JP2002154986A - Insulin secretion regulator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new insulin secretion regulator. SOLUTION: A substance inhibiting an action of IgE-dependent histamine release factor (IgE-dependent HRF) is useful as an insulin secretion promoter and diabetic prophylactic/therapeutic agent. IgE-dependent HRF or a substance having its action is useful as an insulin secretion inhibitor.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an insulin secretagogue containing a substance inhibiting the action of an IgE-dependent histamine releasing factor, an IgE-dependent histamine releasing factor or a substance having the action. The present invention relates to an insulin secretion inhibitor, a method for screening for an insulin secretion regulator, and the like.

[0002]

2. Description of the Related Art Molecular weight 21 k regulated in expression at translation stage
Da tumor-associated protein (translationally controlled t
umor protein p21 (hereinafter, TCTP p21) is derived from mouse erythroleukemia cells as one of tumor-associated proteins.
1983 First identified by Brawerman et al. (Mol. Cell. Biol., 3rd.
Vol. 1197-1203). Thereafter, a human homolog (TCTP p23) was identified from Ehrlich cancer cells (Nucleic Acids Research).
s.), Volume 16, 2350), mouse and human TCT
The P cDNA was cloned in 1988 and 1989, respectively (Nucleic Acid Research (Nuclei).
Acids Res.), Vol. 17, 8367; Nucleic
Acid Research (Nucleic Acids Res.), 16
2350).

[0003] TCTP is a yeast (Yeast, 199
4 years, Volume 10, Suppl A, S63-68), Plants (Plant. Mol. Bi
ol.), 1992, Vol. 19, 501-503) to humans.
resis), 1991, Vol. 18, 150-155). TC
Although TP is induced by cell proliferation, it has been suggested that TP is related to proliferation.
Biology (J. Leuk. Biol), 1995, Vol. 57,
507-512; Biochemistry International (Biochem. Int.), 1989, Vol. 19, 277-.
286), and its function is hardly disclosed. The synthesis of TCTP is based on polypyrim in the 5'-untranslated region (UTR) of mRNA.
It is regulated at the post-transcription level via idine tract, suggesting that the 3′-UTR of mRNA is involved in the regulation of TCTP protein synthesis at the translation stage in vitro (Biomedical, Biochemical,
Actor (Biomed. Biochem. Acta), 1991, 50th
Volume, 1193-1203; Cell Molecular Biological Research (Cell. Mol. Biol. Res.), 1994.
Year, Volume 40, 633-641).

[0004] In addition to tumor cells, TCTP is expressed in normal cells such as keratinocytes, hepatocytes, lymphocytes, platelets, and macrophages (Electrophoresis, 1997, Vol. 18, 150-1).
55; Electrophoresis, 199
2 years, Vol. 13, 992-1001; Electrophoresis, 1992, Vol. 13, 893
-959). In 1995, MacDonald et al. Reported that a factor that promotes histamine release from basophils in the presence of IgE from the culture supernatant of human monocyte cell line U937 (histamine releasing facto).
or HRF) was identified (Science, 199
5 years, Vol. 269, 688-690). This factor was found to be the same protein as human TCTPp23, and it has been reported that TCTP may be an inducer of a late-onset allergic inflammatory response. However, TCTP
There is no report on the relationship between insulin and insulin secretion.

At present, the number of Japanese people suffering from diabetes is about 10 million, including borderline diabetes. The cost of treating cardiovascular disease, renal failure, retinopathy, and neuropathy associated with diabetes is enormous, and the public burden on medical expenses is increasing every year.

[0006] As therapeutic agents for diabetes, insulin preparations,
Insulin secretion stimulants, insulin sensitizers, glucose absorption inhibitors and the like have been used, and have a certain effect in controlling blood sugar levels. However, there is a need to develop more effective drugs, including the complications of diabetes.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide an insulin secretion regulator and a method for screening an insulin secretion regulator.

[0008]

Means for Solving the Problems As a result of intensive studies, the present inventors have found the following points as new functions of TCTP. 1) The pancreatic β cells express TCTP at a high level. 2) When feeding is started, the amount of expression of TCTP in the pancreas increases after 0.5 to 1 hour, and TCTP is secreted into peripheral blood. 3) The secretion of TCTP from MIN6 cells is induced when high glucose load or insulin stimulation is applied to MIN6 cells, a mouse pancreatic β cell line. 4) The effect of recombinant TCTP on MIN6 cells and isolated pancreatic islets of Langerhans inhibits glucose-stimulated insulin secretion. 5) When the neutralizing antibody of TCTP is acted on, the suppression of insulin secretion from mouse isolated pancreatic islets of Langerhans caused by glucose stimulation is released, and in the presence of the neutralizing antibody,
Improving insulin secretion by blocking the action of endogenously secreted TCTP.

[0009] Based on these findings, the present inventor has proposed T
The present inventors have found that CTP is a novel insulin secretion regulator secreted from pancreatic β cells, and as a result of further study, have completed the present invention.

That is, the present invention relates to [1] an insulin secretagogue comprising a substance that inhibits the action of an IgE-dependent histamine releasing factor, and [2] a substance that inhibits the action of an IgE-dependent histamine releasing factor. A prophylactic / therapeutic agent for diabetes, [3] a substance that inhibits the action of IgE-dependent histamine releasing factor, a receptor antagonist of IgE-dependent histamine releasing factor, an antibody against IgE-dependent histamine releasing factor, IgE-dependent The agent according to [1] or [2], which is a substance that suppresses the expression of histamine-releasing factor, a substance that degrades IgE-dependent histamine-releasing factor, or a substance that promotes degradation of IgE-dependent histamine-releasing factor, [4] The agent according to any one of [1] to [3], wherein the IgE-dependent histamine releasing factor is a tumor-associated protein, ] IgE-dependent histamine releasing factor is tumor-associated protein p21, a tumor-associated protein p
The agent according to any one of [1] to [3], which is 23 or tumor-associated protein p26,

[6] The agent according to [1], which is a preventive / therapeutic agent for a disease caused by insufficient insulin secretion, [7] impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy,
The agent according to [1], which is a prophylactic / therapeutic agent for diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy or osteopenia, [8] for mammals A method for promoting insulin secretion, which comprises administering an effective amount of a substance that inhibits the action of an IgE-dependent histamine releasing factor,

[9] a method for preventing or treating a disease caused by insufficient insulin secretion, which comprises administering to a mammal an effective amount of a substance that inhibits the action of an IgE-dependent histamine releasing factor; [10] a mammal Against glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia. , Sexual dysfunction, skin disease, arthropathy or osteopenia prevention and treatment method,

[11] A method for preventing and treating diabetes, which comprises administering to a mammal an effective amount of a substance that inhibits the action of an IgE-dependent histamine releasing factor, [1]
2] use of a substance that inhibits the action of an IgE-dependent histamine releasing factor for producing an insulin secretagogue;
[13] use of a substance that inhibits the action of IgE-dependent histamine releasing factor for producing a prophylactic / therapeutic agent for a disease caused by insufficient insulin secretion, [14] impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, Diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease,
Use of a substance that inhibits the action of an IgE-dependent histamine releasing factor for producing a prophylactic / therapeutic agent for arthropathy or osteopenia; [15] IgE-dependent histamine release for producing a prophylactic / therapeutic agent for diabetes Use of substances that inhibit the action of factors,

[16] an insulin secretion inhibitor comprising an IgE-dependent histamine-releasing factor or a substance having the action thereof, [17] a substance having an IgE-dependent histamine-releasing factor acting as an IgE-dependent histamine-releasing factor Item [16], which is a receptor agonist, a substance that enhances or promotes the action of an IgE-dependent histamine releasing factor, a substance that promotes the expression of an IgE-dependent histamine releasing factor, or a substance that inhibits the degradation of an IgE-dependent histamine releasing factor. The agent of [16] or [17], wherein the IgE-dependent histamine-releasing factor is a tumor-associated protein; [19] the agent of the item [19], wherein the IgE-dependent histamine-releasing factor is a tumor-associated protein p21; Protein p2
The agent according to item [16] or [17], which is 3 or tumor-associated protein p26, [20] the agent according to item [16], which is a preventive or therapeutic agent for a disease caused by excessive insulin secretion,

[21] Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance, unstable diabetes, lipoatrophy, insulin allergy Or the agent according to item [16], which is an agent for preventing or treating insulinoma; [22] administering to a mammal an effective amount of an IgE-dependent histamine releasing factor or a substance having the action thereof. A method for preventing or treating a disease caused by excessive insulin secretion, [23] obesity or hyperlipidemia, comprising administering to a mammal an effective amount of an IgE-dependent histamine releasing factor or a substance having the action thereof. Disease, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance, unstable diabetes, lipoatrophy, insulin Allergic or insulinoma method preventing and treating, [24] the use of substances with IgE-mediated histamine releasing factor or its effects for the production of an agent for the prophylaxis or treatment of diseases caused by insulin hypersecretion,
[25] Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance, unstable diabetes, lipodystrophy, insulin allergy or insulinoma Use of an IgE-dependent histamine releasing factor or a substance having an action thereof for producing a prophylactic / therapeutic agent,

[26] a method for screening for an insulin secretion regulator, which comprises using an IgE-dependent histamine releasing factor and / or an IgE-dependent histamine releasing factor receptor; [27] encoding an IgE-dependent histamine releasing factor [28] a method for screening for an insulin secretion regulatory substance, which comprises using a DNA that encodes an IgE-dependent histamine-releasing factor receptor and / or a DNA that encodes an IgE-dependent histamine-releasing factor receptor; A kit for screening for an insulin secretion regulatory substance comprising a release factor receptor, [29] a DNA encoding an IgE-dependent histamine release factor and / or
D encoding the IgE-dependent histamine releasing factor receptor
A kit for screening for an insulin secretion regulatory substance comprising NA, the screening method according to [30], [26] or [27], or the screening method according to [28] or [29]. Insulin secretion modulator obtainable using a screening kit,

[31] Contains an insulin secretion regulatory substance obtainable by using the screening method of [26] or [27] or the screening kit of [28] or [29]. Medicine
[32] the medicament according to [31], which is an insulin secretion regulator; [33] the drug [3], which is a preventive or therapeutic agent for diabetes;
1) the pharmaceutical according to item [34];
[6] An effective amount of an insulin secretion modulator obtainable by using the screening method according to [27] or the screening kit according to [28] or [29]. A method for regulating insulin secretion, [35] using a screening method according to [26] or [27] or a screening kit according to [28] or [29] for mammals. A method for preventing and treating diabetes, comprising administering an effective amount of an insulin secretion regulator that can be obtained,

[36] A diagnostic agent for abnormal insulin secretion characterized by containing an antibody against IgE-dependent histamine releasing factor, [37] An abnormal insulin secretion characterized by using an antibody against IgE-dependent histamine releasing factor [38] A diagnostic agent for abnormal insulin secretion comprising DNA encoding an IgE-dependent histamine releasing factor, and [39] IgE
The present invention relates to a method for diagnosing abnormal insulin secretion, which comprises using a DNA encoding a dependent histamine releasing factor.

Furthermore, the present invention relates to the present invention, wherein the [40] IgE-dependent histamine releasing factor has a molecular weight of about 21 kDa to 26 kDa.
The agent according to [1], [2] or [16], which is Da; [41] the IgE-dependent histamine-releasing factor is selected from the group consisting of tumor-associated protein p21, tumor-associated protein p23, and tumor-associated protein p26. One or more (preferably about 1 to 30, more preferably about 1 to 9, and still more preferably about 1 to 9 amino acids in the amino acid sequence of the amino acid sequence of the tumor-associated protein p21, the tumor-associated protein p23, or the tumor-associated protein p26. (1 to 5) amino acid sequences, one or two or more amino acids (preferably about 1 to 30) in the amino acid sequence of tumor-associated protein p21, tumor-associated protein p23 or tumor-associated protein p26. More preferably about 1 to 10, more preferably several (1 to 5) amino acids added to the amino acid sequence, tumor-associated protein p21, tumor Related protein p23 or one or more than one amino acid sequence of tumor-associated protein p26 (preferably, about 1 to about 30, more preferably about 1 to 10, more preferably several (1 to 5
The agent according to item [1], [2] or [16], which is a protein comprising an amino acid sequence in which the amino acid sequence of (1) is replaced with another amino acid, or an amino acid sequence obtained by combining the amino acid sequences;

[42] (i) the case where an IgE-dependent histamine releasing factor is brought into contact with its receptor;
The screening method according to [26], wherein a comparison is made between the case where the E-dependent histamine releasing factor and its receptor and a test compound are brought into contact with each other,

[43] (i) a case where a labeled IgE-dependent histamine releasing factor is brought into contact with an IgE-dependent histamine releasing factor receptor; and (ii) a case where a labeled IgE-dependent histamine releasing factor and a test compound are IgE-dependent. IgE-dependent histamine-releasing factor characterized by measuring the amount of binding of a labeled IgE-dependent histamine-releasing factor to an IgE-dependent histamine-releasing factor receptor when brought into contact with an histamine-releasing factor receptor A method for screening a compound or a salt thereof (insulin secretion regulatory substance) that alters the binding property between the compound and an IgE-dependent histamine releasing factor receptor,

[44] (i) When a labeled IgE-dependent histamine releasing factor is brought into contact with cells containing an IgE-dependent histamine releasing factor receptor; Compound I
An IgE-dependent histamine-releasing factor characterized by measuring and comparing the amount of labeled IgE-dependent histamine-releasing factor bound to the cell when the cell is contacted with a cell containing a gE-dependent histamine-releasing factor receptor. A method for screening for a compound or a salt thereof (insulin secretion regulatory substance) that alters the binding property between the compound and an IgE-dependent histamine releasing factor receptor,

[45] (i) contacting a labeled IgE-dependent histamine-releasing factor with a membrane fraction of cells containing an IgE-dependent histamine-releasing factor receptor, and (ii)
When the labeled IgE-dependent histamine releasing factor and the test compound are contacted with the membrane fraction of cells containing the IgE-dependent histamine releasing factor receptor, the labeled I
A compound or a salt thereof that changes the binding between an IgE-dependent histamine-releasing factor and an IgE-dependent histamine-releasing factor receptor, wherein the amount of binding of the gE-dependent histamine-releasing factor to the cell is measured and compared. Insulin secretion modulator) screening method,

[46] (i) contacting a compound that activates an IgE-dependent histamine-releasing factor receptor with cells containing the IgE-dependent histamine-releasing factor receptor; and (ii) IgE-dependent histamine-releasing factor release. When a compound that activates a factor receptor and a test compound are brought into contact with a cell containing the IgE-dependent histamine releasing factor receptor, the cell stimulating activity via the IgE-dependent histamine releasing factor receptor is measured, and compared. I characterized by
a method for screening a compound or a salt thereof (insulin secretion regulatory substance) that changes the binding between a gE-dependent histamine releasing factor and an IgE-dependent histamine releasing factor receptor,

[47] The compound that activates the IgE-dependent histamine releasing factor receptor is a tumor-associated protein p21,
The screening method according to item [46], which is tumor-associated protein p23 or tumor-associated protein p26; [48] an IgE-dependent histamine releasing factor obtainable by the screening method according to item [42] to item [47]. A compound or a salt thereof (insulin secretion regulatory substance) that changes the binding to an IgE-dependent histamine releasing factor receptor, [4
9] IgE-dependent histamine-releasing factor and I obtainable by the screening method of [42] to [47].
Compound or salt thereof that alters binding to gE-dependent histamine releasing factor receptor (insulin secretion regulator)
A medicine characterized by containing

[50] (i) contacting a compound that activates an IgE-dependent histamine releasing factor receptor with an insulin-secreting cell; (ii) a compound that activates an IgE-dependent histamine releasing factor receptor; A method for screening for an insulin secretion regulatory substance, comprising measuring and comparing the amount of secreted insulin when a test compound is brought into contact with insulin-secreting cells, [51]
(I) blood of a non-human mammal, a specific organ (eg, pancreas), a tissue (eg, islet of Langerhans) or a cell (eg, pancreatic β cell, MIN6 cell) isolated from the organ, or (ii) a transformant TCTP or TCT contained in
TCTP by measuring the amount of P receptor mRNA
Or a method for screening a compound that changes the expression level of a TCTP receptor,

[52] Insulin secretion regulator (including a compound which changes the expression level of TCTP or TCTP receptor), which can be obtained by the screening method according to [50] or [51], [53] [50] or an insulin secretion regulator containing an insulin secretion modulator obtainable by the screening method according to [51] (including a compound that changes the expression level of TCTP or TCTP receptor), [54] An antibody against IgE-dependent histamine releasing factor, a test solution and labeled I
reacting gE-dependent histamine releasing factor competitively;
A method for quantifying an IgE-dependent histamine-releasing factor in a test solution, which comprises measuring the ratio of a labeled IgE-dependent histamine-releasing factor bound to the antibody; Reacting the antibody to the insolubilized IgE-dependent histamine-releasing factor and the labeled antibody to the IgE-dependent histamine-releasing factor simultaneously or continuously, and then measuring the activity of the labeling agent on the insolubilized carrier. The present invention provides a method for quantifying an IgE-dependent histamine releasing factor in a test solution, and a method for diagnosing abnormal insulin secretion according to the item [54] or [55].

