JP2009178052A - Method for screening compound for preventing, mitigating or treating pancreatic islet lesion of pancreas - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for screening a compound capable of preventing, mitigating or treating pancreatic islet lesion of the pancreas. <P>SOLUTION: The method for screening the compound for preventing, mitigating or treating the pancreatic islet lesion of the pancreas includes verifying the regulatory effect exhibited by a test compound against the expression of 4F2hc induced by the contact of a human peripheral blood mononucleosis or a human established cell line having properties of a human monocyte or a human macrophage, with a macrophage-activating material. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for screening a compound for preventing, alleviating or treating pancreatic islet lesions, and more particularly, on human peripheral blood mononuclear cells or on human cultured cell established strains having the properties of human monocytes or human macrophages. The present invention relates to a method for screening a compound that prevents, alleviates, or treats pancreatic islet lesions by assaying 4F2hc expressed in. The present invention also relates to a pharmaceutical composition for preventing, alleviating or treating pancreatic islet lesions, and more specifically, human peripheral blood mononuclear cells or human cultured cell established strains having the properties of human monocytes or human macrophages and macrophages The present invention relates to a pharmaceutical composition for preventing, alleviating or treating pancreatic islet lesions comprising a combination of interferon and a compound having a regulatory effect on 4F2hc expression induced by contact with an activator.

Organ tissue damage is caused by a variety of causes including trauma; hypertension; hemodynamic circulatory failure; ischemia-reperfusion injury; metabolic disorders; inflammation due to viral, cancer or bacterial infections; rejection; The radical treatment for the cause includes avoidance of ischemic injury by revascularization to the organ.
In general, cardiovascular or metabolic diseases are treated with antihypertensives or glucose metabolism improvers to avoid cell damage, and immunosuppressive therapy is used to preserve tissues against autoimmune diseases and organ transplant rejection. Treatment and symptomatic treatment are being done. Steroids have various effects and are effective for intractable diseases. However, it is difficult to use effective high doses over a long period of time due to the occurrence of side effects, which may prevent progressive organ tissue damage. Has not reached.
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 also desirable that a molecule that promotes is also expressed.

  Fusion-regulatory protein (FRP) -1, CD98, 4F2hc (4F2 heavy chain) are the same molecules, and are also indicated as FRP-1 / CD98 / 4F2hc molecules. This molecule is known to be expressed in T cells and macrophages during immune stimulation and viral infection, and to promote fusion and form multinucleated cells during viral infection (Non-Patent Document 1), a function associated with β1 integrin, It is involved in various cell functions such as hematopoiesis, apoptosis, lymphocyte proliferation, B and T lymphocyte functions, cell fusion, osteoclastogenesis, mutagenicity, and cell fusion after viral infection. The molecule (FRP-1 / CD98 / 4F2hc molecule) constitutes a heavy chain (H chain) of an amino acid transporter heteromeric amino acid transporter (abbreviated as HAT) (Non-patent Document 2). 3). It is known that this heteromeric amino acid transporter is also expressed on human macrophages (Non-patent Document 4). This heteromeric amino acid transporter has little selectivity for amino acid transport and promotes protein synthesis non-specifically.

  However, there is no known screening method for identifying compounds that prevent, alleviate or treat pancreatic islet lesions in vitro by modulating the expression of FRP-1 / CD98 / 4F2hc molecules.

Critical Review in Immunology (2000); 20: 167-196 Current Drug Metabolism (2001); 2: 339-354 Physiology (2005); 20: 112-124 Am J Physiol Cell Physiol (2001); C1964-C1970954-961

  The present invention provides a screening method for compounds that prevent, alleviate or treat pancreatic islet lesions. Furthermore, the present invention provides a pharmaceutical composition comprising the compound obtained by the screening method and interferon.

  The present inventor found that LAT-2 (L-type amino acid), which is one of the heteromeric amino acid transporters, is compared with a control animal in an animal treated with a compound having an action of selectively reducing and repairing pancreatic islet lesions. We found that mononuclear cells expressing acid transporter 2) molecules localized predominantly in the islet lesions of the pancreas, and that the islet lesions in the pancreas of these animals were significantly reduced.

  Furthermore, the present inventor used human peripheral blood mononuclear cells for stimulation culture with lipopolysaccharide (LPS), or a human cultured cell established strain having the properties of human peripheral blood mononuclear cells, human monocytes or human macrophages (hereinafter referred to as “human peripheral blood mononuclear cells”) In some cases, it is simply called a human cell line.) When interferon is used for stimulation culture, a compound that selectively suppresses and repairs pancreatic islet lesions is added and cultured. As a result, the H chain of the heteromeric amino acid transporter is used. The knowledge which has the effect | action which selectively adjusts the expression of a certain FRP-1 / CD98 / 4F2hc molecule | numerator was acquired.

  That is, the expression of LAT-2, which is one of the heteromeric amino acid transporters, by the mononuclear cell or the established human cultured cell established strain activated the FRP-1 / CD98 / 4F2hc molecule, which is a heavy chain molecule. By adhering and fusing with damaged cells at the time of islet lesions of the pancreas, LAT-2 can widely supply amino acids required by damaged organ tissue cells and promote protein synthesis for repair of cell damage means.

  Then, a compound that selectively suppresses and repairs pancreatic islet lesions selectively induces mononuclear cells expressing LAT-2 to the islet lesion site of the pancreas, and further, this compound is capable of When nuclear cells or the established cell line of human culture are added and cultured with a macrophage activator such as lipopolysaccharide (LPS) or interferon at the same time as the start of stimulation culture, FRP-1 / which is the heavy chain of heteromeric amino acid transporter Since it has an action of selectively adjusting the expression of the CD98 / 4F2hc molecule, it has been found that it can provide a screening method for easily searching for and identifying a corresponding compound. In addition, the present inventor has also obtained knowledge that can provide a pharmaceutical composition for dramatically preventing, alleviating or treating pancreatic islet lesions by using a compound obtained by the screening method and interferon in combination. .

