# Patent

## Title
Method of treatment of diabestes or reduction in pancreatic beta-cells

## Abstract

Disclosed are: an insulin secretion inducer; an insulin secretion-inducing composition; a process for the production of the composition; an accelerator for increasing the number of pancreatic β-cells; a composition for increasing the number of pancreatic β-cells; a process for the production of the composition; and a viral vector for gene therapy. The insulin secretion inducer or the accelerator for increasing the number of pancreatic β-cells comprises a polypeptide having an amino acid sequence encoded by DNA that is known to encode a membrane protein Tm4sf20 (transmembrane 4 L six family member 20) or the like or a fragment of the polypeptide as an active ingredient.

## Background
TECHNICAL FIELD
The present invention relates to an insulin secretion inducer, insulin secretion-inducing composition and a method of manufacturing the same, an accelerator for increasing the number of pancreatic β-cells, a composition for increasing the number of pancreatic β-cells and a method of manufacturing same, in addition to a virus vector for genetic treatment, for use primarily in the treatment of diabetes and of various other diseases.
BACKGROUND ART
In recent years, the alimentary tract has attracted attention not only for absorbing nutrients during meals but also as an internal secretory organ producing gastrointestinal hormones. In addition to ghrelin produced by the stomach, Gastric inhibitory polypeptide: GIP or Glucagon-like peptide-1: GLP-1 secreted from the small intestine have been cloned. Ghrelin has a food intake stimulatory effect and is related to energy metabolism. GLP-1 and GIP are termed incretins and act on pancreatic β-cells in response to food load to induce insulin secretion. GIP is also expressed in adipose tissue and the fact that a GIP-receptor knockout mouse does not experience an increase in body weight even when given high fat foods suggests a connection with obesity.
Thus gastrointestinal hormones are related to energy metabolism and food intake behavior and elucidation of their function is assisting in the development of methods of treating various diseases primarily diabetes. However an overall description of gastrointestinal hormones is not yet clear and many facets remain elusive.
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
Thus the present invention has the object of searching for genes encoding unknown polypeptides from the alimentary tract in order to provide new medicinal uses based on such identified genes.
Means for Solving the Problems
The present inventors have conducted diligent research with respect to the above point and by using an SST method (Signal Sequence Trap Method: Nat. Biotechnol. 1999 May; 17(5): 487-90. A signal sequence trap based on a constitutively active cytokine receptor. Kojima T, Kitamura T.), the present inventors have discovered that a clone CF266 (mCF266) identified using a cDNA fragment isolated from the murine alimentary tract is specifically expressed in the alimentary tract, that there is an insulin secretion effect in a culture supernatant of cells transfected with mCF266 and that mCF266 displays an insulin secretion effect when mCF266 is forcibly expressed in vivo by using a forced expression system employing an adenovirus vector. The present inventors have discovered that the same action is displayed by a synthetic polypeptide which is a fragment of a polypeptide having an amino acid sequence encoded by human CF266 (hCF266) which corresponds to mCF266.
Furthermore the present inventors have discovered that hCF266 has an action of increasing the number of pancreatic β-cell when hCF266 is forcibly expressed in vivo by using a forced expression system employing an adenovirus vector and that the number of pancreatic β-cells is increased in mCF266 transgenic mice. Furthermore the present inventors have discovered the same action is displayed by a synthetic polypeptide which is a fragment of a polypeptide having an amino acid sequence encoded by hCF266. Although the factor causing the increase in the number of pancreatic β-cells is thought to be either a propagation accelerator for pancreatic β-cells or a degradation inhibitor for pancreatic β-cells, in the present specification, both are referred to globally as “accelerator for increasing the number of pancreatic β-cells”.
The present invention was completed using the above insights.
The insulin secretion inducer includes at least one of the following active ingredients (a) to (c):
(a) a polypeptide having an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 1 or SEQ. ID. No. 4;
(b) a polypeptide having an insulin secretion inducing action and having an amino acid sequence in which one or several amino acids have been substituted, deleted and/or added to an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 1 or SEQ. ID. No. 4; and
(c) a fragment of the polypeptide in (a) or (b) having an insulin secretion inducing action.
The insulin secretion inducer according to the present invention is characterized in that a polypeptide having an insulin secretion inducing action and having an amino acid sequence in which one or several amino acids have been substituted, deleted and/or added to an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 4, is a polypeptide having an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 7.
The insulin secretion inducer according to the present invention includes at least one of the following active ingredients (d) to (f):
(d) a polypeptide having an amino acid sequence as set forth in SEQ. ID. No. 3 or SEQ. ID. No. 6;
(e) a polypeptide having an insulin secretion inducing action and having an amino acid sequence in which one or several amino acids have been substituted, deleted and/or added to an amino acid sequence as set forth in SEQ. ID. No. 3 or SEQ. ID. No. 6; and
(f) a fragment of the polypeptide in (d) or (e) having an insulin secretion inducing action.
The insulin secretion inducer according to the present invention is characterized in that the polypeptide having the insulin secretion inducing action and having the amino acid sequence in which one or several amino acids have been substituted, deleted and/or added to an amino acid sequence as set forth in SEQ. ID. No. 6, is a polypeptide having an amino acid sequence as set forth in SEQ. ID. No. 9.
The insulin secretion inducer according to the present invention is characterized in that the fragment of the polypeptide in (d) or (e) having the insulin secretion inducing action is a polypeptide including an amino acid sequence in SEQ. ID. No. 10, SEQ. ID. No. 11 or SEQ. ID. No. 12.