[0027]

BEST MODE FOR CARRYING OUT THE INVENTION Examples of the IgE-dependent histamine releasing factor (IgE-dependent HRF) include tumors of mammals (eg, human, guinea pig, rat, mouse, rabbit, pig, sheep, cow, monkey, etc.). IgE-dependent HRF derived from cells, keratinocytes, liver function, lymphocytes, platelets, macrophages, etc., IgE-dependent HRF isolated from yeast or plants, synthetic IgE-dependent HR
F, a recombinant IgE-dependent HRF produced from an IgE-dependent HRF gene and the like are used. IgE dependence H
Although the molecular weight of RF is not particularly limited, for example, about 21 k
Da to about 30 kDa, preferably about 21 kDa to about 26
kDa, particularly preferably about 26 kDa.

Specifically, the IgE-dependent HRF includes:
Tumor-associated protein (hereinafter referred to as TCTP) p21 (Mol. Cell. Biol.), No. 3
Volume, 1197-1203), TCTP p23 (Nucleic Acids Res.),
Vol. 16, 2350), TCTP p26 (Mol. Cell. Biol.), No. 3
Vol. 1197-1203) and the like.

These TCTP p21 (mouse type), T
CTP p23 (human type) and TCTP p26 (mouse type) are known substances, and can be produced by a method known per se, or commercially available ones can be used. For example, mouse and human TCTPs can be synthesized using genetic engineering techniques, for example, Nucleic Acids Res., Vol. 17, 8367 and Nucleic Acids Research.
s.), vol. 16, 2350, respectively.
A can be manufactured. Furthermore, in the present invention, IgE-dependent H
RF can also be used.

Also, IgE-dependent HRF is TCTP
It may be a protein containing the same or substantially the same amino acid sequence as the amino acid sequence of p21, TCTP p23 or TCTP p26. The amino acid sequence of TCTP p21 is represented by SEQ ID NO: 1, the amino acid sequence of TCTP p23 is represented by SEQ ID NO: 2, and the amino acid sequence of TCTP p26 is represented by SEQ ID NO: 3. TCTP p2
1, As an amino acid sequence substantially the same as the amino acid sequence of TCTP p23 or TCTP p26, for example,
TCTP p21, TCTP p23 or TCTP p
26 amino acid sequences and about 50% or more, preferably about 60%
% Or more, more preferably about 70% or more, still more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.

Examples of proteins containing an amino acid sequence substantially identical to the amino acid sequence of TCTP p21, TCTP p23 or TCTP p26 include, for example, TCTP
containing an amino acid sequence substantially identical to the amino acid sequence of p21, TCTP p23 or TCTP p26;
CTP p21, TCTP p23 or TCTP p2
A protein having substantially the same activity as that of No. 6 is preferred.

The activity substantially the same as that of TCTP p21, TCTP p23 or TCTP p26 includes, for example, ligand binding activity, signal transduction activity, insulin secretion inhibitory activity and the like. Substantially the same indicates that their activities are the same in nature. Therefore, ligand binding activity, signal transduction action,
Activities such as insulin secretion inhibition are equivalent (eg, about 0.01
To 100 times, preferably about 0.5 to 20 times, and more preferably about 0.5 to 2 times), but the quantitative factors such as the degree of activity and the molecular weight of the protein are different. Is also good.

The activities such as ligand binding activity, signal transduction activity and insulin secretion inhibitory activity can be measured according to a method known per se, for example, according to a screening method described later.

Also, IgE-dependent HRF is TCTP
One or more (preferably 1 to 3) in the amino acid sequence of p21, TCTP p23 or TCTP p26
An amino acid sequence in which about 0, more preferably about 1 to 10, and still more preferably several (1 to 5) amino acids have been deleted, TCTP p21, TCTP p23 or TTP
An amino acid sequence in which one or more (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5)) amino acids are added to the amino acid sequence of CTP p26; TCTP p21, TCT
1 in the amino acid sequence of Pp23 or TCTP p26
Or two or more (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to
5)) may be a protein containing an amino acid sequence in which the amino acid is substituted with another amino acid, or an amino acid sequence obtained by combining them.

The IgE-dependent HRF has the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end, according to the convention of peptide labeling. TCTP p21, T
I including CTP p23 or TCTP p26
The gE-dependent HRF has a carboxyl group (-CO
OH), carboxylate (—COO ⁻ ), amide (—C
ONH ₂ ) or an ester.

The IgE-dependent HRF includes all products of the IgE-dependent HRF gene, and is subject to modifications such as splicing variants, phosphorylation, acylation, addition of sugar chains, and protease processing of the gene. All proteins or their partial peptides are also included.

The partial peptide of IgE-dependent HRF is the above-mentioned peptide fragment of IgE-dependent HRF.
Those having substantially the same activity as gE-dependent HRF are included. The molecular weight of the partial peptide, the number of constituent amino acids, and the like are not particularly limited.

The partial peptide can be produced according to a peptide synthesis method known per se or by cleaving an IgE-dependent HRF with an appropriate peptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, I
The target peptide can be produced by condensing a partial peptide or amino acid capable of constituting a gE-dependent HRF with the remaining portion, and removing the protecting group when the product has a protecting group. Known methods for condensation and elimination of protecting groups include, for example, the methods described in (1) to (4) below. M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptide,
Academic Press, NewYork (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Biochemistry Laboratory Course 1, Protein Chemistry IV, 205, (1977) Supervised by Haruaki Yajima, Development of Continuing Drugs Volume 14 Peptide Synthesis Hirokawa Shoten

After the reaction, the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and when obtained by a salt, it can be converted to a free form by a known method. Can be. Polynucleotides encoding IgE-dependent HRFs such as TCTP p21, TCTP p23 or TCTP p26 include the IgE described above.
Nucleotide sequence (DNA or RN)
A, preferably DNA). As the polynucleotide,
It is RNA such as DNA or mRNA encoding IgE-dependent HRF, and may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).

Using a polynucleotide encoding an IgE-dependent HRF, for example, a known experimental medical special edition “New P
CR and its application ”, 15 (7), 1997, or a method analogous thereto, using the mR of IgE-dependent HRF.
NA can be quantified. Examples of the DNA encoding the IgE-dependent HRF include the aforementioned IgE-dependent HRF.
May be any as long as it contains a base sequence encoding In addition, genomic DNA, genomic DN
A library, cDNA derived from the cells and tissues described above,
Any of the above-mentioned cell / tissue-derived cDNA library and synthetic DNA may be used.

The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Also, Reverse Transcriptase Polymerase Chain R is directly used by using a total RNA or mRNA fraction prepared from the cells and tissues described above.
It can also be amplified by eaction (hereinafter abbreviated as RT-PCR method).

Specifically, the DNA encoding TCTP p21 contains, for example, a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by SEQ ID NO: 4, and is substantially similar to TCTP p21. Any DNA may be used as long as it encodes a peptide having the same activity. Examples of the base sequence capable of hybridizing with the base sequence represented by SEQ ID NO: 4 under high stringent conditions include, for example, about 70% or more, preferably about 80% with the base sequence represented by SEQ ID NO: 4
% Or more, more preferably about 90% or more, and still more preferably about 95% or more.

The DNA encoding TCTP p23 contains, for example, a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by SEQ ID NO: 5, and has substantially the same activity as TCTP p23. Any DNA may be used as long as it encodes a peptide. Examples of the base sequence capable of hybridizing with the base sequence represented by SEQ ID NO: 5 under high stringency conditions include, for example, about 70% or more, preferably about 80% or more, with the base sequence represented by SEQ ID NO: 5. Preferably, a nucleotide sequence having a homology of about 90% or more, more preferably about 95% or more is used.

The DNA encoding TCTP p26 contains, for example, a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by SEQ ID NO: 6, and has substantially the same activity as TCTP p26. Any DNA may be used as long as it encodes a peptide. Examples of the base sequence capable of hybridizing with the base sequence represented by SEQ ID NO: 6 under high stringent conditions include, for example, about 70% or more, preferably about 80% or more, with the base sequence represented by SEQ ID NO: 6 Preferably, a nucleotide sequence having a homology of about 90% or more, more preferably about 95% or more is used. Hybridization can be performed by a method known per se or a method analogous thereto, for example, molecular cloning (Mole
cular Cloning) 2nd (J. Sambrook et al., Cold Sprin)
g Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions.

The high stringency conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C. Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable. More specifically, (i) as the DNA encoding TCTP p21 containing the amino acid sequence represented by SEQ ID NO: 1, a DNA containing the base sequence represented by SEQ ID NO: 4 or the like is used. As the DNA encoding TCTP p23 containing the amino acid sequence represented by SEQ ID NO: 2, a DNA containing the base sequence represented by SEQ ID NO: 5 and the like are used, and (iii) SEQ ID NO: 3
TCTP p26 containing the amino acid sequence represented by
As the DNA encoding is used a DNA containing the base sequence represented by SEQ ID NO: 6, and the like.

The DNA encoding the partial peptide of the IgE-dependent HRF may be any DNA as long as it contains the nucleotide sequence encoding the partial peptide of the IgE-dependent HRF. Genome DN
A, any of the genomic DNA library, the cDNA derived from the aforementioned cells / tissues, the cDNA library derived from the aforementioned cells / tissues, and the synthetic DNAs may be used.

Specifically, the DNA encoding the partial peptide of TCTP p21 includes, for example, SEQ ID NO:
A DNA having a partial nucleotide sequence of a DNA containing the nucleotide sequence represented by SEQ ID NO: 4 or a nucleotide sequence hybridizing under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 4; substantially similar to TCTP p21 For example, a DNA having a partial base sequence of a DNA encoding a peptide having the same activity can be used.

The DNA encoding the partial peptide of TCTP p23 is, for example, a DNA having a partial nucleotide sequence of a DNA containing the nucleotide sequence represented by SEQ ID NO: 5.
NA or a partial nucleotide sequence of a DNA containing a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 5 under high stringent conditions and encoding a peptide having substantially the same activity as TCTP p23. DNA or the like is used.

The DNA encoding the partial peptide of TCTP p26 is, for example, a DNA having the partial nucleotide sequence of
NA or a partial nucleotide sequence of a DNA containing a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 6 under high stringent conditions and encoding a peptide having substantially the same activity as TCTP p26. DNA or the like is used.

D capable of hybridizing with the nucleotide sequence represented by SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6
NA is as defined above. The same hybridization method and high stringency conditions as described above are used.

The substance that inhibits the action of IgE-dependent HRF is not particularly limited as long as it can directly or indirectly inhibit the insulin secretion inhibitory action of IgE-dependent HRF. Specifically, substances that inhibit the action of IgE-dependent HRF include receptor antagonists for IgE-dependent HRF, antibodies to IgE-dependent HRF, and IgE-dependent HRF.
A substance that inhibits (suppresses) the action or expression of IgE-dependent HRF, such as a substance that suppresses the expression of E-dependent HRF, a substance that degrades IgE-dependent HRF, and a substance that promotes degradation of IgE-dependent HRF, is used.

An IgE-dependent HRF receptor antagonist is defined as an IgE-dependent HRF receptor that binds to an IgE-dependent HRF receptor.
It refers to a substance that inhibits the binding between E-dependent HRF and an IgE-dependent HRF receptor but does not have an IgE-dependent HRF action, and may be a known substance or a novel substance to be found in the future.

The antibody against IgE-dependent HRF may be either a polyclonal or monoclonal antibody against IgE-dependent HRF.
It can be produced according to a known method for producing an antibody or antiserum using dependent HRF as an antigen. Specifically, it can be produced according to the following method.

[Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The IgE-dependent HRF is administered to a mammal at a site where the antibody can be produced by administration, itself or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of the mammal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats.
Mice and rats are preferably used.

In preparing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, a mouse having an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization. A monoclonal antibody-producing hybridoma can be prepared by fusing the antibody-producing cells contained therein with myeloma cells. The measurement of the antibody titer in the antiserum can be performed, for example, using labeled IgE.
After reacting the dependent HRF with the antiserum, the activity can be measured by measuring the activity of the labeling agent bound to the antibody. The fusion operation is performed by a known method, for example, the method of Kohler and Milstein [Nature, 256, 4
95 (1975)].
Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.

Examples of myeloma cells include NS-1,
P3U1, SP2 / 0 and the like can be mentioned, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is 1: 1 to 2
0: 1 and PEG (preferably PEG100
0-PEG6000) at a concentration of about 10-80%, and incubating at about 20-40 ° C, preferably about 30-37 ° C for about 1-10 minutes, allows efficient cell fusion.

Various methods can be used for screening a monoclonal antibody-producing hybridoma. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which an IgE-dependent HRF antigen is adsorbed directly or together with a carrier. Then, an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (an anti-mouse immunoglobulin antibody is used when the cells used for cell fusion are mice) or protein A is added, and the monoclonal antibody bound to the solid phase is added. Detection method, adding hybridoma culture supernatant to solid phase to which anti-immunoglobulin antibody or protein A is adsorbed, adding IgE-dependent HRF labeled with radioactive substance, enzyme, etc., and detecting monoclonal antibody bound to solid phase And the like.

The monoclonal antibody can be selected according to a method known per se or a method analogous thereto.
Thymidine) in an animal cell culture medium or the like. As a selection and breeding medium, any medium can be used as long as hybridomas can grow. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, 1-10%
Medium containing fetal calf serum (Wako Pure Chemical Industries, Ltd.)
Alternatively, a serum-free medium for hybridoma culture (SFM-10
1, Nissui Pharmaceutical Co., Ltd.) can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(B) Purification of Monoclonal Antibody Separation and purification of the monoclonal antibody can be performed by the same method as in the separation and purification of normal polyclonal antibodies, for example, by separation and purification of immunoglobulin [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, Electrophoresis, adsorption / desorption using an ion exchanger (eg, DEAE), ultracentrifugation, gel filtration, antigen-bound solid phase or active adsorbent such as protein A or protein G to collect only antibody and dissociate the bond Specific purification method for obtaining an antibody).

[Preparation of Polyclonal Antibody] A polyclonal antibody against IgE-dependent HRF can be produced according to a method known per se or a method analogous thereto. For example, a complex of an immune IgE-dependent HRF antigen and a carrier protein is formed, a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody, and an antibody containing IgE-dependent HRF is collected from the immunized animal. The antibody can be produced by separating and purifying the antibody.