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

（式中、Ｒ^１は、水素、置換されていてもよく若しくは介在基で中断されていてもよい鎖状の脂肪族炭化水素基、置換されていてもよい環状の脂肪族炭化水素基、置換されていてもよいアリール基、置換されていてもよい複素環基または置換されていてもよい縮合ヘテロ複素環基を表し、Ｘ^１はハロゲン、シアノ基、置換されていてもよいメルカプト基、置換されていてもよいスルホ基、置換されていてもよいスルホニル基、置換されていてもよい水酸基、置換されていてもよいアミノ基または置換されていてもよいホスホリル基を表し、Ｘ^２は、Ｏ、ＳまたはＮＲ^２を表し、またＲ^２は、水素、酸素、置換されていてもよく若しくは介在基で中断されていてもよい鎖状の脂肪族炭化水素基、置換されていてもよい環状の脂肪族炭化水素基、置換されていてもよいアリール基、置換されていてもよい複素環基または置換されていてもよい縮合ヘテロ複素環基を表す。また、ｎは１〜５の整数を表す。）
で示される化合物、式［ＩＩ］：

（式中、Ｒ^３は置換されていてもよいナフチル基を表し、Ｒ^４は置換されていてもよい炭素数１〜６の鎖状炭化水素基を表す。）
で示される化合物、式［ＩＩＩ］：

（式中、Ｒ^５は、水素または置換されていてもよいアリール基を表す。）
で示される化合物および式［ＩＶ］：

（式中、Ｒ^６は、炭素数３〜６のアルキル基またはベンジル基を表す。）
で示される化合物からなる群から選ばれる化合物である前記（９）または（１０）に記載の医薬組成物、
（１２） 化合物が、２−クロロ−５−オキソテトラヒドロフラン−２−カルボン酸ベンジルエステル、２−フルオロ−５−オキソテトラヒドロフラン−２−カルボン酸ベンジルエステル、（Ｓ）−（＋）−α−メチル−２−ナフタリン−メチル ２−（４−フロロフェノキシ）−５−オキソテトラヒドロフラン−２−カルボン酸エステル、１−（４−フルオロフェノキシ）−３−オキソ−１，３−ジヒドロイソベゾフラン−１−カルボン酸、１−ヒドロキシ−３−オキソ−１，３−ジヒドロイソベゾフラン−１−カルボン酸、２−オキソグルタル酸ブチルエステルおよび２−オキソグルタル酸ベンジルエステルからなる群から選ばれる化合物である前記（９）記載の医薬組成物、
（１３）膵臓の膵島病変を予防、緩和または治療する医薬組成物が、配合剤であることを特徴とする前記（９）〜（１２）のいずれかに記載の医薬組成物、
（１４）膵臓の膵島病変を予防、緩和または治療する医薬組成物が、ヒト末梢血単核球またはヒト単球もしくはヒトマクロファージの性質を有するヒト培養細胞樹立株とマクロファージ活性化物質とが接触して惹起される４Ｆ２ｈｃの発現に対して調節作用を有する化合物を含有してなる薬剤と、インターフェロンとを含有してなる薬剤とからなるキットであることを特徴とする前記（９）に記載の医薬組成物、および
（１５）前記（１）〜（８）のいずれかに記載のスクリーニング方法により選択された膵臓の膵島病変を予防、緩和または治療する化合物と、インターフェロンとを膵臓の膵島病変の予防、緩和または治療を必要とする患者に投与することを特徴とする腎臓の膵臓の膵島病変の予防、緩和または治療方法、
に関する。 That is, the present invention
(1) The test compound shows the expression of 4F2hc induced by contact between human peripheral blood mononuclear cells or human cultured cell established strains having the properties of human monocytes or human macrophages and a macrophage activator. A method for screening a compound for preventing, alleviating or treating pancreatic islet lesions, characterized by assaying a regulatory action;
(2) A test for the regulatory action of a test compound is performed by culturing 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 macrophage activator and a test compound. Then, the amount of 4F2hc expressed on the mononuclear cells or the human monocytes or the established human cell line is compared with the amount of 4F2hc expressed when cultured in the absence of the test compound. The screening method according to (1),
(3) 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 ( 2) The screening method described in the above,
(4) The screening method according to (2), wherein the amount of 4F2hc expressed on a mononuclear cell or a human cultured cell established strain having the properties of human monocytes or human macrophages is calculated by FACScan analysis,
(5) The screening method according to any one of (1) to (3), wherein the macrophage activating substance is interferon or lipopolysaccharide.
(6) The screening method according to any one of (1) to (5) above, wherein the human cultured cell established strain having the properties of human monocytes or human macrophages is the human leukemia cultured cell strain THP-1.
(7) (a) Human peripheral blood mononuclear cells are cultured in human AB type serum in the presence of lipopolysaccharide and a test compound, and (b) human peripheral blood mononuclear cells are recovered from the resulting culture solution. (C) a 4F2hc antibody is reacted with the collected human peripheral blood mononuclear cells, (d) the amount of 4F2hc is calculated by FACScan analysis, and (e) the 4F2hc amount in the absence of the test compound is calculated. A method for screening a compound capable of preventing, alleviating or treating pancreatic islet lesions, characterized by detecting an increase or decrease in the amount of 4F2hc compared to the amount;
(8) (a) a cultured human cell line having human monocyte or human macrophage properties in fetal bovine serum is cultured in the presence of lipopolysaccharide or interferon and a test compound, and (b) a culture solution obtained. (C) a 4F2hc antibody is reacted with the recovered human cultured cell established strain, and (d) the amount of 4F2hc is calculated by FACScan analysis. (E) a compound capable of preventing, alleviating or treating pancreatic islet lesions characterized by detecting an increase or decrease in the amount of 4F2hc by comparing the amount of 4F2hc with the amount of 4F2hc in the absence of the test compound Screening method,
(9) a compound having a regulatory action on the expression of 4F2hc 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 pancreatic islet lesions in combination with interferon,
(10) The pharmaceutical composition according to (9), wherein the compound is a compound selected by the screening method according to any one of (1) to (8),
(11) The compound is of the formula [I]:

(Wherein R ¹ represents hydrogen, a chained aliphatic hydrocarbon group which may be substituted or interrupted by an intervening group, a cyclic aliphatic hydrocarbon group which may be substituted, substituted Represents an optionally substituted aryl group, an optionally substituted heterocyclic group or an optionally substituted fused heterocyclic group, and X ¹ represents a halogen, a cyano group, an optionally substituted mercapto group, a substituted Represents an optionally substituted sulfo group, an optionally substituted sulfonyl group, an optionally substituted hydroxyl group, an optionally substituted amino group or an optionally substituted phosphoryl group, and X ² represents O , S or NR ² , and R ² represents hydrogen, oxygen, a chained aliphatic hydrocarbon group which may be substituted or interrupted by an intervening group, an optionally substituted cyclic group Aliphatic hydrocarbons , Optionally substituted aryl group, an optionally substituted heterocyclic group or an optionally substituted condensed heterocyclic heterocyclic group. Further, n represents an integer of 1-5.)
A compound of formula [II]:

