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JP6841404B2 - peptide - Google Patents

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Publication number
JP6841404B2
JP6841404B2 JP2016163465A JP2016163465A JP6841404B2 JP 6841404 B2 JP6841404 B2 JP 6841404B2 JP 2016163465 A JP2016163465 A JP 2016163465A JP 2016163465 A JP2016163465 A JP 2016163465A JP 6841404 B2 JP6841404 B2 JP 6841404B2
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peptide
colorectal cancer
present
inflammatory bowel
bowel disease
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JP2018030807A (en
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裕二 内藤
裕二 内藤
内山 和彦
和彦 内山
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KYOTO PREFECTURAL UNIVERSITY OF MEDICINE
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Description

本発明は、ペプチドに関する。また、本発明は、炎症性腸疾患の予防又は治療剤、大腸がんの治療剤に関する。 The present invention relates to peptides. The present invention also relates to a prophylactic or therapeutic agent for inflammatory bowel disease and a therapeutic agent for colorectal cancer.

潰瘍性大腸炎やクローン病を代表とする炎症性腸疾患(Inflammatory bowel disease:以下IBD)は原因不明の慢性再燃性消化管炎症疾患である。近年、IBDの病態における免疫機構や腸内細菌の関与などが明らかとなっており、それらを標的とする様々な治療が提唱されている。その治療効果の目標は臨床的寛解ではなく、内視鏡的に確認する「粘膜治癒」が重要であり粘膜治癒が得られれば、臨床的寛解の維持、手術率の低下につながることが示されている。IBDに対しては近年多くの治療薬が臨床応用されており、治療成績も向上しているが、同時に治療抵抗性で手術を余儀なくされる症例も依然として多く存在している。また、炎症性腸疾患の患者数は年々増加の一途をたどっており、内科治療に抵抗性で手術にいたる症例も増加している。したがって、既存の内科治療に加え、粘膜治癒を促すことによる長期寛解維持効果を発揮する治療の開発が急務である。 Inflammatory bowel disease (IBD), represented by ulcerative colitis and Crohn's disease, is a chronic relapsed gastrointestinal inflammatory disease of unknown cause. In recent years, the immune system and the involvement of intestinal bacteria in the pathophysiology of IBD have been clarified, and various treatments targeting them have been proposed. It has been shown that the goal of the therapeutic effect is not clinical remission, but endoscopically confirmed "mucosal healing" is important, and if mucosal healing is obtained, it will lead to maintenance of clinical remission and reduction of surgical rate. ing. In recent years, many therapeutic agents have been clinically applied to IBD, and the treatment results have improved, but at the same time, there are still many cases in which surgery is unavoidable due to treatment resistance. In addition, the number of patients with inflammatory bowel disease is steadily increasing year by year, and the number of patients who are resistant to medical treatment and undergo surgery is also increasing. Therefore, in addition to the existing medical treatments, there is an urgent need to develop a treatment that exerts a long-term remission maintenance effect by promoting mucosal healing.

一方、進行した大腸癌に対しては分子標的薬の開発などで治療効果は向上しているが、いまだに死亡率は増加傾向にある。進行大腸癌に対する内科治療は、複数の治療を繰り返して実施する過程で治療抵抗性を示し、治療の継続が不可能になってしまう。したがって、既存の抗癌剤治療に加え、別のメカニズムによる大腸癌化学療法の開発が急務である。 On the other hand, for advanced colorectal cancer, the therapeutic effect has been improved by the development of molecular-targeted drugs, but the mortality rate is still on the rise. Medical treatment for advanced colorectal cancer shows resistance to treatment in the process of repeatedly performing a plurality of treatments, making it impossible to continue the treatment. Therefore, there is an urgent need to develop colorectal cancer chemotherapy by another mechanism in addition to the existing anticancer drug treatment.

本発明は、炎症性腸疾患、大腸がんの治療薬を提供することを目的とする。 An object of the present invention is to provide a therapeutic agent for inflammatory bowel disease and colorectal cancer.

本発明者は、Wnt5aの部分ペプチドが炎症性腸疾患、大腸がんに有効であることを見出した。 The present inventor has found that a partial peptide of Wnt5a is effective for inflammatory bowel disease and colorectal cancer.

本発明は、以下のペプチド、炎症性腸疾患の予防又は治療剤、大腸がん治療剤を提供するものである。
項1. 下記のアミノ酸配列(I)
QLADFRKVGDALKEKYDSAAAMR (I)
を含む、ペプチド。
項2. 下記のアミノ酸配列(Ia)
R−QLADFRKVGDALKEKYDSAAAMR (Ia)
(式中、Rは下記の13種
GVSGSCSLKTCWL、
VSGSCSLKTCWL、
SGSCSLKTCWL、
GSCSLKTCWL、
SCSLKTCWL、
CSLKTCWL、
SLKTCWL、
LKTCWL、
KTCWL、
TCWL、
CWL、
WL、又は
L
のいずれかである)
を含む、項1に記載のペプチド。
項3. GVSGSCSLKTCWLQLADFRKVGDALKEKYDSAAAMRで表される、項1又は2に記載のペプチド
項4. 項1,2又は3のペプチドを有効成分として含む炎症性腸疾患の予防又は治療剤。
項5. 項1,2又は3のペプチドを有効成分として含む大腸がん治療剤。
The present invention provides the following peptides, prophylactic or therapeutic agents for inflammatory bowel disease, and therapeutic agents for colorectal cancer.
Item 1. The following amino acid sequence (I)
QLADFRKVGDALKEKYDSAAAMR (I)
Including peptides.
Item 2. The following amino acid sequence (Ia)
R 1- QLADFRKVGDALKEKYDSAAAMR (Ia)
(In the formula, R 1 is the following 13 types
GVSGSCSLKTCWL,
VSGSCSLKTCWL,
SGSCSLKTCWL,
GSCSLKTCWL,
SCSLKTCWL,
CSLKTCWL,
SLKTCWL,
LKTCWL,
KTCWL,
TCWL,
CWL,
WL or
L
Is one of)
Item 2. The peptide according to Item 1.
Item 3. Item 4. Peptide item 1 or 2 represented by GVSGSCSLKTCWLQLADFRKVGDALKEKYDSAAAMR. A prophylactic or therapeutic agent for inflammatory bowel disease containing the peptides of Items 1, 2 or 3 as an active ingredient.
Item 5. A therapeutic agent for colorectal cancer containing the peptides of Items 1, 2 or 3 as an active ingredient.