Regarding the complex of an immunizing antigen and a carrier protein used for immunizing a mammal, the type of the carrier protein and the mixing ratio between the carrier and the hapten are determined by the antibody against the hapten immunized by crosslinking the carrier. Any material may be cross-linked at any ratio, if possible, for example, bovine serum albumin,
Bovine thyroglobulin, keyhole, limpet, hemocyanin, etc. in a weight ratio of about 0.1 to 2 with respect to 1 hapten
A method of coupling at a rate of 0, preferably about 1 to 5 is used.

For coupling the hapten and the carrier, various condensing agents can be used. For example, glutaraldehyde, carbodiimide, maleimide active ester, active ester reagent containing a thiol group or a dithiopyridyl group and the like are used.

The condensed product is administered to a warm-blooded animal at a site capable of producing an antibody by itself or together with a carrier and a diluent. In order to enhance antibody production ability at the time of administration,
Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered. The administration can usually be performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.

The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of the mammal immunized by the above-mentioned method. The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the serum described above. The separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.

The IgE-dependent HRF expression inhibitor is
The substance is not particularly limited as long as it suppresses the expression of the IgE-dependent HRF.
Gene expression inhibitors, IgE-dependent HRF gene promoter inhibitors, IgE-dependent HRF mRNA expression inhibitors, IgE-dependent HRF mRNA translation inhibitors, IgE-dependent HRF secretion inhibitors, etc. are used. Specifically, an antisense DNA against the DNA encoding the above IgE-dependent HRF is preferably used.

Antisense DNA having a base sequence complementary to or substantially complementary to the DNA encoding IgE-dependent HRF or a part thereof includes IgE-dependent HRF.
Any antisense DNA may be used as long as it has a base sequence complementary to or substantially complementary to the DNA encoding RF or a part thereof and has an action capable of suppressing the expression of the DNA. Good.

The nucleotide sequence substantially complementary to the DNA encoding the IgE-dependent HRF is, for example, a nucleotide sequence complementary to the DNA encoding the IgE-dependent HRF (that is, the complementary strand of the DNA of the present invention). About 70% or more, preferably about 80% or more,
More preferably about 90% or more, most preferably about 95%
Base sequences having the above homology are exemplified. In particular, of the entire base sequence of the complementary strand of the DNA encoding the IgE-dependent HRF, the complementary sequence of the base sequence of the portion encoding the N-terminal site of the IgE-dependent HRF (for example, the base sequence near the start codon, etc.) 70% or more, preferably about 8
Antisense DNA having a homology of 0% or more, more preferably about 90% or more, and most preferably about 95% or more is suitable. These antisense DNAs have the known D
It can be manufactured using an NA synthesizer or the like.

As a substance that degrades IgE-dependent HRF, for example, an IgE-dependent HRF-degrading enzyme is used. As a substance that promotes the degradation of IgE-dependent HRF, for example, an activity-promoting substance of an IgE-dependent HRF-degrading enzyme is used.

The substance having an IgE-dependent HRF action is not particularly limited as long as it has an IgE-dependent HRF action (eg, insulin secretion inhibitory action).
Specifically, the substance having the action of IgE-dependent HRF includes a receptor agonist of IgE-dependent HRF (Ig-dependent HRF).
E-dependent HRF itself) or a DNA encoding the same, a substance that enhances or promotes the action of IgE-dependent HRF, a substance that promotes expression of IgE-dependent HRF, IgE
Substances that inhibit the degradation of dependent HRF are used.

An IgE-dependent HRF receptor agonist refers to a substance that binds to an IgE-dependent HRF receptor and has the same action as an IgE-dependent HRF. It may be. Specifically, the above-mentioned TCTP p21, TCTP
p23, TCTP p26 and the like are used. TCTP
Ig such as p21, TCTP p23, TCTP p26
As the DNA encoding the E-dependent HRF, the same DNA as described above is used.

As the substance that enhances or promotes the action of IgE-dependent HRF, a substance that directly or indirectly enhances or promotes the insulin secretion inhibitory action of IgE-dependent HRF is used. Specifically, IgE-dependent HR
Examples of the substance include a substance that promotes the binding of F to an IgE-dependent HRF receptor, a substance that activates an IgE-dependent HRF, and a substance that enhances the stability of an IgE-dependent HRF.

The substances promoting IgE-dependent HRF expression include:
The substance is not particularly limited as long as it promotes the expression of the above-mentioned IgE-dependent HRF.
Gene expression promoter, IgE-dependent HRF gene promoter activator, IgE-dependent HRF mRNA
, An expression promoter for IgE-dependent HRF mRNA, a promoter for secretion of IgE-dependent HRF, and the like. As a substance that inhibits degradation of IgE-dependent HRF, for example, an inhibitor of an IgE-dependent HRF-degrading enzyme is used.

The above-mentioned IgE-dependent HRF, a substance that inhibits the action of IgE-dependent HRF, and a substance that has the action of IgE-dependent HRF may form a salt, and are physiologically acceptable with an acid or a base. And especially preferred are physiologically acceptable acid addition salts. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,
For example, salts with methanesulfonic acid and benzenesulfonic acid) are used.

IgE-dependent HRF is a novel inhibitor of insulin secretion and is also known as a proinflammatory factor. Therefore, a substance that inhibits the action of IgE-dependent HRF is useful as an insulin secretagogue or a preventive or therapeutic agent for diabetes.

Specifically, a pharmaceutical composition containing a substance that inhibits the action of IgE-dependent HRF can be used for a disease caused by impaired insulin secretion (secretion inhibition), such as diabetes, impaired glucose tolerance, ketosis, acidosis, It can be used as a preventive / therapeutic agent for diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy, osteopenia and the like.

In particular, a pharmaceutical composition containing IgE-dependent HRF or a substance that inhibits its action is useful for preventing diabetes.
Useful as a therapeutic. Diabetes includes insulin dependent (type I) diabetes, non-insulin dependent (type II) diabetes and the like.

On the other hand, IgE-dependent HRF or a substance having an action thereof is useful as an insulin secretion inhibitor or an insulin synthesis inhibitor. Insulin secretion inhibition includes inhibiting normal secretion of insulin, lowering abnormal secretion of insulin to a normal value, and the like. The degree of secretion inhibition is, for example, about 10 to 100%, preferably about 30 to 100%.

Specifically, a pharmaceutical composition containing IgE-dependent HRF or a substance having the action thereof is useful for treating diseases caused by excessive insulin secretion, such as obesity, hyperlipidemia,
Can be used as a preventive or therapeutic agent for type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, etc. it can.

The above pharmaceutical composition can be safely used for mammals (eg, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, human, etc.). The above pharmaceutical composition can be prepared according to a method known per se which is generally used in a method for producing a pharmaceutical preparation.
A substance that inhibits the action of E-dependent HRF or an IgE-dependent HRF or a substance that has the action, as it is, or mixed with a pharmacologically acceptable carrier, for example,
Tablets (including sugar-coated tablets, film-coated tablets), powders, granules, capsules, (including soft capsules),
It can be safely administered orally or parenterally (eg, topically, rectally, intravenously, etc.) as a pharmaceutical preparation such as a liquid, injection, suppository, sustained release agent and the like.

When a DNA encoding an IgE-dependent HRF or an antisense DNA is used as the above-mentioned therapeutic or prophylactic agent, the DNA may be used alone or in a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector or the like. After insertion into a suitable vector, it can be administered to humans or warm-blooded animals according to conventional means. The antisense DNA can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and administered with a gene gun or a catheter such as a hydrogel catheter.

The content of the substance inhibiting the action of IgE-dependent HRF or the substance having IgE-dependent HRF or the action thereof in the pharmaceutical composition is about 0.01 to about 100% by weight of the whole preparation.

The content of the substance inhibiting the action of IgE-dependent HRF or the ingredient other than IgE-dependent HRF or the substance having the action in the pharmaceutical composition is about 10 to about 99.9% by weight of the whole preparation. is there.

The pharmaceutical composition containing the substance inhibiting the action of IgE-dependent HRF or the substance having the action of IgE-dependent HRF of the present invention can be used for other therapeutic agents for diabetes, diabetic complications, Antihypertensive, antihypertensive,
It can be used in combination with a drug such as an anti-obesity drug, a diuretic, a chemotherapeutic drug, or an immunotherapy drug (hereinafter sometimes abbreviated as a concomitant drug). At this time, the administration time of the preparation of the present invention and the concomitant drug is not limited, and they may be administered to the administration subject simultaneously or at an interval. The dose of the concomitant drug can be appropriately determined based on the clinically used dose. In addition, the compounding ratio of the substance that inhibits the action of IgE-dependent HRF or the substance that has IgE-dependent HRF or the action thereof and the concomitant drug used in the pharmaceutical composition of the present invention depends on the administration subject, administration route, target disease, and symptom. , Can be appropriately selected depending on the combination and the like. For example, when the administration subject is a human, the concomitant drug may be used in an amount of 0.01 to 100 parts by weight per 1 part by weight of the substance that inhibits the action of IgE-dependent HRF.

Other therapeutic agents for diabetes include insulin preparations (eg, animal insulin preparations extracted from bovine and porcine pancreas; human insulin preparations genetically synthesized using Escherichia coli and yeast; insulin zinc; protamine insulin Zinc; a fragment or derivative of insulin (eg, INS-1 etc.), an insulin sensitivity enhancer (eg, pioglitazone hydrochloride, troglitazone, rosiglitazone or maleate thereof, JTT-501, M
CC-555, YM-440, GI-262570, K
RP-297, FK-614, CS-011, etc.), α-
Glucosidase inhibitors (eg, voglibose, acarbose, miglitol, emiglitate, etc.), biguanides (eg, phenformin, metformin, buformin, etc.), sulfonylureas (eg, tolbutamide, glibenclamide, gliclazide, chlorpropamide, tolazamide, acetohexamide) Glyclopyramide, glimepiride, etc.) and other insulin secretagogues (eg, repaglinide, senaglinide, mitiglinide or its calcium salt hydrate, GLP-1, nateglinide, etc.), dipeptidyl peptidase IV inhibitors (eg, NVP-DPP) −
278, PT-100, P32 / 98, etc.), β3 agonists (eg, CL-316243, SR-58611-11)
A, UL-TG-307, AJ-9677, AZ401
40), amylin agonists (eg, pramlintide), phosphotyrosine phosphatase inhibitors (eg, vanadic acid), gluconeogenesis inhibitors (eg, glycogen phosphorylase inhibitors, glucose-6-phosphatase inhibitors, glucagon antagonists, etc.) , SGLT (sodium-glucose
cotransporter) inhibitors (eg, T-1095, etc.) and the like.

Examples of the therapeutic agent for diabetic complications include aldose reductase inhibitors (eg, tolrestat, epalrestat, zenarestat, zopolrestat, fidarestat (SNK-860), and minalrestat (ARI).
-509), CT-112, etc.), neurotrophic factors (eg, N
GF, NT-3, etc.), AGE inhibitors (eg, ALT-94)
5, pimagedine, pyratoxatin, N-phenacylthiazolium bromide (ALT-766), EXO-226
And the like, active oxygen scavengers (eg, thioctic acid, etc.), cerebral vasodilators (eg, thioprid, etc.) and the like.

As antihyperlipidemic agents, statin compounds which are cholesterol synthesis inhibitors (eg, pravastatin,
Simvastatin, lovastatin, atorvastatin, fluvastatin, cerivastatin or a salt thereof (eg,
Sodium salts, etc.), fibrate compounds having a squalene synthase inhibitor or triglyceride lowering action (eg, bezafibrate, clofibrate, simfibrate, clinofibrate, etc.).

Examples of antihypertensive agents include angiotensin converting enzyme inhibitors (eg, captopril, enalapril, delapril, etc.), angiotensin II antagonists (eg, losartan,
Candesartan, cilexetil, etc.), calcium antagonists (eg, manidipine, nifedipine, amlodipine, efonidipine, nicardipine, etc.), clonidine and the like.

Examples of the antiobesity agent include central antiobesity agents (eg, dexfenfluamine, fenfluramine, phentermine, sibutramine, ampepramone, dexamphetamine, mazindol, phenylpropanolamine, clobenzolex, etc.), Pancreatic lipase inhibitors (eg, orlistat etc.), β3 agonists (eg, CL
-316243, SR-58611-A, UL-TG-
307, AJ-9677, AZ40140, etc.), peptidic appetite suppressants (eg, leptin, CNTF (ciliary neurotrophic factor), etc.), cholecystokinin agonists (eg, lynch tryp, FPL-15849, etc.) and the like. .

Examples of diuretics include xanthine derivatives (eg, sodium theobromine salicylate, calcium theobromine salicylate, etc.), thiazide-based preparations (eg, ethiazide, cyclopentiazide, trichlormethiazide, hydrochlorothiazide, hydroflumethiazide, benzylhydrochlorothiazide, penflutizide) , Polythiazide, methyclothiazide, etc.), anti-aldosterone preparations (eg, spironolactone, triamterene, etc.), carbonic anhydrase inhibitors (eg, acetazolamide, etc.), chlorobenzenesulfonamide preparations (eg, chlorthalidone, mefluside, indapamide, etc.), azosemide , Isosorbide,
Examples include ethacrynic acid, piretanide, bumetanide, furosemide and the like.

Examples of chemotherapeutic agents include alkylating agents (eg, cyclophosphamide, ifosfamide, etc.), antimetabolites (eg, methotrexate, 5-fluorouracil, etc.), anticancer antibiotics (eg, mitomycin, adriamycin) And the like, plant-derived anticancer agents (eg, vincristine, vindesine, taxol, etc.), cisplatin, carboplatin, etopoxide and the like.
Of these, furtulon or neofurturon, which is a 5-fluorouracil derivative, is preferred.

Examples of the immunotherapeutic agent include a microorganism or bacterial component (eg, muramyl dipeptide derivative, picibanil, etc.), a polysaccharide having an immunopotentiating activity (eg, lentinan,
Schizophyllan, krestin, etc.), cytokines obtained by genetic engineering techniques (eg, interferon, interleukin (IL), etc.), colony stimulating factors (eg, granulocyte colony stimulating factor, erythropoietin, etc.). -1, IL-2, IL-12 and the like are preferable.

Furthermore, drugs which have been shown to improve cachexia in animal models and clinically, ie, cyclooxygenase inhibitors (eg, indomethacin etc.) [Cancer Research, Vol. 49, 5935-59]
39, 1989], progesterone derivatives (eg, megesterol acetate) [Journal of Clinical Oncolog
y), Vol. 12, pp. 213-225, 1994], carbohydrate steroids (eg, dexamethasone, etc.), metoclopramide drugs, tetrahydrocannabinol drugs (all references are as described above), fat metabolism improvers ( For example, eicosapentaenoic acid, etc.) [British Journal of Cancer, No. 68
314-318, 1993], growth hormone,
IGF-1 or TN, a factor that induces cachexia
Antibodies against F-α, LIF, IL-6, and oncostatin M can also be used in combination with the preparation of the present invention.

Further, glycation inhibitors (eg, ALT-711 etc.), nerve regeneration promoters (eg, Y-128, VX853, prosaptide etc.), antidepressants (eg, desipramine, amitriptyline, imipramine), antiepileptic drugs (Eg, lamotrigine), antiarrhythmic drug (eg, mexiletine), acetylcholine receptor ligand (eg, ABT-594), endothelin receptor antagonist (eg, ABT
-627), monoamine uptake inhibitors (eg, tramadol),
Narcotic analgesics (eg, morphine), GABA receptor agonists (eg,
Gabapentin), α2 receptor agonist (eg, clonidine), local analgesic (eg, capsaicin), protein kinase C inhibitor (eg, LY-333531), anxiolytic (eg, benzothiazepine), phosphodiesterase inhibitor (Eg, sildenafil), dopamine receptor agonist (eg, apomorphine) and the like can also be used in combination with the preparation of the present invention.

