WO2020158491A1

WO2020158491A1 - Insulin secretion promoting peptide

Info

Publication number: WO2020158491A1
Application number: PCT/JP2020/001791
Authority: WO
Inventors: 久美子佐伯; 雅子岡; 和典松村; 亜峰朱; 豊隆森
Original assignee: 国立研究開発法人国立国際医療研究センター; 株式会社Ｉｄファーマ
Priority date: 2019-01-29
Filing date: 2020-01-21
Publication date: 2020-08-06
Also published as: JP2022049715A

Abstract

The present invention provides a peptide that promotes insulin secretion, and a use for said peptide. A search for insulin secretion promoting factors present in a culture supernatant of brown adipocytes (BA-SUP) derived from human pluripotent stem cells led to the identification of a bioactive peptide that promotes insulin secretion. Through the development of a peptide that imitates said peptide, it was discovered that a short-chain linear peptide capable of forming an α-helix structure exhibits strong insulin secretion promoting activity. Moreover, it was discovered that by heat-treating said peptide under acidic conditions or freeze-drying the peptide after dissolving in a buffer solution, the peptide becomes an active form having an increased insulin secretion promoting effect. Said peptide exhibited an effect of promoting insulin secretion using pancreatic beta cells, and of reducing the blood sugar level in vivo. The peptide group according to the present invention is expected to be applicable to the development of a therapeutic medication for glucose metabolism disorders.

Description

Insulin secretagogue peptide

The present invention relates to a short-chain insulin secretagogue peptide and internal medicine therapy for the purpose of correcting hyperglycemic condition due to insufficient insulin secretion.

The number of people with obesity and overweight is increasing worldwide, and in the 2017 “World Burden Study”, one in three people in the world is obese or overweight (Non-patent Document 1). Obesity and overweight increase the risk of developing life-threatening diseases such as type 2 diabetes, ischemic heart disease, cerebrovascular disease, and cancer. Furthermore, those who have experienced obesity or overweight in the past have a higher risk of death than those who have maintained normal weight (Non-patent Document 2). In addition, the higher the blood sugar level of a pregnant woman, the greater the endless burden on the baby (Non-Patent Document 3), and the higher the incidence of cerebral palsy in the offspring of mothers who were obese or overweight in early pregnancy (Non-patent Document 3). It has been reported that a child of an obese or overweight pregnant woman is prone to malformation (Non-Patent Document 5). As described above, obesity and overweight have serious effects on the health of the individual and the next generation.

With the increase in obese/overweight patients, the greatest threat is the increase in the number of people with type 2 diabetes. The prevalence of diabetes is estimated to be 9.5 million in Japan and 420 million worldwide. According to a 2013 Chinese survey, the prevalence of diabetes in adults was estimated to be 10.9%, and the prevalence of prediabetes was estimated to be 35.7% (Non-Patent Document 6). It is reported to be twice as high (Non-Patent Document 7). In addition, according to a US survey from 1983 to 2011, the number of people suffering from ischemic heart disease was reduced by about 20%, but the number of people suffering from diabetes is increasing (Non-Patent Document 8). In Mexico, about one in four people has been diagnosed with diabetes (Non-Patent Document 9).

Type 2 diabetes has a high incidence of complications. For example, in a follow-up study of 2018 people diagnosed with diabetes by the age of 20 for about 8 years, the prevalence of complications was 32% among type 1 diabetic patients and type 2 diabetes. It was 72% in diabetic patients (Non-Patent Document 10).

As mentioned above, the increase in the number of obese/overweight patients and the associated increase in the prevalence of type 2 diabetes is an urgent task that requires global countermeasures.

However, it is not easy for obese/overweight sufferers to lose weight and maintain a reduced weight. In addition, it was also reported in a survey in the United States from 1988 to 2014 that the proportion of “obesity/overweight adults trying to lose weight” was decreasing (Non-Patent Document 11).

Currently, it cannot be said that the treatment of type 2 diabetes is sufficiently effective, and the treatment satisfaction of patients is low. According to a February 2017 U.S. report, the proportion of glycated hemoglobin (HbA1c) 6% or less at 5 years was 29% in diabetic patients who underwent bariatric surgery (gastric bypass group), but only for medical treatment. Only 5% in the group (Non-Patent Document 12), bariatric surgery outweighs medical treatment in controlling blood glucose in diabetic patients. However, bariatric surgery is a invasive procedure, and development of an excellent therapeutic agent for type 2 diabetes that surpasses current therapeutic agents is desired.

In addition, a meta-analysis of the prospective observational study reported in 2016 showed that the risk of cardiovascular disease increased from 5.7% in HbA1c (Non-patent document 13), and the complete onset of complications in type 2 diabetes was reported. From the point of view of prevention, it means that the current medical treatment is insufficient in 95% of cases.

Currently, high research costs and medical costs are invested in early diagnosis and treatment of complications, but what should be prioritized above all is to provide an excellent therapeutic drug capable of controlling blood glucose below 5.7% HbA1c. It is to develop.

In fact, on January 30, 2017, the world's number one selling drug for diabetes has announced that the pharmaceutical company is aiming to discover a new treatment for type 2 diabetes in partnership with Oxford University in the United Kingdom. .. The company has also announced that it will set up a new research center on the premises of the university and will spend £115 million over the next 10 years. This is an example that clearly demonstrates the inadequacy of current medical treatment for type 2 diabetes.

WO2012/147853

An object of the present invention is to provide an “insulin secretagogue-promoting short-chain peptide” present in the culture supernatant of brown adipocytes (hereinafter, BA-SUP). Another object of the present invention is to provide a means for obtaining information on the amino sequence of the peptide.

Another object of the present invention is to provide an organic chemically synthesized product of this peptide. Another object of the present invention is to provide a physicochemical treatment method for converting the synthetic product into an “active structural isomer” that exerts a higher insulin secretagogue effect, and information regarding the method.

Another object of the present invention is to provide an “active mutant peptide” that stably exhibits insulin secretagogue activity.

Another object of the present invention is to provide a peptide preparation that enables therapeutic use in diabetes, specifically, for example, in patients with type 2 diabetes who have poor blood glucose control with existing insulin secretagogues. To do.

As mentioned above, the development of new therapeutic agents that enable strict blood glucose control (HbA1c ≤ 5.7%) in type 2 diabetes is an important and highly urgent drug discovery business. However, even if the drug discovery research is repeated with a novel strategy based on the conventional understanding of the pathological condition of type 2 diabetes, a new drug that achieves the above target cannot be developed. In order to develop excellent therapeutic agents that are both safe and effective, it is necessary to fundamentally rethink the pathophysiology of type 2 diabetes. From this point of view, “brown adipose tissue (BAT)” should be noted.

Brown fat cells produce heat by burning the neutral fat accumulated in the cells when the demand for body heat production increases in cold environments. Although research on rodents had preceded, it was reported in 2009 that human adults also had brown adipocytes (Non-patent Documents 14 to 17).

Studies using genetically modified mice have demonstrated that brown adipocytes have the effects of "preventing obesity," "improving glucose metabolism," "improving lipid metabolism," and "improving leptin sensitivity." Moreover, contributions to "prevention of middle-aged overweight" and "improvement of glucose metabolism" have been shown from epidemiologic studies using human cancer screening and human dock data, and studies in which healthy volunteers participated.

Initially, the improvement of glucose metabolism by brown adipocytes was considered to be a secondary effect associated with fat burning, whereas brown adipocyte-deficient mice exhibited severe obesity and diabetes, whereas brown adipocyte thermogenic capacity was increased. Since the mice deficient only in Nd showed neither obesity nor impaired glucose metabolism, it was suggested that the improvement of glucose metabolism by brown adipocytes is "an action mediated by a soluble factor (hormone)". Since then, research has been promoted worldwide toward the discovery of "a sugar metabolism-improving hormone specifically produced by brown adipocytes", but until now, such a factor has not been identified. There are two reasons.

One is that the hormone search was performed by gene cloning technology. This technique is effective when there is a one-to-one correlation between the gene expression and the expression of the protein encoded by the gene, but most peptide hormones produced by endocrine cells process precursor proteins (specific amino acid sites are identified). Is a short-chain peptide (about 3 to 20 amino acids) produced by the step of cleaving with peptidase and cutting out from the precursor). Therefore, the effectiveness of gene cloning technology is poor.

Another reason is that brown adipose tissue (BAT) was collected from the living body of the mouse, and the brown adipocytes separated and collected from this were used as the material. BAT is a digestive enzyme produced by exocrine pancreatic gland cells, which expresses a strong group of degrading enzymes such as chymotrypsin group, trypsin group, and RNase1, RNase1. The quality of brown adipocytes begins to deteriorate rapidly from the moment it is taken. Therefore, it is extremely difficult to obtain the hormone group produced by BAT in an uninjured state (Non-Patent Document 18).

On the other hand, it is impossible from an ethical point of view to obtain BAT from a living human body and use it for research. In addition, there is no technique for culturing and cryopreserving brown adipocytes recovered from mouse BAT or human BAT.

As described above, the discovery of "a sugar metabolism-improving hormone specifically produced by brown adipocytes" requires the establishment of a strategy that enables the preparation of "high-quality brown adipocytes" without being attacked by proteolytic enzymes. It is a prerequisite. Moreover, the "high-quality brown adipocytes" used as a material exhibit a glucose metabolism improving action independent of the heat-producing ability, are stable and can be prepared at any time, and have a glucose metabolism improving action in the culture supernatant. To be recognized is necessary. For example, if the "technology for producing brown adipocytes from human pluripotent stem cells" is applied, stable preparation of high-quality brown adipocyte culture supernatant is possible only by a general-purpose cell culture technique.

The present inventors have previously developed "technology for inducing brown adipocyte differentiation of human pluripotent stem cells" (Patent Document 1, Non-Patent Documents 19, 20). When the present inventors conducted an experiment in which BA-SUP prepared from this human pluripotent stem cell-derived brown adipocyte was administered to mice, it was found that the fasting blood glucose level measured 16 hours later decreased. From this result, it is suggested that there is a factor having an insulin secretagogue action in BA-SUP. In addition, considering that the half-life in blood of known hormones such as insulin and GLP-1 that have a hypoglycemic effect is about 3 hours, the physiological effects of BA-SUP are shown 16 hours after administration. The factor was considered to be a "new glucose metabolism improving hormone".

In addition, the present inventors, when BA-SUP was added to the pancreatic beta cell line, regardless of the glucose concentration of the culture medium, that is, high glucose solution (16.8 mM; 302 mg/dL) low glucose solution (2.8 mM). 50 mg/dL) also found that the amount of insulin secretion increased. This means that there is a factor in BA-SUP that promotes the "basal secretion" of insulin in pancreatic beta cells, and the existing insulinotropic hormone (incretin) is present only in a hyperglycemic environment. In contrast to promoting insulin secretion.
In addition, the insulin secretagogue-activating factor in BA-SUP was inactivated by treatment with the peptide-degrading enzyme trypsin, and the value of about 800 Da was obtained by HPLC using a gel filtration column (Superdex 75 5/150GL; GE). Since the factor was detected in the molecular weight fraction, it was revealed that the factor was "a short chain peptide having 10 or less amino acids and larger than a dipeptide or a tripeptide". Since a molecule having such characteristics does not exist in the known sugar metabolism-improving hormone, it was revealed that "a novel insulin secretagogue peptide" exists in BA-SUP.

Therefore, we tried to purify the factor by applying HPLC, but as shown below, it was found that it has a "unique property" that was difficult to assume for general peptides, and a general peptide purification protocol was used. Was found to be inapplicable.

We tried to purify the factor by applying reversed-phase chromatography (HPLC using a hydrophobic column), which is commonly used for peptide purification, but no matter what kind of hydrophobic column was used and what mobile phase was used. However, under all conditions examined, the factor was recovered in the flow through (non-binding fraction) along with a large amount of contaminants.
From the above, it was concluded that it is impossible to purify the factor by reverse phase chromatography.

At the same time, the results of the previous section show that ZipTip (Merck KGaA, Germany) filled with hydrophobic resin, which is commonly used in the desalting process of samples, is used for the desalination concentration of BA-SUP for the purification of the factor. It also means that it cannot be used.

We tried to purify the factor by normal phase chromatography using an ion exchange column, but the factor was deactivated in the fractionation work using the “strong cation exchange column” and “strong anion exchange column”, In the fractionation work using the “cation exchange column” and “weak anion exchange column”, the factor was collected in the flow through together with a large amount of contaminants.
As described above, it was concluded that the factor cannot be purified even by normal phase chromatography using an ion exchange column.

From the above, it was concluded that only HPLC using a "gel filtration column" is effective for purifying the factor. The gel filtration column is one that dilutes and elutes the molecular population in the order of the molecular weight as a guide by the principle of the molecular sieve, and has a lower resolution than the hydrophobic column and the ion exchange column. However, the matrix packed in the column includes chemically synthesized polymers other than the agarose/dextran type used in Superdex75/150GL (GE), and it is possible to use multiple types of gel filtration columns for the purpose. It was considered possible to increase the purity of the molecule.

In addition, since the factor is inactivated by trypsin treatment, when mass spectrometrically analyzing the active fraction of HPLC on a gel filtration column, analysis is performed before and after trypsin treatment, and a "peak disappeared by trypsin treatment (target molecule It is considered that the peak derived from the target molecule can be distinguished from the contaminant-derived peak group by identifying the "peak corresponding to the target molecule" and "the peak appearing in the trypsin treatment (the peak corresponding to the trypsin-treated fragment of the target molecule)". It was

Also, as mentioned above, since the factor does not bind to the hydrophobic column at all, it was strongly suggested that it is composed of only hydrophilic amino acids.