(In the formula, R ³ represents an optionally substituted naphthyl group, and R ⁴ represents an optionally substituted chain hydrocarbon group having 1 to 6 carbon atoms.)
A compound of formula [III]:

(In the formula, R ⁵ represents hydrogen or an optionally substituted aryl group.)
And a compound of formula [IV]:

(In the formula, R ⁶ represents an alkyl group having 3 to 6 carbon atoms or a benzyl group.)
The pharmaceutical composition according to the above (9) or (10), which is a compound selected from the group consisting of compounds represented by:
(12) The compound is 2-chloro-5-oxotetrahydrofuran-2-carboxylic acid benzyl ester, 2-fluoro-5-oxotetrahydrofuran-2-carboxylic acid benzyl ester, (S)-(+)-α-methyl- 2-Naphthalene-methyl 2- (4-fluorophenoxy) -5-oxotetrahydrofuran-2-carboxylic acid ester, 1- (4-fluorophenoxy) -3-oxo-1,3-dihydroisobezofuran-1-carboxylic acid 1-hydroxy-3-oxo-1,3-dihydroisobezofuran-1-carboxylic acid, 2-oxoglutaric acid butyl ester and 2-oxoglutaric acid benzyl ester, Pharmaceutical composition,
(13) The pharmaceutical composition according to any one of (9) to (12) above, wherein the pharmaceutical composition for preventing, alleviating or treating pancreatic islet lesions is a combination drug,
(14) A pharmaceutical composition for preventing, alleviating or treating pancreatic islet lesions is obtained by contacting human peripheral blood mononuclear cells or human cultured cell established strains having the properties of human monocytes or human macrophages with a macrophage activator. The medicament according to (9) above, which is a kit comprising a drug containing a compound having a regulatory action on the expression of 4F2hc induced by an agent, and a drug containing interferon. A composition, and (15) a compound for preventing, alleviating or treating pancreatic islet lesions selected by the screening method according to any one of (1) to (8) above, and interferon for preventing pancreatic islet lesions A method for the prevention, alleviation or treatment of pancreatic islet lesions of the kidney pancreas, characterized by administration to a patient in need of relief or treatment,
About.

  According to the screening method of the present invention, a compound capable of preventing, alleviating or treating pancreatic islet lesions can be easily searched and identified. In addition, according to the present invention, a pharmaceutical compound for preventing, alleviating or treating pancreatic islet lesions can be provided.

  According to the method for screening a compound capable of preventing, alleviating or treating pancreatic islet lesions of the present invention, human peripheral blood mononuclear cells or human cultured cell established strains having the properties of human monocytes or human macrophages are contacted with a macrophage activator. The regulatory effect of the test compound on the expression of 4F2hc induced in this way is assayed.

  The test of the regulatory effect exhibited by the test compound comprises culturing human peripheral blood mononuclear cells or human cultured cell established strains having the properties of human monocytes or human macrophages and a macrophage activator in the presence of the test compound, This is carried out by comparing the amount of 4F2hc expressed on the mononuclear cells or the human cultured cell established strain with the amount of 4F2hc expressed when cultured in the absence of the test compound.

  Human peripheral mononuclear cells (peripheral blood mononuclear cells; abbreviated as PBMC) used in this screening method can be obtained from human peripheral blood by a method known per se. For example, a method for separating mononuclear cells from human peripheral blood is an aqueous solution of 5.7% w / v Ficoll 400 and 9.0% w / v sodium diatrizoate having a specific gravity. And centrifuge method using Ficoll pack (Ficoll-Paque, registered trademark, Pharmacia Fine Chemicals) adjusted to 1.077 g / mL. More specifically, the above method comprises the steps of: (a) placing a predetermined amount of Ficoll pack on the bottom of the test tube; (b) pipetting a neat or diluted blood sample carefully onto the Ficoll pack. (C) Ficoll pack blood prepared in (b) so that a blood component having a specific gravity greater than the specific gravity of the Ficoll pack proceeds into the Ficoll pack or passes through the Ficoll pack. Centrifuging the preparation at about 400-500 × g for about 30-40 minutes, and (d) collecting the mononuclear cell layer separated above the Ficoll pack with a pipette.

  The human cultured cell established strain having the properties of human monocytes or human macrophages used in the present invention refers to those showing a stimulus response by a macrophage activator similar to human normal monocytes or macrophages. Examples of such a human cultured cell established strain include a human leukemia cultured cell strain and the like, and specifically a human leukemia cultured cell strain THP-1. The human leukemia cell line THP-1 can be purchased and used from the Human Science Foundation, Health Science Research Resources Bank, (Sennan City, Osaka). The THP-1 is a cultured cell line having both macrophage-like cell activities established from the peripheral blood of human acute monocytic leukemia patients.

  On the other hand, the macrophage activator used in the screening method of the present invention is a human peripheral blood mononuclear cell (hereinafter also abbreviated as PBMC) or a heteromeric amino acid transporter in the human cultured cell line. Asc-1 molecule, LAT-1 (L-type amino acid transporter 1), LAT-2 (L-type amino acid transporter 2) (these have FRP-1 / CD98 / 4F2hc molecule in the H chain), etc. Although it will not specifically limit if it is a substance which expresses, Preferably it is lipopolysaccharide (LPS) or interferon. Examples of interferon include interferon α, interferon β, and interferon γ, and interferon γ is preferable.

  The lipopolysaccharide can be prepared using a method known per se. For example, the method of extracting from microorganisms and performing the process which removes toxicity as needed is mentioned. For extraction from microorganisms, for example, a hot phenol extraction method (Westphal & Jann., Methods Carbohydr. Chem. 5, 83-89 (1965)) or a microorganism is treated with proteinase K in the presence of sodium lauryl sulfate (SDS). The method etc. can be mentioned. Moreover, what was synthesize | combined chemically may be used and a commercially available thing can also be used suitably. In the present invention, when a solution is prepared using an appropriate solvent, preferably RPMI 1640 solution, the concentration is about 60 to 100 μg / mL, preferably about 70 to 90 μg / mL, more preferably about 80 μg / mL. It is preferable to use those having a high concentration. As the lipopolysaccharide, a commercially available product (for example, LPS derived from Escherichia coli manufactured by Sigma, catalog number L2654) can be preferably used. On the other hand, as human interferon, human recombinant interferon produced by genetic engineering technology can be preferably used.