The present invention provides use of at least one of the following (a) to (c) in the preparation of an insulin secretion inducer:
(a) a polypeptide having an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 1 or SEQ. ID. No. 4;
(b) a polypeptide having an insulin secretion inducing action and having an amino acid sequence in which one or several amino acids have been substituted, deleted and/or added to an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 1 or SEQ. ID. No. 4; and
(c) a fragment of the polypeptide in (a) or (b) having an insulin secretion inducing action.
The present invention provides use of at least one of the following (d) to (f) in the preparation of an insulin secretion inducer:
(d) a polypeptide having an amino acid sequence as set forth in SEQ. ID. No. 3 or SEQ. ID. No. 6;
(e) a polypeptide having an insulin secretion inducing action and having an amino acid sequence in which one or several amino acids have been substituted, deleted and/or added to an amino acid sequence as set forth in SEQ. ID. No. 3 or SEQ. ID. No. 6; and
(f) a fragment of the polypeptide in (d) or (e) having an insulin secretion inducing action.
The insulin secretion-inducing composition according to the present invention includes at least one active ingredient being a polypeptide having an amino acid sequence as set forth in SEQ. ID. No. 3, a polypeptide having an amino acid sequence as set forth in SEQ. ID. No. 6, a polypeptide having an amino acid sequence as set forth in SEQ. ID. No. 9, a polypeptide including an amino acid sequence as set forth in SEQ. ID. No. 10, a polypeptide including an amino acid sequence as set forth in SEQ. ID. No. 11 or a polypeptide including an amino acid sequence as set forth in SEQ. ID. No. 12.
A method of preparing an insulin secretion-inducing composition, the method including the steps of integrating DNA as an exogenous gene into culturable cells, the DNA being DNA having a base sequence as set forth in at least one of SEQ. ID. No. 1, SEQ. ID. No. 4 and SEQ. ID. No. 7, or being DNA capable of hybridizing under stringent conditions with a strand complementary to DNA having the base sequence as set forth in at least one of SEQ. ID. No. 1, SEQ. ID. No. 4 and SEQ. ID. No. 7, culturing the cells and expressing the gene.
The viral vector employed for inducing insulin secretion according to the genetic treatment of the present invention is characterized in that DNA is integrated as an exogenous gene into a viral vector enabling expression of the exogenous gene, the DNA being DNA having a base sequence as set forth in at least one of SEQ. ID. No. 1, SEQ. ID. No. 4 and SEQ. ID. No. 7, or being DNA capable of hybridizing under stringent conditions with a strand complementary to DNA having the base sequence as set forth in at least one of SEQ. ID. No. 1, SEQ. ID. No. 4 and SEQ. ID. No. 7.
The viral vector inducing insulin secretion according to the genetic treatment of the present invention is characterized in that it is an adenovirus vector.
An accelerator for increasing the number of pancreatic β-cells according to the present invention includes at least one of the following active ingredients (a) to (c):
(a) a polypeptide having an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 1 or SEQ. ID. No. 4;
(b) a polypeptide having an action of increasing the number of pancreatic β-cells and having an amino acid sequence in which one or several amino acids have been substituted, deleted and/or added to an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 1 or SEQ. ID. No. 4; and
(c) a fragment of the polypeptide in (a) or (b) having an action of increasing the number of pancreatic β-cells.
The accelerator for increasing the number of pancreatic β-cells according to the present invention is characterized in that the polypeptide having the action of increasing the number of pancreatic β-cells and having the amino acid sequence in which one or a plurality of amino acids has been substituted, deleted and/or added to the amino acid sequence encoded by DNA having the base sequence as set forth in SEQ. ID. No. 4, is a polypeptide having an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 7.
The accelerator for increasing the number of pancreatic β-cells according to the present invention includes at least one of the following active ingredients (d) to (f):
(d) a polypeptide having an amino acid sequence as set forth in SEQ. ID. No. 3 or SEQ. ID. No. 6;
(e) a polypeptide having an action of increasing the number of pancreatic β-cells and having an amino acid sequence in which one or several amino acids have been substituted, deleted and/or added to an amino acid sequence as set forth in SEQ. ID. No. 3 or SEQ. ID. No. 6; and
(f) a fragment of the polypeptide in (d) or (e) having an action of increasing the number of pancreatic β-cells.
The accelerator for increasing the number of pancreatic β-cells according to the present invention is characterized in that the polypeptide having an action of increasing the number of pancreatic β-cells and having the amino acid sequence in which one or several amino acids have been substituted, deleted and/or added to the amino acid sequence as set forth in SEQ. ID. No. 6, is a polypeptide having an amino acid sequence as set forth in SEQ. ID. No. 9.
The accelerator for increasing the number of pancreatic β-cells according to the present invention is characterized in that a fragment of the polypeptide in (d) or (e) having the action of increasing the number of pancreatic β-cells is a polypeptide including an amino acid sequence as set forth in SEQ. ID. No. 10, SEQ. ID. No. 11 or SEQ. ID. No. 12.
The present invention provides use of at least one of the following (a) to (c) in the preparation of an accelerator for increasing the number of pancreatic β-cells:
(a) a polypeptide having an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 1 or SEQ. ID. No. 4;
(b) a polypeptide having an action of increasing the number of pancreatic β-cells and having an amino acid sequence in which one or several amino acids have been substituted, deleted and/or added to an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 1 or SEQ. ID. No. 4; and
(c) a fragment of the polypeptide in (a) or (b) having an action of increasing the number of pancreatic β-cells.