As mentioned above, a general peptide purification protocol is not effective for purifying the “new insulin secretagogue peptide” in BA-SUP, but as described above, it is difficult to purify by an ion exchange column, but by gel filtration. The present inventors have conducted diligent studies to obtain candidates for the amino acid sequence of the target peptide by paying attention to the fact that they can be purified and that they are inactivated by trypsin treatment.

Also, regarding the amino acid sequence candidates obtained in the previous section, by searching the human gene/protein database, we have selected those that may be derived from the gene product encoded by the human genome. Whether or not the selected candidate peptide was the target peptide was judged by organically synthesizing a peptide having the amino acid sequence and evaluating the insulin secretagogue activity.

When purifying a target molecule from BA-SUP containing a large amount of impurities by HPLC, a step of "desalting and concentrating" BA-SUP is required. Since “desalting” is difficult to carry out with an ion exchange column as described above, a column packed with a gel filtration carrier, for example, a PD-10 column (manufactured by GE Healthcare) is effective. The PD-10 column is a disposable column packed with a dextran gel filtration carrier, and separates the target substance and salts by utilizing the difference in molecular weight. Regarding "concentration", general-purpose freeze-drying that is effective for peptide concentration is effective.

Human ES cell-derived brown adipocytes prepared using the method described in Reference Example 1 were cultured for 16 hours in the "buffer containing salts and a low concentration of glucose (KRTB buffer)" described in Reference Example 2 for BA. -SUP was prepared, desalted and concentrated with PD-10 column and freeze-dryer, then dissolved in KRTB buffer, fractionated by HPLC using Superdex75/5/150GL (mobile phase: KRTB buffer), and reference example Insulin secretagogue activity was measured by the method described in 2 above, the fraction with the highest activity was collected, dissolved in 0.05% acetonitrile/0.1% acetic acid after desalting and concentration, and used with GS-320 HQ column (Shodex). HPLC fractionation (mobile phase: 0.05% acetonitrile/0.1% acetic acid), desalting and concentrating them, and then dissolving them in KRTB buffer to measure insulin secretagogue activity, which was derived from the peptide in the fraction with the highest activity. The UV absorbance at 260 nm was below the detection limit, and most of the impurities present in BA-SUP were removed.

Mass spectrometry was performed on the desalted and concentrated product of the above-mentioned fraction (LC-TOF/MS, positive ion mode ESI method). In addition, it is divided into two (A, B) in advance, A is as it is, B is subjected to mass spectrometry after trypsin treatment, 1) Peaks that are detected in A but not in B (a ₁ , a ₂ , a) ₃ ,...), 2) The peaks (b ₁ , b ₂ , b ₃ , ...) that are not detected in A but are detected in B are extracted, and for these, a _n =b _m +b _l +19 ( Linear), or a combination (a _n , b _m , b _l ) such that a _n =b _m +b _l +37 (ring) was selected, and there was no match for the former, but there was no match for the latter. Only one set was obtained. Therefore, by matching the human genome database, the amino acid sequence of configurable a _n is extracted on the assumption that a peptide fragment derived from the encoded protein in human genes. Further, the peptide of interest as described above from the be comprised of hydrophilic amino acids is suggested, all amino acids constituting a _n was also added condition that a hydrophilic amino acid.
As a result, the amino-terminal (N-terminal) and carboxyl-terminal of "threonine-lysine-glutamic acid-aspartic acid-glycine-arginine-glutamine (TKEDGRQ)" (SEQ ID NO: 1) were identified as candidates for insulin secretagogue peptides present in BA-SUP. A "lactam cyclic peptide" having a peptide bond at the (C-terminal) was obtained. Hereinafter, this is referred to as circ-TKEDGRQ.

From a chemo-thermodynamic point of view, it is envisioned that short-chain peptides form multiple "structural isomers" in aqueous solution, which are in thermodynamic dynamic or static equilibrium. However, the structural isomer showing the desired physiological activity is considered to be a specific one among them. Therefore, in the activity evaluation test using the organic chemically synthesized peptide product, it is assumed that the biological activity is not detected unless the organic chemically synthesized peptide stably and stably forms the “active structural isomer”. To be done.
That is, it is expected that it will be difficult to detect physiological activity with high reproducibility even if the insulin secretagogue activity is evaluated using an organic chemical synthetic product circ-TKEDGRQ aqueous solution. In that case, information on the higher-order structural features of the "active structural isomer" is obtained by some method, and a processing technology for converting the organic chemical synthetic product circ-TKEDGRQ into the "active structural isomer" is developed. , Or it is effective to produce an “active mutant peptide” that mimics the higher-order structure of the active structural isomer.

In order to obtain information on the higher-order structure characteristics of the "active structural isomer", various short-chain peptides are subjected to various physicochemical treatments, and the higher-order structure (α helix, Pi helix, 3 ₁₀ helix, 2 ₇ ribbon, random coil, one of the β such sheets) is correlated with insulinotropic activity, it is useful to obtain data suggest. The key to obtaining the "insulin secretagogue peptide" is to prepare a mutant peptide that mimics the higher-order structure of the "active structural isomer" based on the obtained information.
Although conventional mutant analysis focused on specific amino acids to design mutants (eg, substituting serine with alanine or glutamic acid mimics non-phosphorylated or phosphorylated, respectively). On the other hand, in the variant analysis focusing on “structural isomers”, it is useful to design a variant in which multiple amino acids are simultaneously substituted, focusing on the “higher-order structure” formed by a specific amino acid sequence. ..

The present invention has the following configurations based on the above findings.
(A) Information on the amino acid sequence of the insulin secretagogue-active peptide predicted from the results of mass spectrometry of the fractionated sample of BA-SUP
(B) Information on physicochemical treatment by which an organic chemical synthetic product of a peptide having the amino acid sequence of (A) induces "conversion to an active structural isomer" to exert an insulin secretagogue action.
(C) An active variant peptide of the peptide having the amino acid sequence of (A)

The "active variant" of (C) is not limited to those shown in the examples of the present specification, and it is possible to reproduce the characteristics of the higher-order structure of the "active structural isomer" shown in the present specification. Includes variants that can be easily inferred from known information.

The present invention also provides medical therapy using the peptide of the present invention. One example of the medical therapy using the novel insulin secretagogue peptide of the present invention is medical therapy for diabetes including type 2 diabetes, and in particular, conventional medical therapy using an insulin secretagogue has poor blood glucose control. Is a medical treatment for type 2 diabetes mellitus.

Further, a further example of the medical therapy using the novel insulin secretagogue peptide of the present invention is intended for early (mild) type 2 diabetes mellitus, borderline diabetes mellitus, or untreated type 2 diabetes mellitus. It is a medical treatment that was done. Borderline diabetes cases include, for example, cases in which the blood glucose level rises at any time but the fasting blood glucose is in the normal range. In the present invention, borderline diabetes is also included in diabetes.

Further, another example of the medical therapy using the novel insulin secretion-promoting peptide of the present invention is medical therapy for a case of prediabetes.

That is, the present invention relates to a peptide that exhibits insulin secretagogue activity, its use, and the like, and more specifically includes the invention described in each of the claims. An invention consisting of an arbitrary combination of two or more inventions described in claims which refer to the same claim is also an invention intended in the present specification. That is, the present invention includes the following inventions.
[1] A peptide comprising an amino acid sequence capable of forming an α-helix structure, wherein the peptide and/or its active form has insulinotropic activity.
[2] The peptide according to [1], which consists of several to more than ten amino acids.
[3] The peptide according to [1] or [2], which comprises an amino acid having a hydroxyl group and/or a hydrophilic amino acid and an amino acid sequence of 5 or more residues capable of forming the helix.
[4] The peptide according to [3], wherein the amino acid sequence having 5 or more residues forming the α-helix structure is composed of α-helix-forming amino acids.
[5] The peptide according to [4], wherein the α-helix forming amino acid is selected from the group consisting of methionine, alanine, leucine, glutamic acid and lysine.
[6] The peptide according to [5], wherein the amino acid sequence of 5 residues or more forming the α-helix structure contains 5 alanines (AAAAA/SEQ ID NO: 3).
[7] The peptide according to any one of [3] to [6], wherein the amino acid having a hydroxyl group and/or the hydrophilic amino acid is an amino acid selected from threonine, serine and tyrosine.
[8] The peptide according to any one of [3] to [6], wherein the amino acid having a hydroxyl group and/or the hydrophilic amino acid is threonine or serine.
[9] The peptide according to any one of [3] to [8], which contains a hydrophilic neutral amino acid following the amino acid sequence of 5 residues or more forming the α-helix structure.
[10] The peptide according to [9], wherein the hydrophilic neutral amino acid is glutamine.
[11] Threonine-alanine-alanine-alanine-alanine-alanine-glutamine (TAAAAAQ/SEQ ID NO:4), serine-alanine-alanine-alanine-alanine-alanine-glutamine (SAAAAAQ/SEQ ID NO:12), or threonine-alanine- A peptide comprising the amino acid sequence of alanine-alanine-alanine-alanine-glutamine-glycine-glycine (TAAAAAQGG/SEQ ID NO: 11), wherein the peptide and/or its active form has insulinotropic activity.
[12] A peptide comprising an amino acid sequence of threonine-lysine-glutamic acid-aspartic acid-glycine-arginine-glutamine (TKEDGRQ/SEQ ID NO: 1), the peptide and/or its active form having insulinotropic activity. peptide.
[13] A threonine-lysine-glutamic acid-aspartic acid-glycine-arginine-glutamine (TKEDGRQ/SEQ ID NO: 1) amino acid at the amino terminal and carboxyl terminal is a cyclic peptide in which a lactam ring is formed by a peptide bond, [12] The described peptides.
[14] The active form is a peptide that has been treated in the step of heating and cooling under acidic conditions and/or the step of dissolving in a buffer and freeze-drying, [1] to [13] peptide.
[15] The peptide according to any one of [1] to [14], in which another compound is separably linked.
[16] A composition comprising the peptide according to any one of [1] to [15] and a pharmaceutically acceptable carrier.
[17] The composition according to [16], which is a pharmaceutical composition.
[18] The composition according to [16] or [17], which is used for promoting insulin secretion and/or promoting glucose metabolism.
[19] The composition according to any of [16] to [18], which is used for treating or preventing diabetes or prediabetes.
[20] A method for treating or preventing diabetes or prediabetes, which comprises the step of administering the peptide according to any one of [1] to [15].

It should be noted that any technical matter described in this specification and any combination thereof are intended in this specification. Further, in these inventions, inventions excluding any matters described in the present specification or any combination thereof are also intended in the present specification. Further, regarding the present invention, a certain aspect described in the specification not only discloses the aspect but also an invention excluding the aspect from the invention disclosed in a higher order including the aspect. It is disclosed.

The present invention can provide materials for basic research on pancreatic β cells, including the discovery of an unidentified insulin secretion mechanism.

According to the present invention, it is possible to provide a material for animal studies relating to glucose metabolism control, including the discovery of an unidentified blood glucose control mechanism.

According to the present invention, cases of diabetes and pre-diabetes, for example, untreated type 2 diabetes, early type (mild) type 2 diabetes, and poor glycemic control by conventional medical therapy using insulin secretagogues. It is possible to provide effective medical therapy for cases of type 2 diabetes and type 1 diabetes (in particular, slowly progressive type 1 diabetes).