  As a medium used for culturing human peripheral blood mononuclear cells or human cultured cell established strains, RPMI medium is preferable. This RPMI medium is described in Goding, J. et al. W. (1980) J.M. Immunol. Methods 39, 285, JAMA 199 (1957) 519. A commercially available product (manufactured by Sigma) may also be used.

Culture of human peripheral blood mononuclear cells can be preferably carried out at about 37 ° C. in the presence of human AB serum, or in the presence of fetal bovine serum. “Test compound”, “lipopolysaccharide or interferon”, “human AB serum or fetal bovine serum” and “human peripheral blood mononuclear cell or human cultured cell established strain” are used in advance in combination of two or all of any combination. After mixing, it may be added to the medium, or each may be added to the medium alone. The culture time for human peripheral blood mononuclear cells or human cultured cell established strains 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, human peripheral blood mononuclear cells or human cultured cell established strains are collected, and FRP-1 / CD98 / 4F2hc molecules expressed on the mononuclear cells or human cultured cell established strains are reacted with antibodies against the molecules. FRP-1 / CD98 / 4F2hc expressed on the mononuclear cells or the human cultured cell established strain by analyzing the human peripheral blood mononuclear cell or human cultured cell established strain to which the antibody is bound, for example, by FACScan The amount of molecules can be calculated.

The antibody against the FRP-1 / CD98 / 4F2hc molecule may be a monoclonal antibody or a polyclonal antibody. A monoclonal antibody can be prepared as follows, for example, according to the method described in J Virology (1992); 66: 5999-6007. That is, BALB / c mice are immunized with a human epithelial cell line FL, the spleens of the mice are collected, and cell fusion with SP ^2/0 -AG-14 myeloma cells is performed. For screening to detect hybridoma cells, a fusion-enhancing assay using FL or Hela cells infected with Newcastle disease virus is used. Thereby, hybridoma cells producing mouse anti-human monoclonal antibodies 4-5-1 and 6-1-3 can be detected. The hybridoma cells are statically cultured in a MEM (Minimum essential medium) medium supplemented with 5% fetal bovine serum. The culture supernatant can be used for FACScan at the stock concentration. In addition, HBJ-127 hybridoma cells (see Jpn J Cancer Res. (Gann) (1985); 76: 336) obtained using human bladder cancer culture T24 as an immunogen were added with 5% fetal bovine serum in the same manner. And static culture in the MEM culture solution. The culture supernatant can be used for FACScan at the stock concentration. The properties of the monoclonal antibodies 4-5-1 and 6-1-3 and HBJ-127 are described in Critical Reviews in Immunology, volume 20 / issue 3, 2000, 167-196. A polyclonal antibody against the FRP-1 / CD98 / 4F2hc molecule synthesizes a peptide corresponding to amino acid residues 164 to 175 of human 4F2hc (HKNQKDDDVAQTD (SEQ ID NO: 1)), and introduces a cysteine residue at the C-terminus thereof. It can be obtained by coupling KLH (keyhole-limpet hemocyanin), immunizing a rabbit, and purifying on an affinity column bound with the same peptide (human 4F2hc 164-175).

FACScan allows the above-described monoclonal antibody or polyclonal antibody to act on the collected human peripheral blood mononuclear cells or human cultured cell established strain, and then causes a fluorescent antibody (eg, FITC-goat IgG) as a secondary antibody to act on the antibody. Human peripheral blood mononuclear cells or human cultured cell established strains stained with a liquid flow, passing through the focal point of the laser light, and measuring the fluorescence emitted by individual cells, human peripheral blood mononuclear cells Alternatively, it is carried out by measuring the amount of FRP-1 / CD98 / 4F2hc molecule expressed in a cultured human cell line. FACScan can also measure proteins in the cytoplasm by making holes in the cell membrane. The case where the protein in the cytoplasm is also measured will be described below. First, the cells are fixed. That is, about 4% formaldehyde solution is added to human peripheral blood mononuclear cells or human cultured cell established strains 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 established strains after immobilization treatment, and left at about 4 ° C. for about 30 minutes. For the membrane permeation 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 4F2hc polyclonal antibody, which is a primary antibody, is reacted with human peripheral blood mononuclear cells or human cultured cell established strain cells 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 cultured cell established strains stained with the above-mentioned fluorescent-labeled antibody are flowed on a liquid flow, passed through the focal point of laser light, and measured for fluorescence emitted by individual cells. The amount of 4F2hc molecule expressed in blood mononuclear cells or human cultured cell established strains is measured.

  By detecting the increase or decrease in FRP-1 / CD98 / 4F2hc molecular weight by comparing the FRP-1 / CD98 / 4F2hc molecular weight measured above with the amount of 4F2hc in the absence of the test compound , The regulatory effect of the test compound on the expression of the FRP-1 / CD98 / 4F2hc molecule can be assayed. Here, by identifying and selecting a compound that increases the amount of 4F2hc decreased due to the islet lesion of the pancreas and decreases the molecular weight of FRP-1 / CD98 / 4F2hc increased due to the pancreatic islet lesion, Compounds that can prevent, alleviate or treat lesions can be screened.

  Another aspect of the present invention relates to the expression of 4F2hc 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 pancreatic islet lesions comprising a combination of a compound having a regulatory action (hereinafter also referred to as an active ingredient compound) and interferon. By using the active ingredient compound and interferon in combination, the effect of preventing, alleviating or treating pancreatic islet lesions can be enhanced compared to the use of the compound alone.