The present invention provides use of at least one of the following (d) to (f) in the preparation of an accelerator for increasing the number of pancreatic β-cells:
(d) a polypeptide having an amino acid sequence having a base sequence as set forth in SEQ. ID. No. 3 or SEQ. ID. No. 6;
(e) a polypeptide having an action of increasing the number of pancreatic β-cells and having an amino acid sequence in which one or several amino acids have been substituted, deleted and/or added to an amino acid sequence as set forth in SEQ. ID. No. 3 or SEQ. ID. No. 6; and
(f) a fragment of the polypeptide in (d) or (e) having an action of increasing the number of pancreatic β-cells.
A composition for increasing the number of pancreatic β-cells according to the present invention includes at least one active ingredient being a polypeptide having an amino acid sequence as set forth in SEQ. ID. No. 3, a polypeptide having an amino acid sequence as set forth in SEQ. ID. No. 6, a polypeptide having an amino acid sequence as set forth in SEQ. ID. No. 9, a polypeptide including an amino acid sequence as set forth in SEQ. ID. No. 10, a polypeptide including an amino acid sequence as set forth in SEQ. ID. No. 11 or a polypeptide including an amino acid sequence as set forth in SEQ. ID. No. 12.
A method of preparing a composition for increasing the number of pancreatic β-cells according to the present invention includes the steps of integrating DNA as an exogenous gene into culturable cells, the DNA being DNA having a base sequence as set forth in at least one of SEQ. ID. No. 1, SEQ. ID. No. 4 and SEQ. ID. No. 7, or being DNA capable of hybridizing under stringent conditions with a strand complementary to DNA having the base sequence as set forth in at least one of SEQ. ID. No. 1, SEQ. ID. No. 4 and SEQ. ID. No. 7, culturing the cells and expressing the gene.
A viral vector for increasing the number of pancreatic β-cells according to the genetic treatment of the present invention is characterized in that DNA is integrated as an exogenous gene into a viral vector enabling expression of the exogenous gene, the DNA being DNA having a base sequence as set forth in at least one of SEQ. ID. No. 1, SEQ. ID. No. 4 and SEQ. ID. No. 7, or being DNA capable of hybridizing under stringent conditions with a strand complementary to DNA having the base sequence as set forth in at least one of SEQ. ID. No. 1, SEQ. ID. No. 4 and SEQ. ID. No. 7.
The viral vector for increasing the number of pancreatic β-cells according to the genetic treatment of the present invention is characterized in that it is an adenovirus vector.
Effects of the Invention
According to the present invention, an insulin secretion inducer, insulin secretion-inducing composition and a method of manufacturing the same, an accelerator for increasing the number of pancreatic β-cells, a composition for increasing the number of pancreatic β-cells and a method of manufacturing same, and a virus vector for genetic treatment, for use primarily in the treatment of diabetes, and of various other diseases are provided.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a northern blot showing a distribution of expression of mCF266, using mCF266 cDNA as a probe;
FIG. 2 is a graph showing induction of insulin secretion with respect to a culture cell line of murine pancreatic β-cells in a culture supernatant of cells transfected with CF266;
FIG. 3A and FIG. 3B are graphs showing induction of insulin secretion with respect to a model mouse for type-2 diabetes using a recombinant adenovirus expressing mCF266;
FIG. 4 is a graph showing induction of insulin secretion with respect to islets of Langerhans from a murine pancreas due to the action of a fragment of a polypeptide having an amino acid sequence encoded by hCF266(27V);
FIG. 5 is a graph showing reduction in blood glucose level with respect to a model mouse for type-1 diabetes using a recombinant adenovirus expressing hCF266(27V);
FIG. 6 is a graph showing an increase in the number of pancreatic β-cells with respect to a model mouse for type-1 diabetes using a recombinant adenovirus expressing hCF266(27V);
FIG. 7A and FIG. 7B are graphs showing an increase in the number and area of pancreatic β-cells by mCF266 using a transgenic mouse; and
FIG. 8 is a graph showing an increase in the number of pancreatic β-cells with respect to a model mouse for type-1 diabetes due to the action of a fragment of a polypeptide having an amino acid sequence encoded by hCF266(27V).



PREFERRED MODE FOR CARRYING OUT THE INVENTION
The insulin secretion inducer according to the present invention includes at least one of the following active ingredients (a) to (c):
(a) a polypeptide having an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 1 or SEQ. ID. No. 4;
(b) a polypeptide having an insulin secretion inducing action and having an amino acid sequence in which one or several amino acids has been substituted, deleted and/or added to an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 1 or SEQ. ID. No. 4; and
(c) a fragment of the polypeptide in (a) or (b) having an insulin secretion inducing action.
The accelerator for increasing the number of pancreatic β-cells according to the present invention includes at least one of the following active ingredients (a) to (c):
(a) a polypeptide having an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 1 or SEQ. ID. No. 4;
(b) a polypeptide having an action of increasing the number of pancreatic β-cells and having an amino acid sequence in which one or several amino acids have been substituted, deleted and/or added to an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 1 or SEQ. ID. No. 4; and
(c) a fragment of the polypeptide in (a) or (b) having an action of increasing the number of pancreatic β-cells.
mCF266 acquired from a murine alimentary tract by the present inventors is DNA having the base sequence as set forth in SEQ. ID. No. 1. This DNA has a known overall length of 1505 bp and is DNA encoding a membrane protein Tm4sf20 (Transmembrane 4L six family member 20) expressed in murine muscle tissue (NCBI: LOCUS NM 025453). The protein coding sequence (CDS) of mCF266 is 41 . . . 721 and encodes a polypeptide having a 266 amino acid sequence as set forth in SEQ. ID. No. 3 (refer to SEQ. ID. No. 2). However, there have been no reports to date of this polypeptide having an insulin secretion inducing action or action of increasing the number of pancreatic β-cells.