FIG. 3 is a view showing “insulin secretion promoting action of circ-TKED GRQ (SEQ ID NO: 1): effect of thermal acid treatment”. FIG. 3 is a diagram showing “insulin secretion promoting action of circ-TKED GRQ (SEQ ID NO: 1): influence of solvent during lyophilization treatment”. It is a figure which shows "the effect of a linear peptide (with freeze-drying process)." The results using the peptide of TKEDGRQ (SEQ ID NO: 1) are shown. It is a figure which shows "the effect of linear deletion mutant peptide (with freeze-drying process)." The peptides of KEDGRQ (amino acids 2 to 7 of SEQ ID NO: 1), TKEDGR (amino acids 1 to 6 of SEQ ID NO: 1), TKEDG (amino acids 1 to 5 of SEQ ID NO: 1), and TKEDGRQ (SEQ ID NO: 1) were used. The results are shown. FIG. 2 is a diagram showing “a higher-order structure prediction of amino acid sequence TKEDGRQ (SEQ ID NO: 1)”. It is a figure which shows "the effect of alanine stretch (1): no freeze-drying process". The result using the peptide of TAAAAAQ (SEQ ID NO: 4) containing an alanine stretch (AAAAA/SEQ ID NO: 3) is shown. It is a figure which shows "the effect of alanine stretch (2): with freeze-drying process". The result using the peptide of TAAAAAQ (SEQ ID NO: 4) is shown. It is a figure which shows "the effect of alanine stretch (3): no freeze-drying process". The results obtained using peptides of TAAAAAQ (SEQ ID NO: 4), AAAAAAA (SEQ ID NO: 5) and TKEDGRQ (SEQ ID NO: 1) are shown. It is a figure which shows "the effect of alanine stretch (4): with freeze-drying process". The results obtained using peptides of TAAAAAQ (SEQ ID NO: 4), AAAAAAA (SEQ ID NO: 5) and TKEDGRQ (SEQ ID NO: 1) are shown. It is a figure which shows "the influence of N-terminal biotinylation of TAAAAAQ (sequence number 4)." The results using TKEDGRQ (SEQ ID NO: 1) as a comparison target are also shown. It is a figure which shows "the effect of MAAAAAQ (sequence number 7): There is no freeze-drying." The results using TAAAAAQ (SEQ ID NO: 4) as a comparison target are also shown. It is a figure which shows "the effect of TAAAAA (sequence number 10): No freeze-drying process". It is a figure which shows "the effect of TAAAAA (sequence number 10): Freeze-drying processing". The results using TAAAAAQ (SEQ ID NO: 4) and TKEDGRQ (SEQ ID NO: 1) as comparison targets are also shown. It is a figure which shows "the effect of TAAAAAQGG (sequence number 11): No freeze-drying process". The results of using TAAAAAQ (SEQ ID NO: 4) and AAAAAAA (SEQ ID NO: 5) as comparison targets are also shown. It is a figure which shows "the effect of SAAAAAQ (sequence number 12): (1) no freeze-drying process, (2) freeze-drying process". The results of using TAAAAAQ (SEQ ID NO: 4) and TAAAAAQGG (SEQ ID NO: 11) as comparison targets are also shown. It is a figure which shows "the effect of YAAAAAQ (sequence number 13), DAAAAAQ (sequence number 14), KAAAAAQ (sequence number 15), TAAAAAE (sequence number 16): No freeze-drying process. The results using TAAAAAQGG (SEQ ID NO: 11) are also shown as comparison targets. It is a figure which shows "the effect of YAAAAAQ (sequence number 13), DAAAAAQ (sequence number 14), KAAAAAQ (sequence number 15), TAAAAAE (sequence number 16): freeze-drying processing. The results using TAAAAAQGG (SEQ ID NO: 11) are also shown as comparison targets. FIG. 4 is a diagram showing “a decrease in blood glucose level by administration of TAAAAAQ (SEQ ID NO: 4) to diet-induced obese mice”. The results using KEDGRQ (amino acids 2 to 7 of SEQ ID NO: 1) are also shown as a comparison target. FIG. 3 is a diagram showing “a decrease in blood glucose level and promotion of insulin secretion by administration of TAAAAAQ (SEQ ID NO: 4) to ob/ob mice”. The results of using TKEDGRQ (SEQ ID NO: 1) as a comparison target are also shown. It is a figure which shows "the effect of GlpTKEDGR (sequence number 22) and GlpTKEDGR (sequence number 23): No freeze-drying process." FIG. 8 is a diagram showing “improved glucose tolerance by administration of TAAAAAQGG (SEQ ID NO: 11) to insulin deficient mice (borderline diabetes)”. The results using GlpTKEdGR (SEQ ID NO:23) are also shown as comparison targets. FIG. 2 is a diagram showing “improved glucose tolerance by administration of TAAAAAQGG (SEQ ID NO: 11) to insulin deficient mice (type 1 diabetes)”. The results using GlpTKEdGR (SEQ ID NO:23) are also shown as comparison targets.

The present invention relates to a peptide having an amino acid sequence capable of forming an α-helix structure, the peptide and/or its active form having insulinotropic activity and its use. In the present invention, it was revealed that a short-chain peptide capable of forming an α-helix-like helix has an action of acting on pancreatic β-cells to promote insulin secretion. In addition, the peptide exerts the action of lowering the blood glucose level when administered to a hyperglycemic mouse. Therefore, the peptide of the present invention is useful for promoting insulin secretion, lowering blood glucose level, and/or promoting glucose metabolism.

In the present invention, the α-helix structure means an α-helix or a helix similar thereto (α-helix-like helix) structure. For example, in the present invention, the α-helix structure may be a helix structure formed by the interaction of amino acids 4 residues apart (that is, 2 amino acids located across 3 residues). In the present invention, the α-helix structure includes the typical α-helix shown above in FIG. 5 and the helix shown below in FIG. In the present invention, the α-helix structure has, for example, about 3.6 amino acid residues, for example, amino acid 3.2 to 4.0 residues, 3.3 to 3.9 residues, 3.4 to 3.8 residues, or 3.5 to 3.7 residues, such as an average of about 3.5 residues or Included are helical or cyclic structures that rotate about 3.6 residues. For example, a helix having about 2 turns about 7 residues, for example, about 1.5 to 2.5 turns, preferably 1.6 to 2.4 turns, 1.7 to 2.3 turns, 1.8 to 2.2 turns, or 1.9 to 2.1 turns is included in the present invention in the α-helix structure. To be done.

The insulin secretagogue activity of a peptide can be determined by administering the peptide to cells having insulin-producing ability such as pancreatic β cells. Although cells having insulin-producing ability may be appropriately selected, for example, pancreatic beta cell line MIN6 cells (Miyazaki et al., Endocrinology 127: 126-132, 1990), rat pancreatic islets and the like can be used. The cells are cultured, a peptide is added, and the insulin concentration in the culture supernatant is measured. If the insulin concentration is significantly increased, the peptide is considered to have insulinotropic activity.

Examples of the peptide of the present invention include insulin secretagogue-active peptides identified by mass spectrometry of the active fraction of BA-SUP. The amino acid sequence of the “insulin secretagogue active peptide identified by mass spectrometry of the active fraction of BA-SUP” according to the present invention is threonine-lysine-glutamic acid-aspartic acid-glycine-arginine-glutamine (TKEDGRQ/SEQ ID NO: 1). ) Is a lactam cyclic peptide having an amino terminus (N terminus) and a carboxyl terminus (C terminus) bonded to each other (hereinafter, referred to as circ-TKEDGRQ). Therefore, a cyclic peptide containing the amino acid sequence of TKEDGRQ (SEQ ID NO: 1) is suitable as the peptide of the present invention.

Also, the above-mentioned lactam cyclic peptide "circ-TKEDGRQ" can be converted into "active structural isomers" that exert a strong insulin secretagogue action by being subjected to physicochemical treatment. As the physicochemical treatment for converting into an “active structural isomer” that exerts an insulin secretagogue action, for example, a step of dissolving in a HEPES buffer solution having a pH of about 10 mM and a pH of about 7.6 and then freeze-drying is preferable.

Further, as the physicochemical treatment for converting the above-mentioned lactam cyclic peptide into the “active structural isomer” that exerts a strong insulin secretagogue action, for example, about 0.1% formic acid and about 140 mM sodium chloride are included. A step of dissolving the peptide in the solution, heating at about 60° C. for about 1 hour, and then gradually cooling to about room temperature is also suitable.

The present invention also relates to an “active mutant peptide” in which the above-mentioned lactam cyclic peptide has been subjected to amino acid substitution so as to stably exert an insulin secretagogue action. The peptide is a peptide capable of forming a three-dimensional structure which is presumed to be formed by the freeze-drying step and/or the thermal acid treatment step, and may be a cyclic peptide or a linear peptide. Good.

The polypeptide of the present invention will be described in more detail below.

In the present invention, the peptide means a short-chain polypeptide, and the length thereof is not particularly limited, but it is typically several to ten and several. In the present invention, the peptides include those called oligopeptides or oligomers. Further, in the present invention, the peptides include those having a linear shape, a cyclic shape, a branched shape and the like.

In the present invention, the term "several" may be a number greater than 2 or 3, for example, about 5 to 15, preferably 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 ~8, 5-7, 6-12, 6-11, 6-10, 6-9, or 6-8, more preferably 6-10, 7-10, Or 7 to 9 pieces. In the present invention, the number of ten and several may be, for example, about 11 to 19, preferably 12 to 18, 12 to 17, 12 to 16, 12 to 15, 12 to 14, or 12. Can be up to 13. The length of the peptide of the present invention is not limited to the following, but is, for example, 20 amino acids or less, or less than 20 amino acids, for example, 5 to 19 amino acids, preferably 5 to 17 amino acids, 5 to 16 amino acids, 6 to 15 amino acids, 6-12 amino acids, 6-11 amino acids, 6-11 amino acids, 6-10 amino acids, 7-10 amino acids, or about 10 amino acids, more preferably 9 or less, for example 6-9 amino acids , More preferably 7-9 amino acids, for example 7 amino acids, 8 amino acids, or 9 amino acids. A heptapeptide and a peptide obtained by adding about 1 or 2 amino acids to the heptapeptide are particularly suitable as the peptide of the present invention. Amino acids can be added, for example, at the C-terminus.

The amino acid sequence capable of forming an α-helix structure contained in the peptide of the present invention can be appropriately selected. The amino acid sequence preferably contains an amino acid that easily forms an α-helix structure (this is referred to as an α-helix forming amino acid), but may include other amino acids. Amino acids that are difficult to form an α-helix structure (for example, proline, glycine, tyrosine, and serine) may be included in the peptide of the present invention, but are included in the amino acid sequence that can form an α-helix structure. Preferably not. Examples of the α-helix forming amino acid include methionine, alanine, leucine, glutamic acid and lysine. In the present invention, the amino acids in the amino acid sequence capable of forming an α-helix structure, α-helix-forming amino acids occupy, for example, 50% or more, preferably 60% or more, more preferably 80% or more, more preferably 90% or more. You can stay. More preferably, the amino acid sequence capable of forming an α-helix structure is composed of only α-helix forming amino acids.

The length of the amino acid sequence capable of forming an α-helix structure may be appropriately selected, but is, for example, 4 amino acids or more, preferably 5 amino acids or more, more preferably 6 amino acids or more, and further preferably 7 amino acids or more. For example, an amino acid sequence capable of forming an α-helix structure consisting of 5, 6, 7, or 8 amino acids can be preferably used. For example, a peptide containing an amino acid sequence capable of forming an α-helix structure consisting of 5 amino acids is suitable as the peptide of the present invention.

In the present invention, the α-helix structure formed by the amino acid sequence preferably has a relatively small radius of gyration. In order to reduce the radius of gyration, amino acids with small side chains can be selected. For example, the average molecular weight of the side chains of amino acids that form the amino acid sequence capable of forming an α-helix structure is preferably smaller than the average molecular weight of the side chains of 20 types of natural amino acids, for example, 20 types of natural amino acids. The average molecular weight of the side chain is preferably 80% or less, 70% or less, 60% or less, or 50% or less. More specifically, the molecular weight of the side chain of amino acids constituting the amino acid sequence capable of forming an α-helix structure is, for example, 74 or less of the majority (for example, 50% or more, preferably 60% or more, more preferably 80% or more). % Or more), for example, the molecular weight of the side chain is 73 or less, 72 or less, 70 or less, 68 or less, 65 or less, 62 or less, 60 or less, 40 or less, 30 or less, or 20 or less amino acids, α It is preferable to occupy a majority of amino acids (for example, 50% or more, preferably 60% or more, more preferably 80% or more, 90% or more, or 100%) constituting an amino acid sequence capable of forming a helix structure. For example, the peptide of the present invention containing an amino acid sequence capable of forming an α-helix structure consisting of 5 amino acids has a side chain molecular weight of 74 or less, 73 or less, 72 or less, 70 or less, 68 or less, 65 or less, 62 or less, 60 or less. Hereinafter, 40 or less, 30 or less, or 20 or less amino acids may be contained in 3 or more, 4 or more, or 5 of 5 amino acids.

Further, in the peptide of the present invention, it is preferable to include alanine in the amino acid sequence forming the α-helix structure, for example. The ratio of alanine may be appropriately selected, but is, for example, 30% or more, preferably 40% or more, 50% or more, 60% or more, more preferably 80% or more, or 100%. For example, the peptide of the present invention containing an amino acid sequence capable of forming an α-helix structure consisting of 5 amino acids may contain 3 or more, 4 or more, or 5 alanines in 5 amino acids. Preferably, the peptide of the present invention contains, as an amino acid sequence forming an α-helix structure, 3 consecutive alanines or more, more preferably 4 consecutive alanines or more. Five consecutive alanines (AAAAA/SEQ ID NO: 3) are suitable as an amino acid sequence capable of forming an α-helix structure.

Further, in the peptide of the present invention, an amino acid having a hydroxyl group and/or a hydrophilic amino acid is preferably linked to the N-terminal side of an amino acid sequence capable of forming an α-helix structure. That is, the peptide of the present invention is preferably a peptide containing an amino acid sequence having an α-helix structure, following an amino acid having a hydroxyl group and/or a hydrophilic amino acid. The amino acid having a hydroxyl group and/or the hydrophilic amino acid is preferably the N-terminal amino acid of the peptide.

Preferred examples of the amino acid having the hydroxyl group and/or the hydrophilic amino acid include threonine, serine, and tyrosine, but are not limited thereto. Examples of the hydrophilic amino acid include arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine, cysteine, histidine, and methionine, and particularly arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine. preferable. Further, the amino acid may be a hydrophilic neutral amino acid (for example, threonine, serine, asparagine, glutamine). Particularly preferred amino acids are hydrophilic amino acids having a hydroxyl group, such as threonine or serine.

Also, the peptide of the present invention is preferably one in which a hydrophilic amino acid is linked to the C-terminal side of an amino acid sequence capable of forming an α-helix structure. That is, the peptide of the present invention is preferably a peptide containing a hydrophilic amino acid following an amino acid sequence capable of forming an α-helix structure. The amino acid may be appropriately selected, but hydrophilic neutral amino acids such as threonine, serine, glutamine and asparagine are preferred, and glutamine is more preferred.