（式中、Ｒ^１は、水素、置換されていてもよく若しくは介在基で中断されていてもよい鎖状の脂肪族炭化水素基、置換されていてもよい環状の脂肪族炭化水素基、置換されていてもよいアリール基、置換されていてもよい複素環基または置換されていてもよい縮合ヘテロ複素環基を表し、Ｘ^１はハロゲン、シアノ基、置換されていてもよいメルカプト基、置換されていてもよいスルホ基、置換されていてもよいスルホニル基、置換されていてもよい水酸基、置換されていてもよいアミノ基または置換されていてもよいホスホリル基を表し、Ｘ^２は、Ｏ、ＳまたはＮＲ^２を表し、またＲ^２は、水素、酸素、置換されていてもよく若しくは介在基で中断されていてもよい鎖状の脂肪族炭化水素基、置換されていてもよい環状の脂肪族炭化水素基、置換されていてもよいアリール基、置換されていてもよい複素環基または置換されていてもよい縮合ヘテロ複素環基を表す。また、ｎは１〜５の整数を表す。）
で示される化合物、式［ＩＩ］：

（式中、Ｒ^３は置換されていてもよいナフチル基を表し、Ｒ^４は置換されていてもよい炭素数１〜６の鎖状炭化水素基を表す。）
で示される化合物、式［ＩＩＩ］：

（式中、Ｒ^５は、水素または置換されていてもよいアリール基を表す。）
で示される化合物または式［ＩＶ］：

（式中、Ｒ^６は、炭素数３〜６のアルキル基またはベンジル基を表す。）
で示される化合物等が挙げられる。具体的には、２−クロロ−５−オキソテトラヒドロフラン−２−カルボン酸ベンジルエステル（下記化合物（６−１））、２−フルオロ−５−オキソテトラヒドロフラン−２−カルボン酸ベンジルエステル（下記化合物（６−２））、（Ｓ）−（＋）−α−メチル−２−ナフタリン−メチル ２−（４−フロロフェノキシ）−５−オキソテトラヒドロフラン−２−カルボン酸エステル（下記化合物（３−２））、１−（４−フルオロフェノキシ）−３−オキソ−１，３−ジヒドロイソベゾフラン−１−カルボン酸（下記化合物（７−６））、１−ヒドロキシ−３−オキソ−１，３−ジヒドロイソベゾフラン−１−カルボン酸（下記化合物（ａ））、２−オキソグルタル酸ブチルエステル（下記化合物（ｂ））、２−オキソグルタル酸ベンジルエステル（下記化合物（ｃ））等が挙げられる。

Examples of the active ingredient compounds include compounds that prevent, alleviate or treat pancreatic islet lesions selected by the screening method. Examples of such compounds include compounds described in WO 01/72730, for example, the following formula [I]:

(Wherein R ¹ represents hydrogen, a chained aliphatic hydrocarbon group which may be substituted or interrupted by an intervening group, a cyclic aliphatic hydrocarbon group which may be substituted, substituted Represents an optionally substituted aryl group, an optionally substituted heterocyclic group or an optionally substituted fused heterocyclic group, and X ¹ represents a halogen, a cyano group, an optionally substituted mercapto group, a substituted Represents an optionally substituted sulfo group, an optionally substituted sulfonyl group, an optionally substituted hydroxyl group, an optionally substituted amino group or an optionally substituted phosphoryl group, and X ² represents O , S or NR ² , and R ² represents hydrogen, oxygen, a chained aliphatic hydrocarbon group which may be substituted or interrupted by an intervening group, an optionally substituted cyclic group Aliphatic hydrocarbons , Optionally substituted aryl group, an optionally substituted heterocyclic group or an optionally substituted condensed heterocyclic heterocyclic group. Further, n represents an integer of 1-5.)
A compound of formula [II]:

(In the formula, R ³ represents an optionally substituted naphthyl group, and R ⁴ represents an optionally substituted chain hydrocarbon group having 1 to 6 carbon atoms.)
A compound of formula [III]:

(In the formula, R ⁵ represents hydrogen or an optionally substituted aryl group.)
Or a compound of formula [IV]:

(In the formula, R ⁶ represents an alkyl group having 3 to 6 carbon atoms or a benzyl group.)
And the like. Specifically, 2-chloro-5-oxotetrahydrofuran-2-carboxylic acid benzyl ester (the following compound (6-1)), 2-fluoro-5-oxotetrahydrofuran-2-carboxylic acid benzyl ester (the following compound (6) -2)), (S)-(+)-α-methyl-2-naphthalene-methyl 2- (4-fluorophenoxy) -5-oxotetrahydrofuran-2-carboxylic acid ester (the following compound (3-2)) 1- (4-fluorophenoxy) -3-oxo-1,3-dihydroisobezofuran-1-carboxylic acid (the following compound (7-6)), 1-hydroxy-3-oxo-1,3-dihydroisobe Zofran-1-carboxylic acid (the following compound (a)), 2-oxoglutaric acid butyl ester (the following compound (b)), 2-oxoglutarate benzyl ester Ether (the following compound (c)), and the like.

  The pharmaceutical composition of the present invention may be a combination of the active ingredient compound and interferon, 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 interferon may be administered separately or simultaneously. The molar ratio of the active ingredient compound to interferon is usually 1: 0.001 to 1: 0.5, preferably 1: 0.02 to 1: 0.1.

  EXAMPLES Hereinafter, although an experiment example is given and this invention is demonstrated concretely, this invention is not restrict | limited by these experiment examples. In this experimental example, the compound (6-2) was used.

[Experiment 1]
(experimental method)
(1) Animal experimental model SDT (Spontaneously Diabetic Torii) rats, which are type 2 diabetes onset model animals, were used to evaluate whether a compound exerts an effect on prevention, alleviation or treatment in pancreatic islet lesions ( Int. J. Exp. Diabetes Res., 1 (2000) pp. 89-100 and Biochemical and Biophysical Research Communication, 314 (2004), pp. 870-877). Male SDT rats develop hyperglycemia without obesity from 20 weeks of age and develop diabetes at 100% at 40 weeks of age. SDT rats are commercially available and can be purchased from Clare Japan.
A known compound (6-2) as a treatment group was administered to SDT rats (male, 10 weeks of age) at a dose of 10 mg / kg / day by subcutaneous injection every day except Sunday. As a control group, a 5% gum arabic physiological saline solution as a vehicle was administered by subcutaneous injection every day except Sunday as in the treatment group. The administration observation period was continued up to 44 weeks of age by continuing subcutaneous injection of the same dose every day after the onset of diabetes.