Furthermore the present inventors have confirmed that human CF266 (hCF266) which corresponds to mCF266 has the same action as mCF266. hCF266 is DNA having the base sequence as set forth in SEQ. ID. No. 4 and has a known length of 2308 bp as DNA encoding human TM4SF20 (NCBI: LOCUS NM 024795). The CDS of hCF266 is 38 . . . 727 and encodes a polypeptide having a 229 amino acid sequence as set forth in SEQ. ID. No. 6 (refer to SEQ. ID. No. 5). However there have been no reports to date of this polypeptide having an insulin secretion inducing action or action of increasing the number of pancreatic β-cells.
The insulin secretion inducer and accelerator for increasing the number of pancreatic β-cells according to the present invention have an active ingredient being a polypeptide having an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 1 or SEQ. ID. No. 4.
As long as an insulin secretion inducing action and an action for increasing the number of pancreatic β-cells is present, the insulin secretion inducer and accelerator for increasing the number of pancreatic β-cells according to the present invention may also include an active ingredient being a polypeptide having an amino acid sequence in which one or several amino acids have been substituted, deleted and/or added to an amino acid sequence of the polypeptide above. It is known that a polypeptide having an amino acid sequence modified by substitution, deletion and/or addition of one or several amino acids from a given amino acid sequence may retain its physiological activity (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, M. J. & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500, Wang, A. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413).
When one or several amino acids are substituted by another amino acid, it is desirable that the properties of the amino acid side chains before and after substitution are conserved. The properties of amino acid side chains include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having side chains containing hydroxyl groups (S, T, V), amino acids having side chains containing sulfur atoms (C, M), amino acids having side chains containing amides (D, N, E, Q), amino acids having side chains containing bases (R, K, H) and amino acids having side chains containing aromatic series (H, F, Y, W) (the letters in the brackets represent single letter symbols for amino acids).
More precisely, a polypeptide having an insulin secretion inducing action and an action of increasing the number of pancreatic β-cells and having an amino acid sequence in which one or several amino acids have been substituted, deleted and/or added to an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 4 for example includes a polypeptide having a 229 amino acid base sequence as set forth in SEQ. ID. No. 9 which is encoded by DNA having a base sequence as set forth in SEQ. ID. No. 7 based on a single nucleotide polymorphism (SNPs) of hCF266 (refer to SEQ. ID. No. 8). The difference between the polypeptide having the amino acid sequence set forth in SEQ. ID. No. 6 and polypeptide having the amino acid sequence set forth in SEQ. ID. No. 9 is that the 27.sup.th amino acid in the former is valine (hCF266(27V)) whereas the latter has alanine (hCF266(27A)).
As long as an insulin secretion inducing action and an action of increasing the number of pancreatic β-cells is present, the insulin secretion inducer and the accelerator for increasing the number of pancreatic β-cells according to the present invention may include an active ingredient being a fragment of the above polypeptides.
More precisely, a fragment of a polypeptide having an insulin secretion inducing action and an action of increasing the number of pancreatic β-cells for example includes a polypeptide including at least a 19 amino acid sequence ALYCMLISIQALLKGPLMC (refer to SEQ. ID. No. 10) corresponding to the 98-116th amino acids of the polypeptide having the amino acid sequence as set forth in SEQ. ID. No. 6, a polypeptide including at least a 19 amino acid sequence CNNTRGMFLSSLFSVITVI (refer to SEQ. ID. No. 11) corresponding to the 78-96th amino acids of the same polypeptide or a polypeptide including at least a 19 amino acid sequence TSNDTMASGWRASSFHFDS (refer to SEQ. ID. No. 12) corresponding to the 161-179th amino acids of the same polypeptide.
The polypeptide having an insulin secretion inducing action and action of increasing the number of pancreatic β-cells or a fragment of such a polypeptide may be produced by chemical synthesis or may be obtained using recombinant technologies. For example the polypeptide may be obtained from a culture supernatant by incorporating DNA having a base sequence as set forth in SEQ. ID. No. 1, SEQ. ID. No. 4 or SEQ. ID. No. 7, or DNA capable of hybridizing under stringent conditions with a strand complementary to DNA having the base sequence as set forth in SEQ. ID. No. 1, SEQ. ID. No. 4 or SEQ. ID. No. 7 as an exogenous gene into a culturable host cell and culturing the cell in order to enable genetic expression.
The host cell may be a suitable known cell such as a bacterium, yeast cell, insect cell or animal cell. Animal cells include HEK293 cells, HEK293T cells, CHO-K1 cells and COS cells.
Herein the term “DNA capable of hybridizing under stringent conditions” means DNA obtained by using the object DNA as a probe and employing a method including colony hybridization, plaque hybridization or southern blot hybridization. For example, DNA and the like may be identified by using a filter fixing DNA originating from a colony or plaque, and in the presence of 0.7-1.0 M sodium chloride, hybridization is performed at 65° C. Thereafter, 0.1-2×SSC solution (1×SSC composition: 150 mM sodium chloride, 15 mM sodium citrate) is used to wash the filter under conditions of 65° C. (if necessary, refer to Molecular Cloning: A Laboratory Manual, 2nd, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989. or similar text). Homology between the base sequence of DNA used as a probe and the base sequence of DNA capable of hybridizing under stringent conditions is preferably at least 80%, more preferably at least 90%, still more preferably at least 93%, particularly preferably at least 95% and most preferably at least 98%.