That is, the peptide of the present invention has an amino acid having a hydroxyl group at the N-terminal and/or a hydrophilic amino acid, which is subsequently linked with an amino acid sequence capable of forming an α-helix structure, followed by a hydrophilic neutral amino acid. Peptides containing linked sequences are preferred. In other words, an amino acid sequence capable of forming an α-helix structure has a structure in which an amino acid having a hydroxyl group and/or a hydrophilic amino acid and a hydrophilic neutral amino acid are sandwiched, that is, an amino acid having a hydroxyl group and Preference is given to a peptide in which an amino acid sequence capable of forming an α-helix structure is located between a hydrophilic amino acid and a hydrophilic neutral amino acid.
More specifically, there are amino acids having a hydroxyl group such as threonine, serine, and tyrosine at the N-terminus and/or hydrophilic amino acids, and subsequently, for example, α containing 3 or more, 4 or more, or 5 alanines. A peptide containing a sequence in which amino acid sequences capable of forming a helix structure are linked, followed by a hydrophilic neutral amino acid such as glutamine is particularly suitable as the peptide of the present invention. The amino acid sequence capable of forming the α-helix structure may be, for example, a sequence consisting of 5 amino acids, and a preferable sequence includes 5 consecutive alanines (AAAAA/SEQ ID NO: 3).

The above-mentioned hydrophilic neutral amino acid such as glutamine may be the C-terminal amino acid of the peptide, but an amino acid or the like may be further added to the C-terminal side as long as it retains insulin secretagogue activity. For example, a few amino acids, specifically, about 1, 2, 3, 4, or 5 amino acids can be added as long as the activity is maintained. Although the amino acid to be added is not particularly limited, examples thereof include glycine, and for example, one to which one glycine (G) or two glycines (GG) are added is also suitable as the peptide of the present invention.

More specifically, examples of the peptide of the present invention include threonine-alanine-alanine-alanine-alanine-alanine-glutamine (TAAAAAQ/SEQ ID NO: 4), serine-alanine-alanine-alanine-alanine-alanine-glutamine (SAAAAAQ/ SEQ ID NO: 12), and the amino acid sequences of threonine-alanine-alanine-alanine-alanine-alanine-glutamine-glycine-glycine (TAAAAAQGG/SEQ ID NO: 11), and also TAAAAAQG (SEQ ID NO: 19), SAAAAAQG (SEQ ID NO: 19). 20), and also amino acid sequences such as SAAAAAQGG (SEQ ID NO: 21). Peptides containing these amino acid sequences are preferable in the present invention, and peptides having the amino acid sequences are particularly preferable in the present invention.

The present invention also provides a peptide in which one or several amino acids are substituted, deleted, added, and/or inserted in the peptide specifically exemplified above, wherein the peptide and/or its active form promotes insulin secretion. It relates to active peptides. The number of amino acids modified is, for example, 1 to 3, preferably 1 to 2, and most preferably 1.

The present invention also relates to a peptide in which the amino acids of the peptides specifically exemplified above are conservatively substituted, and the peptide and/or its active form has insulinotropic activity. Conservative substitutions of such amino acids are well known to those skilled in the art. Examples of groups of amino acids corresponding to conservative substitutions include basic amino acids (eg, lysine, arginine, histidine), acidic amino acids (eg, aspartic acid, glutamic acid), uncharged polar amino acids (eg, glycine, asparagine, glutamine, serine, Threonine, tyrosine, cysteine), non-polar amino acids (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta branched amino acids (e.g. threonine, valine, isoleucine), and aromatic amino acids (e.g. tyrosine, phenylalanine). , Tryptophan, histidine) and the like. Also, for example, the value of BLOSUM62 matrix (Henikoff, S. and Henikoff, J. G. (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) is 2 or less, more preferably 1 or less, and more preferably Substitutions can be given to 0 amino acids. The number of amino acid substitutions is, for example, 1 to 3, preferably 1 to 2, and more preferably 1.

Needless to say, the peptides of the present invention do not include known peptides known to have insulinotropic activity. The peptide of the present invention, for example, a peptide known to have insulinotropic activity, for example, a dipeptide or tripeptide known to have insulinotropic activity, specifically, JP2018-123065, It does not include dipeptides or tripeptides as described in JP-A-2016-034930 and WO2012/109561. Further, the peptides of the present invention do not include known inlectins (GLP-1, GIP) and GLP-1 analogs (known diabetes drugs). That is, the peptide of the present invention is a peptide which is not known to have insulinotropic activity.

The peptide of the present invention may be a natural peptide or a synthetic peptide. The amino acids that compose the peptide may be natural amino acids or non-natural amino acids. The peptide or amino acid may or may not be modified. The modification may be naturally modified or artificially modified. Modifications include modifications such as peptide backbones, amino acid side chains, amino (N) termini, or carboxyl (C) termini. Modifications include acetylation, acylation, ADP ribosylation, amidation, cyclization, disulfide bond formation, methylation, demethylation, pyroglutamine oxidation, γ-carboxylation, glycosylation, hydroxylation, iodination, myristoyl. Oxidization, oxidation, phosphorylation, ubiquitination and the like, but are not limited thereto. The modification may be addition of a compound such as flavin, nucleotide, nucleotide derivative, lipid, lipid derivative, or phosphatidylinositol.

Further, the peptides of the present invention include derivatives, modified products and analogs. Such derivatives include molecules having a modified form of the functional group of the peptide of the present invention by modification, addition, mutation, substitution or deletion. The modification of the functional group is carried out for the purpose of, for example, protecting the functional group existing in the polypeptide (for example, replacing the functional group with a protecting group), controlling the stability or tissue translocation of the peptide, or controlling the activity of the peptide. Can be broken. For example, the peptides of the present invention include those in which any of the N-terminal, C-terminal, and side chain functional groups of amino acids constituting the polypeptide is modified with other substituents such as a protecting group. .. Examples of the substituent include, but are not limited to, various alkyl groups, acyl groups, amides, phosphoric acid groups, amino groups, carboxyl groups and ester groups.

Further, the peptides of the present invention include multimers such as dimers, trimers, and tetramers in which peptides are bound to each other, branched molecules, and cyclized molecules. Also, the peptide may be bound to a carrier. For example, the peptide of the present invention may be linked to polyethylene glycol (PEG), dextran, or another polymer.

The amino acids that compose the peptide of the present invention may be L-form and/or D-form. Use of D-form amino acids is useful for reducing degradation by peptidases. Moreover, the amino acid is not limited to the natural amino acid as described above, and may be a non-natural amino acid. Further, the peptide bond of the peptide of the present invention may be appropriately replaced with a covalent bond other than the peptide bond. Substitution with a non-peptide bond can reduce the sensitivity to peptidases and improve the duration of drug efficacy. Examples of non-peptide bonds include, but are not limited to, imino bonds, ester bonds, ketomethylene bonds, α-aza bonds, carba bonds, hydroxyethylene bonds, thioamide bonds, olefinic double bonds and the like.

The polypeptide of the present invention also includes a salt thereof. Such salts are derived from acids or bases. Specifically, for example, salts with inorganic acids such as hydrochlorides, phosphates, hydrobromides, sulfates, nitrates, acetates, lactates, formates, butyrates, glycolates and propiones. Salts with organic acids such as acid salts, fumarates, maleates, succinates, tartrates, citrates, malates, oxalates, benzoates, methanesulfonates, benzenesulfonates Or ammonium salts, alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, salts with organic bases, salts with amino acids such as arginine and lysine, and the like.

The present invention also relates to a nucleic acid encoding the peptide of the present invention and a vector carrying the nucleic acid. The nucleic acids and vectors of the present invention are useful for expressing and/or producing the peptides of the present invention. The nucleic acid of the present invention may be DNA or RNA. As the vector, a desired vector such as a plasmid, a viral vector, a non-viral vector (for example, liposome) can be used. Viral vectors include, but are not limited to, adenovirus vectors, retrovirus vectors (including lentivirus vectors), paramyxovirus vectors (including Sendai virus vectors), and the like.

For example, the peptide of the present invention shows significant insulin secretagogue activity as it is and/or when it is converted to an active form by an appropriate treatment. Since the activity of a peptide can be changed by the change of its higher-order structure, it is possible to impart or increase the insulin secretagogue activity by converting the peptide to an active form by appropriate treatment. Generally, as a method for changing the higher-order structure of a peptide, acid treatment, alkali treatment, heat treatment, transition metal addition, a combination thereof and the like can be mentioned, and activation may be carried out by any of these treatments. Examples of the acid treatment include, but are not limited to, treatment with an acidic solvent such as formic acid, acetic acid, citric acid and phosphoric acid. Further, the pH in the acid treatment can be appropriately selected. Examples of the alkali treatment include alkaline solvents such as ammonia solution and ammonium chloride solution. Examples of the metal to be added include sodium, zinc, lithium, manganese, and the like, and salts such as sodium chloride, zinc chloride, zinc sulfate, lithium chloride, and manganese chloride can be appropriately added.

For example, when the peptide of the present invention is a cyclic peptide, it was found that it is possible to impart or increase insulin secretagogue activity by combining acid treatment and heat treatment. In particular, the activity can be increased by a hot acid treatment in which salts are added and heated after the acid treatment.

That is, the present invention is
(1) a step of acid-treating the peptide of the present invention,
(2) adding a salt to the peptide of the present invention, and (3) heating the peptide of the present invention,
And a peptide having an increased insulin secretagogue activity, which is produced by the treatment with a hot acid containing The present invention also relates to a method for producing a peptide having an insulin secretagogue-promoting activity imparted or increased, including the steps described above, and a method for imparting or increasing the insulin secretion-promoting activity to the peptide of the present invention, comprising the steps described above.

The acid treatment may be, for example, treatment with an acidic solvent having a pH of about 2.0 to 6.4. For example, pH 2.5 to 6.0, pH 2.8 to 5.5, pH 3.0 to 5.0, pH 3.0 to 4.5, pH 3.0 to 4.0, pH It can be 3.2 to 3.8, for example about pH 3.5. The term “about” as used herein generally means, for example, ±15%, more preferably ±10%.

The acidic solvent is not particularly limited as long as the above pH can be achieved, but examples thereof include acidic solvents such as formic acid, acetic acid, citric acid, and phosphoric acid, and particularly preferably treatment with formic acid or acetic acid. ..

The concentration of formic acid or acetic acid may be adjusted appropriately, for example, 0.01 to 1% (v/v), preferably 0.02 to 0.8% (v/v), 0.03 to 0.5% (v/v), 0.05 to 0.3% (V/v), 0.08 to 0.2% (v/v), for example, about 0.1% (v/v). The treatment time may be adjusted appropriately.

After the acid treatment, salts are added to the peptide. The salt to be added may be appropriately selected, and examples thereof include sodium salt and zinc salt, and specific examples thereof include sodium chloride and zinc chloride, but are not limited thereto.

The concentration of salts may be adjusted appropriately. For example, for sodium salts, the final concentration is 10 mM to 1 M, preferably 20 to 800 mM, 30 to 600 mM, 50 to 500 mM, 80 to 300 mM, 100 to 200 It can be mM, 110-180 mM, for example about 140 mM. The final concentration of zinc salt can be 1 μM to 1 M, preferably 10 μM to 100 mM, 300 μM to 30 mM, for example, about 10 mM.

Heat the peptide after adding the salts. The heating temperature may be appropriately selected, and examples thereof include 37 to 90°C, preferably 45 to 85°C, 50 to 85°C, or 55 to 80°C. For example, when 0.1% formic acid is used as the acidic solvent, , 37-90°C, preferably 45-80°C, 50-75°C, 55-70°C, 58-65°C, for example about 60°C. When 0.1% acetic acid is used, the temperature is 37 to 90°C, preferably 45 to 85°C, 50 to 85°C, 60 to 85°C, for example, about 80°C. The heating time may be adjusted appropriately. Specifically, it can be appropriately selected, for example, from 10 minutes to overnight, and is appropriately in the range of, for example, 20 minutes to 10 hours, 30 minutes to 5 hours, 40 minutes to 3 hours, or 50 minutes to 2 hours. It can be selected, for example, about 1 hour. However, these are merely examples, and those skilled in the art may appropriately set the optimum conditions according to the salt or the like to be used. After heating, it can be cooled slowly, for example.

It was also found that the peptide of the present invention can impart or increase the insulin secretagogue activity by the process of freeze-drying regardless of its form, for example, cyclic peptide or linear peptide. In particular, the activity can be increased by a treatment of lyophilization after dissolution in a buffer solution.

The pH of the buffer solution may be appropriately selected, for example, pH 6.2 to 8.2, pH 6.5 to 8.0, pH 7.0 to 8.0, pH 7.2 to 8.0, pH 7.4 to 8.0, pH 6.7 to 7.9, pH 6.8 to 7.9 , PH 7.0 to 7.8, pH 7.2 to 7.8, pH 7.3 to 7.8, or pH 7.5 to 7.8 can be appropriately selected, for example, about pH 7.6. A buffering agent is also appropriately selected, and examples thereof include HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid) and TRIS (tris(hydroxymethyl)aminomethane). In the present invention, HEPES is preferably used.

The concentration of the buffering agent in the buffer solution is appropriately selected, but is, for example, 0.1 to 500 mM, preferably 0.2 to 300 mM, 0.5 to 200 mM, 1 to 100 mM, 2 to 80 mM, 5 to 50 mM, or 8 It can be appropriately selected within a range of up to 30 mM, for example, it can be about 10 mM.