(2) Observation items and methods 1) Non-fasting blood glucose: Every 2 weeks, non-fasting blood glucose is converted into a simple blood glucose meter Antisense II (Daiichi Sankyo Co., Ltd.) by the glucose oxidation method. Measured.
2) Body weight: Body weight was measured at the time of compound administration.
3) Plasma insulin concentration: At 40 weeks of age, plasma insulin concentration was measured simultaneously with non-fasting blood glucose using a commercially available kit, Levis Insulin-Rat (U type, manufactured by Shiba Goat Co., Ltd.).
4) Collection of pancreatic tissue: The rats were killed by ether anesthesia at the age of 44 weeks after the administration of the compound was completed, the pancreas and duodenum were removed, the duodenum was removed and the pancreas weight was measured. The tissue was fixed with 10% neutral formalin, embedded in paraffin, sliced into a thickness of 4 microns, and subjected to morphological evaluation and immunopathological examination.
5) Immunopathological examination of the number of LAT-2 positive cells fused with insulin positive cells: Double immunization based on the protocol of the Pentana HX System Benchmark (Pentana Japan, Tokyo) including the Pentana left-wing automatic immunostaining device Staining was performed. Tissue sections were used without heat treatment at 100 ° C. for 1 hour after deparaffinization. As blocking, 10% goat serum (Histfine SAB-PO (R) kit, manufactured by Nichirei Bioscience Co., Ltd.) was used. As a primary antibody, an antibody against insulin (polyclonal guinea-pig anti-swine insulin antibody (Code A0564, manufactured by DakoCytomation)) was used and reacted at room temperature for 1 hour. As a secondary antibody, a biotin-labeled anti-rabbit IgG antibody (goat) (Histofine SAB-PO (R) kit, manufactured by Nichirei Bioscience) was reacted at room temperature for 30 minutes, and then a simple stain DAB solution The color was developed using a Histofine SAB-PO (R) kit, manufactured by Nichirei Bioscience Co., Ltd. Subsequently, the affinity-purified anti-rat LAT-2 polyclonal antibody (rabbit) was reacted at an optimal concentration of 50 times for 1 hour at room temperature. Similarly, as a secondary antibody, biotin-labeled anti-rabbit IgG antibody (goat) (Histfine SAB-PO (R) kit, manufactured by Nichirei Bioscience) was reacted at room temperature for 30 minutes, and then BlueMap Using TM KIT (manufactured by Pentona), color was developed by reacting at room temperature for 32 minutes. Nuclear Fast Red was used as a counter stain. An affinity purified anti-rat LAT-2 polyclonal antibody (rabbit) was prepared as follows. That is, for the production of an anti-LAT-2 peptide polyclonal antibody (rabbit), a peptide corresponding to an oligopeptide (PVKDPDSEEQP (SEQ ID NO: 2)) corresponding to the amino acid residue of 521-531 of LAT-2 was synthesized, and A cysteine residue was introduced at the C-terminus, and keyhole limpet hemocyanine was bound as an adjuvant to the C-terminal cysteine residue to immunize rabbits. Serum was collected on days 7 and 14 after immunization. The formation was concentrated by an adsorption dissociation operation using an affinity column to which an oligopeptide corresponding to the amino acid residue of 521-531 of LAT-2 was bound to obtain an affinity purified antibody having a protein amount of 0.5 mg / ml.

上記表１から、化合物（６−２）投与群では対照群に比べて、４０週齢時において、体重が重く、非空腹時血糖値が低く、インスリン基礎分泌量が高い傾向であった。 (result)
The results are as shown in Tables 1 to 3 below.

From Table 1 above, the compound (6-2) administration group tended to be heavier, had a lower non-fasting blood glucose level, and had a higher insulin basal secretion at the age of 40 weeks than the control group.

＊１：分葉間に位置する膵島組織の残存率の程度は、正常大１個を５点、中程度減少１個を３点、高度減少１個を１点とし、それぞれの膵島組織の数を掛けて算出した点数の合計で評価した。

* 1: The degree of survival of the islet tissue located between the lobes is 5 points for normal size, 3 points for moderate decrease, and 1 point for 1 decrease in altitude. The total score calculated by multiplying by was evaluated.

  From Table 2 above, in the compound (6-2) administration group compared to the control group, the insulin-positive cell population that appears significantly in the pancreatic islet tissue that is excised at 44 weeks of age and that appears in the lobule It can be seen that there are significantly more.

From Table 3 above, in the compound (6-2) administration group, more insulin-LAT-2 positive fusion cells present in the islet tissue located in the lobule were preserved more than in the control group, and It can be seen that the number of insulin-LAT-2 positive fusion cells present in the islet tissue located in FIG.
(Discussion)
From the above results, it can be seen that the compound (6-2) has an action of preventing, alleviating or treating pancreatic islet lesions.

[Experimental example 2]
(experimental method)
1) Isolation of human peripheral blood mononuclear cells (abbreviated as PBMC):
A small amount of heparin was filled from normal human peripheral blood into a syringe and about 30 ml was collected and immediately mixed with an equal amount of 0.9% physiological saline (with 1 mM EDTA added). A 50 ml tube was overlaid with 12 ml histopack (Sigam-Aldrich, HISTOPAQUE-1119, Cat No. 1119-1) and 10 ml Ficoll pack (Amersham Bioscience, Ficoll-Paque Plus), and the blood was layered gently. Centrifugation was performed at 400 g for 20 minutes at room temperature, and 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 washed by centrifugation at 250 g for 10 minutes at 4 ° C. The supernatant was discarded and the same operation was performed twice. About 8 ml of the collected plasma was added to the pellet, mixed, and allowed to stand at 37 ° C. for 10 minutes. Centrifugal washing was performed at 250 g for 10 minutes at 4 ° C., RPMI 1640 without serum (2 mM L glutamine, 5 μg / ml gentamicin, hereinafter abbreviated as culture medium RPMI) was added to the pellet, and centrifugal washing was performed in the same manner. The final cell number of PBMC was adjusted to 2 × 10 ⁶ / ml.

2) Preparation of lipopolysaccharide (LPS):
1 mg of LPS derived from E. coli (Sigma, Catalog No. L2654) was diluted with 5 ml of the culture solution RPMI, and passed through 0.2 μl of a Millipore filter, and used as LPS.

3) Test compound dilution:
Dissolve the test compound in a sterilized DMSO (dimethyl sulfoxide) stock solution, proceed with dilution with the culture medium RPMI, and add the test compound to a final DMSO concentration of 10 ⁻⁵ (v / v). After dilution adjustment, the activity was examined at a test compound concentration of 100 nM or less.

4) Culture:
To each well of the microplate (Falcon 3046), 1000 μl of PBMC solution, the same amount of LPS, 250 μl of test compound solution, 250 μl of human AB serum were added, and 5% CO ₂ -95% air at 37 ° C. for 6 days. In culture.

5) Recovery of cultured PBMC:
After completion of the culture, PBMCs were collected using rubber with adherent cells. The culture solution was washed once with a serum-free Hanks solution (5 μl / ml gentamicin added), and the concentration of the number of cells was adjusted to 4 × 10 ⁶ / ml with the same solution.