The separation and purification of the polypeptide or fragment thereof may be performed using methods normally used in peptide chemistry including for example ion-exchange resin, partition chromatography, gel chromatography, and reverse phase chromatography.
The insulin secretion inducer and accelerator for increasing the number of pancreatic β-cells according to the present invention may be administered as an injectable preparation (subcutaneously, intracutaneously, intramuscularly, intravenously, intraperitoneally or the like), as a preparation administered transdermally, transmucosally, or pernasally or as a preparation administered orally (tablet, capsule, granules, liquid, suspension or the like). In response to the requirements of a method of administration or form of preparation, the insulin secretion inducer and accelerator for increasing the number of pancreatic β-cells according to the present invention may include suitable additives such as suspending agents, solubilization agent, stabilizers, tonicity agents, preserving agents, absorption preventing agents, surface active agents, dilution agents, excipients, pH adjusting agents and anti-oxidizing agents. The method and used amounts may be suitably determined with respect to the gender, age, body weight and malady of a patient.
For example, the polypeptide being the active ingredient, or a fragment thereof, may as required be converted to a pharmaceutically acceptable alkali metal salt such as sodium salt, organic salt such as an acetate or inorganic salt such as a hydrochloride and may be formed as a pharmaceutical formulation in the form of a freeze-dried article after sterilization. During use, induction of insulin secretion and acceleration of an increase in pancreatic β-cells are enabled by intravenous administration in an injectable form by dissolution into saline or the like.
The polypeptide being the active ingredient, or the fragment thereof, of an insulin secretion inducer or an accelerator for increasing the number of pancreatic β-cells according to the present invention may be used alone as a pure substance purified to a high degree or may be a mixture of a plurality of types of substances and may be used in various aspects as an insulin secretion inducer composition or a composition for increasing the number of pancreatic β-cells.
A viral vector for inducing insulin secretion or a viral vector for increasing the number of pancreatic β-cells in accordance with the genetic therapy of the present invention is characterized by incorporating DNA having a base sequence set forth in at least one of SEQ. ID. No. 1, SEQ. ID. No. 4 and SEQ. ID. No. 7, or DNA capable of hybridizing under stringent conditions with a strand complementary to DNA having the base sequence as set forth in at least one of SEQ. ID. No. 1, SEQ. ID. No. 4 and SEQ. ID. No. 7 as an exogenous gene in a viral vector enabling expression of the exogenous gene. An actual example of a viral vector includes an adenovirus vector constructed by linking DNA having a base sequence as set forth in any one of SEQ. ID. No. 1, SEQ. ID. No. 4 and SEQ. ID. No. 7 with a CAG promoter. This type of viral vector for example enables induction of insulin secretion and increase in the number of pancreatic β-cells by intravenous injection for example.
Since the insulin secretion inducer, insulin secretion inducer composition and the viral vector for inducing insulin secretion by genetic therapy according to the present invention display an excellent insulin secretion action in vivo, it is effective for diseases associated with reduction of insulin secretion capability and in particular for treatment of type-2 diabetes. In other words, use of the insulin secretion inducer, insulin secretion inducer composition and the viral vector for inducing insulin secretion by genetic therapy provides a method of treatment for diseases associated with reduction of insulin secretion capability and in particular for treatment of type-2 diabetes.
Since the accelerator for increasing the number of pancreatic β-cells, composition for increasing the number of pancreatic β-cells and the viral vector for increasing the number of pancreatic β-cells by genetic therapy according to the present invention display an excellent action for increasing the number of pancreatic β-cells in vivo, it is effective for diseases associated with reduction or necrosis in pancreatic β-cells and in particular for treatment of type-1 diabetes. In other words, use of the accelerator for increasing the number of pancreatic β-cells, composition for increasing the number of pancreatic β-cells and the viral vector for increasing the number of pancreatic β-cells by genetic therapy provide a method of treatment for diseases associated with reduction or necrosis in pancreatic β-cells and in particular for treatment of type-1 diabetes.
EXAMPLES
The present invention will be described in further detail with reference to the embodiments. However interpretation of the present invention is not limited by the following description.
Reference Example 1
Site Expression Distribution of mCF266
In reference example 1, the distribution of expression by site of mRNA of mCF266 acquired from the alimentary tract of a mouse using an SST method was examined.
Firstly various organs and tissues (cerebrum, heart, lungs, liver, pancreas, spleen, kidney, small intestine, white adipose tissue, brown adipose tissue and muscle) were removed from a mouse and total RNA was extracted in accordance with the instruction manual attached to TRIzol (Invitrogen). Then hybridization was performed by preparing a membrane having 10 μg/lane according to a defined method and using a probe of cDNA (mRNA clone) formed from mCF266 labeled with [α-32P] dCTP. The results are shown in FIG. 1. As clearly shown by FIG. 1, mCF266 is specifically expressed in the small intestine.
EMBODIMENT 1
Action of Culture Supernatant of CF266 Transfected Cells on Cultured Cell Strain MIN6 from Murine Pancreatic β-Cells
In Embodiment 1, an action was confirmed with respect to MIN6 cells using a culture supernatant of a HEK293T cell strain originating from fetal kidney epithelial cells transfected with CF266.
Firstly, HEK293T cells subcultured in DMEM media enriched with 5% FCS and an antibiotic (penicillin 100 U/mL, streptomycin 10 mg/mL) were plated onto a 10 cm dish at a concentration of 1×106 cells/dish. On the following day, HEK292T cells were transfected with an expression vector for mCF266 (pCAGGS-mCF266) using a FuGENE6 (Roche) and mCF266 was forcibly expressed. After 24 hours, the media was exchanged for Opti-MEM media and after a further 24 hours, a culture supernatant was recovered.