Also, an intercalator may be added to the peptide of the present invention. By adding a substance that intercalates to the peptide chain, the structure of the peptide of the present invention can be converted to the active form, and the insulin secretagogue activity can be imparted or increased. After adding the intercalator, the peptide of the present invention may be lyophilized. Lyophilization may be performed according to a well-known procedure. In the present invention, the peptide having an amino acid sequence capable of forming an α-helix structure also includes a peptide that forms an α-helix structure by treatment with hot acid, dissolution in a buffer solution and freeze-drying, or addition of an intercalator. To be done.

The present invention also relates to the above-mentioned peptide of the present invention, wherein the desired other compound is cleavably linked. The compound may or may not inhibit the insulin secretagogue activity possessed by the peptide of the present invention, as long as it can be cleaved and separated from the peptide. That is, the peptides to which the compound is bound include those having insulinotropic activity and those having no insulinotropic activity. A peptide having no insulin secretagogue activity can be converted into an active form by cleaving the compound from the peptide.

The compound is not particularly limited, but is a fusion peptide in which an amino acid or a polypeptide (which may be a short-chain polypeptide called a peptide) is adopted as another compound and is fused with the peptide of the present invention. (Or a fusion polypeptide). If the fused boundary portion is cleaved so that the two can be separated from each other, the peptide of the present invention (which can exert insulinotropic activity) can be produced by the cleaving. For that purpose, for example, another polypeptide may be added so that the boundary becomes a protease cleavage site. The other peptide to be fused is not particularly limited, and examples thereof include any polypeptide from a short peptide having a few residues such as a tag to a long polypeptide such as a protein. Specific examples thereof include, but are not limited to, His tag, HA tag, Myc tag, FLAG, GFP, maltose binding protein, glutathione S-transferase (GST), alkaline phosphatase, HRP and other enzymes. Alternatively, an antibody fragment (eg, Fc fragment) or the like may be fused. Further, a leader sequence, a secretory signal, a preprotein or proprotein sequence, etc. may be fused.

The present invention also provides the use of the peptide of the present invention for promoting insulin secretion and/or promoting glucose metabolism. The invention also provides the use of the peptides of the invention for lowering blood glucose levels. The present invention also provides the use of the peptide of the present invention in the manufacture of a medicament or reagent for promoting insulin secretion and/or promoting glucose metabolism. The invention also provides the use of the peptides of the invention in the manufacture of a medicament or reagent for lowering blood glucose levels. The present invention also provides an insulin secretagogue and a sugar metabolism enhancer containing the peptide of the present invention. The present invention also provides a blood glucose lowering agent containing the peptide of the present invention. The present invention also provides the use of the peptide of the present invention for the treatment or prevention of diabetes or pre-diabetes, the use of the peptide of the present invention in the manufacture of a medicament for the treatment or prevention of diabetes or pre-diabetes, and the invention Provided are therapeutic and prophylactic agents for diabetes or pre-diabetes containing peptides. In the present invention, diabetes includes borderline diabetes as described above.
Further, the peptide of the present invention specifically promotes insulin secretion in a hyperglycemic state to lower the blood glucose level, and does not promote insulin secretion and induces hypoglycemia in a state where the blood glucose is not high (Examples 11 to 13). Therefore, the insulin secretagogue and the glucose metabolism promoter of the present invention are useful as a hyperglycemic state-specific insulin secretagogue and a glucose metabolism promoter, respectively, and the hypoglycemic agent of the present invention is a hyperglycemic condition-specific. It is useful as an effective hypoglycemic agent.

The peptide of the present invention acts on pancreatic β cells to promote insulin secretion, thereby promoting glucose metabolism and exerting an effect of lowering blood glucose level. Therefore, the peptide of the present invention can be used as a research tool for the insulin secretion control mechanism of pancreatic β-cells and as a therapeutic tool for obesity, hyperlipidemia and the like. Such uses include in vitro use and in vivo use, ex vivo use. In particular, the peptide of the present invention can be used for the treatment and prevention of various pathological conditions and symptoms that require improved glucose metabolism.

The peptide of the present invention is also useful for various cosmetic purposes for the purpose of preventing or reducing an increase in body weight and body fat, reducing weight (including partial weight loss), improving body shape, and the like.

For example, the present invention provides a medical or cosmetic method for the prevention or treatment of obesity, which method comprises administering a peptide.

Similarly, the present invention provides a medical therapy for improving insulin resistance and/or preventing or treating diabetes or pre-diabetes, which comprises administering a peptide of the present invention.

In addition, the medical therapy of the present invention prevents or treats obesity, improves insulin resistance, prevents or treats diabetes, prevents or treats hyperlipidemia, treats to promote hematopoiesis, and creates a coronary bypass circulation. Medical treatment in combination with any of the following surgery, which comprises the administration of a peptide of the invention.

Further, the present invention is a composition for preventing and/or treating various diseases showing metabolic disorders such as obesity and/or overweight, metabolic syndrome, type 2 diabetes and type 1 diabetes, which comprises the peptide of the present invention as an active ingredient. I will provide a. The present invention also provides a cosmetic composition containing the peptide of the present invention as an active ingredient for the purpose of preventing or reducing an increase in body weight or body fat, thinning (including partial thinning), improving body shape and the like.

In particular, the peptide of the present invention exerts a strong action of promoting insulin secretion and lowering the blood glucose level when the blood glucose level is high at any time (diabetes type), and promotes insulin secretion when the blood glucose level is not high at any time (normal type). The activity is expected to be relatively low (see Example 11). Also, in cases of onset of borderline diabetes, there is no decrease in fasting blood glucose, which has a normal blood glucose level, and it is possible to specifically suppress hyperglycemia during glucose load and bring it closer to the normal value, resulting in hypoglycemia. Does not occur (Examples 12 to 13). Therefore, the peptide of the present invention is considered to be advantageous in that it exhibits an effect only in a hyperglycemic state in vivo, that is, it is highly safe without inducing hypoglycemia.

Further, by enhancing the basal insulin secretion by the peptide of the present invention, it is possible to lower the blood glucose at any time and improve the ability to cope with the glucose load accompanying meals (glucose tolerance). It is also considered that the peptide of the present invention is used to enhance basal insulin secretion in the pathological condition in which the basal insulin secretory ability is decreased with the progress of type 2 diabetes. The peptide of the present invention can also be used to improve the insulin secretory function of residual pancreatic β cells in type 1 diabetes. Thus, the peptide drug of the present invention provides an effective glycemic control strategy for a wide range of cases of type 2 diabetes and type 1 diabetes (in particular, slowly progressive type 1 diabetes).

The peptide of the present invention can be formed into a composition as appropriate. That is, the present invention relates to a composition comprising the peptide of the present invention (including the above-mentioned fusion polypeptide and the like) and a pharmaceutically acceptable carrier or medium. The composition is particularly useful as a pharmaceutical composition, and can be used, for example, for promoting insulin secretion, promoting glucose metabolism, and/or improving glucose metabolism, and for treating or preventing diabetes or prediabetes. it can. The composition of the present invention is also useful as a cosmetic composition. The formulation can be carried out by a known pharmaceutical method. A pharmacologically acceptable carrier or medium is appropriately selected, and examples thereof include water (eg, sterile water), physiological saline (eg, phosphate buffered saline), glycol, ethanol, glycerol, lactose, sucrose, calcium phosphate, Examples include oils such as gelatin, dextran, agar, pectin, olive oil, peanut oil, and sesame oil.Emulsifiers, suspending agents, surfactants, buffers, flavoring agents, diluents, preservatives, stabilizers, shaping agents. Agents, vehicles, preservatives, sustained-release agents and the like.

For formulation as an injection, it can be formulated using a carrier such as distilled water for injection. Examples of the aqueous solution for injection include an aqueous solution containing other components such as physiological saline, glucose, D-sorbitol, D-mannose, D-mannitol and sodium chloride. In addition, alcohol, propylene glycol, polyethylene glycol, nonionic surfactant and the like may be contained.

The pharmaceutical composition and cosmetic composition of the present invention are preferably administered by parenteral administration. For example, it can be formulated as an injection, a nasal agent, a pulmonary agent, a transdermal agent and the like. Administration may be systemic or local, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like. The details of the administration method can be appropriately selected depending on the age and symptoms of the patient. The dose is, for example, in the range of 0.0001 mg to 1000 mg per 1 kg of body weight, for example, 0.0005 to 500 mg, 0.001 to 400 mg, 0.002 to 300 mg per 1 kg of body weight, calculated as a dry peptide. It can be set to 0.005-200 mg, but is not limited to this. Alternatively, for example, the dose may be set to 0.001 to 100,000 mg per patient, for example, 0.002 to 70000 mg, 0.003 to 50000 mg, 0.005 to 40,000 mg, 0.01 to 20000 mg, but is not limited thereto. Those skilled in the art can appropriately determine an appropriate dose and administration method in consideration of the body weight, age, symptoms, etc. of the patient.

The peptide of the present invention can be administered to a desired subject. The administration subject can be appropriately selected, for example, mammals, specifically, rodents such as mice, rats, guinea pigs, non-rodent animals such as cows, pigs, goats, rabbits, desired primates, Non-human primates such as monkeys, and humans are included.

The embodiments of the present invention will be described below based on the examples shown in the drawings. Note that the embodiments are not limited to the following examples, and the design can be appropriately changed by using a conventionally known technique such as the above-described document without departing from the spirit of the present invention.

All documents cited in this specification are incorporated herein by reference.

[Example 1] Relationship between physicochemical treatment of circ-TKEDGRQ and insulin secretagogue activity:
A lactam cyclic peptide circ-TKEDGRQ in which the N-terminal and the C-terminal of the peptide having the amino acid sequence "threonine-lysine-glutamic acid-aspartic acid-glycine-arginine-glutamine" (SEQ ID NO: 1) were peptide-bonded was synthesized organically. (Eurofin Genomics Co., Ltd.). This was dissolved in pure water to prepare a 5 mg/ml (6.1 mM) stock solution. This was added to mouse pancreatic beta cell line MIN6 (Miyazaki et al., Endocrinology 127: 126-132, 1990) at various concentrations, and the insulin secretagogue activity was evaluated by the method described in Reference Example 2.
As a result, the expected high level of insulin secretagogue activity was not detected (FIG. 1A, N=1). Since no high level of activity was detected even after repeated experiments, it was concluded that "active structural isomers" were not formed at a high ratio by simply dissolving the organic chemical synthesis product circ-TKEDGRQ in water. ..

Next, the method for converting the organic chemically synthesized product circ-TKEDGRQ into active structural isomers at a high rate was examined. Generally, "acid treatment", "alkali treatment", "heat treatment", and "transition metal addition" are known as methods for changing the higher-order structure of a short-chain peptide. Therefore, the conditions were examined using "pH", "temperature", and "types and concentrations of coexisting metal salts" as parameters. Specifically, formic acid, acetic acid, citric acid, and phosphoric acid are used as an acidic solvent (pH 3.5-pH 7.4), ammonium chloride solution is used as an alkaline solvent (pH 10-11), and ammonium acetate solution is used as a neutral solvent. Make a solvent, add sodium chloride, zinc chloride, zinc sulfate, lithium chloride, or manganese chloride to it, and use a heat block, PCR device, water bath, or incubator at 40°C to 90°C for 10 minutes. A sample which was heated for 60 minutes and desalted on a PD-10 column and then concentrated was evaluated for insulin secretagogue activity.
As a result, 1 μL of an organic chemical synthesis product circ-TKEDGRQ in water (1 mM) was diluted with 0.1% formic acid to 1000 μL (final concentration 1 μM), and then 140 mM final concentration of sodium chloride was added. Incubated in a heat block at ℃ for 1 hour, allowed to stand at room temperature for heat removal, and then desalted and concentrated on a PD-10 column. When added to mouse pancreatic beta cell line MIN6 at various concentrations, insulin was dose-dependently administered. A secretagogue activity was observed (FIG. 1B, N=1). When the robustness related to the detection of insulin secretagogue activity was examined by repeating the experiment, the probability of successful detection of the activity was about 20%. That is, it was considered that the stability of the experimental system in the conversion of structural isomers cannot be said to be extremely high in the treatments focused on the parameters of “acid treatment”, “alkali treatment”, “heat treatment”, and “metal salt”. The reason for this is that the conformational transformation of short-chain peptides is limited to stochastic events under the above parameters (Note: active structural isomerism due to coexistence of multiple structural isomers in chemical thermodynamic equilibrium). It was considered that the efficiency of structural isomer conversion is greatly affected by unidentified factors other than the above parameters).

As mentioned above, it was found that the organic chemically synthesized product circ-TKEDGRQ has an insulin secretagogue promoting activity, and it is possible to increase the activity by thermal acid treatment in which salts are added and heated after acid treatment. However, it could not be said that this method could convert the organic chemically synthesized product circ-TKEDGRQ into the “active structural isomer” with extremely high reproducibility.