6) Preparation of monoclonal antibodies against FRP-1 / CD98 / 4F2hc (mouse anti-human monoclonal antibodies 4-5-1, 6-1-3 and HBJ-127):
Mouse anti-human monoclonal antibodies 4-5-1 and 6-1-3 were prepared according to the method described in J Virology (1992); 66: 5999-6007. That is, BALB / c mice were immunized with a human epithelial cell line FL, mouse spleens were collected, and cell fusion was performed with SP ^2/0 -AG-14 myeloma cells. For screening for detecting hybridoma cells, a fusion-enhancing assay using FL cells or Hela cells infected with Newcastle disease virus was used. As a result, hybridoma cells producing mouse anti-human monoclonal antibodies 4-5-1 and 6-1-3 were detected.
The hybridoma cells were statically cultured in a MEM medium supplemented with 5% fetal bovine serum. The culture supernatant was used for FACScan at the stock concentration.
HBJ-127 hybridoma cells obtained using human bladder cancer culture T24 as an immunogen (see Jpn J Cancer Res. (Gann) (1985); 76: 336) were similarly added to MEM supplemented with 5% fetal bovine serum. Static culture was performed in the culture solution. The culture supernatant was used for FACScan at the stock concentration.
The properties of mouse anti-human monoclonal antibodies 4-5-1, 6-1-3 and HBJ-127 obtained above are described in Critical Reviews in Immunology (2000); 20: 167-196.

7) Analysis by FACScan:
100 μl of three types of antibodies (namely, mouse anti-human monoclonal antibodies 4-5-1, 6-1-3 and HBJ-127) were placed in a tube for FACScan, and the same amount of test cells were added. PBS (Ca ⁺⁺ ) was placed as a control. The mixture was allowed to stand at 37 ° C. for 10 minutes and at 4 ° C. for 20 minutes, centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, 4 ml of PBS (Ca ⁺⁺ ) was added and centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. 50 μl of the secondary antibody, FITC-goat IgG (anti-mouse whole IgG, Cappel Cat No. 55493) was added to the tube and allowed to stand at 4 ° C. for 30 minutes. Again, 4 ml of PBS (Ca ⁺⁺ ) was added, centrifuged at 1500 rpm for 5 minutes to remove the supernatant, stirred, and 300 μl to 500 μl of 1% formalin-PBS solution was added and floated. Approximately 10,000 cells were measured, and the migration rate of the monocyte macrophage fraction was compared with the control. If there was a difference of 10% or more, it was determined to be significant.

(result)
The results are as shown in Table 4 below.

(Discussion)
From Table 4 above, compound (6-2) activated the molecule for FRP-1 / CD98 / 4F2hc, an amino acid transporter expressed in the macrophage fraction of human PBMC stimulated with LPS and cultured, Increased.
Although not shown in the table, when different normal human PBMC was used, conversely, in the example where the expression of the molecule was increased to 90% or more, compound (6-2) was selectively 5% or less. It showed the effect of reducing the expression.

[Experiment 3]
(experimental method)
In the same manner as in Experiment 2 above 1) to 5), test compound (6-2) (final concentration 10 nM) was added to human peripheral blood mononuclear cells simultaneously with the addition of LPS (final concentration 80 μg / ml). For 6 days. Furthermore, using a rabbit anti-human 4F2hc polyclonal antibody (see Biol Pharm Bull 30: 415-422, 2007) as a primary antibody, a larger fraction than monocyte macrophages in human PBMC was similarly obtained. The peak value of expressed HAT was measured by FACScan using a commercially available cell fixation / cell membrane permeabilization kit (trade name: BD Cytofix / Cytoperm ^™ Kit, catalog No. 554714, BD Bioscience).
In addition, the rabbit anti-human rBAT (related to b ^{0, +} -type amino acid transporter) polyclonal antibody was used as a control, with no test compound added, and the amino group end of human rBAT (MAEDKSKRDSIEMSMKGC (SEQ ID NO: 3)). A product obtained by immunizing a rabbit by coupling a synthetic peptide consisting of a carboxyl group terminal (CHLQMLEVVPEKDPE (SEQ ID NO: 4)) of mouse b ^{0, +} AT (BAT1) to a carrier KLH (keyhole-limpet hemocyanin) used.

(result)
The results are as shown in Table 5 below.

(Discussion)
From Table 5 above, test compound (6-2) selectively reduced the amount of 4F2hc in the heavy chain of HAT without affecting the amount of rBAT. Therefore, since the test compound (6-2) has an effect of reducing pancreatic islet lesions, a compound that reduces pancreatic islet lesions by selecting a compound having an effect of regulating the expression of 4F2hc amount in the same manner Was confirmed to be screened.

[Experimental Example 4]
(experimental method)
In Experimental Example 3, human leukemia cultured cell line THP-1 was used instead of human peripheral blood mononuclear cells, the addition concentration of the test compound (6-2) was 100 nM, and bovine instead of human AB serum. It is the same except that fetal serum is used, and the peak value of HAT expressed in a larger fraction (Large-cell) is higher than that in the human leukemia cultured cell line THP-1 not stimulated by LPS. Asked. In addition, the test compound to which no test compound was added was used as a control, and the peak value of HAT was measured using a commercially available cell fixation / cell membrane permeabilization kit (trade name: BD Cytofix / Cytoperm ^™ Kit, catalog No. 554714, BD By FACScan using Bioscience).

(result)
The results are as shown in Table 6 below.

(Discussion)
From Table 6 above, test compound (6-2) significantly changed the amount of 4F2hc, which is a heavy chain of HAT. Therefore, since the test compound (6-2) has an effect of reducing pancreatic islet lesions, a compound having an effect of regulating the expression of 4F2hc in the same manner using the human leukemia cultured cell line THP-1. It was confirmed that a compound that reduces pancreatic islet lesions can be easily screened by selecting.

[Experimental Example 5]
(experimental method)
In Experimental Example 3, human leukemia cultured cell line THP-1 was used in place of human peripheral blood mononuclear cells, test compound (6-2) was added at a final concentration of 100 nM, and fetal bovine in place of human AB serum. Serum was used in the same manner except that human recombinant interferon-gamma (Serotec PHP050) (final concentration 100 ng / ml) was used instead of LPS.Rabbit anti-human 4F2hc polyclonal was used as the primary antibody. Using an antibody (see Biol Pharm Bull 30: 415-422, 2007), a larger fraction (Large) is also compared with that in the human leukemia cell line THP-1 that has not been stimulated with LPS. The peak value of HAT expressed in -cell) was determined, and the test compound without addition of the test compound (DMSO only) was used as a control (control), and the rabbit anti-human rBAT poly Polyclonal antibody was obtained in the same manner as in Experimental Example 2.

