与しない対照群（ｎ＝４）では、閉塞解除後６日目の腎機能が平均０．４６５（１／血漿クレアチンｍｇ^−１・ｄｌ）であった。また、有意差検定はグループ１ｖｓグループ２で行った。(3) Results The results are as shown in Table 5 below.

Note) In the control group (n = 4) to which neither compound [II] nor TNF-α was administered, the renal function on the 6th day after the release of the obstruction averaged 0.465 (1 / plasma creatine mg ⁻¹ · dl). . In addition, the significant difference test was performed in group 1 vs. group 2.

The renal function shows recovery from the third day after the release of the obstruction, but on the sixth day after the release of the obstruction, the group 1 (compound [II] single administration group) has a renal function of 0.465 to 0.706 as compared with the control group It can be seen that Compound [II] has an effect of improving renal function. In addition, when Group 1 and Group 2 (compound [II] and TNF-α combined administration group) were compared, Group 2 showed recovery from renal dysfunction on both day 3 after the release of obstruction and day 6 after the release of obstruction. A good tendency was shown (significant difference test on the 6th day after release of the occlusion: p = 0.086).
From the above results, in the combined administration group of compound [II] and rat recombinant TNF-α for preventing, alleviating or treating renal tubular lesions in the kidney, the combined administration was carried out prophylactically from 5 days before the creation of ureteral obstruction, and then continued. Even after the ureteral obstruction, the renal tubule lesions are prevented more effectively than the compound [II] alone administration group by continuing the administration for 20 days including the obstruction period of 14 days and the deocclusion period of 6 days. It was confirmed that it could be alleviated or treated.

  The method of the present invention is useful in the pharmaceutical industry because it can screen for preventive / mitigated / therapeutic agents for renal tubular lesions by a simple method.

Claims (17)

  To test the regulatory effect of a test compound on the expression of rBAT induced by contact of peripheral blood mononuclear cells or cultured cell established strains having the properties of monocytes or macrophages with a macrophage activator A method for screening a compound for preventing, alleviating or treating renal tubular lesions of the kidney.   Modulatory effect of the test compound on the expression of rBAT induced by contact of human peripheral blood mononuclear cells or human cultured cell established strains having the properties of human monocytes or human macrophages and macrophage activator A screening method for a compound for preventing, alleviating or treating renal tubular lesions of the kidney, characterized in that   In the presence of a test compound, a test of a regulatory action exhibited by the test compound was conducted in the presence of a human peripheral blood mononuclear cell activated by a macrophage activator or a human cultured cell established strain having the properties of a human monocyte or human macrophage. Culturing or culturing a human cultured blood cell line having the properties of human peripheral blood mononuclear cells or human monocytes or human macrophages in the presence of a macrophage activator and a test compound, and the mononuclear cells or the human The screening method according to claim 2, which is carried out by comparing the amount of rBAT expressed on the cultured cell established strain with the amount of rBAT expressed when cultured in the absence of the test compound.   The culture of human peripheral blood mononuclear cells is carried out in the presence of human AB type serum, or the culture of established human cell lines having the properties of human monocytes or human macrophages is carried out in the presence of fetal bovine serum. The screening method according to Item.   The amount of rBAT expressed on human peripheral blood mononuclear cells or human cultured cell established strains having the properties of human monocytes or human macrophages is calculated by FACScan analysis. 5. The screening method according to item 4.   The macrophage activator for human peripheral blood mononuclear cells is mitomycin C-treated human peripheral blood mononuclear cells, and the macrophage activator for human cultured cell established strains having the properties of human monocytes or human macrophages is human recombinant TNF. The screening method according to any one of claims 2 to 5.   The screening method according to any one of claims 2 to 6, wherein the human cultured cell established strain having the properties of human monocytes or human macrophages is a human lymphoma cultured cell strain U937.   (1) (a) culturing a mixed culture of human peripheral blood mononuclear cells and mitomycin C-treated human peripheral blood mononuclear cells in the presence of a test compound, or (b) human peripheral blood mononuclear cells Is cultured in the presence of mitomycin C-treated human peripheral blood mononuclear cells and the test compound, (2) human peripheral blood mononuclear cells are recovered from the resulting culture medium, and (3) the recovered human peripheral blood mononuclear cells The anti-rBAT antibody is reacted with the sphere, (4) the rBAT amount is calculated by FACScan analysis, and (5) the rBAT amount is increased or decreased compared with the rBAT amount in the absence of the test compound. A method for screening a compound for preventing, alleviating or treating renal tubular lesions, characterized by detecting   The screening method according to claim 8, wherein the culture in the presence of the test compound is carried out in human AB serum.   (1) (a) a cultured human cell line having the properties of human monocytes or human macrophages is cultured in the presence of human recombinant TNF and a test compound, or (b) human monocytes treated with human recombinant TNF Alternatively, a human cultured cell established strain having the properties of human macrophages is cultured in the presence of a test compound, (2) the human cultured cell established strain is recovered from the obtained culture medium, and (3) the recovered human cultured cells An anti-rBAT antibody is reacted with the established strain, (4) the rBAT amount is calculated by FACScan analysis, and (5) the rBAT amount is increased by comparing the rBAT amount with the rBAT amount in the absence of the test compound or A method for screening a compound for preventing, alleviating or treating renal tubular lesions, characterized by detecting a decrease.   The screening method according to claim 10, wherein the culture in the presence of the test compound is carried out in fetal bovine serum.   A human peripheral blood mononuclear cell, or a compound having a regulatory action on the expression of rBAT induced by contact of a human cultured cell established strain having the properties of human monocytes or human macrophages with a macrophage activator, and human A pharmaceutical composition for preventing, alleviating or treating renal tubular lesions in combination with recombinant TNF.   The pharmaceutical composition according to claim 11, wherein the compound is a compound selected by the screening method according to any one of claims 2 to 11.   14. The pharmaceutical composition according to claim 12 or 13, wherein the compound is 1-chloro-3-oxo-1,3-dihydroisobenzofuran-1-carboxylic acid benzyl ester.   The pharmaceutical composition according to any one of claims 12 to 14, wherein the pharmaceutical composition for preventing, alleviating or treating renal tubular lesions is a combination drug.   A pharmaceutical composition for preventing, alleviating or treating renal tubular lesions is caused by contact between human peripheral blood mononuclear cells or human cultured cell established strains having the properties of human monocytes or human macrophages and macrophage activator 13. The kit according to claim 12, which comprises a drug containing a compound having a regulatory effect on the expression of rBAT, and a drug containing human recombinant TNF. Pharmaceutical composition.   A compound that prevents, alleviates or treats renal tubular lesions selected by the screening method according to any one of claims 2 to 11, and human recombinant TNF is used to prevent renal tubular lesions. A method for preventing, alleviating or treating renal tubular lesions of the kidney, which comprises administering to a patient in need of alleviation or treatment.