By using the preparation of the present invention and the concomitant drug together, for example, the effect of enhancing the action of the preparation of the present invention or the concomitant drug; the effect of reducing the dose of the preparation of the present invention or the concomitant drug;
Excellent effects such as the side effect reduction effect of the preparation of the present invention or the concomitant drug can be obtained.

The pharmacologically acceptable carriers that may be used in the production of the above-mentioned pharmaceutical composition include various organic or inorganic carrier materials commonly used as pharmaceutical materials, such as excipients and lubricants in solid preparations. Examples include powders, binders and disintegrants, or solvents in liquid preparations, dissolution aids, suspending agents, tonicity agents, buffers and soothing agents. Further, if necessary, usual additives such as preservatives, antioxidants, coloring agents, sweeteners, adsorbents, and wetting agents can be used in appropriate amounts. Examples of the excipient include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, and the like. Examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like. Examples of the binder include crystalline cellulose, sucrose, D
-Mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, sodium carboxymethylcellulose and the like. Disintegrants include, for example, starch, carboxymethylcellulose, carboxymethylcellulose calcium, sodium carboxymethyl starch,
L-hydroxypropyl cellulose and the like. As the solvent, for example, water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil,
Olive oil and the like. Examples of the solubilizer include polyethylene glycol, propylene glycol, D
-Mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like. Examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; for example, polyvinyl alcohol, polyvinylpyrrolidone, Examples include hydrophilic polymers such as sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose. Examples of the tonicity agent include glucose, D-sorbitol, sodium chloride,
Glycerin, D-mannitol and the like can be mentioned. Examples of the buffer include buffers such as phosphate, acetate, carbonate, and citrate. Examples of the soothing agent include benzyl alcohol and the like. As preservatives, for example, paraoxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol,
Dehydroacetic acid, sorbic acid and the like. Examples of the antioxidant include sulfite, ascorbic acid, α-tocopherol and the like.

The dose of the pharmaceutical composition of the present invention varies depending on the administration subject, administration route, disease, symptom and the like. About 60 kg)
About 0.01 to about 100 mg / kg body weight of a daily active ingredient (a substance that inhibits the action of IgE-dependent HRF);
Preferably, about 0.01 to about 30 mg / kg body weight, more preferably about 1 to about 20 mg / kg body weight may be orally administered once or several times a day.

Since IgE-dependent HRF is a novel inhibitor of insulin secretion, the IgE-dependent HRF and / or its receptor, the D encoding the IgE-dependent HRF,
DNA encoding NA and / or its receptor is useful for screening for insulin secretion modulators.

As insulin secretion regulators, IgE
Compounds that change the binding between the dependent HRF and the IgE-dependent HRF receptor (eg, agonists, antagonists, etc.), and compounds that change the expression level of IgE-dependent HRF or IgE-dependent HRF receptor, and the like. (1) Screening method for compound (agonist, antagonist, etc.) that changes binding between IgE-dependent HRF and IgE-dependent HRF receptor Compound that changes binding between IgE-dependent HRF and IgE-dependent HRF receptor (A) IgE-dependent HR
Cell stimulating activity via F receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorus Oxidation, c
Compounds having an activity of promoting or suppressing the activation of fos, the decrease of pH, etc. (so-called Ig)
E-dependent HRF receptor agonist), (b) a compound having no cell stimulating activity (so-called IgE-dependent HRF
Receptor antagonists), (c) a compound that enhances the binding force between IgE-dependent HRF and IgE-dependent HRF receptor, or (d) IgE-dependent HRF and IgE-dependent H
Compounds that reduce the binding strength to the RF receptor are included.

That is, the present invention relates to [1] (i) IgE
Contacting dependent HRF with its receptor and (i
i) a compound or a compound that alters the binding between IgE-dependent HRF and an IgE-dependent HRF receptor, which is characterized by comparing IgE-dependent HRF with its receptor and a test compound. A method for screening a salt is provided. In the screening method of the present invention, for example, in the cases (i) and (ii), for example, the amount of IgE-dependent HRF binding to an IgE-dependent HRF receptor, the cell stimulating activity, and the like are measured and compared. And

More specifically, the present invention relates to [2] (i)
Contacting a labeled IgE-dependent HRF with an IgE-dependent HRF receptor; and (ii) a labeled IgE-dependent HRF.
When an RF and a test compound are brought into contact with an IgE-dependent HRF receptor, the amount of labeled IgE-dependent HRF bound to the IgE-dependent HRF receptor is measured and compared with an IgE-dependent HRF. A method for screening a compound or a salt thereof that changes the binding to an IgE-dependent HRF receptor, [3] (i) labeled I
When the gE-dependent HRF is brought into contact with the cells containing the IgE-dependent HRF receptor, and (ii) when the labeled IgE-dependent HRF and the test compound are brought into contact with the cells containing the IgE-dependent HRF receptor. Labeled I
measuring the amount of binding of gE-dependent HRF to the cell and comparing the amount, and a method for screening a compound or a salt thereof that changes the binding between IgE-dependent HRF and an IgE-dependent HRF receptor, [4] (I) labeled I
contacting the gE-dependent HRF with the membrane fraction of cells containing the IgE-dependent HRF receptor, and (ii) labeled I
gE-dependent HRF and IgE-dependent HR
IgE-dependent HRF and IgE-dependent HRF characterized by measuring and comparing the amount of labeled IgE-dependent HRF bound to the cell when contacted with the membrane fraction of an F receptor-containing cell. A method for screening a compound or a salt thereof that changes the binding to a receptor, and [5] (i) a compound that activates an IgE-dependent HRF receptor is brought into contact with a cell containing the IgE-dependent HRF receptor. And when (ii) the compound that activates the IgE-dependent HRF receptor and the test compound are contacted with cells containing the IgE-dependent HRF receptor.
Cell-stimulating activity through the dependent HRF receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein Of IgE-dependent HR and IgE-dependent HR characterized by measuring and comparing phosphorylation of c-fos, activation of c-fos, activity of promoting or suppressing pH, etc.).
Provided is a method for screening a compound or a salt thereof that changes the binding to an F receptor.

A specific description of the screening method of the present invention will be given below. As the IgE-dependent HRF, those similar to the aforementioned IgE-dependent HRF and its partial peptides are used. Compounds that activate IgE-dependent HRF receptor include, for example, TCTPp21, TCTP
p23, TCTP p26 and the like are used. The IgE-dependent HRF receptor may be any as long as it contains an IgE-dependent HRF receptor.
Cells containing IgE-dependent HRF (eg, pancreatic β-cells, MIN6 cells, etc.), mammalian organs (eg, pancreas)
Cell membrane fractions and the like are preferred.

However, since it is extremely difficult to obtain human-derived organs in particular, human-derived organs expressed in large amounts using recombinants were used for screening.
A gE-dependent HRF or its receptor is suitable.

The production of the IgE-dependent HRF receptor is preferably carried out by expressing the DNA of the IgE-dependent HRF receptor in mammalian cells or insect cells. D encoding the desired IgE-dependent HRF receptor moiety
Complementary DNA is used for the NA fragment, but is not necessarily limited thereto. For example, a gene fragment or a synthetic DNA may be used. In order to introduce a DNA fragment encoding the IgE-dependent HRF receptor into host animal cells and express them efficiently, the DNA fragment must be expressed in a nuclear polyhedrosis virus belonging to a baculovirus using an insect as a host. virus; NPV) polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter, human heat shock promoter, cytomegalovirus promoter, SRα promoter and the like.

The quantity and quality of the expressed receptor can be examined by a method known per se. For example, literature [Namb
i, P. Et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267, 19555-19559
P., 1992].

Therefore, in the screening method of the present invention, the one containing an IgE-dependent HRF receptor may be an IgE-dependent HRF receptor purified according to a method known per se, or an IgE-dependent HR receptor.
Cells containing the F receptor may be used, or the membrane fraction of cells containing the IgE-dependent HRF receptor may be used.

In the screening method of the present invention,
When using cells containing the gE-dependent HRF receptor,
The cells may be fixed with glutaraldehyde, formalin, or the like. The immobilization method can be performed according to a method known per se.

The cell containing the IgE-dependent HRF receptor refers to a host cell expressing the IgE-dependent HRF receptor. Examples of the host cell include Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells and the like. Is preferred.

As the cell membrane fraction, after crushing the cells,
It refers to a fraction containing a large amount of cell membrane obtained by a method known per se. As a method for disrupting cells, Potter-El
Examples include a method of crushing cells with a vehjem-type homogenizer, crushing with a Waring blender or a polytron (manufactured by Kinematica), crushing with ultrasonic waves, crushing by ejecting cells from a thin nozzle while applying pressure with a French press, and the like. For fractionation of cell membranes, fractionation by centrifugal force, such as fractionation centrifugation and density gradient centrifugation, is mainly used. For example, the cell lysate is fed at a low speed (500 r
pm to 3000 rpm) for a short time (usually about 1 minute to 10 minutes).
Min) and centrifuged, and the supernatant was further spun at high speed (15,000 rpm to 3
(0000 rpm) for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The expressed Ig was contained in the membrane fraction.
It is rich in E-dependent HRF receptors and membrane components such as cell-derived phospholipids and membrane proteins.

The amount of the receptor protein in the cells containing the IgE-dependent HRF receptor and in the membrane fraction was 1 / cell.
It is preferably from 0 ³ to 10 ⁸ molecules, and more preferably from 10 ⁵ to 10 ⁷ molecules. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which makes it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. .

IgE-dependent HRF and IgE-dependent HRF
In order to carry out the above [1] to [4] for screening for a compound that changes the binding to the receptor, for example,
IgE labeled with appropriate IgE-dependent HRF receptor fraction
A dependent HRF is required.

As the IgE-dependent HRF receptor fraction,
A natural IgE-dependent HRF receptor fraction or a recombinant IgE-dependent HRF receptor fraction having an activity equivalent thereto is desirable. Here, “equivalent activity” refers to equivalent ligand binding activity, signal transduction action, and the like.

Examples of the labeled IgE-dependent HRF include a labeled IgE-dependent HRF and a labeled IgE-dependent HR.
An F analog compound or the like is used. For example, [ ³ H],
Ig labeled with [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc.
E-dependent HRF or the like is used.

Specifically, IgE-dependent HRF and IgE
To screen for a compound that alters the binding to the dependent HRF receptor, cells or membrane fractions containing the IgE-dependent HRF receptor are first suspended in a buffer suitable for screening. Prepare an IgE-dependent HRF receptor. PH 4 buffer
Phosphate buffer of 10 to 10 (preferably pH 6 to 8),
IgE-dependent HRF such as Tris-HCl buffer and I
Any buffer that does not inhibit the binding to the gE-dependent HRF receptor may be used. For the purpose of reducing non-specific binding, a surfactant such as CHAPS, Tween-80 ^™ (Kao-Atlas), digitonin, and deoxycholate can be added to the buffer. Furthermore, IgE-dependent HRF and IgE-dependent H
For the purpose of suppressing the degradation of the RF receptor, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Research Laboratories), and pepstatin can also be added. 0.0
A fixed amount (50 ml) is added to 1 ml to 10 ml of the receptor solution.
(00 cpm to 500,000 cpm) of labeled IgE-dependent HRF is added, and 10 ^-4 M to ^{10 -10} M of the test compound is simultaneously present. A reaction tube to which a large excess of unlabeled IgE-dependent HRF is added to determine the amount of non-specific binding (NSB) is also prepared. The reaction is carried out at about 0 ° C. to 50 ° C., preferably about 4 ° C. to 37 ° C., for about 20 minutes to 24 hours.
Preferably, it is performed for about 30 minutes to 3 hours. After the reaction, the mixture is filtered through a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter.
When the count (B ₀ -NSB) obtained by subtracting the non-specific binding amount (NSB) from the count (B ₀ ) when there is no antagonistic substance is defined as 100%, the specific binding amount (B-NSB) is, for example, , 50% or less can be selected as a candidate substance having competitive inhibitory ability.

IgE-dependent HRF and IgE-dependent HRF
In order to carry out the method of the above [5] for screening a compound that changes the binding to the receptor, for example, Ig
Cell-stimulating activity through the E-dependent HRF receptor, for example,
Arachidonic acid release, acetylcholine release, intracellular Ca ²⁺
Known activities to promote or suppress release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, decrease in pH, etc. Or a commercially available measurement kit.

Specifically, cells containing an IgE-dependent HRF receptor are first cultured in a multiwell plate or the like. Before performing the screening, replace the cells with a fresh medium or an appropriate buffer that is not toxic to cells in advance, add the test compound, etc., incubate for a certain period of time, extract the cells or collect the supernatant,
The products produced are quantified according to the respective method. When the production of a substance (for example, arachidonic acid or the like) as an indicator of cell stimulating activity is difficult due to a degrading enzyme contained in a cell, an assay may be performed by adding an inhibitor against the degrading enzyme. In addition, the activity such as cAMP production suppression can be detected as a production suppression effect on cells whose basic production amount has been increased by forskolin or the like.

For screening by measuring the cell stimulating activity, cells expressing an appropriate IgE-dependent HRF receptor are required. As the cells expressing the IgE-dependent HRF receptor, a cell line having a natural IgE-dependent HRF receptor, a cell line expressing a recombinant IgE-dependent HRF, and the like are preferable.

The test compounds include, for example, peptides,
Proteins, non-peptidic compounds, synthetic compounds, fermentation products,
Cell extracts, plant extracts, animal tissue extracts and the like are used, and these compounds may be novel compounds or known compounds.

IgE-dependent HRF and IgE-dependent HRF
Screening kits for compounds or salts thereof that alter the binding to the receptor include IgE-dependent HRF, IgE
And a cell containing the IgE-dependent HRF receptor, a cell containing the IgE-dependent HRF receptor, and a membrane fraction of a cell containing the IgE-dependent HRF receptor.

Examples of the screening kit of the present invention include the following. 1. Screening reagent Measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco)
One containing 05% bovine serum albumin (manufactured by Sigma). Sterilized by filtration through a 0.45 μm filter, 4 ° C
Or may be prepared at the time of use. IgE-dependent HRF receptor preparation MIN6 cells containing the IgE-dependent HRF receptor were
Passage into a 12-well plate at 5 × 10 ⁵ / well, 37 ° C.
Cultured for 2 days in 5% CO ₂ and 95% air. Labeled IgE-dependent HRF An aqueous solution of IgE-dependent HRF labeled with commercially available [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc., is stored at 4 ° C. or −20 ° C. Before use, dilute to 1 μM with a measurement buffer. IgE-dependent HRF standard solution IgE-dependent HRF was dissolved in PBS containing 0.1% bovine serum albumin (manufactured by Sigma) to a concentration of 1 mM,
Store at -20 ° C.

[0120] 2. Measurement method IgE-dependent HRF receptor-containing MIN6 cells cultured on a 12-well tissue culture plate were mixed with 1 ml of a measurement buffer solution.
After washing twice, 490 μl of measurement buffer is added to each well. After adding 5 μl of a test compound solution of 10 ⁻³ to 10 ⁻¹⁰ M, 5 μl of labeled IgE-dependent HRF was added, and the mixture was added at room temperature.
Let react for hours. To determine the amount of non-specific binding, 5 μl of 10 ⁻³ M IgE-dependent HRF was used instead of the test compound.
I will add it. Remove the reaction solution and wash three times with 1 ml of washing buffer. 0.2N labeled IgE-dependent HRF bound to cells
Dissolve in NaOH-1% SDS and mix with 4 ml of liquid scintillator A (Wako Pure Chemical Industries). Liquid scintillation counter (Beckman)
Radioactivity was measured using Percent Maximum Binding
(PMB) is obtained by the following equation. PMB = [(B−NSB) / (B ₀ −NSB)] × 10
0 PMB: Percent Maximum Binding B: Value when a sample is added NSB: Non-specific Binding (Non-specific binding amount) B ₀ : Maximum binding amount

The compound obtained by using the screening method or the screening kit of the present invention or a salt thereof has an effect of changing the binding between IgE-dependent HRF and an IgE-dependent HRF receptor, etc., so that insulin secretion can be prevented. Is a compound that regulates, specifically,
(A) IgE-dependent HRF receptor agonist, (B) I
gE-dependent HRF receptor antagonist, (c) IgE
Compound that enhances the binding between HRF-dependent HRF and IgE-dependent HRF receptor or (d) IgE-dependent HRF and Ig
It is a compound that reduces the binding force to the E-dependent HRF receptor.