(result)
The results are as shown in Table 7 below.

(Discussion)
From Table 7 above, test compound (6-2) selectively reduced the amount of 4F2hc in the heavy chain of HAT without affecting the amount of rBAT. Therefore, since the test compound (6-2) has an effect of reducing pancreatic islet lesions, a compound that reduces pancreatic islet lesions by selecting a compound having an effect of regulating the expression of 4F2hc amount in the same manner It was confirmed that screening can be easily performed.

  According to the method of the present invention, since a pancreatic islet lesion therapeutic drug can be screened by a simple method, it is useful in the pharmaceutical industry.

Claims (15)

  A test compound exhibits a regulatory action on the expression of 4F2hc 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 a macrophage activator. A screening method for a compound for preventing, alleviating or treating pancreatic islet lesions, characterized by comprising assaying.   The test of the regulatory action exhibited by the test compound was carried out by culturing 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 macrophage activator and a test compound, The amount of 4F2hc expressed on mononuclear cells or the human monocytes or human cultured cell established strains is compared with the amount of 4F2hc expressed when cultured in the absence of the test compound. The screening method described.   The human peripheral blood mononuclear cells are cultured in the presence of human AB type serum, or a human cultured cell established strain having the properties of human monocytes or human macrophages is cultured in the presence of fetal bovine serum. Screening method.   The screening method according to claim 2, wherein the amount of 4F2hc expressed on a mononuclear cell or a human cultured cell established strain having the properties of human monocytes or human macrophages is calculated by FACScan analysis.   The screening method according to claim 1, wherein the macrophage activator is interferon or lipopolysaccharide.   The screening method according to claim 1, wherein the human cultured cell established strain having the properties of human monocytes or human macrophages is a human leukemia cultured cell strain THP-1.   (1) Human peripheral blood mononuclear cells are cultured in human AB type serum in the presence of lipopolysaccharide and a test compound. (2) Human peripheral blood mononuclear cells are recovered from the resulting culture solution, (3 4) An antibody of 4F2hc is reacted with the collected human peripheral blood mononuclear cells, (4) the amount of 4F2hc is calculated by FACScan analysis, and (5) the amount of 4F2hc is compared with the amount of 4F2hc in the absence of the test compound And a method for screening a compound for preventing, alleviating or treating pancreatic islet lesions, comprising detecting an increase or decrease in the amount of 4F2hc.   (1) In human fetal bovine serum, a human cultured cell established strain having the properties of human monocytes or human macrophages is cultured in the presence of lipopolysaccharide or interferon and a test compound. (2) Human cultured cell established strains having the properties of spheres or human macrophages were collected, (3) 4F2hc antibody was reacted with the collected human cultured cell established strains, and (4) the amount of 4F2hc was calculated by FACScan analysis (5 A method for screening a compound for preventing, alleviating or treating pancreatic islet lesions, comprising detecting the increase or decrease in the amount of 4F2hc by comparing the amount of 4F2hc with the amount of 4F2hc in the absence of the test compound .   A compound having a regulatory effect on the expression of 4F2hc 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, and interferon, A pharmaceutical composition for preventing, alleviating or treating pancreatic islet lesions in combination.   The pharmaceutical composition according to claim 9, wherein the compound is a compound selected by the screening method according to claim 1. The compound is of the formula [I]:

(Wherein R ¹ represents hydrogen, a chained aliphatic hydrocarbon group which may be substituted or interrupted by an intervening group, a cyclic aliphatic hydrocarbon group which may be substituted, substituted Represents an optionally substituted aryl group, an optionally substituted heterocyclic group or an optionally substituted fused heterocyclic group, and X ¹ represents a halogen, a cyano group, an optionally substituted mercapto group, a substituted Represents an optionally substituted sulfo group, an optionally substituted sulfonyl group, an optionally substituted hydroxyl group, an optionally substituted amino group or an optionally substituted phosphoryl group, and X ² represents O , S or NR ² , and R ² represents hydrogen, oxygen, a chained aliphatic hydrocarbon group which may be substituted or interrupted by an intervening group, an optionally substituted cyclic group Aliphatic hydrocarbons , Optionally substituted aryl group, an optionally substituted heterocyclic group or an optionally substituted condensed heterocyclic heterocyclic group. Further, n represents an integer of 1-5.)
A compound of formula [II]:

(In the formula, R ³ represents an optionally substituted naphthyl group, and R ⁴ represents an optionally substituted chain hydrocarbon group having 1 to 6 carbon atoms.)
A compound of formula [III]:

(In the formula, R ⁵ represents hydrogen or an optionally substituted aryl group.)
And a compound of formula [IV]:

(In the formula, R ⁶ represents an alkyl group having 3 to 6 carbon atoms or a benzyl group.)
The pharmaceutical composition according to claim 9 or 10, which is a compound selected from the group consisting of compounds represented by:   The compound is 2-chloro-5-oxotetrahydrofuran-2-carboxylic acid benzyl ester, 2-fluoro-5-oxotetrahydrofuran-2-carboxylic acid benzyl ester, (S)-(+)-α-methyl-2-naphthalene -Methyl 2- (4-fluorophenoxy) -5-oxotetrahydrofuran-2-carboxylic acid ester, 1- (4-fluorophenoxy) -3-oxo-1,3-dihydroisobezofuran-1-carboxylic acid, 1- The pharmaceutical composition according to claim 9, which is a compound selected from the group consisting of hydroxy-3-oxo-1,3-dihydroisobezofuran-1-carboxylic acid, 2-oxoglutarate butyl ester and 2-oxoglutarate benzyl ester.   The pharmaceutical composition according to any one of claims 9 to 12, wherein the pharmaceutical composition for preventing, alleviating or treating pancreatic islet lesions is a combination drug.   A pharmaceutical composition for preventing, alleviating or treating pancreatic islet 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 a macrophage activator. The pharmaceutical composition according to claim 9, which is a kit comprising a drug comprising a compound having a regulatory effect on the expression of 4F2hc and a drug comprising interferon.   A compound for preventing, alleviating or treating pancreatic islet lesions selected by the screening method according to claim 1 and interferon are administered to a patient in need of prevention, alleviation or treatment of pancreatic islet lesions, To prevent, alleviate or treat pancreatic islet lesions.