However, in the process of examining the above-mentioned conditions, it was found that there is a tendency to increase the conversion efficiency to the active structural isomer in the working process called "lyophilization". Therefore, organic chemistry was applied to various aqueous buffers (TRIS buffer, HEPES buffer, BIS-TRIS buffer, MES buffer, phosphate buffer) and organic solvents (methanol, ethanol, isopropanol, diethyl ether, acetonitrile). The synthetic product circ-TKEDGRQ was dissolved, and when a solvent containing a salt was used, desalting was carried out on a PD-10 column, and then the freeze-dried product was used to evaluate the insulin secretagogue activity.
As a result, 2 μl of an aqueous solution of the circ-TKEDGRQ organic chemical synthesis product (6.1 mM) diluted to 100 μM was 100 times diluted with 10 mM TRIS buffer (pH 8.0) or 10 mM HEPES buffer (pH 7.4). The insulin secretagogue activity was detected when the insulin secretagogue activity was evaluated by using each of them diluted to 200 μl, lyophilized, and dissolved in 200 μl of KRTB (final concentration 1 μM) (FIG. 2, N = 1). When the robustness regarding the detection of insulin secretagogue activity was examined by repeating the experiment, the probability that the activity was successfully detected was about 60%, and it was found that the robustness can be improved by the treatment with the buffer solution.

In order to evaluate the importance of circ-TKEDGRQ having a lactam cyclic structure in the insulin secretagogue activity, a linear peptide (threonine-lysine-glutamic acid-aspartic acid-glycine-arginine-glutamine: TKEDGRQ) (SEQ ID NO: 1 Was organically synthesized, and freeze-dried by the above method using HEPES buffer (pH 7.6) to evaluate the insulin secretagogue activity.
As a result, the activity as high as that of the cyclic peptide was not detected even when a high concentration (240 μM) of the linear peptide was used (FIG. 3, N=3). The results did not change even after repeated experiments, and it was concluded that the lactam ring structure is important for circ-TKEDGRQ to exert a high insulin secretagogue activity.

In order to confirm the results of the preceding paragraph, a linear peptide shorter than "threonine-lysine-glutamic acid-aspartic acid-glycine-arginine-glutamine (TKEDGRQ)" (SEQ ID NO: 1), "lysine-glutamic acid-aspartic acid-glycine- "Arginine-Glutamine (KEDGRQ)" (2-7 of SEQ ID NO: 1), "Threonine-Lysine-Glutamic acid-Aspartic acid-Glycine-Arginine (TKEDGR)" (1-6 of SEQ ID NO: 1), "Threonine-Lysine-Glutamic acid" -Aspartic acid-glycine (TKEDG)" (1-5 of SEQ ID NO: 1) was synthesized organically and lyophilized by the above method using HEPES buffer (pH 7.6) to promote insulin secretion. The activity was evaluated.
As a result, no activity as high as that of the cyclic peptide was detected with any of the linear peptides (FIG. 4, N=3), and in order for circ-TKEDGRQ to exert a high level of insulin secretagogue activity, lactam was required. The ring structure was determined to be the key.

As described above, circ-TKEDGRQ, which is a peptide in which the N-terminal and C-terminal of TKEDGRQ (SEQ ID NO: 1) form a lactam ring, exhibits a high level of insulin secretagogue activity when it forms a specific conformation, It was shown that it is possible to obtain an active peptide with improved robustness by treatment with thermal acid and/or treatment with a buffer solution. However, it was suggested that it is difficult to convert into an “active structural isomer” with extremely high stability and high probability by the treatment using “pH”, “temperature” and “metal salt” as parameters.

[Example 2] Insulin secretion promoting action of TAAAAAQ (SEQ ID NO: 4) in pancreatic β cells:
From Example 1, the seven amino acid sequence "TKEDGRQ" (SEQ ID NO: 1) is 1) formed a lactam cyclic peptide by peptide-bonding the N-terminus and the C-terminus, and 2) is lyophilized in HEPES buffer. It was suggested that the higher order structure is changed to exert a high level of insulin secretagogue activity by the two steps of. However, since lactam cyclic peptides are time-consuming and expensive to synthesize, it would be a great advantage if linear peptides exhibiting similar activities could be obtained. In considering what kind of linear peptide has insulinotropic activity, considering that the linear peptide has a smaller molecular size due to cyclization, first, an amino acid having a smaller side chain is constructed. It is effective to select them as members. Next, we examined what kind of conformational change would occur when the process of "lyophilize in HEPES buffer" was applied to the cyclic peptide TKEDGRQ.

Free software (QARRK ONLINE, https://zhanglab.ccmb.med.umich.edu/QUARK2/) (D. Xu and Y. Zhang., Proteins) , 2012, 80, 1715-1735; D. Xu and Y. Zhang., Proteins, 2013, 81: 229-239), a sequence in which TKEDGR was connected in tandem was input to predict the higher-order structure. It seems that the pitch is smaller than (the result of inputting DDAAAAAAAAAAKK (SEQ ID NO: 17) (Perutz, PNAS 99: 5596-5600, 2002), which is reported to stably form α-helix on Fig. 5). The structure was obtained (Fig. 5, bottom). Here, the HEPES molecule is known to intercalate into a Z-type DNA double helix to broaden the angle of helical stacking (de Rosa et al., PNAS 107: 9088-9092, 2010), It is expected that the pitch of the helix will be increased when HEPES intercalates.
Based on the above, when the two steps of 1) cyclization and 2) lyophilization in HEPES buffer are applied to the linear peptide TKEDGR (SEQ ID NO: 1), it is similar to the α-helix formed by amino acids with small side chains. It was speculated that the probability of adopting the higher-order structure described above would increase.

Amino acids that easily form α-helix are, for example, alanine and methionine (Scholtz et al., Annu. Rev.Biophys. Biomol.Struct, Vol. 21, pages 95-118, 1992), of which the side chain is small. It is alanine that is thought to form an α-helix with a small radius. That is, a short-chain peptide with continuous alanine (alanine stretch peptide) was assumed as a candidate for “a linear peptide that mimics the higher-order structure of active circ-TKEDGRQ”. However, since alanine is a hydrophobic amino acid, it is also known that alanine stretch peptide easily forms a β sheet and self-aggregates (Ma B&Nussinov R. Protein Science 11:2335-2350, 2002).

Therefore, of the 7 amino acids of TKEDGRQ, the NAA (threonine) and C-terminus (glutamine) were left unchanged, while the internal 5 amino acids were replaced with alanine (AAAAA/SEQ ID NO: 3), which is a peptide TAAAAAQ (SEQ ID NO: 4). ) Was considered as a candidate for "a linear peptide that mimics the higher-order structure of active circ-TKEDGRQ", and this was chemically synthesized to evaluate the insulin secretagogue activity.
Specifically, the chemically-synthesized product TAAAAAQ was dissolved in pure water (5 mg/ml; 8.32 mM) and added in various amounts to 50 μl of an assay system using the pancreatic β-cell line MIN6 to enhance the insulin secretion promoting ability. Evaluation was carried out (final concentration 5.6 μM, 90 μM, 350 μM). As a result, an insulin secretagogue action was detected in a dose-dependent manner (Fig. 6). Furthermore, it was examined whether freeze-drying in HEPES buffer enhances the action of TAAAAAQ to promote insulin secretion. Specifically, 2 μl of an organic chemical synthetic product TAAAAAQ aqueous solution (5 mg/ml) was diluted to 1/100 with 10 mM HEPES buffer (pH 7.6) to make 200 μl, which was freeze-dried to 200 μl. Insulin secretagogue evaluation was performed using the redissolved KRTB of the above (final concentration 83 μM). As a result, the insulin secretagogue activity was detected at a lower concentration than that in the untreated case (Fig. 6) (Fig. 7). These results were reproduced with high robustness even after repeated experiments.

From the above, it was revealed that TAAAAAQ exerts insulin secretagogue activity even without treatment, and exerts insulin secretagogue activity at a lower concentration after freeze-drying treatment in HEPES buffer.

[Example 3] Evaluation of AAAAAAA on insulin secretagogue action:
From Example 2, it was confirmed that TAAAAAQ (SEQ ID NO: 4) has insulinotropic activity, but it is unclear whether T (threonine) at the N terminus and Q (glutamine) at the C terminus are essential for the activity. .. Therefore, a linear peptide AAAAAAA (SEQ ID NO: 5) in which all seven amino acids were converted to alanine was organically synthesized to evaluate the insulin secretagogue activity.
As a result, 2 μl of an aqueous solution of the chemically synthesized product AAAAAAA (5 mg/ml) was used to evaluate the insulin secretagogue activity using the pancreatic β cell line MIN6 (final concentration 10 μg/50 μl), but no significant insulin secretagogue activity was observed. It was not detected (FIG. 8, N=3). No significant insulin secretagogue activity was detected even after repeated experiments, and it was found that AAAAAAA does not have significant insulin secretagogue activity as it is.

Next, we investigated the possibility of conversion into active structural isomers by freeze-drying in HEPES buffer. That is, 2 μl of an organic chemical compound AAAAAAA solution (5 mg/ml) was diluted 100-fold with 10 mM HEPES buffer (pH 7.6) to 200 μl, freeze-dried, and redissolved in 200 μl KRTB. The insulin secretion-promoting activity was evaluated using the prepared product (final concentration 2.5 μg/50 μl; 97 μM). As a result, a remarkable insulin secretagogue activity was detected (Fig. 9, N=3). However, the success rate of conversion of active structural isomers was evaluated by repeating the experiment, and it was about 60%. Therefore, conversion of AAAAAA to active structural isomers by freeze-drying in HEPES buffer was extremely high. It was judged that the phenomenon cannot be said to be reproduced with sex.

As described above, AAAAAAA does not show any significant insulin secretagogue activity when untreated, but since it can exert a high level of insulin secretagogue activity after freeze-drying treatment in HEPES buffer, the peptide N in Example 2 It was confirmed that the T at the terminal and the Q at the C terminal are not essential for the activity, and that the active form has some instability.

[Example 4] Evaluation of biotin-TAAAAAQ on insulin secretagogue action:
Biotin, which specifically binds to Avidin, is widely used for various purposes as a modifier for the N-terminal of peptides. From Example 3, it was suggested that the N-terminal amino acid (threonine) contributes to the formation of active structural isomers of insulin secretagogue activity, but biotin modification to the N-terminal of the peptide contributes to insulin secretagogue activity. Knowing what kind of effect it will have is important information for carrying out the task of identifying the TAAAAAQ (SEQ ID NO: 4) receptor and binding partner. Therefore, a peptide biotin-TAAAAAQ in which biotin was added to the amino group of T (threonine) at the N terminus of TAAAAAQ was organically synthesized and the insulin secretagogue activity was evaluated in the same manner as in Example 2 (FIG. 4).
As a result, it was revealed that biotin-TAAAAAQ did not have a significant insulin secretagogue activity (FIG. 10, N=3). Further, no significant insulin secretagogue activity was detected even after repeated experiments, indicating that biotin-TAAAAAQ does not have significant insulin secretagogue activity.

From the above, it was shown that the N-terminal of TAAAAAQ may be important for the insulin secretagogue activity, and it is not preferable to use a probe with biotin added to the N-terminal to identify the binding partner of TAAAAAQ.

[Example 5] Evaluation of insulin secretion promoting action of AAAAAAQ, MAAAAAQ, GAAAAAQ, CAAAAAQ:
In order for the linear heptapeptide to have an insulinotropic activity, an amino acid such as T (threonine) is present at the N-terminal and an Q (glutamine) is present at the C-terminal with the alanine stretch AAAAA (SEQ ID NO: 3) sandwiched between them. It is suggested that the presence of amino acids such as is effective, and that the N-terminal amino group is preferably not modified.
Next, for the purpose of investigating which amino acid at the N-terminus exerts insulin secretagogue activity, T (threonine) at the N-terminus is replaced with A (alanine), which is an amino acid that easily forms an α-helix. Or peptide substituted with M (methionine) (AAAAAAQ (SEQ ID NO:6), MAAAAAQ (SEQ ID NO:7)), peptide substituted with G (glycine) with the smallest molecular weight GAAAAAQ (SEQ ID NO:8), dimer The peptide CAAAAAQ (SEQ ID NO: 9) substituted for C (cysteine), which is an amino acid involved in formation, was investigated for its insulin secretagogue activity.

Organochemically synthesized product AAAAAAQ formed a precipitate when dissolved in pure water (5 mg/ml), and the precipitate did not disappear even after ultrasonic treatment. Therefore, the ability to promote insulin secretion using the pancreatic β cell line MIN6 could not be properly evaluated.

The organic chemically synthesized product MAAAAAQ could be dissolved in pure water (5 mg/ml). Therefore, the insulin secretion promoting ability was evaluated in the same manner as in Example 3 (final concentration: 10 μg/50 μl). However, no significant insulin secretagogue activity was detected (Fig. 11, N=3). This result was the same even when the experiment was repeated.

Organochemically synthesized product GAAAAAQ formed a precipitate when dissolved in pure water (5 mg/ml). On the other hand, the organic chemical compound CAAAAAQ could be dissolved in pure water (5 mg/ml, 8.26 mM), but a precipitate was formed during storage at 4°C. Therefore, in both cases, the ability to promote insulin secretion using the pancreatic β-cell line MIN6 could not be properly evaluated.

As mentioned above, it was revealed that AAAAAAQ, GAAAAAQ, CAAAAAQ are insoluble or sparingly soluble in water, and MAAAAAQ is soluble in water but has no significant insulin secretagogue activity. In other words, it was suggested that the peptide is capable of forming an α-helix, but it is also important that the peptide retains solubility in the insulin secretagogue activity of TAAAAAQ. Therefore, it is considered that the N-terminal amino acid is preferably a hydrophilic amino acid such as T (threonine) or an amino acid having a hydroxyl group.