The compound includes a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like. These compounds may be novel compounds or known compounds.

An agonist for the IgE-dependent HRF receptor and a compound that enhances the binding strength between the IgE-dependent HRF and the IgE-dependent HRF receptor include IgE-dependent HR.
Since F has an action similar to that of F (eg, an insulin secretion inhibitory action), it is useful as a safe and low-toxic drug for the above-mentioned diseases caused by excessive insulin secretion.

On the other hand, an antagonist to an IgE-dependent HRF receptor and a compound capable of reducing the binding force between the IgE-dependent HRF and the IgE-dependent HRF receptor include IgE-dependent HRF receptors.
Since the action of E-dependent HRF can be inhibited, it is useful as a safe and low-toxicity medicine against diseases and diabetes caused by the above-mentioned insulin secretion deficiency (secretion inhibition).

(2) Method for Screening Compounds that Alter the Expression of IgE-Dependent HRF or IgE-Dependent HRF Receptor DNA encoding IgE-dependent HRF and / or DNA encoding IgE-dependent HRF receptor are When used as a probe, it can be used for screening for a compound that changes the expression level of IgE-dependent HRF or IgE-dependent HRF receptor.

That is, the present invention relates to, for example, an IgE-dependent HRF or IgE-dependent HGF or IgE contained in (i) blood of a non-human mammal, a specific organ, a tissue or cell isolated from an organ or (ii) a transformant. IgE-dependent HRF by measuring the amount of mRNA of the sexual HRF receptor
Alternatively, the present invention provides a method for screening a compound that changes the expression level of an IgE-dependent HRF receptor.

More specifically, (i) blood of a non-human mammal, a specific organ, a tissue or cell isolated from an organ, or (ii) a transformant in the presence or absence of a test compound The present invention provides a method for screening a compound that changes the expression level of IgE-dependent HRF or IgE-dependent HRF receptor by measuring and comparing the amount of mRNA of IgE-dependent HRF or IgE-dependent HRF receptor contained in . IgE-dependent HRF or Ig
The measurement of the mRNA amount of the E-dependent HRF receptor is specifically performed as follows.

(I) Normal or disease model non-human mammals (eg, mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteriosclerotic rabbits) , Cancer-bearing mice, etc.), drugs (eg, anti-dementia drugs, blood pressure lowering drugs,
An anticancer drug, an antiobesity drug, etc.) or a physical stress (eg, immersion stress, electric shock, light / dark, low temperature, etc.) is given, and after a certain period of time, blood or a specific organ (eg, pancreas, etc.) Separate tissues (eg, islets of Langerhans) or cells (eg, pancreatic β cells, MIN6 cells) are obtained. The mRNA of IgE-dependent HRF or IgE-dependent HRF receptor contained in the obtained blood or the like can be obtained, for example, from cells or the like by a conventional method.
The NA can be extracted and quantified by using, for example, a technique such as TaqMan PCR, and can also be analyzed by performing a Northern blot by a known method. (Ii) A transformant expressing the IgE-dependent HRF receptor is prepared, and the IgE-dependent HRF contained in the transformant is prepared.
The mRNA of the receptor can be quantified and analyzed in the same manner.

More specifically, screening for a substance that alters the expression level of IgE-dependent HRF or IgE-dependent HRF receptor can be performed by (i) using a drug or a physical A certain time before giving stress etc. (30 minutes to 24 hours ago, preferably 30 minutes to 12 hours ago, more preferably 1 hour ago
6 hours before) or after a certain time (after 30 minutes to 3 days, preferably after 1 hour to 2 days, more preferably after 1 hour)
The test compound is administered at the same time as the drug or physical stress, and after a lapse of a certain time after administration (30 hours).
Minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), the IgE contained in the cells.
-Dependent HRF or IgE-dependent HRF receptor mRN
It can be performed by quantifying and analyzing the amount of A,
(Ii) When culturing the transformant according to a conventional method, the test compound is mixed in a medium, and after culturing for a certain period of time (1 to 7 days, preferably 1 to 3 days, more preferably 2 to 3 days) Days after), the IgE-dependent HR contained in the transformant
It can be carried out by quantifying and analyzing the amount of F receptor mRNA. The substance obtained by using the screening method of the present invention is a substance having an action of changing the expression level of IgE-dependent HRF or IgE-dependent HRF receptor. Specifically, (a) IgE-dependent HRF or By increasing the expression level of the IgE-dependent HRF receptor, the cell stimulating activity via the IgE-dependent HRF receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular cGMP
(B) IgE-dependent substances that enhance or inhibit the production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. It is a substance that attenuates the cell stimulating activity by decreasing the expression level of HRF or IgE-dependent HRF receptor.

Examples of the substance include peptides, proteins, non-peptidic compounds, synthetic compounds, and fermentation products. These compounds may be novel compounds or known compounds.

The substance for enhancing the cell stimulating activity is Ig
Since it has an action similar to that of E-dependent HRF (eg, insulin secretion inhibitory action), it is useful as a safe and low toxic drug for the above-mentioned diseases caused by excessive insulin secretion.

The substance that attenuates the cell stimulating activity is Ig
Since the action of E-dependent HRF can be inhibited, it is useful as a safe and low-toxicity medicine against diseases and diabetes caused by the above-mentioned insulin secretion deficiency (secretion inhibition).

When a substance obtained by using the above-described screening method of the present invention is used as a pharmaceutical composition, it can be carried out according to a conventional method. For example, a substance or Ig that inhibits the above-described action of IgE-dependent HRF
Tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as in the case of a drug containing E-dependent HRF or a substance having the action thereof.

The preventive / therapeutic agent for diseases or diabetes caused by insulin secretion deficiency (secretion inhibition) containing the substance for enhancing the cell stimulating activity or the substance for decreasing the cell stimulating activity is the above-mentioned diabetic. It can be used in combination with agents such as therapeutic agents, therapeutic agents for diabetic complications, therapeutic agents for hyperlipidemia, antihypertensive agents, antiobesity agents, diuretics, chemotherapeutic agents, and immunotherapeutic agents.

The preparations thus obtained are safe and have low toxicity, and are therefore suitable for mammals (eg, humans, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can be administered.

The dosage of a substance having an effect of changing the expression level of IgE-dependent HRF or IgE-dependent HRF receptor obtained by using the screening method of the present invention is as follows:
Although there are differences depending on the administration subject, target organ, symptoms, administration method and the like, in the case of oral administration, generally, for example, when a compound that reduces the cell stimulating activity is used for the treatment of diabetes, the patient (as 60 kg) Is about 0.1 to 100 mg, preferably about 1.0 to 50 mg per day,
More preferably, it is about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptoms, administration method, and the like.
In the form of injection, usually diabetic patients (as 60kg)
In the above, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

Antibodies to IgE-dependent HRF are Ig
Since it has a binding property to E-dependent HRF, the IgE-dependent HRF concentration in a living body can be quantified with high sensitivity, which is useful for diagnosis of abnormal insulin secretion.

The diagnostic agent for abnormal insulin secretion comprising an antibody against IgE-dependent HRF is an IgE-dependent HRF.
In addition to the antibody against RF, general additives necessary for quantification of the antigen may be contained.

A method for quantifying IgE-dependent HRF is described in, for example,
It can be used in combination with a competitive method, a sandwich immunoassay and the like. That is, the IgE-dependent HRF concentration in the sample can be measured by bringing the sample into contact with the receptor protein of the present invention.

Specifically, (i) an antibody against IgE-dependent HRF (HRF antibody) is allowed to react competitively with a test solution and labeled IgE-dependent HRF, and the labeled A method for quantifying IgE-dependent HRF in a test solution, which comprises measuring the proportion of the detected IgE-dependent HRF; and (ii) HR insolubilized on the test solution and a carrier.
Quantitative determination of IgE-dependent HRF in a test solution, wherein the F antibody and another labeled HRF antibody are reacted simultaneously or successively, and then the activity of the labeling agent on the insolubilized carrier is measured. Method is used. In the quantification method (ii), one antibody is an antibody that recognizes the N-terminus of the IgE-dependent HRF, and the other antibody is an IgE-dependent HRF.
It is desirable that the antibody reacts with the C-terminus.

In addition to the quantification of IgE-dependent HRF using a monoclonal antibody against IgE-dependent HRF, detection by tissue staining or the like is also possible. For these purposes, the antibody molecule itself may be used, and F (ab ′) ₂ , Fab ′,
Alternatively, the Fab fraction may be used.

[0142] The quantification method using the HRF antibody is not particularly limited, and the amount of the antigen in the liquid to be measured (for example,
Measuring the amount of the antibody, antigen or antibody-antigen complex corresponding to the amount of peptide) by chemical or physical means and calculating this from a standard curve prepared using a standard solution containing a known amount of antigen. If so, any measurement method may be used. For example, nephelometry, a competitive method, an immunometric method, and a sandwich method are preferably used, and in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.

As a labeling agent used in a measuring method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Examples of radioisotopes include [ ¹²⁵ I], [ ¹³¹ I], [ ³ H],
[ ¹⁴ C] and the like are used. As the above-mentioned enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For the insolubilization of an antigen or an antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing a peptide or an enzyme may be used. As the carrier, agarose, dextran, insoluble polysaccharides such as cellulose, polystyrene,
Synthetic resins such as polyacrylamide and silicon, and glass are examples.

In the sandwich method, insoluble HR
After reacting the test solution with the F monoclonal antibody (primary reaction), and further reacting with another labeled HRF monoclonal antibody (secondary reaction), the activity of the labeling agent on the insolubilized carrier is measured to measure the activity. IgE-dependent H in test solution
The amount of RF can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You may.

IgE-dependent HRF by sandwich method
In the measurement method described above, as the HRF monoclonal antibody used in the primary reaction and the secondary reaction, an antibody having a different site for binding IgE-dependent HRF is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is, for example, an antibody used in the secondary reaction is IgE-dependent HR.
When recognizing the C-terminal of F, as the antibody used in the primary reaction, an antibody that recognizes, for example, an N-terminal other than the C-terminal is preferably used.

The HRF monoclonal antibody can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephelometry and the like.

In the competition method, after an antigen in a test solution and a labeled antigen are allowed to react competitively with an antibody, an unreacted labeled antigen (F) and a labeled antigen (B) bound to the antibody are used. Is separated (B / F separation), and the labeling amount of either B or F is measured,
The amount of antigen in the test solution is quantified. In this reaction method, a liquid phase method using a soluble antibody as an antibody, B / F separation using polyethylene glycol, a second antibody against the antibody, or the like,
An immobilized antibody is used as the first antibody, or an immobilized method using a soluble first antibody and using the immobilized antibody as the second antibody is used.

In the immunometric method, an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of a labeled antibody, and then the solid phase and the liquid phase are separated. The antigen therein is reacted with an excessive amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.

In nephelometry, the amount of insoluble sediment resulting from the antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.

In applying these individual immunoassays to the quantification method of the present invention, no special conditions, operations, and the like need to be set. What is necessary is just to construct the measuring system of the peptide of this invention, adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like.

For example, "Radioimmunoassay" edited by Hiroshi Irie (Kodansha, published in 1974), "Radioimmunoassay" edited by Hiroshi Irie (published in Kodansha, 1979), "Enzyme immunoassay" edited by Eiji Ishikawa et al. Shoin, published in 1978), Eiji Ishikawa et al., “Enzyme Immunoassay” (2nd edition) (Medical Shoin, published in 1982), Eiji Ishikawa, et al., “Enzyme Immunoassay” (3rd edition) (Medical) Shoin, published in 1987), "Me
thods in ENZYMOLOGY '' Vol. 70 (Immunochemical Techniq
ues (Part A)), ibid.Vol. 73 (Immunochemical Techniq
ues (Part B)), Ibid.Vol. 74 (Immunochemical Techniq
ues (Part C)), ibid.Vol. 84 (Immunochemical Techniq
ues (Part D: Selected Immunoassays)), ibid, Vol. 9
2 (Immunochemical Techniques (Part E: Monoclonal An
tibodies and General Immunoassay Methods)), ibid
Vol. 121 (Immunochemical Techniques (Part I: Hybrid
oma Technology and Monoclonal Antibodies))
Academic Press). As described above, IgE-dependent HRF can be quantified with high sensitivity by using the HRF antibody.

Furthermore, when an increase in the concentration of IgE-dependent HRF is detected using an HRF antibody, for example,
Diseases such as diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy, osteopenia, or will be affected in the future It can be diagnosed that the probability is high.

Furthermore, IgE-dependent H
By quantifying the concentration of RF, the IgE-dependent HR
When a decrease in the concentration of F is detected, for example, obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance, instability Diabetes, lipoatrophy, insulin allergy,
It can be diagnosed as a disease, such as insulinoma, or highly likely to be affected in the future.

The HRF antibody can be used for detecting IgE-dependent HRF present in a subject such as a body fluid or a tissue. In addition, for preparation of an antibody column used for purifying IgE-dependent HRF, detection of IgE-dependent HRF in each fraction during purification, analysis of the behavior of IgE-dependent HRF in test cells, and the like. Can be used.

DNA encoding IgE-dependent HRF
For example, by using as a probe, a human or warm-blooded animal (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat,
Can detect an abnormality (gene abnormality) in DNA or mRNA encoding IgE-dependent HRF in dogs, monkeys, etc.).
It is useful as a gene diagnostic agent for A damage, mutation or decreased expression, and increase or excessive expression of the DNA or mRNA.

The above-mentioned genetic diagnosis using DNA encoding IgE-dependent HRF can be performed, for example, by the known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, p. 874-879 (1)
Procedings of the National Academy of Scien (989), Procedings of the National Academy of Scien
ces of the United States of America), Vol. 86,
2766-2770 (1989)).

For example, when overexpression of IgE-dependent HRF is detected by Northern hybridization, for example, diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy,
It can be diagnosed as having a high possibility of hyperlipidemia, sexual dysfunction, skin disease, arthropathy, osteopenia, etc., or having a high possibility of suffering in the future.

When IgE-dependent decrease in HRF expression is detected by Northern hybridization, for example, obesity, hyperlipidemia, type 2 diabetes, hypoglycemia,
Hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy,
It can be diagnosed as being likely to be edema, insulin resistance, unstable diabetes, lipoatrophy, insulin allergy, insulinoma or the like, or is likely to be affected in the future.

[0160]

The present invention will be further described by the following examples and experimental examples, which are merely implementations and do not limit the present invention and do not depart from the scope of the present invention. It may be changed in a range.