Next, MIN6 cells subcultured in DMEM media (high glucose, Invitrogen) enriched with 15% FCS, an antibiotic (penicillin 100 U/mL, streptomycin 10 mg/mL) and 2-mercaptoethanol were plated onto a 24-well plate at a concentration of 3×105 cells/well. On the following day, 0.5 mL/well KRBH buffer solution (2.8 mM glucose) was added and pre-culturing was conducted for 30 minutes. Then 0.5 mL/well of a mixed solution of the above culture supernatant and KRBH buffer solution (1:1(v/v)) was added and stimulated for one hour. Then glucose-stimulated insulin secretion (GSIS) was measured for the MIN6 cells. The measurement was performed by measuring insulin in the culture solution using an ELISA method (using a REBIS Insulin Kit (Shibayagi): same hereafter). The insulin value was normalized using the total protein amount of MIN6 cells.
In the same manner, respective expression vectors for DNA (hCF266(27V)) having a base sequence as set forth in SEQ. ID. No. 4 cloned from a human alimentary tract cDNA library (BD Biosciences) and DNA (hCF266(27A)) having a base sequence as set forth in SEQ. ID. No. 7 acquired by using a known method to introduce a mutation into DNA (hCF266(27V)) were transfected into HEK293T cells and subjected respectively to forcible expression. The culture supernatant was used to measure glucose-stimulated insulin secretion (GSIS) in MIN6 cells in the same manner as that described above.
The results are shown in FIG. 2. In the figure, “Mock” shows the results when adding a culture supernatant obtained by using the same method as above to transfect an empty vector (pCAGGS) into HEK293T cells. As clearly shown by FIG. 2, the culture supernatant of cells forcibly expressing CF266 induce glucose-stimulated insulin secretion with respect to MIN6 cells and displays an excellent action when hCF266(27V) is forcibly expressed.
EMBODIMENT 2
Action of mCF266 with Respect to a Type-2 Diabetes Model KK/Ay Mouse
In the second embodiment, an adenoviral vector expressing mCF266 was used to confirm the action of mCF266 with respect to a KK/Ay mouse which is a type-2 diabetes model mouse.
Firstly an adenoviral vector (prepared using ViraPower which is a tradename of Invitrogen) constructed by linking mCF266 to a CAG promoter was intravenously injected at a concentration of 6×109 PFU/mL into the caudal vein of a 18-week old KK/Ay mouse (body weight approximately 47-51 g) given a high-fat high-sucrose diet. After four days, an intravenous glucose tolerance test (i.v. GTT) was performed by periodically taking blood and measuring the blood-glucose level of the serum and the insulin level.
The results are shown in FIG. 3A and FIG. 3B. In the figures, “Ad GFP” shows the results when using an adenoviral vector constructed by linking DNA encoding GFP in place of mCF266. As clearly shown by FIG. 3A and FIG. 3B, when mCF266 is expressed in vivo in the type-2 diabetes model mouse, in comparison with the control expressing GFP, although the blood-glucose level displays only a slightly low tendency (FIG. 3A), the insulin value is significantly higher (FIG. 3B). Consequently expression of mCF266 is shown to induce insulin secretion.
EMBODIMENT 3
Action of Polypeptides Contained in Culture Supernatant of Cells Transfected with hCF266(27V) on Islets of Langerhans from Murine Pancreas
In the third embodiment, a polypeptide contained in the culture supernatant of HEK293T cells transfected with hCF266(27V) was formed by chemical synthesis and the action of the synthetic polypeptide was confirmed with respect to islets of Langerhans from a murine pancreas.
Firstly, using the same method as the first embodiment, an expression vector for hCF266(27V) was transfected into HEK293T cells, hCF266(27V) was forcibly expressed and the culture supernatant recovered. Then an anion exchange column (POROS HQ column; ABI) was used to fractionate the recovered culture supernatant.
Next, islets of Langerhans from a pancreas isolated from a C57BL/6 mouse were plated into a 24-well plate at a concentration of 10 islets/well. After culturing for 2 hours in RPMI1640 media (10% FCS; Invitrogen), 0.5 mL/well of KRBH buffer solution (2.8 mM glucose or 20 mM glucose) was added and pre-culturing was performed for 30 minutes. Then 0.5 mL/well of a mixed solution of the above culture supernatant and KRBH buffer solution (1:1(v/v)) was added and stimulated for one hour. Then glucose-stimulated insulin secretion (GSIS) was measured for the islets of Langerhans from a murine pancreas. The measurement was performed by using an ELISA method to measure the insulin in the culture solution. The fractions confirmed to have insulin secretion inducing properties were analyzed using a mass spectrometer (TOF-MAS) and three types of polypeptides predicted to have a 19 amino acid sequence from a specifically observed peak mass were chemically synthesized. The three polypeptides are ALYCMLISIQALLKGPLMC (polypeptide A: refer to SEQ. ID. No. 10), CNNTRGMFLSSLFSVITVI (polypeptide B: refer to SEQ. ID. No. 11) and TSNDTMASGWRASSFHFDS (polypeptide C: refer to SEQ. ID. No. 12).
Then 0.5 mL/well of a solution dissolving the respective three polypeptides A-C in KRBH buffer solution at a concentration of 10 nM was added to islets of Langerhans from a murine pancreas pre-cultured for 30 minutes using the same method as above and then stimulated for 30 minutes. Then glucose-stimulated insulin secretion was measured for the islets of Langerhans from a murine pancreas using the same method as above. The insulin value was normalized using total DNA amount for the islets of Langerhans from a murine pancreas.