[Example 6] Evaluation of insulin secretion promoting action of TAAAAAA:
From Example 3, the importance of amino acids such as T (threonine) at the N-terminus and amino acids such as Q (glutamine) at the C-terminus for efficient formation of active peptides having insulinotropic activity is shown in Example 4, Example 5 suggested the importance of N-terminal hydrophilic amino acids or amino acids having a hydroxyl group.
Subsequently, in order to examine the importance of Q at the C terminus, the insulin secretagogue activity of the peptide TAAAAAA (SEQ ID NO: 10) in which it was replaced with A (alanine) was evaluated. Specifically, 2 μl of organic chemically synthesized TAAAAAA dissolved in pure water (5 mg/ml) was added to the assay system using pancreatic β cell line MIN6 to evaluate the insulin secretagogue ability (final Concentration 10 μg/50 μl).
As a result, TAAAAA showed insulin secretagogue activity (Fig. 12, N=3). However, the activity was lower than that of TAAAAAQ. Repeating the experiment did not change the results.

Next, the possibility of conversion into active structural isomers by freeze-drying in HEPES buffer was examined. That is, 2 μl of an organic chemical synthesis product TAAAAAA (5 mg/ml) was diluted 100-fold with 10 mM HEPES buffer (pH 7.6) to 200 μl, freeze-dried, and redissolved in 200 μl KRTB. The insulin secretion-promoting activity was evaluated using the prepared product (final concentration 2.5 μg/50 μl).
As a result, no significant insulin secretagogue activity was detected (FIG. 13, N=3). Even when the experiment was repeated, no significant insulin secretagogue activity was detected.

Above, it was concluded that TAAAAAA has an insulinotropic activity, but its activity is lower than TAAAAAQ. This result shows the importance of hydrophilic amino acids such as C-terminal Q (glutamine) in TAAAAAQ. From Example 1, TKEDGRQ (SEQ ID NO: 1) (FIGS. 3 and 4) and KEDGRQ (amino acids 2 to 7 of SEQ ID NO: 1) (FIG. 4), which are terminal Q (glutamine) peptides, have high insulin secretion. Since it had no stimulatory activity, C-terminal Q (glutamine) was considered to be important for insulin secretagogue activity in a peptide having a specific amino acid sequence located on the N-terminal side.

[Example 7] Effect of TAAAAAQGG on insulin secretion promotion in pancreatic beta cells:
To identify a TAAAAAQ receptor or binding partner, it is useful to add a tag to TAAAAAQ, but as shown in Example 4, addition of biotin to the N-terminus abolishes insulin secretagogue activity. .. Therefore, in order to further investigate the addition to the C terminus, the peptide TAAAAAQGG (SEQ ID NO: 11), in which "glycylglycine (GG)" commonly used as a spacer for linking tags is added to the C terminus, They were synthesized and evaluated for insulin secretagogue activity in the same manner as in FIG.
As a result, TAAAAAQGG showed insulin secretagogue activity equivalent to that of TAAAAAQ (Fig. 14). This result was reproduced even after repeated experiments. As described above, it was revealed that the addition of the spacer sequence GG to the C terminus does not affect the insulin secretagogue activity. The results also indicate that it is not essential that the C-terminal amino acid be Q (glutamine).

[Example 8] Insulin secretion promoting action of pancreatic β cells by SAAAAAQ:
From Examples 4 to 7, it was shown that amino acids such as N-terminal T (threonine) are important for high-level insulin secretagogue activity. In particular, T is "a hydrophilic amino acid having a hydroxyl group". Therefore, S (serine), which is also a “hydrophilic amino acid having a hydroxyl group”, may have the same effect. Therefore, a peptide SAAAAAQ (SEQ ID NO: 12) in which N at the N-terminal was replaced by S at the S-position was organically synthesized, and the insulin secretagogue activity was evaluated in the same manner as in FIGS. 12 and 13.
As a result, SAAAAAQ was TAAAAAQ both when untreated (Fig. 15(1), N=3) and when freeze-dried in HEPES buffer (Fig. 15(2), N=3). And TAAAAAQGG showed insulin secretagogue activity. These results were reproduced even after repeated experiments.
As described above, SAAAAAQ was found to have insulinotropic activity. This suggests that an amino acid having similar properties to T and S, for example, a hydrophilic amino acid having a hydroxyl group such as T and S is particularly preferable as the N-terminal amino acid.

[Example 9] Evaluation of YAAAAAQ, and DAAAAAQ, KAAAAAQ, and TAAAAAE on insulin secretion promoting action:
Although it was suggested from Examples 2 and 8 that the N-terminal "amino acid having a hydroxyl group" is preferable for the insulin secretagogue activity, amino acids having a hydroxyl group are classified as hydrophilic amino acids. In addition to T (threonine) and S (serine), there is Y (tyrosine), which is classified as a hydrophobic amino acid. Therefore, a peptide YAAAAAQ (SEQ ID NO: 13) in which T in TAAAAAQ was replaced with Y was synthesized organically, and the insulin secretagogue activity was evaluated in the same manner as in FIGS. 12 and 13.
As a result, untreated YAAAAAQ did not show high insulin secretagogue activity (Fig. 16). On the other hand, a relatively high insulin secretagogue activity was detected after no lyophilization treatment in HEPES buffer (Fig. 17), but the titer was lower than that of TAAAAAQGG.

From the above, it was found that TAAAAAQ is preferably an amino acid having a hydroxyl group at the N-terminus, but more preferably a hydrophilic amino acid having a hydroxyl group, for the insulin secretion promoting activity of TAAAAAQ.

Next, in order for TAAAAAQ to exert insulin secretagogue activity, it was clarified whether the N-terminal needs to be a "hydrophilic amino acid having a hydroxyl group", or whether "hydrophilic amino acid" has a similar effect. In order to do so, the effect of "amino acids having charged side chains (acidic amino acids, basic amino acids)" was investigated. Specifically, the peptide DAAAAAQ (SEQ ID NO: 14) in which T at the N-terminus is replaced with D (aspartic acid), which is a hydrophilic amino acid having a carboxyl side chain, and K (lysine), which is a hydrophilic amino acid having an amino side chain, is used. The peptide KAAAAAQ (SEQ ID NO: 15) substituted with was chemically synthesized and evaluated for insulin secretagogue activity in the same manner as in FIGS. 11 and 12.
In addition, regarding C-terminal Q (glutamine), it is necessary to investigate whether this needs to be Q, or whether E (glutamic acid), which is a “hydrophilic amino acid” having a similar structure, has a similar effect. , TAAAAAQ (SEQ ID NO: 16), a peptide in which the C-terminal Q of TAAAAAQ was replaced with E, was synthesized organically and the insulin secretagogue activity was evaluated in the same manner as in FIGS. 11 and 12.
As a result, it was confirmed that KAAAAAQ did not show a high insulin secretagogue activity without treatment, whereas DAAAAAQ and TAAAAAE showed a relatively high insulin secretagogue activity (Fig. 16). Further, YAAAAAQ, DAAAAAQ, KAAAAAQ, and TAAAAAE all showed recovery of insulin secretagogue activity by performing no freeze-drying treatment in HEPES buffer (Fig. 17). However, the activity of each peptide was lower than that of TAAAAAQGG.

As mentioned above, in order for the linear heptapeptide to exert the insulin secretagogue activity, 1) N-terminal is a hydrophilic amino acid (T, S) having a hydroxyl group, 2) The central part is an α-helix such as five alanines It has been found that it is preferable that it is a stretch consisting of amino acids capable of forming 3) and that 3) its C-terminal side is a hydrophilic neutral amino acid such as Q having no charged side chain. Further, it was revealed from Example 7 that an amino acid can be further added to the C-terminus.

[Example 10] Blood glucose reduction by TAAAAAQ administration in diet-induced obese mice Seven-week-old C57BL/6 mice were loaded with a high-fat diet (D12492, Research Diets) for 19 weeks. At the age of 26 weeks, 2 μl of TAAAAAQ aqueous solution (8.32 mM) was diluted with 200 μl of 10 mM HEPES buffer (pH 7.6) (100-fold dilution), and the lyophilized sample was dissolved in 150 μl of saline and intraperitoneally injected. (10 μg/animal). Thereafter, the animals were not fed and blood was collected 2 hours later to measure blood glucose level and serum insulin level. As a control, the same treatment was performed using TKEDGRQ (FIG. 3 and FIG. 4) in which a high insulin secretagogue activity was not detected in the in vitro test. In addition, both TKEDGRQ (SEQ ID NO: 1) and TAAAAAQ (SEQ ID NO: 4) were administered to three C57BL/6 mice, and the blood glucose levels of “before administration” and “after 2 hours after administration” were compared.
As a result, as shown in FIG. 18, the TKEDGRQ-administered mice showed a tendency for a decrease in blood glucose level along with “fasting for 2 hours”, and the TAAAAAQ-administered mice showed a marked decrease in blood glucose level.
As described above, both TKEDGRQ and TAAAAAQ showed a hypoglycemic tendency in diet-induced obese mice, but TAAAAAQ was shown to exert a remarkable hypoglycemic effect.

[Example 11] Blood glucose reduction by TAAAAAQ administration in ob/ob mice 13-week-old ob/ob mice were loaded with a high-fat diet (D12492, Research Diets) for 12 weeks. At the age of 25 weeks, 2 μl of TAAAAAQ aqueous solution (8.32 mM) was diluted with 200 μl of 10 mM HEPES buffer (pH 7.6) (100-fold dilution), and the freeze-dried sample was dissolved in 150 μl of saline and intraperitoneal. (10 μg/animal). Thereafter, the animals were not fed and blood was collected 2 hours later to measure blood glucose level and serum insulin level. As a control, the same treatment was performed using KEDGRQ (amino acids 2 to 7 of SEQ ID NO: 1) (FIG. 4) in which a high insulin secretagogue activity was not detected in the in vitro test. Both KEDGRQ and TAAAAAQ (SEQ ID NO: 4) were administered to two ob/ob mice to compare the blood glucose level and the serum insulin level “before administration” and “2 hours after administration”.
As shown in FIG. 19, the KEDGRQ-administered mice showed a slight decrease in blood glucose level (160 mg/dL→149 mg/ml) regardless of the blood glucose level (pre-dose blood glucose) at any time. dL, 126 mg/dL → 119 mg/dL), serum insulin level decreased to about half (2.22 OD _450nm → 1.10 OD _450nm , 1.27 OD _450nm → 0.61 OD _450nm ) reflecting the non-fed state for 2 hours. .. On the other hand, one TAAAAAQ-administered mouse had a high blood glucose level (pre-dose blood glucose) of 199 mg/dL at any time (diabetes type), but the TAAAAAQ administration significantly lowered the blood glucose level to 131 mg/dL. Further, the serum insulin level showed a low decrease rate (2.47 OD _450nm → 1.92 OD _450nm ) despite the non-feeding state for 2 hours, showing a value of 78% at the time of feeding. The other animal had a normal blood glucose level (pre-dose blood glucose level) of 134 mg/dL, which was normal, and 125 mg/dL even after TAAAAAQ administration, and serum insulin also decreased reflecting the non-fed state ( 1.88 OD _450nm → 0.53 OD _450nm ).
Based on the above, TAAAAAQ has the effect of promoting insulin secretion by lowering the blood glucose level when the blood glucose level is high (diabetic type), but promoting insulin secretion when the blood glucose level is not high (normal type). Not to be shown. This result strongly suggests that the insulin secretagogue action of TAAAAAQ is exerted only in the hyperglycemic state in vivo, that is, it is highly safe and does not induce hypoglycemia.

[Example 12] Improvement of glucose tolerance by administration of TAAAAAQGG in mice with borderline diabetes caused by single administration of low-dose streptozotocin Single-dose streptozotocin (90-100 mg/Kg) of streptozotocin was administered to 6-week old C57BL/6 mice. STZ) (Nacalai Tesque, Inc., 32238-91, used by dissolving in water) was intravenously injected into the tail, and thereafter, blood was collected about twice a week to measure the blood glucose level at any time. Three to five weeks later, an oral glucose tolerance test (OGTT) was conducted using 6 mice whose blood glucose exceeded 180 mg/dL at any time and confirmed to have impaired glucose metabolism. It was Before OGTT, 4 μl of TAAAAAQGG (SEQ ID NO: 11) aqueous solution (5 mg/ml) was diluted to 150 μl of saline and intraperitoneally administered to 3 mice (20 μg/mouse). As a control, 4 μl of the mutant peptide (SEQ ID NO: 22, GlpTKEdGR) aqueous solution (5 mg/ml) diluted to 150 μl of saline was intraperitoneally administered to the remaining 3 mice (( 20 μg/animal). Here, “Glp” in GlpTKEdGR represents pyroglutamic acid (Note: TKEDGRQ of SEQ ID NO: 1 has been moved from the C-terminal Q to the N-terminal. Q becomes pyroglutamic acid due to the inevitable oxidation reaction during peptide synthesis. ). In addition, “d” represents “D-type aspartic acid” (Note: the amino acids that make up proteins in living organisms are L-type, but aspartic acid is a very small amount, but D-type is mixed). In addition, GlpTKEdGR and GlpTKEDGR (SEQ ID NO: 23) were tested in the same manner as in FIG. 8 in an insulin secretion promoting ability evaluation test performed by adding 2 μl of an aqueous solution (5 mg/ml) to the pancreatic β cell line MIN6 ( It has been confirmed that there is no insulin secretagogue activity (final concentration 10 μg/50 μl) (FIG. 20, N=3). After administration of the peptide, the mice were fasted, and 16 hours later, blood was collected from the tail vein to measure the fasting blood glucose level. As a result, it was confirmed that the fasting blood glucose level of all 6 animals was less than 110 mg/dL. In other words, it was confirmed that the fasting blood glucose was in the normal range although the blood glucose level was increased at any time due to impaired insulin secretion function, and the condition of borderline diabetes was exhibited. Therefore, 0.2 g/kg glucose solution was orally administered using a sonde, and blood glucose levels were measured after 15, 30, 60, 120 minutes (oral glucose tolerance test).
As a result, at all time points, the blood sugar level in the TAAAAAQGG administration group was lower than that in the control peptide administration group, and particularly, the blood glucose level 15 minutes after glucose loading was decreased (FIG. 21).
From the above, TAAAAAQGG was shown to have an effect of improving glucose tolerance in mice with borderline diabetes caused by impaired insulin secretion.