The gene cloning using Escherichia coli was performed by molecular cloning (Molecular clonin).
The method described in g) was followed. In the present specification and drawings, when bases and amino acids are indicated by abbreviations,
IUPAC-IUB Commission on Biochemical Nome
It is based on the abbreviation by nclature or the abbreviation commonly used in the art, and examples thereof are described below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine mRNA: messenger ribonucleic acid Gly: glycine Ala: alanine Val: valine Leu: leucine Ile: isoleucine Ser: Serine Serine: Serine Serine: Serine Serine Cysteine Met: methionine Glu: glutamic acid Asp: aspartic acid Lys: lysine Arg: arginine His: histidine Phe: phenylalanine Tyr: tyrosine Trp: tryptophan Pro: proline Asn: amino acid sequence of asparagine G: Asparagine G amino acid Show. [SEQ ID NO: 2] This shows the amino acid sequence of TCTP p23. [SEQ ID NO: 3] This shows the amino acid sequence of TCTP p26. [SEQ ID NO: 4] DNA encoding TCTP p21
Shows the nucleotide sequence of [SEQ ID NO: 5] DNA encoding TCTP p23
Shows the nucleotide sequence of [SEQ ID NO: 6] DNA encoding TCTP p26
Shows the nucleotide sequence of

Example 1 (1) Compound A that inhibits the action of IgE-dependent HRF 10 mg (2) Lactose 60 mg (3) Corn starch 35 mg (4) Gelatin 3 mg (5) Magnesium stearate 2 mg Compound A 10 mg and lactose 60 mg and Corn starch 3
5 mg of the mixture is granulated through a 1 mm mesh sieve using 0.03 ml of a 10% aqueous gelatin solution (3 mg as gelatin), dried at 40 ° C. and sieved again. The granules thus obtained are mixed with 2 mg of magnesium stearate,
Compress. The resulting core tablet is coated with a sugar coating in an aqueous suspension of sucrose, titanium dioxide, talc and gum arabic. The coated tablets are polished with beeswax to obtain coated tablets.

Example 2 (1) Compound A that inhibits the action of IgE-dependent HRF 10 mg (2) Lactose 70 mg (3) Corn starch 50 mg (4) Soluble starch 7 mg (5) Magnesium stearate 3 mg Compound A 10 mg and stearic acid 3 mg of magnesium are granulated with 0.07 ml of an aqueous solution of soluble starch (7 mg as soluble starch), dried and mixed with 70 mg of lactose and 50 mg of corn starch. The mixture is compressed to give tablets.

Example 3 (1) Compound A that inhibits the action of IgE-dependent HRF 5 mg (2) Salt 20 mg (3) Distilled water The total amount was 2 ml. Compound A 5 mg and salt 20 mg were dissolved in distilled water.
Add water to bring the total volume to 2 ml. The solution is filtered and filled under aseptic conditions into 2 ml ampules. After sterilizing the ampoule,
Seal to obtain solution for injection.

Example 4 (1) Compound B having an action of IgE-dependent HRF 10 mg (2) Lactose 60 mg (3) Corn starch 35 mg (4) Gelatin 3 mg (5) Magnesium stearate 2 mg Compound B 10 mg, lactose 60 mg and corn starch 3
5 mg of the mixture is granulated through a 1 mm mesh sieve using 0.03 ml of a 10% aqueous gelatin solution (3 mg as gelatin), dried at 40 ° C. and sieved again. The granules thus obtained are mixed with 2 mg of magnesium stearate,
Compress. The resulting core tablet is coated with a sugar coating in an aqueous suspension of sucrose, titanium dioxide, talc and gum arabic. The coated tablets are polished with beeswax to obtain coated tablets.

Example 5 (1) Compound B having an action of IgE-dependent HRF 10 mg (2) Lactose 70 mg (3) Corn starch 50 mg (4) Soluble starch 7 mg (5) Magnesium stearate 3 mg Compound B 10 mg and magnesium stearate 3 mg are granulated with 0.07 ml of an aqueous solution of soluble starch (7 mg as soluble starch), dried and mixed with 70 mg of lactose and 50 mg of corn starch. The mixture is compressed to give tablets.

Example 6 (1) Compound B having an action of IgE-dependent HRF 5 mg (2) Salt 20 mg (3) Distilled water The total amount was 2 ml. Compound B 5 mg and salt 20 mg were dissolved in distilled water.
Add water to bring the total volume to 2 ml. The solution is filtered and filled under aseptic conditions into 2 ml ampules. After sterilizing the ampoule,
Seal to obtain solution for injection. Specifically, the fourth embodiment
In Examples 6 to 6, TCTP p21, T
Use CTP p23 or TCTP p26.

Experimental Example 1 (1) Experimental Materials Male C57BL / 6 and male C3H / HeN mice (Japan SLC, Shizuoka), Wistar male rats (Japan SLC, Shizuoka) and Hartley male guinea pigs (Japan SLC, Shizuoka) The pancreatic tissue was removed. Changes in TCTP p21 in the pancreas, liver and serum following feeding were determined by male C3H / HeN mice and C57BL /
Six mice were used. Mouse pancreatic β cell line (MIN6 cells)
Is an immobilized fetal calf serum (FCS, Flow, McLea
n, VA) 15%, 100 IU / ml penicillin, 100 μg /
3 ml using DMEM containing ml streptomycin, 5 μM 2-mercaptoethanol and 25 mM glucose.
The cells were cultured at 7 ° C. in 5% CO ₂ -95% air. In the experiment, cells of passages 18 to 23 were used. Isolation of mouse pancreatic islets of Langerhans followed the method of Lacy et al. C3H / He
Hank's balanced sa in the pancreas of N or C57BL / 6 mice
lt solution (HBSS) was injected, and the pancreas was inflated and then removed. After cutting with scissors, 37 ° C for 10 minutes,
Incubation was carried out with HBSS containing 2.5 mg / ml collagenase S-1 (Nitta Gelatin, Osaka). HBSS was added to the digested pancreatic cells, and centrifugal washing was performed three times at 400 g for 1 minute. Finally, after removing the supernatant, Dulbecco's m
odified Eagle's medium (DMEM) was added to perform centrifugal washing. The cells were dispersed in a dish containing DMEM, and the pancreatic islets of Langerhans were collected using a 2 μl pipetteman under a microscope. The isolated islet of Langerhans is DM
The cells were cultured in EM at 37 ° C. in 5% CO ₂ -95% air.

(2) Preparation of anti-TCTP p21 antibody and immunohistochemical staining of pancreatic tissue TCTP p21 obtained by microsequencing
A peptide was synthesized based on the nucleotide sequence of the N-terminal 20 amino acids of the above, and a rabbit was immunized with the peptide as an antigen to prepare antiserum. Agarose beads on which the synthetic peptide used as the antigen was immobilized were prepared, and the obtained polyclonal antibody was affinity-purified. A paraffin-embedded tissue of the resected tissue of human pancreatic cancer was cut into a 4 μm-thick section using a microtome, placed on a slide glass, and air-dried. After deparaffinization with xylene, the alcohol concentration was gradually reduced to hydrate and washed with distilled water for 5 minutes. Phosphate-buffered sa
Goat normal serum diluted 100-fold with line (PBS) was added dropwise, and allowed to stand at room temperature for 20 minutes to perform blocking. The cells were reacted with the above-mentioned anti-TCTP p21 antibody (100-fold dilution) at room temperature for 1 hour. The bound antibody was combined with a peroxidase-labeled anti-rabbit IgGF (ab ') ² antibody,
Biotin peroxidase staining kit (Vectastain A
(BC kit). At the same time, hematoxin staining was performed. C57BL / 6 mouse (6-8 weeks old)
From the pancreas was fixed with a 3% formalin solution and embedded in paraffin. The embedded tissue was cut into 4 μm-thick sections using a microtome, placed on a slide glass, and air-dried. After deparaffinization with xylene, the alcohol concentration was gradually reduced to hydrate and washed with distilled water for 5 minutes. P
Goat normal serum diluted 100-fold with BS was added dropwise, and
The mixture was allowed to stand still for minutes, and blocking was performed. Anti-insulin antibody (100-fold dilution, SANBIO Amsterdam, Nethe
rlands) for 2 hours at room temperature. After washing three times for 5 minutes with PBS, the mixture was reacted for 1.5 hours at room temperature using a rhodamine-labeled anti-mouse IgGF (ab ') ² antibody (100-fold diluted, Amersham Japan, Tokyo) as a secondary antibody. Washed 4 times with PBS for 10 minutes. Further, an anti-TCTP p21 antibody (100-fold dilution) was used as a primary antibody and reacted at room temperature for 2 hours. After washing 3 times with PBS for 5 minutes,
Fluorescein isothiocyanate (FIT) as a secondary antibody
C) Using a labeled anti-rabbit IgGF (ab ') ² antibody (100-fold dilution, Santa Cruz, Santa Cruz, CA), the reaction was carried out at room temperature for 1.5 hours. After washing three times with PBS for 5 minutes, the cells were sealed with PermaFluor ^™ Aqueous Mounting Medium (Turner, Tokyo, Japan) and observed with a confocal laser microscope.

(3) Recombinant TCTP p21 (r
Production of TCTP p21) Based on the method already reported (Journal of Immunol. (J. Immunol.), Vol. 161, 6356), human TC
TP p21 cDNA was incorporated into a glutathione-S-transferase fusion protein expression vector and expressed in E. coli. The fusion protein was treated with thrombin (40 units) to dissociate glutathione-S-transferase, and rTCTP p21 was purified by ion exchange chromatography.

(4) Measurement of TCTP p21 in Pancreatic Tissue, Cells, Culture Solution and Serum The isolated pancreatic and hepatic tissues were 2 mM EDTA, 2 mM E
GTA, 1 mM phenylmethylsulfonyl fluoride (PM
SF) and pH 7.4 containing 0.1 mM leupeptin.
mM Tris-HCl buffer was added and homogenized with a Teflon (registered trademark) potter. The obtained homogenate was centrifuged at 10500 g at 4 ° C. for 15 minutes to prepare a soluble protein. MI cultured in 35 mm diameter culture dish
In N6 cells, 2 mM EDTA, 2 mM EGTA, 1 mM
PH 7.4 containing PMSF and 0.1 mM leupeptin
The cells were detached with a rubber policeman after adding 0 mM Tris-HCl buffer, and the cells were homogenized with a Teflon (registered trademark) potter. The resulting homogenate
Centrifugation was performed at 10500 g at 4 ° C. for 15 minutes to prepare a soluble protein. The medium was collected in a 1.5 ml microtube and centrifuged at 900 g for 5 minutes. The supernatant is centrifuged (10,000 NMWL filter tube; Millipore, Bedfo)
rd, MA). Blood collected from the mouse heart and tail vein was placed in a BD tube containing an anticoagulant and centrifuged at 5000 rpm to obtain a serum containing no platelets. The extracted protein (50 μg), serum 4 μl, and a 50-fold concentrated culture solution (20 μl) were separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. After the transfer, the PVDF membrane was blocked with 4% purified milk casein for 1 hour at room temperature, and washed with PBS containing 0.05% Tween 20 (PBS-T). Next, the PVDF membrane was reacted with a primary antibody (3,000-fold diluted anti-TCTP p21 antibody) at room temperature for 1 hour. After washing with PBS-T, a secondary antibody [horse radish peroxidase labeled anti-rabbit IgGF (ab ') ² diluted 6,000-fold] (Amersham Japan, Tokyo)
And 1 hour at room temperature. After washing the PVDF membrane with PBS-T, the bound primary antibody was detected using an ECL detection kit (Amersham Japan, Tokyo).

(5) Measurement of insulin secretion from MIN6 cells and isolated pancreatic islets of Langerhans MIN6 cells have a high glucose content of D containing 15% FCS.
After culturing for 4 days in 24-well and 96-well plates in MEM,
Used for experiments. After replacing with DMEM containing 2.8 mM glucose and 15% FCS for 1.5 hours, DME containing 25 mM glucose and 15% FCS was exchanged.
M and glucose stimulation was performed for various times.
Cells to which different concentrations of rTCTP p21 were added for various times also stimulated glucose in the same manner, and the amount of insulin secreted into the culture solution was measured by enzyme-linked enzyme.
immunosorbent assay (ELISA) kit (Levis
Insulin Kid, Shiba Goat, Gunma). Cells were lysed with 1N NaOH and used for protein quantification. The isolated pancreatic islets of Langerhans were placed in DMEM containing 2.8 mM glucose and 15% FCS at different concentrations, and different concentrations of rTCTP p21 were added for various periods of time. The amount of released insulin was measured in the same manner as described above. All incubations were performed at 37 ° C., 5% CO ₂ -95% air.

(6) TCTP p21 in pancreatic β cells
Protein expression TCTP in mouse, rat and guinea pig tissues
The expression of p21 protein was examined by Western blotting using anti-TCTP p21 antibody (FIG. 1). Although the expression level in each tissue was different depending on the animal species, high levels of TCTP p21 protein expression were commonly observed in the pancreas. An approximately 30 kDa protein having a higher molecular weight than the 26 kDa TCTP p21 protein is N-glycosylated TCTP p21. TCTP in mouse pancreatic tissue
The expression of p21 and insulin was confirmed by fluorescent antibody double immunohistochemical staining. TCTP p21 was expressed in insulin-secreting cells (β cells) in the islets of Langerhans of the pancreas (FIG. 2). In addition, immunohistochemical staining of the normal tissue portion of the resected specimen of human pancreatic cancer using an anti-TCTP antibody confirmed that TCTP was expressed in β cells of the islets of Langerhans.

(7) Induction of TCTP p21 in Pancreatic β Cells by Glucose Stimulation Mouse pancreatic β cells (MIN6 cells) were transformed with low glucose (2.
After incubation in DMEM (8 mM) for 1.5 hours, DMEM was exchanged for high glucose (25 mM), and the change in TCTP p21 protein content over time was examined by Western blotting (FIG. 3A). Assuming that TCTP p21 level before high glucose stimulation was 1, TCTP induced in MIN6 cells by glucose stimulation
The p21 level densitometer values are shown below.

[0175]

[Table 1]

At the same time, TCTP secreted into the culture solution
Examination of the amount of p21 revealed that TCTP p21 was not detected until 1 hour after stimulation, but a low level of TCTP p21 was detected between 1 and 4 hours after stimulation (FIG. 3B).

(8) Inhibition of TCTP p21 Expression Level in MIN6 Cells by Insulin and Secretory Action 10 nM insulin was added to MIN6 cells under high glucose and low glucose, and intracellular TCTP p21 was added.
Changes in the amount of 21 protein were examined over time by Western blotting (FIGS. 4A, B). TCT before insulin stimulation
Assuming that P p21 level is 1, the densitometer value of TCTP p21 level in MIN6 cells after insulin stimulation is shown below.

[0178]

[Table 2]

[0179]

[Table 3]

At the same time, TCTP secreted into the culture solution
When the amount of p21 was examined, no secretion of TCTP p21 was detected within the detection range under high glucose, but under low glucose, TCTP corresponding to the amount reduced in cells was detected.
p21 secretion was confirmed (FIG. 4C).

(9) Inhibition of insulin secretion of MIN6 cells by rTCTP p21 MIN6 cells incubated for 8 hours with rTCTP p21 at 0.01 to 2 μg / ml were incubated for 1.5 hours under low glucose (2.8 mM), The medium was exchanged for a high glucose (25 mM) culture medium, and glucose was stimulated for 1 hour to measure secreted insulin.
The amount of insulin secreted in one hour under low glucose was defined as the basal secretion. During this time, rTCTP p21
Was always added to the reaction solution. The amount of secreted insulin is shown below.

[0182]

[Table 4]

Next, the amount of insulin secreted for 1 hour when 1 μg / ml rTCTP p21 was added 3.5, 5.5, 9.5 and 13.5 hours before glucose stimulation is shown.

[0184]

[Table 5]

Further, 8 μg / ml of rTCTP p21 was used.
Shown are data obtained by incubating time-treated MIN6 cells under low glucose for 1.5 hours and measuring insulin secretion from glucose-stimulated cells over time compared to untreated cells.

