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Method of screening remedy for diabetes

a screening remedy and diabetes technology, applied in the field of diabetes screening remedy, can solve the problems of reducing blood glucose, increasing the number of patients in accordance, and affecting the effect of insulin preparations, so as to promote insulin secretion, promote insulin secretion, and promote insulin secretion

Inactive Publication Date: 2003-09-25
ASTELLAS PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011] With the aim of solving the aforementioned problems, the present inventors have conducted intensive studies and, as a result, found that an amount of insulin secreted is increased by activation under a high glucose concentration by overexpressing a pancreas-specifically expressing "polypeptide having an amino acid sequence of SEQ ID NO: 2" in pancreatic .beta. cells and that, by contrast, an amount of insulin secreted is not changed by activation under a low glucose concentration, and thus found that the polypeptide and the cell expressing the polypeptide may be used as a screening tool for an agent for treating diabetes having an activity promoting insulin secretion specifically under a high glucose concentration and capable of controlling blood glucose within a normal range. Further, the inventors succeeded in providing a novel screening method for an agent for treating diabetes by use of the screening tool. The inventors have confirmed that activating substances obtained by screening known compounds not known to have an activity of treating diabetes, by use of the screening method, exhibit an activity of increasing an amount of insulin and an activity of decreasing blood glucose in rat plasma when glucose is administered, and suppress an increase of a blood glucose level in a diabetes model rat when glucose is administered, and thus clarified the utility of the screening method of the present invention. Further, the inventors established a process for manufacturing a pharmaceutical composition for treating diabetes using an analysis of the polypeptide activation, and completed the present invention.
[0075] As the marker sequence, a sequence for easily carrying out a confirmation of polypeptide expression, a confirmation of intracellular localization thereof, a purification thereof, or the like may be used. As the sequence, there may be mentioned, for example, a FLAG epitope, a hexa-histidine tag, a hemagglutinin tag, or a myc epitope, etc.

Problems solved by technology

It is considered that 95% or more of Japanese patients with diabetes are NIDDM, and there is a problem in that the number of patients increases in accordance with changes of life-style.
Further, symptoms in the digestive system such as loss of appetite occur.
The insulin preparations certainly decrease blood glucose.
As described above, conventionally used agents for promoting insulin secretion and insulin preparations have these problems.

Method used

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  • Method of screening remedy for diabetes
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Examples

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example 1

Isolation of Polynucleotide Encoding Polypeptide Consisting of Amino Acid Sequence of SEQ ID NO: 2

[0162] A full-length cDNA encoding the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 was prepared by a reverse transcriptase-polymerase chain reaction (RT-PCR), using a human genomic DNA (TOYOBO) as a template, in accordance with the following procedures.

[0163] An oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 3 was used as a forward primer, and an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 4 was used as a reverse primer. At each of the 5'-termini of the primers, an XbaI recognition sequence added thereto existed, respectively. The RT-PCR was carried out, using a polymerase (Pyrobest DNA polymerase; Takara-shuzo) in the presence of 5% dimethylsulfoxide (DMSO), by repeating a cycle composed of treatments at 98.degree. C. for 10 seconds, at 58.degree. C. for 30 seconds, and at 72.degree. C. for 2 minutes, 34 times. As a result,...

example 2

Confirmation of Expression Distribution of mRNA of Polypeptide Consisting of Amino Acid Sequence of SEQ ID NO: 2

[0169] An expression distribution of the polynucleotide encoding the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 was analyzed by an RT-PCR method in accordance with the following procedures.

[0170] In a first step, a poly A+ RNA (5 .mu.g; Clontech) prepared from human organs, more particularly, brain, i.e., amygdala, caudate nucleus, hippocampus, corpus callosum, substantia nigra, and cerebellum, spinal cord, pituitary gland, heart, placenta, lung, trachea, liver, kidney, pancreas, small intestine, stomach, spleen, bone marrow, thymus, thyloid gland, salivary gland, adrenal gland, mammary gland, prostate, testis, and ovary, was reacted with a DNase (DNase; Nippon Gene) at 37.degree. C. for 15 minutes. A part (4 .mu.g) of a resulting poly A+ RNA treated with the DNase was used for a reaction with a reverse transcriptase (MMLV Reverse Transcriptase; Clon...

example 3

Isolation and Confirmation of Expression Distribution of Rat Polynucleotide Corresponding to that Encoding Polypeptide Consisting of Amino Acid Sequence of SEQ ID NO: 2

[0172] A rat polynucleotide corresponding to the human polynucleotide encoding the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 and isolated in Example 1 was obtained by the following procedures.

[0173] A PCR was first carried out, using a rat genomic DNA (rat genomic DNA; Clontech) as a template and the oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 11 and the oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 12, each used in Example 2, as a primer set. In the PCR, a DNA polymerase (Pyrobest DNA polymerase; Takara-shuzo) was used, and a cycle composed of treatments at 98.degree. C. for 10 seconds, at 57.degree. C. for 30 seconds, and at 72.degree. C. for 1 minute was repeated 34 times in the presence of 5% DMSO. Then, a further PCR was carried out, using resu...

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Abstract

A convenient screening tool and a convenient screening method for obtaining an agent for treating diabetes, a pharmaceutical composition for treating diabetes, and a process for manufacturing the pharmaceutical composition are disclosed. The screening tool is a G protein-coupled receptor, a variation functionally equivalent thereto, or a homologous polypeptide thereof which promotes insulin secretion under a high glucose concentration by activation, or cells transformed with an expression vector comprising a polynucleotide encoding the above polypeptide and expressing the polypeptide.

Description

[0001] The present invention relates to a method for screening agents for the treatment of diabetes.[0002] Diabetes is a disease with a persistent hyperglycemia, and it is considered that many environmental factors and genetic factors cause diabetes. A main factor regulating blood glucose is insulin, and it is known that a deficiency of insulin or a redundant presence of various factors inhibiting the activities of insulin (such as genetic factors, lack of exercise, fatness, stress, or the like) cause hyperglycemia.[0003] There are two major types of diabetes, which are classified into an insulin dependent diabetes mellitus (IDDM) caused by a decreased pancreatic insulin secretion due to an autoimmune disease or the like, and a noninsulin dependent diabetes mellitus (NIDDM) caused by a decreased pancreatic insulin secretion due to an exhausted pancreas with a continuous hypersecretion of insulin. It is considered that 95% or more of Japanese patients with diabetes are NIDDM, and the...

Claims

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Application Information

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IPC IPC(8): A61K38/00A61P3/10A61P43/00C07K14/705C07K14/72C12N1/15C12N1/19C12N1/21C12N15/12
CPCA61K38/00C07K14/723C07K14/705A61P3/10A61P43/00A61P5/50
Inventor OHISHI, TAKAHIDETAKASAKI, JUNMATSUMOTO, MITSUYUKISAITO, TETSUKAMOHARA, MASAZUMISOGA, TAKATOSHIYOSHIDA, SHIGERUUEDA, YOSHITAKA
Owner ASTELLAS PHARMA INC
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