[Example 13] Restoration of insulin secretion by administration of TAAAAAQGG in mice that developed diabetes by single administration of low-dose streptozotocin In the same manner as in Example 12, 6-week-old C57BL/6 mice were treated with a low dose (100 mg/Kg). STZ was intravenously injected into the tail, and blood was collected twice/week to monitor the blood glucose level at any time. In the second half of the second week, 4 μl of a TAAAAAQGG (SEQ ID NO: 11) aqueous solution ( 5 mg/ml) was diluted in 150 μl of saline and administered intraperitoneally (20 μg/animal). Thereafter, the animals were fasted, and blood was sampled at 0 hours, 2 hours, and 6 hours, and blood glucose levels and serum insulin levels were measured.
As a result, the blood glucose level exceeded 250 mg/dL at 0 hours and the serum insulin level was below the measurement limit, but the serum insulin level recovered to a measurable level after 2 hours. The value was obtained by diluting 4 μl of the mutant peptide (GlpTKEdGR) (SEQ ID NO: 23) aqueous solution (5 mg/ml) into 150 μl of saline and intraperitoneally administered to the borderline diabetic mouse obtained in Example 12 ( (20 μg/animal), that is, it was found to recover to a level equivalent to that when a low dose of the control peptide was administered to a mouse that had not yet developed diabetes. After that, in the TAAAAAQGG-administered group, the serum insulin level was maintained at 6 hours, and the blood glucose level was further lowered due to fasting. However, it did not cause hypoglycemia, and its value was comparable to that obtained when the control peptide was administered to mice that had not yet developed diabetes and they were fasted for 6 hours.
From the above, it was shown that TAAAAAQGG has an action of improving glucose tolerance by recovering insulin secretion in a mouse in which diabetes was caused by insufficient insulin secretion.

[Reference Example 1] Induction of brown adipocytes from human embryonic stem cells:
Human embryonic stem cells (KhES-3, Suemori et al., Biochem Biophys Res Commun 345:926-932, 2006; Cellosaurus ID CVCL_B233; Cellosaurus ID CVCL_B233; RIKEN Cell Bank ID HES0003) were provided by Institute for Regenerative Medicine, Kyoto University. I used the one. KhES-3 is 20% Knockout Serum Replacement (KSR) (Life Technologies, Inc.), 5ng/ml FGF2, 1% non-essential amino acids solution, 100μM 2-mercaptethanol, on MEF after X-ray irradiation treatment. Maintenance culture was performed using DMEM/F12 (Life Technologies, Inc.) medium containing 2 mM L-glutamine.

When inducing differentiation into brown adipocytes, first, the MEF is separated and removed from KhES-3, so the suspension of pluripotent stem cells recovered by the stripping solution treatment is allowed to stand in a centrifuge tube for about 30 seconds. Only pluripotent stem cells were selectively sedimented at.

Induction of differentiation into brown adipocytes was performed in the following two steps.
(1) Process A
Precipitate consisting of pluripotent stem cells was added to 4 ml of medium for cell aggregate preparation (5 mg/ml BSA, 1% volume synthetic lipid solution, 1% volume x100 ITS-A, 450 μM MTG, 2 mM L-Glutamine, 5%). %DM PFHII, IMDM/F12 medium containing 50 μg/ml ascorbic acid, 20 ng/ml BMP4, 5 ng/ml VEGF, 20 ng/ml SCF, 2.5 ng/ml Flt3L, 2.5 ng/ml IL6, 5 ng/ml IGF2-containing medium), the cells were transferred to a 6-well MPC-coated culture dish, and cultured at 37°C in a 5% CO ₂ incubator. Thereafter, the culture was continued for 8 to 10 days while changing the half amount of the medium every 3 days. For medium exchange, tilt the entire MPC-coated culture dish about 30 degrees and leave it for about 1 minute, and after confirming that the cell aggregates have completely settled, gently aspirate only the culture supernatant with a half volume pipette, The same amount of fresh medium for producing cell aggregates was added, and the cell aggregates were uniformly dispersed by gently shaking the entire MPC-coated culture dish.

(2) Process B
Transfer the pluripotent stem cell-derived cell aggregate prepared above to a 10 ml centrifuge tube and let stand for 30 seconds to 1 minute to sediment the cell components, then remove the supernatant and remove 3 ml. Brown adipocyte induction medium (5 mg/ml BSA, 1% volume synthetic lipid solution, 1% volume x100 ITS-A, 450 μM MTG, 2 mM L-Glutamine, 5% volume PFHII, IMDM containing 50 μg/ml ascorbic acid) /F12 medium, containing 10 ng/ml BMP7, 5 ng/ml VEGF, 20 ng/ml SCF, 2.5 ng/ml Flt3L, 2.5 ng/ml IL6, 5 ng/ml IGF2), and add at 1100 rpm for 5 minutes. It was centrifuged. This was transferred to a cell culture dish previously reacted with 0.1% porcine gelatin aqueous solution for 10 minutes at room temperature, and cultured at 37° C. in a 5% CO ₂ incubator. Thereafter, the cells were cultured for 1 week while changing the medium every 3 days.

As a result, cells with multivesicular lipid droplets (a large number of yolky, glossy spheres) in the cytoplasm were obtained. In addition, it was confirmed by oil red O staining (a test for making neutral fat develop a red color) that these spherical objects were lipid droplets.

In addition, by RT-PCR, expression of genes characteristic of brown adipocytes (UCP1, PRDM16, PGC1α, Cyt-c, CIDE-A, ELOVL3, PPARα, EVA1, NTRK3) was induced by electrophoresis. confirmed. Furthermore, it was also confirmed that the expression of PPARγ and adiponectin, which are common markers for brown adipocytes and white adipocytes, was induced. On the other hand, it was also confirmed that the expression of resistin, phosphoserine transaminase 1 (PSAT1), and endothelin receptor alpha (EDNRA), which are markers for white adipocytes, was not induced.
Note that resistin is a gene that not only causes insulin resistance but is also associated with canceration and arteriosclerosis. The fact that the expression of resistin was not induced in human pluripotent stem cell-derived brown adipocytes means that not only drug discovery research using human pluripotent stem cell-derived brown adipocytes but also human pluripotent stem cell-derived brown adipocytes This is an extremely important point in considering the safety of cell therapy using saccharin.

Also, electron microscopic observation confirmed the presence of multivesicular lipid droplets, which are the fine structure characteristic of brown adipocytes, and mitochondria that were long and vertically fused. Furthermore, the cristae that developed like a ladder characteristic of brown adipocytes were confirmed inside the mitochondria.

[Reference Example 2] Insulin secretion promoting action on pancreatic beta cells of human ESC-derived BA supernatant or synthetic peptide prepared with "buffer containing only salts and low concentration glucose":
Human ESC-derived BA SUP was prepared as follows. That is, BA was induced to be differentiated from the human ESC maintained in culture on MEF by the method described in Reference Example 1, and the differentiation medium was removed from the mature BA on Day 10, and then Krebs-Ringer-containing 2.8 mM glucose was added. TRIS-Bicarbonate (KRTB) buffer (NaCl: 119 mM, KCl: 4.74 mM, CaCl 2 1.19 mM, MgCl 2 1.19 mM, KH 2 PO 4: 1.19 mM, NaHCO 3: 25 mM, TRIS (pH7.4): 10 mM, Glucose 2.8 mM) was added, and the supernatant was recovered after culturing for 16 hours in a carbon dioxide gas culture device (37°C, 5% CO ₂ ) (hereinafter, the recovered supernatant is referred to as BA-SUP). .. On the other hand, a 96-well plate using the recommended method (Miyazaki et al., Endocrinology 127: 126-132, 1990) was MIN6 cells, a mouse pancreatic beta cell line (distributed by Junichi Miyazaki, specially appointed professor of the University-Industry Collaboration Division of Osaka University (current)) It was cultured in. After washing this with PBS buffer, culturing for 1 hour in KRH buffer containing 2.8 mM glucose (low glucose treatment), removing the cell supernatant, and then adding BA-SUP or control SUP, and culturing with carbon dioxide The cells were cultured in the device (37°C, 5% CO ₂ ) for 2 hours. In the peptide addition experiment, a synthetic peptide was dissolved in the above KRTB instead of BA-SUP and added (control is KRTB). Then, the supernatant of the MIN6 cells was collected, and the insulin concentration was measured by the ELISA method (ultrasensitive mouse insulin measurement kit, Morinaga Institute of Bioscience).

According to the present invention, it becomes possible to provide a short-chain peptide drug having an action of promoting basal insulin secretion, which has never been available until now. When the basal insulin secretion is increased, the blood glucose is lowered at any time, so that the ability to cope with the glucose load accompanying meals (glucose tolerance) is also improved. Furthermore, since a decrease in basal insulin secretion ability is a pathological condition seen in advanced cases of type 2 diabetes, it is possible to exert an effective hypoglycemic effect even in cases where conventional insulin secretagogues have become ineffective as the disease progresses. There is expected. That is, it is possible to provide an effective blood glucose control strategy for a wide range of type 2 diabetes cases. In addition, pancreatic β cells remain in about 1/3 of the cases of type 1 diabetes, and improvement of insulin secretory function of residual pancreatic β cells has attracted attention as a new therapeutic strategy for type 1 diabetes. Therefore, the short-chain peptide drug according to the present invention provides an effective glycemic control strategy even in cases of type 1 diabetes (particularly slowly progressive type 1 diabetes).
Since the synthesis of insulin secretagogue short-chain peptides according to the present invention can be carried out in many countries and regions in the world, it can be applied to the giant plant industry, is practical and has high industrial utility value.

Claims

A peptide containing an amino acid sequence capable of forming an α-helix structure, and the peptide and/or its active form has insulinotropic activity.
The peptide according to claim 1, which consists of several to a dozen or more amino acids.
The peptide according to claim 1 or 2, which contains an amino acid sequence having 5 or more residues capable of forming the helix, following an amino acid having a hydroxyl group and/or a hydrophilic amino acid.
The peptide according to claim 3, wherein the amino acid sequence of 5 or more residues forming the α-helix structure is composed of α-helix-forming amino acids.
The peptide according to claim 4, wherein the α-helix forming amino acid is selected from the group consisting of methionine, alanine, leucine, glutamic acid and lysine.
The peptide according to claim 5, wherein the amino acid sequence of 5 or more residues forming the α-helix structure contains 5 alanines (AAAAA/SEQ ID NO: 3).
The peptide according to any one of claims 3 to 6, wherein the amino acid having a hydroxyl group and/or the hydrophilic amino acid is an amino acid selected from threonine, serine and tyrosine.
The peptide according to any one of claims 3 to 6, wherein the amino acid having a hydroxyl group and/or the hydrophilic amino acid is threonine or serine.
The peptide according to any one of claims 3 to 8, which contains a hydrophilic neutral amino acid following an amino acid sequence of 5 residues or more forming the α-helix structure.
The peptide according to claim 9, wherein the hydrophilic neutral amino acid is glutamine.
Threonine-alanine-alanine-alanine-alanine-alanine-glutamine (TAAAAAQ/SEQ ID NO:4), serine-alanine-alanine-alanine-alanine-alanine-glutamine (SAAAAAQ/SEQ ID NO:12), or threonine-alanine-alanine-alanine A peptide comprising an amino acid sequence of -alanine-alanine-glutamine-glycine-glycine (TAAAAAQGG/SEQ ID NO: 11), wherein the peptide and/or its active form has insulinotropic activity.
A peptide containing the amino acid sequence of threonine-lysine-glutamic acid-aspartic acid-glycine-arginine-glutamine (TKEDGRQ/SEQ ID NO: 1), and the peptide and/or its active form have insulinotropic activity.
The peptide according to claim 12, which is a cyclic peptide in which amino acids at the amino terminal and the carboxyl terminal of threonine-lysine-glutamic acid-aspartic acid-glycine-arginine-glutamine (TKEDGRQ/SEQ ID NO: 1) form a lactam ring by a peptide bond. ..
The peptide according to any one of claims 1 to 13, wherein the active form is a peptide that has been treated in the step of heating and cooling under acidic conditions and/or the step of dissolving in a buffer solution and freeze-drying.
The peptide according to any one of claims 1 to 14, wherein another compound is separably linked.
A composition comprising the peptide according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier.
The composition according to claim 16, which is a pharmaceutical composition.
The composition according to claim 16 or 17, which is used for promoting insulin secretion and/or promoting glucose metabolism.
The composition according to any one of claims 16 to 18, which is used for treating or preventing diabetes or prediabetes.
A method for treating or preventing diabetes or pre-diabetes, which comprises the step of administering the peptide according to any one of claims 1 to 15.