[0186]

[Table 6]

Recombinant TCT prepared from Escherichia coli
The P p21 protein (rTCTP p21) contains a small amount of E. coli-derived endotoxin (LPS) (1).
The μg / ml rTCTP p21 reaction contains about 10 ng / ml LPS). To confirm that the action of rTCTP p21 was not due to contaminating LPS, various concentrations of E. coli K-235 LPS (Sigma) were added to MIN6 cells and allowed to act for 8 hours. 2 shows insulin secretion from this cell.

[0188]

[Table 7]

In addition, polymyxin B (200 ng / ml)
RTCTP p21 treatment at 60 ℃ for 60 minutes
The inhibitory action of TCTP p21 on insulin secretion by glucose stimulation was not eliminated.

(10) Inhibition of insulin secretion from mouse isolated pancreatic islets of Langerhans by rTCTP p21 (10-1) Pancreatic islets of Langerhans were isolated from C3H / HeN mice, and were isolated in DMEM of high glucose for 0.01 to 1 day.
5 μg / ml rTCTP p21 was added to 5 medium size islets of Langerhans and allowed to act for 8 hours, followed by incubation with low glucose (2.8 mM) for 1.5 hours. This islet of Langerhans is converted to high glucose (25 mM).
And the medium was stimulated with glucose for 1 hour.
The amount of insulin secreted in one hour under low glucose was defined as the basal secretion. During this time, rTCTP p21
Was always added to the reaction solution. The amount of secreted insulin is shown below.

[0191]

[Table 8]

Pancreatic islets of Langerhans were also isolated from C57BL / 6 mice to confirm the effect of rTCTP p21. Five separated medium size islets of Langerhans were treated with 1 μg / ml of rTCTP p21 for various times, and then stimulated with glucose to measure the amount of insulin secreted per hour.

[0193]

[Table 9]

(10-2) Pancreatic islets of Langerhans were isolated from SD rats, and 0.5 to 2 was isolated in high glucose DMEM.
μg / ml of rTCTP p21 was added to about 5 medium-sized islets of Langerhans, allowed to act for 3 hours, and then incubated with low glucose (2.8 mM) for 1 hour.
The islets of Langerhans were exchanged for a culture solution of high glucose (25 mM), and glucose stimulation was performed for 1 hour. still,
The amount of insulin secreted per hour under low glucose was defined as the basal secretion. During this time, rTCTP p21 was always added to the reaction solution. The amount of secreted insulin is shown below.

[0195]

[Table 10]

(10-3) that the insulin secretion inhibitory effect of rTCTP p21 is not due to cell injury;
Also, to confirm that this inhibition is reversible,
The following experiment was performed. First, 5 medium-sized pancreatic islets of Langerhans isolated from C57BL / 6 mice were
Incubated in DMEM containing 2.8 mM glucose for hours. At the end of the 1 hour incubation, 1
mg / ml of rTCTP p21 was added and 30 minutes later,
The islets of Langerhans were stimulated with DMEM containing 25 mM glucose for 1 hour in the presence of mg / ml rTCTP p21 (Table 1).
1). Then wash the islets of Langerhans with DMEM
After removing rTCTP p21 and incubating again with DMEM containing 2.8 mM glucose for 30 minutes (Table 11), the islets of Langerhans were stimulated with DMEM containing 25 mM glucose for 1 hour (Table 11), during each incubation during which. Was measured for the amount of insulin secreted. The experiment was performed three times.

[0197]

[Table 11]

(11) T in mouse pancreas by food intake
Induction of CTP p21 C3H / HeN mice fasted for 24 hours were fed a solid diet, TCTP p21 levels in the pancreas were measured over time by Western blotting, and TCTP p21 before feeding was started.
TCTP p21 in the pancreas after feeding, with the level as 1
The decintometer values of the levels are shown below (see FIG. 5).

[0199]

[Table 12]

C57BL / 6 mice were fed a solid diet daily for 2 hours (8:00 to 10:00) for 7 days, immediately before the 8th day of diet, 1, 2, 4, 8 and 12 hours after initiation. After sacrifice, peripheral blood was collected, and the liver and pancreas were removed. The amount of TCTP p21 in plasma (fraction excluding platelets) and in the liver and pancreas was measured by Western blotting, and the TCTP p21 level before feeding was set to 1, and the TCTP p21 level after feeding was determined as a densitometer. The values are shown below. The data was shown as the average of two animals (see FIG. 6).

[0201]

[Table 13]

Experimental Example 2 Insulin Secretion-Promoting Effect of Anti-rTCTP p21 Neutralizing Antibody (1) Concentration-Dependent Effect of Purified Neutralizing Antibody Rabbit was immunized with rTCTP p21 and anti-rTCTP p2
One antibody was prepared. The obtained antiserum was affinity-purified to prepare antibody A. Antibody neutralizing activity was measured using pancreatic islets of Langerhans isolated from C57BL / 6 mice. 1.5 medium-sized Langerhans islands for 1.5 hours 2.
After incubation with DMEM containing 8 mM glucose, the amount of insulin released after stimulation with DMEM containing 25 mM glucose for 1 hour was measured. 1 μg / ml rTCTP
p21 was added immediately after the start of incubation with DMEM containing 2.8 mM glucose for 1.5 hours, and was continuously present during high glucose stimulation. Antibody treatment is rTCTP p
What reacted with 21 and antibody A at room temperature for 30 minutes was added. Table 14 shows the results.

[0203]

[Table 14]

(2) Endogenous rTCT using purified neutralizing antibody
P p21 inhibitory effect The neutralizing activity of antibody A against endogenous TCTP p21 was measured using pancreatic islets of Langerhans isolated from C57BL / 6 mice. Five medium-sized islets of Langerhans were incubated with DMEM containing 2.8 mM glucose for 1.5 hours, and then stimulated with DMEM containing 25 mM glucose for 1 hour to measure the amount of insulin released. As a control, 1 μg / ml of normal rabbit IgG was used. Table 15 shows the results.

[0205]

[Table 15]

Experimental Example 3 (1) TCT from MIN6 cells stimulated by glucose
Pp21 induction Mouse pancreatic β cell line (MIN6 cells) was immortalized
% (FCS, Flow Inc., McLean, VA), 37 ° C., 5% CO ₂ -95% using DMEM containing 100 IU / ml penicillin, 100 mg / ml streptomycin, 5 μM 2-mercaptoethanol and 25 mM glucose The cells were cultured in air. In the experiment, cells of passages 18 to 23 were used. 2.5, 5, 1
Culture for 4 hours at 0, and 25 mM glucose concentration,
The level of TCTP p21 in cells was measured by Western blotting using a TP p21 antibody. As a result, MIN
6 cells were measured by TCT in proportion to the glucose concentration in the culture.
Induced Pp21 (FIG. 7)

(2) Induction of TCTP p21 from Pancreatic Tissue by Glucose Stimulation and Feeding (2-1) 3 g / kg of C57BL / 6 male mice (12-16 weeks old) fasted overnight using an oral probe Glucose of the body weight was administered intragastrically, and the pancreas and serum were collected over time to collect TCTP p.
The amount of 21 was measured by Western blotting (FIG. 8). In addition, the same measurement was performed for mice fed a solid diet (FIG. 9). As a result, similar to that observed in MIN6 cells,
Oral administration of glucose to mice results in transient TCTP p peaking in pancreatic tissue within 1-2 hours within 1-2 minutes.
Induction of TCTP was found to be consistent with TCTP in blood.
p21 levels also increased. Feeding also caused stronger increases in pancreatic tissue and blood TCTP p21 levels than with glucose alone.

(2-2) A neutralizing antibody (antibody A) of TCTP p21 or normal rabbit IgG (0.8 mg) was intraperitoneally administered to C57BL / 6 male mice (12-16 weeks old) which had been fasted overnight. Two hours later, 2 g / kg body weight of glucose was intraperitoneally administered, and the amount of insulin in plasma was measured by ELISA. As a result, when a neutralizing antibody is administered, 30, 60, after glucose administration,
Insulin secretion at 120 minutes was significantly higher (Fig. 1
0). In FIG. 10, 〇 shows the result when normal rabbit IgG was administered, and ■ shows the result when neutralizing antibody was administered.

(2-3) Physiological saline containing 30 μg of rTCTP p21 in the tail vein of C57BL / 6 mice fasted overnight
Administer 50 μl or 50 μl of saline (vehicle) and immediately intraperitoneally 2 g / kg body weight glucose
After 30 minutes, insulin and blood glucose levels were measured. as a result,
rTCTP p21 significantly inhibited insulin secretion due to glucose administration and significantly increased blood glucose levels (FIG. 1).
1).

(3) Insulin Secretion Inhibition by rTCTP p21 MIN6 cells were incubated for 1.5 hours in DMEM containing 2.8 mM glucose and 15% FCS, and then exchanged with DMEM containing 25 mM glucose and 15% FCS to stimulate high glucose stimulation. Was performed. 1 μg of rTCTP 30 minutes before high glucose stimulation
p21 or the same amount of physiological saline (vehicle) was added. 1 and 2 hours after glucose stimulation, total RN
A was extracted and the mRNA levels of β-actin and proinsulin were measured by RT-PCR. Similarly, cell proteins were extracted before and one hour after the glucose stimulation, and the intracellular insulin content was measured by Western blotting. As a result, r
TCTP p21 inhibited glucose-stimulated insulin secretion from MIN6 cells, while rTCTP p21 did not affect insulin mRNA expression (FIG. 1).
2) Since the insulin content in the cells was not changed (FIG. 13), it was considered that the insulin secretion process was inhibited. Values are mean ± SD, n = 4.

(4) Inhibition of insulin secretion by overexpression of TCTP p21 The pcDNA3.1 expression plasmid vector incorporating human TCTP p21 cDNA or the pcDNA3.1 expression plasmid vector alone was subjected to MI by cationic liposome (lipofectoAMINE) method.
It was introduced into N6 cells to overexpress TCTP p21. High glucose stimulation was performed on cells into which only the vector had been introduced and cells overexpressing TCTP p21, and the amount of insulin secreted for 60 minutes was measured. As a result, cells into which only the vector was introduced (the vector in FIG. 14) and TCTP
T of cells overexpressing p21 (TCTP in FIG. 14)
The CTP p21 content was confirmed by Western blotting,
Comparing the insulin secretion ability of those cells, TCT
In the cells overexpressing Pp21, insulin secretion ability was lost (FIG. 14). Values are mean ± SD, n = 6.

(5) Measurement of Fasting TCTP p21 Levels Male 16-week-old C57BL / 6 mice were treated with 2% 10% glucose water.
The mice were allowed to drink for 4 weeks, fasted overnight with the mice for 4 weeks, and blood was collected from the tail vein of the mice, and the TCTP p21 level in the serum was measured by Western blotting. As a result, fasting TCTP p21 levels increased in mice whose body weight was increased by 10-15% over that of control mice due to chronic glucose load (FIG. 1).
5).

(6) Measurement of Insulin and Blood Glucose Levels Using Neutralizing Antibodies to TCTP p21 Male C57BL / 6 mice fed with 10% glucose water for 4 weeks were fed at night, and then fasted at 8:00 am. 10% glucose water was also changed to tap water. Neutralizing antibody (0.8 in total IgG)
mg) or normal rabbit IgG (0.8 mg)
The administration was performed intraperitoneally at 4 and 7 hours, respectively. Blood insulin levels and blood glucose levels were measured before and 4 and 10 hours after the start of fasting. The value of each individual after the start of fasting was set to 100%, and the subsequent changes were shown. Values are shown as mean ± SEM. Control IgG administration group 6 animals, neutralizing antibody administration group 10 animals.
As a result, mice with chronic glucose load
Administration of the CTP p21 neutralizing antibody significantly increased fasting insulin (FIG. 16) and significantly reduced fasting blood glucose (FIG. 17). In FIGS. 16 and 17, □ shows the results when normal rabbit IgG was administered, and ■ shows the results when neutralizing antibodies were administered.

(7) TCTP in serum of GK rat in fasting state
Measurement of p21 amount 3 of GK (Goto-Kakizaki) rat and control rat
One week old fasting serum was collected, and TCTP p2 in serum was analyzed by Western blotting using an anti-TCTP p21 antibody.
1 was measured. GK rats are Wistar rats (Jcl: Wist
ar rats) were subjected to an oral glucose tolerance test (OGTT), and rats with low glucose tolerance were selected and bred repeatedly to establish a spontaneous diabetes model rat (Goto, Y. et a
l., Spontaneous produced by selective breeding of
normal Wistar rats. Proc. Jap. Acad. Ser. B51: 80-8
5, 1975). In GK rats, TC of 30 kDa is already over 3 weeks old
The amount of TP p21 was increasing. The TCTP p21 in serum was measured in the same manner for each of four GK rats and four control rats at the age of 3, 12, and 20 weeks, and compared with the densitometer value. The amount is 3.6 ± 3 weeks old
0.5 times (mean ± SD, n = 4), 2.2 ± 0.5 times (n =
4), at 20 weeks of age, it increased by 2.3 ± 0.4 times (n = 4).

[0215]

The substance that inhibits the action of IgE-dependent HRF is useful as an insulin secretagogue, a prophylactic / therapeutic agent for diabetes, and the like, and the IgE-dependent HRF or a substance having the action is useful as an insulin secretion inhibitor.

[0216]