The results are shown in FIG. 4. In the figure, “Cont.” shows the results of performing experiments using the method above in addition to stimulating by addition of only KRBH buffer solution. “hCF266(27V)” shows the results for experiments conducted using the same method as above in addition to causing stimulation by addition of a mixed solution of the above culture supernatant of HEK293T cells transfected with hCF266(27V) and KRBH buffer solution (1:1(v/v)). Furthermore “GLP-1” shows the results for experiments conducted using the same method as above in addition to causing stimulation by addition of a solution dissolving human GLP-1 (Peptide Institute Inc.) at a concentration of 10 nM in KRBH buffer solution. As clearly shown by FIG. 4, the three types of polypeptide A-C induce glucose-stimulated insulin secretion with respect to islets of Langerhans from a murine pancreas.
EMBODIMENT 4
Action of hCF266(27V) on a Type-1 Diabetes STZ Model Mouse
In the fourth embodiment, an adenoviral vector expressing hCF266(27V) was used to confirm the action of hCF266(27V) with respect to an STZ model mouse which is a type-1 diabetes model mouse.
Firstly a 7-week old C57BL/6 mouse (body weight approximately 18-20 g) was fasted for 18 hours from day 0 and then streptozocin dissolved in saline (100 mg/kg body weight) was administered into the interperitoneal region and free feeding was immediately allowed. From the following day, after fasted for 18 hours the same amount of streptozocin was re-administered on the second day into the interperitoneal region and free feeding was immediately allowed. The serum blood-glucose level was measured on the seventh day. A fasting blood glucose level of greater than or equal to 150 mg/dL was determined to be diabetes and the experiment described hereafter was conducted. On the same day, a recombinant adenovirus (prepared using ViraPower which is a tradename of Invitrogen) constructed by linking hCF266(27V) to a CAG promoter was intravenously injected at a concentration of 1×109 PFU/mL into the caudal vein of a STZ mouse determined to be suffering diabetes. On the twelfth day, an oral glucose tolerance test (OGTT) was performed by periodically taking blood and measuring the blood-glucose level of the serum and the insulin level.
The results are shown in FIG. 5. In the figure, “Ad (−)” shows the results of using an adenoviral vector not incorporating an exogenous gene. “Ad GFP” shows the results of using an adenoviral vector constructed by linking DNA encoding GFP in place of hCF266(27V). As clearly shown by FIG. 5, when hCF266(27V) is expressed in vivo in the type-1 diabetes model mouse, in comparison with the control expressing GFP or expressing nothing, the blood glucose level is significantly reduced. Consequently expression of hCF266(27V) is shown to suppress increases in the blood glucose level.
The pancreas of the STZ mice was removed and after fixing the tissue with paraformaldehyde, the tissue sample preparation office at University of Tsukuba was employed to prepare a paraffin-embedded section. After the section was removed from paraffin, antigen activation was performed for 30 minutes at room temperature using 0.1% Triton X-100 and then blocking was performed for one hour at room temperature using 5% skim milk. After blocking, a primary antibody reaction was performed overnight at 4° C. using polyclonal anti-insulin guinea pig antibody (Daco) and polyclonal anti-glucagon rabbit antibody (Daco). The section was washed using Tris-buffered saline containing 0.1% Tween 20 (TBST). A secondary antibody reaction was performed in a darkroom for one hour at room temperature using polyclonal sheep anti-guinea pig IgG conjugated FITC antibody (Daco) and polyclonal goat anti-rabbit IgG conjugated Cy3 antibody (Daco). The section was washed using TBST. The section was thereafter mounted on a fluorescence photobleaching prevention agent (VECTASHILD Mounting Medium; Vector) and observed through a fluorescence microscope (BZ-8000; KEYENCE) to measure the surface ratio (β-cells/α-cells) of β-cells to α-cells.
The results are shown in FIG. 6. As clearly shown by FIG. 6, when hCF266(27V) is expressed in vivo in the type-1 diabetes model mouse, in comparison with the control expressing GFP or expressing nothing, the surface ratio (β-cells/α-cells) of β cells to α cells is significantly increased. Consequently expression of hCF266(27V) has been shown to increase the number of β-cells.
EMBODIMENT 5
Action of mCF266 in Transgenic Mice
In the fifth embodiment, the action of mCF266 in transgenic mice was confirmed.
Firstly, an mCF266 expression vector (pCAGGS-mCF266) was constructed by linking mCF266 to a CAG promoter and linearized. The University of Tsukuba Laboratory Animal Resource Center was employed to produce a transgenic mouse. The introduced gene was confirmed using PCR and southern blotting and mating for five generations with C57BL/6 mice was conducted.
The pancreas of the transgenic mice was removed and after fixing the tissue with paraformaldehyde, the tissue sample preparation office at University of Tsukuba was employed to prepare a paraffin-embedded section. After the section was removed from paraffin, antigen activation was performed for 30 minutes at room temperature using 0.1% Triton X-100 and then blocking was performed for one hour at room temperature using 5% skim milk. After blocking, a primary antibody reaction was performed overnight at 4° C. using polyclonal anti-insulin guinea pig antibody (Daco) and polyclonal anti-glucagon rabbit antibody (Daco). The section was washed using Tris-buffered saline containing 0.1% Tween20 (TBST). A secondary antibody reaction was performed in a darkroom for one hour at room temperature using polyclonal sheep anti-guinea pig IgG conjugated FITC antibody (Daco) and polyclonal goat anti-rabbit IgG conjugated Cy3 antibody (Daco). The section was washed using TBST. The section was thereafter mounted on a fluorescence photobleaching prevention agent (VECTASHILD Mounting Medium; Vector) and observed through a fluorescence microscope (BZ-8000; KEYENCE) to measure the surface area of islets of Langerhans stained by insulin relative to the pancreas surface area on the section. The number of islets of Langerhans was normalized with reference to a wild-type mouse taking a value of 1.