[Sequence List] <110> Takeda Chemical Industries, Ltd. <120> An Insulin Secretion Inhibitor <130> A4843 <150> JP 2000-280153 <151> 2000-09-11 <160> 6 <210> 1 <211> 172 <212> PRT <213> Mouse <400> 1 Met Ile Ile Try Arg Asp Leu Ile Ser His Asp Glu Leu Phe Ser Asp 1 5 10 15 Ile Try Lys Ile Arg Glu Ile Ala Asp Gly Leu Cys Leu Glu Val Glu 20 25 30 Gly Lys Met Val Ser Arg Thr Glu Gly Ala Ile Asp Asp Ser Leu Ile 35 40 45 Gly Gly Asn Ala Ser Ala Glu Gly Pro Glu Gly Glu Gly Thr Glu Ser 50 55 60 Thr Val Val Thr Gly Val Asp Ile Val Met Asn His His Leu Gln Glu 65 70 75 80 Thr Ser Phe Thr Lys Glu Ala Tyr Lys Lys Tyr Ile Lys Asp Tyr Met 85 90 95 Lys Ser Leu Lys Gly Lys Leu Glu Glu Gln Lys Pro Glu Arg Val Lys 100 105 110 Pro Phe Met Thr Gly Ala Ala Glu Gln Ile Lys His Ile Leu Ala Asn 115 120 125 Phe Asn Asn Tyr Gln Phe Phe Ile Gly Glu Asn Met Asn Pro Asp Gly 130 135 140 Met Val Ala Leu Leu Asp Tyr Arg Glu Asp Gly Val Thr Pro Phe Met 145 150 155 160 Ile Phe Phe Lys Asp Gly Leu Glu Met Glu Lys Cys 165 170 <210> 2 <211> 172 <212> PRT <213> Human <400> 2 Met Ile Ile Try Arg Asp Leu Ile Ser His Asp Glu Met Phe Ser Asp 1 5 10 15 Ile Try Lys Ile Arg Glu Ile Ala Asp Gly Leu Cys Leu Glu Val Glu 20 25 30 Gly Lys Met Val Ser Arg Thr Glu Gly Asn Ile Asp Asp Ser Leu Ile 35 40 45 Gly Gly Asn Ala Ser Ala Glu Gly Pro Glu Gly Glu Gly Thr Glu Ser 50 55 60 Thr Val Ile Thr Gly Val Asp Ile Val Met Asn His His Leu Gln Glu 65 70 75 80 Thr Ser Phe Thr Lys Glu Ala Tyr Lys Lys Tyr Ile Lys Asp Tyr Met 85 90 95 Lys Ser Leu Lys Gly Lys Leu Glu Glu Gln Arg Pro Glu Arg Val Lys 100 105 110 Pro Phe Met Thr Gly Ala Ala Glu Gln Ile Lys His Ile Leu Ala Asn 115 120 125 Phe Lys Asn Tyr Gln Phe Phe Ile Gly Glu Asn Met Asn Pro Asp Gly 130 135 140 Met Val Ala Leu Leu Asp Tyr Arg Glu Asp Gly Val Thr Pro Tyr Met 145 150 155 160 Ile Phe Phe Lys Asp Gly Leu Glu Met Glu Lys Cys 165 170 <210> 3 <211> 172 <212> PRT <213> Mouse <400> 3 Met Ile Ile Try Arg Asp Leu Ile Ser His Asp Glu Leu Phe Ser Asp 1 5 10 15 Ile Try Lys Ile Arg Glu Ile Ala Asp Gly Leu Cys Leu Glu Val Glu 20 25 30 Gly Lys Met Val Ser Arg Thr Glu Gly Ala Ile Asp Asp Ser Leu Ile 35 40 45 Gly Gly Asn Ala Ser Ala Glu Gly Pro Glu Gly Glu Gly Thr Glu Ser 50 55 60 Thr Val Val Thr Gly Val Asp Ile Val Met Asn His His Leu Gln Glu 65 70 75 80 Thr Ser Phe Thr Lys Glu Ala Tyr Lys Lys Tyr Ile Lys Asp Tyr Met 85 90 95 Lys Ser Leu Lys Gly Lys Leu Glu Glu Gln Lys Pro Glu Arg Val Lys 100 105 110 Pro Phe Met Thr Gly Ala Ala Glu Gln Ile Lys His Ile Leu Ala Asn 115 120 125 Phe Asn Asn Tyr Gln Phe Phe Ile Gly Glu Asn Met Asn Pro Asp Gly 130 135 140 Met Val Ala Leu Leu Asp Tyr Arg Glu Asp Gly Val Thr Pro Phe Met 145 150 155 160 Ile Phe Phe Lys Asp Gly Leu Glu Met Glu Lys Cys 165 170 <210> 4 <211> 516 <212> DNA <213> Mouse <400 > 4 atgatcatct accgggacct catcagccat gacgagctcat tctccgacat ctacaagatc 60 cgggagatcg cggacgggct gtgcctggag gtggagggca agatggtcag tagaacagag 120 ggtgccatcg atgactcgct catcgg gaggag gagcaggag gagcaggag gagcaggag cc tt gacattgtca tgaaccatca cttacaagaa 240 accagcttca caaaagaggc ttacaaaaag tacatcaaag actacatgaa atcactcaaa 300 ggcaaacttg aagagcagaa accagaaaga gtaaagcctt ttatgactgg agctgcagag 360 cagattaagc acatccttgc taatttcaat aactaccagt tttttattgg tgaaaacatg 420 aatccagatg gtatggttgc tctcctggac taccgtgaag atggtgtgac tccattcatg 480 attttcttta aggatggctt agagatggag aaatgt 516 <210> 5 <211> 516 <212> DNA <213> Human <400> 5 atgattatct accgggacct catcagccac gatgagatgt tctccgacat ctacaagatc 60 cgggagatcg cggacgggtt gtgcctggag gtggagggga agatggtcag taggacagaa 120 ggtaacattg atgactcgct cattggtgga aatgcctccg ctgaaggccc cgagggcgaa 180 ggtaccgaaa gcacagtaat cactggtgtc gatattgtca tgaaccatca cctgcaggaa 240 acaagtttca caaaagaagc ctacaagaag tacatcaaag attacatgaa atcaatcaaa 300 gggaaacttg aagaacagag accagaaaga gtaaaacctt ttatgacagg ggctgcagaa 360 caaatcaagc acatccttgc taatttcaaa aactaccagt tctttattgg tgaaaacatg 420 aatccagatg gcatggttgc tctattggac taccgtgagg atggtgtgac cccatatatg 480 attttcttta aggatggttt agaaatg gaa aaatgt 516 <210> 6 <211> 516 <212> DNA <213> Mouse <400> 6 atgatcatct accgggacct catcagccat gacgagctgt tctccgacat ctacaagatc 60 cgggagatcg cggacgggct gtgcctggag gtggagggca agatggtcag tagaacagag 120 ggtgccatcg atgactcgct catcggtgga aatgcttccg ctgaaggtcc ggagggcgaa 180 ggtaccgaaa gcacagtagt caccggtgtt gacattgtca tgaaccatca cttacaagaa 240 accagcttca caaaagaggc ttacaaaaag tacatcaaag actacatgaa atcactcaaa 300 ggcaaacttg aagagcagaa accagaaaga gtaaagcctt ttatgactgg agctgcagag 360 cagattaagc acatccttgc taatttcaat aactaccagt tttttattgg tgaaaacatg 420 aatccagatg gtatggttgc tctcctggac taccgtgaag atggtgtgac tccattcatg 480 attttcttta aggatggctt agagatggag aaatgt 516

[Brief description of the drawings]

FIG. 1 shows the expression of TCTPp21 protein in mouse (A), rat (B) and guinea pig (C) tissues by anti-T
The result of having investigated by the western blotting method using CTP p21 antibody is shown.

FIG. 2 shows the results of confirming the expression of TCTP p21 and insulin in mouse pancreatic tissue by fluorescent antibody double immunohistochemical staining.

FIG. 3 shows the results of a time-course study of changes in the amount of TCTP p21 protein in pancreatic β cells and in the culture supernatant when stimulated with glucose.

FIG. 4. Addition of 10 nM insulin to MIN6 cells under high glucose (25 mM glucose) and low glucose (2.8 mM glucose), and intracellular TCTP
The result of having investigated the change of p21 protein content with time by Western blotting is shown. A is under high glucose (25m
(M glucose), B shows the amount of TCTP p21 protein in the culture supernatant under low glucose (2.8 mM glucose), and C shows the amount under low glucose (2.8 mM glucose).

FIG. 5 shows the results of a time-course Western blot analysis of the amount of TCTP p21 protein in the pancreas fed to mice that had been fasted overnight.

FIG. 6 shows plasma (A) and pancreas (B) of a mouse that has taken a diet
2 shows the results of time-course examination of the amount of TCTPp21 protein in the liver (C) by Western blotting.

FIG. 7 shows the results of examining the expression level of TCTP p21 protein in cells when MIN6 cells were cultured with glucose at each concentration for 4 hours.

FIG. 8: TCT of pancreatic tissue (left panel) and serum (right panel) upon oral administration of glucose to mice fasted overnight
The result of having investigated the change of Pp21 amount is shown.

FIG. 9: TCTP p21 in pancreatic tissue (left panel) and serum (right panel) when mice fed an overnight fast were fed a solid diet.
The results of examining the change in the amount are shown.

FIG. 10 shows the results of examining the effects of neutralizing antibodies on insulin secretion when glucose was intraperitoneally administered to mice fasted overnight. 〇 shows the result when normal rabbit IgG was administered, and ■ shows the result when neutralizing antibody was administered.

FIG. 11 shows the results of examining the effects of recombinant TCTP p21 on insulin secretion and blood glucose levels when glucose was intraperitoneally administered to mice fasted overnight. The vehicle demonstrated the effect of administering saline, rT
CTP shows the effect of administering recombinant TCTP p21.

FIG. 12. Recombinant TCTP p in MIN6 cells.
The result of having examined the amount of proinsulin mRNA at the time of adding 21 is shown. Vehicle shows the effect when physiological saline was added, and rTCTP shows the effect when recombinant TCTP p21 was added. (H) shows the time after glucose stimulation.

FIG. 13. Recombinant TCTP p21 was converted to MIN6
3 shows the results of examining changes in intracellular insulin content when added to cells. Vehicle shows the effect when physiological saline was added, and rTCTP shows the effect when recombinant TCTP p21 was added. The horizontal axis indicates glucose concentration, and the vertical axis indicates insulin concentration.

FIG. 14 shows the results of examining the insulin secretion inhibitory effect of TCTP p21 overexpression. The vector shows the results of cells transfected with the vector alone, while TCTP shows the results of TCTP p
The results for cells overexpressing 21 are shown. The horizontal axis indicates glucose concentration and the vertical axis indicates insulin secretion.

FIG. 15: TCTP p21 in serum of fasting mice
The result of measuring the level is shown. Normal chow uses 4 tap water
2 wks high glucose of mice that had been drinking for a week, 4 wks high gl of mice that had been drinking 10% glucose water for 2 weeks
ucose shows the results of mice that had been drinking 10% glucose water for 4 weeks.

FIG. 16 shows the results obtained by administering a TCTP p21 neutralizing antibody to a mouse that had been drinking 10% glucose water for 4 weeks and examining the change over time in insulin levels. The horizontal axis indicates the time after administration of the neutralizing antibody, and the vertical axis indicates the ratio of the blood insulin level. □
Shows the results when normal rabbit IgG was administered, and ■ shows the results when neutralizing antibodies were administered.

FIG. 17 shows the results obtained by administering a TCTP p21 neutralizing antibody to a mouse that had been drinking 10% glucose water for 4 weeks and examining the change over time in blood glucose level. The horizontal axis indicates the time after administration of the neutralizing antibody, and the vertical axis indicates the ratio of the blood glucose level. □ indicates normal rabbit IgG
And ■ shows the results when the neutralizing antibody was administered.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 3/10 A61P 9/10 9/10 13/12 13/12 15/00 15/00 17/00 17 / 00 19/02 19/02 19/10 19/10 25/00 25/00 35/00 35/00 A61K 37/04

Claims (39)

[Claims] 1. An insulin secretagogue comprising a substance that inhibits the action of an IgE-dependent histamine releasing factor. 2. A prophylactic / therapeutic agent for diabetes comprising a substance which inhibits the action of an IgE-dependent histamine releasing factor. 3. A substance that inhibits the action of an IgE-dependent histamine-releasing factor is a receptor antagonist of an IgE-dependent histamine-releasing factor, an antibody against the IgE-dependent histamine-releasing factor, a substance that suppresses the expression of an IgE-dependent histamine-releasing factor, 3. The agent according to claim 1, which is a substance that degrades a histamine-releasing factor or a substance that promotes the decomposition of an IgE-dependent histamine releasing factor. 4. The agent according to claim 1, wherein the IgE-dependent histamine releasing factor is a tumor-associated protein. 5. The agent according to claim 1, wherein the IgE-dependent histamine releasing factor is tumor-associated protein p21, tumor-associated protein p23 or tumor-associated protein p26. 6. The agent according to claim 1, which is an agent for preventing or treating a disease caused by insufficient insulin secretion. 7. A preventive or therapeutic agent for impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy or osteopenia. The agent according to claim 1. 8. A method for promoting insulin secretion, comprising administering to a mammal an effective amount of a substance that inhibits the action of an IgE-dependent histamine releasing factor. 9. A method for preventing or treating a disease caused by insufficient insulin secretion, which comprises administering to a mammal an effective amount of a substance that inhibits the action of an IgE-dependent histamine releasing factor. 10. A method for administering an effective amount of a substance that inhibits the action of an IgE-dependent histamine releasing factor to a mammal, comprising impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, A method for preventing and treating diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy or osteopenia. 11. A method for preventing and treating diabetes, comprising administering to a mammal an effective amount of a substance that inhibits the action of an IgE-dependent histamine releasing factor. 12. Use of a substance that inhibits the action of an IgE-dependent histamine releasing factor for producing an insulin secretagogue. 13. Use of a substance that inhibits the action of an IgE-dependent histamine releasing factor for producing a prophylactic / therapeutic agent for a disease caused by insufficient insulin secretion. 14. Production of a preventive / therapeutic agent for impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy or osteopenia. The use of a substance that inhibits the action of an IgE-dependent histamine releasing factor. 15. Use of a substance that inhibits the action of an IgE-dependent histamine releasing factor for producing an agent for preventing or treating diabetes. 16. An insulin secretion inhibitor comprising an IgE-dependent histamine releasing factor or a substance having the action thereof. 17. A substance having an action of an IgE-dependent histamine-releasing factor, a receptor agonist of an IgE-dependent histamine-releasing factor, a substance enhancing or promoting the action of an IgE-dependent histamine-releasing factor, or an IgE-dependent histamine-releasing factor. 17. The agent according to claim 16, which is a substance that inhibits the degradation of an expression promoting substance or an IgE-dependent histamine releasing factor. 18. The agent according to claim 16, wherein the IgE-dependent histamine releasing factor is a tumor-associated protein. 19. The agent according to claim 16, wherein the IgE-dependent histamine releasing factor is tumor-associated protein p21, tumor-associated protein p23 or tumor-associated protein p26. 20. The agent according to claim 16, which is a prophylactic / therapeutic agent for a disease caused by excessive insulin secretion. 21. Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance, unstable diabetes, lipodystrophy, insulin allergy or 17. The agent according to claim 16, which is an agent for preventing or treating insulinoma. 22. A method for preventing or treating a disease caused by excessive insulin secretion, which comprises administering to a mammal an effective amount of an IgE-dependent histamine releasing factor or a substance having an action thereof. 23. An obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, which comprises administering to a mammal an effective amount of an IgE-dependent histamine releasing factor or a substance having the action thereof. , Diabetic neuropathy, diabetic nephropathy,
A method for preventing or treating diabetic retinopathy, edema, insulin resistance, unstable diabetes, lipodystrophy, insulin allergy or insulinoma. 24. Use of an IgE-dependent histamine releasing factor or a substance having an action thereof for the manufacture of a prophylactic / therapeutic agent for a disease caused by excessive insulin secretion. 25. Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance, unstable diabetes, lipodystrophy, insulin allergy or Use of an IgE-dependent histamine releasing factor or a substance having an action thereof for producing a prophylactic / therapeutic agent for insulinoma. 26. A method for screening for a substance regulating insulin secretion, comprising using an IgE-dependent histamine releasing factor and / or an IgE-dependent histamine releasing factor receptor. 27. A method for screening for an insulin secretion regulatory substance, comprising using a DNA encoding an IgE-dependent histamine releasing factor and / or a DNA encoding an IgE-dependent histamine releasing factor receptor. 28. A kit for screening an insulin secretion regulatory substance, comprising an IgE-dependent histamine releasing factor and / or an IgE-dependent histamine releasing factor receptor. 29. A screening kit for an insulin secretion regulatory substance, comprising a DNA encoding an IgE-dependent histamine releasing factor and / or a DNA encoding an IgE-dependent histamine releasing factor receptor. 30. An insulin secretion regulatory substance obtainable by using the screening method according to claim 26 or 27 or the screening kit according to claim 28 or 29. 31. A medicament comprising an insulin secretion regulatory substance obtainable by using the screening method according to claim 26 or 27 or the screening kit according to claim 28 or 29. 32. The method according to claim 3, which is an insulin secretion regulator.
The medicament according to 1. 33. The method according to claim 3, which is an agent for preventing or treating diabetes.
The medicament according to 1. 34. An effective amount of an insulin secretion regulatory substance obtainable by using the screening method according to claim 26 or 27 or the screening kit according to claim 28 or 29 to a mammal. Insulin secretion regulation method. 35. A mammal, comprising administering an effective amount of an insulin secretion regulatory substance obtainable by using the screening method according to claim 26 or 27 or the screening kit according to claim 28 or 29. Diabetes prevention and treatment method. 36. A diagnostic agent for abnormal insulin secretion, comprising an antibody against IgE-dependent histamine releasing factor. 37. A method for diagnosing abnormal insulin secretion, comprising using an antibody against an IgE-dependent histamine releasing factor. 38. An agent for diagnosing abnormal insulin secretion, comprising a DNA encoding an IgE-dependent histamine releasing factor. 39. A method for diagnosing abnormal insulin secretion, which comprises using a DNA encoding an IgE-dependent histamine releasing factor.