The results are shown in FIG. 7A and FIG. 7B. In the figures, “WT” shows the results when a wild-type mouse is used. As clearly shown by FIG. 7A and FIG. 7B, in comparison with a wild-type mouse, the surface area (FIG. 7A) and the number (FIG. 7B) of β-cells is conspicuously increased depending on whether the sample is hetero (+/−) or homo (+/+). Thus expression of mCF266 has been shown to increase the number of β-cells.
EMBODIMENT 6
Action of Polypeptide Contained in Culture Supernatant of hCF266(27V) Transfected Cells on Type-1 Diabetes STZ Model Mice
In the sixth embodiment, the action of synthetic polypeptides A-C on Type-1 diabetes STZ model mice was confirmed in the same manner as the third embodiment.
Firstly a 7-week old C57BL/6 mouse (body weight approximately 18-20 g) was fasted for 18 hours from day 0 and then streptozocin dissolved in saline (100 mg/kg body weight) was fasted for 18 hours from day 0 and then streptozocin dissolved in saline (100 mg/kg body weight) was administered into the interperitoneal region and free feeding was immediately allowed. From the following day, after fasted for 18 hours the same amount of streptozocin was re-administered on the second day into the interperitoneal region and free feeding was immediately allowed. The serum blood-glucose level was measured on the seventh day. A fasting blood glucose level of greater than or equal to 150 mg/dL was determined as diabetes and the experiment described hereafter was conducted. STZ mice determined to have diabetes were administered into the interperitoneal region twice daily the polypeptide A-C (25 nmol/kg body weight) dissolved in saline for eight weeks from the seventh day.
The pancreas of the STZ mice was removed and after fixing the tissue with paraformaldehyde, the tissue sample preparation office at University of Tsukuba was employed to prepare a paraffin-embedded section. After the section was removed from paraffin, antigen activation was performed for 30 minutes at room temperature using 0.1% Triton X-100 and then blocking was performed for one hour at room temperature using 5% skim milk. After blocking, a primary antibody reaction was performed overnight at 4° C. using polyclonal anti-insulin guinea pig antibody (Daco) and polyclonal anti-glucagon rabbit antibody (Daco). The section was washed using Tris-buffered saline containing 0.1% Tween 20 (TBST). A secondary antibody reaction was performed in a darkroom for one hour at room temperature using polyclonal sheep anti-guinea pig IgG conjugated FITC antibody (Daco) and polyclonal goat anti-rabbit IgG conjugated Cy3 antibody (Daco). The section was washed using TBST. The section was thereafter mounted on a fluorescence photobleaching prevention agent (VECTASHILD Mounting Medium; Vector) and observed through a fluorescence microscope (BZ-8000; KEYENCE) to measure the surface ratio (β-cells/α-cells) of β-cells to α-cells.
The results are shown in FIG. 8. In the figures, “Cont.” shows the results of an experiment conducted using the same method as that described above in addition to using physiological saline solution not containing the polypeptides A-C. As clearly shown by FIG. 8, a mouse administered with the polypeptide A-C displays a significant increase in the surface ratio (β-cells/α-cells) β-cells to α-cells. That effect is excellent when polypeptide A is administered.
INDUSTRIAL APPLICABILITY
The present invention may be applied to industry in that it provides an insulin secretion inducer, insulin secretion-inducing composition and a method of manufacturing the same, an accelerator for increasing the number of pancreatic β-cells, a composition for increasing the number of pancreatic β-cells and a method of manufacturing same, in addition to a virus vector for genetic treatment, for use primarily in the treatment of diabetes and of various other diseases.

## Claims

1. A method of treatment of diabetes or reduction in pancreatic β-cells, said method comprising administering a pharmaceutical composition to a subject in need thereof, the pharmaceutical composition including at least one of the following active ingredients (a) to (f):
(a) a polypeptide having an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 1 or SEQ. ID. No. 4;
(b) a polypeptide having an insulin secretion inducing action and/or an action of increasing the number of pancreatic β-cells, and having an amino acid sequence in which one amino acid has been substituted, deleted and/or added to an amino acid sequence encoded by DNA having a base sequence as set forth in SEQ. ID. No. 1 or SEQ. ID. No. 4;
(c) a fragment of the polypeptide in (a) or (b) having an insulin secretion inducing action and/or an action of increasing the number of pancreatic β-cells, said fragment comprising the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:12;
(d) a polypeptide having an amino acid sequence as set forth in SEQ. ID. No. 3 or SEQ. ID. No. 6;
(e) a polypeptide having an insulin secretion inducing action and/or an action of increasing the number of pancreatic β-cells, and having an amino acid sequence in which one amino acid has been substituted, deleted and/or added to an amino acid sequence as set forth in SEQ. ID. No. 3 or SEQ. ID. No. 6; and
(f) a fragment of the polypeptide in (d) or (e) having an insulin secretion inducing action and/or an action of increasing the number of pancreatic β-cells, said fragment comprising the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:12.

2. The method of claim 1 wherein the active ingredient is a fragment of a polypeptide having an amino acid sequence as set forth in SEQ. ID. NO. 3 or SEQ. ID. No. 6, said fragment consisting of the amino acid sequence set forth in SEQ. ID. NO. 10, SEQ. ID. No. 11 or SEQ. ID. No. 12.