本発明のペプチドは、大腸粘膜細胞の増殖を促進し、大腸粘膜の創傷治癒に有効であり、炎症性腸疾患の予防又は治療剤として有用である。 The peptide of the present invention promotes the proliferation of large intestine mucosal cells, is effective for wound healing of the large intestine mucosa, and is useful as a preventive or therapeutic agent for inflammatory bowel disease.

また、本発明のペプチドは、大腸がん細胞の増殖を抑制し、大腸がんの治療剤として有用である。 In addition, the peptide of the present invention suppresses the growth of colorectal cancer cells and is useful as a therapeutic agent for colorectal cancer.

大腸がんの発症は、炎症性腸疾患により促進すると言われており、本発明のペプチドは、潰瘍性大腸炎の治癒促進により、大腸がんを予防でき、かつ大腸がんの治療効果を有し、強力な大腸保護作用を有する。 It is said that the onset of colorectal cancer is promoted by inflammatory bowel disease, and the peptide of the present invention can prevent colorectal cancer by promoting the healing of ulcerative colitis and has a therapeutic effect on colorectal cancer. It has a strong large intestine protective effect.

本発明のペプチドは、大腸粘膜細胞の増殖を促進し、かつ、大腸がんの増殖を抑制するという、相反する作用を併せ持つという格別の効果を奏するものである。 The peptide of the present invention has a special effect of promoting the growth of colon mucosal cells and suppressing the growth of colon cancer, which have contradictory actions.

本発明のペプチドとコントロールによる大腸がんの発育抑制の比較。Comparison of growth suppression of colorectal cancer by the peptide and control of the present invention.

本発明のペプチドは、ヒトWnt5a由来であり、23アミノ酸のペプチド(I)を必須配列として含み、さらにN末端側にR基で表される1〜13個のアミノ酸もしくはペプチドを連結したものであってもよい。これらの23〜36個のアミノ酸からなるペプチドは、共通して式(I)の23個のアミノ酸配列を保有している。 The peptide of the present invention is derived from human Wnt5a, contains a peptide (I) of 23 amino acids as an essential sequence, and further has 1 to 13 amino acids or peptides represented by R groups linked to the N-terminal side. You may. These peptides consisting of 23 to 36 amino acids commonly carry the 23 amino acid sequences of formula (I).

23個と36個のアミノ酸配列の配列番号は、以下の通りである。
配列番号1: QLADFRKVGDALKEKYDSAAAMR
配列番号2: GVSGSCSLKTCWLQLADFRKVGDALKEKYDSAAAMR
ヒト及びマウスのWnt5aの塩基配列及びアミノ酸配列は公知であり、データベースに下記のアクセッション番号で登録されている。
マウスのWnt5a : NM_009524
ヒトのWnt5a : NM_003392
ヒトとマウスのWnt5aの配列番号1,2に対応するアミノ酸配列は同一であり、本発明のペプチドは、ヒト及びマウスを含む哺乳動物に対し、炎症性腸疾患の予防又は治療剤、並びに、大腸がん治療剤として有用である。
The SEQ ID NOs of the 23 and 36 amino acid sequences are as follows.
SEQ ID NO: 1: QLADFRKVGDALKEKYDSAAAMR
SEQ ID NO: 2: GVSGSCSLKTCWLQLADFRKVGDALKEKYDSAAAMR
The nucleotide and amino acid sequences of human and mouse Wnt5a are known and are registered in the database with the following accession numbers.
Mouse Wnt5a: NM_009524
Human Wnt5a: NM_003392
The amino acid sequences corresponding to SEQ ID NOs: 1 and 2 of Wnt5a in humans and mice are the same, and the peptides of the present invention can be used as a prophylactic or therapeutic agent for inflammatory bowel disease in mammals including humans and mice, and the large intestine. It is useful as a cancer therapeutic agent.

配列番号1で示される23アミノ酸のペプチド(I)(QLADFRKVGDALKEKYDSAAAMR)は、エピトープを含んでおり、腸粘膜創傷治癒作用及び大腸がんの増殖抑制作用に必須の部分である。また、本発明のペプチドは水溶性である。 The 23-amino acid peptide (I) (QLADFRKVGDALKEKYDSAAAMR) shown in SEQ ID NO: 1 contains an epitope and is an essential part for intestinal mucosal wound healing action and colon cancer growth inhibitory action. Moreover, the peptide of the present invention is water-soluble.

本発明の式(I)又は(Ia)のペプチドは、固相合成により製造してもよく、これらのペプチドをコードするDNAを大腸菌などの適当な宿主に導入して遺伝し工学的な手法により製造してもよい。また、式(I)又は(Ia)のペプチドのN末端は、メチル化もしくはアセチル化されていてもよく、C末端はメチルエステル、エチルエステル、プロピルエステル、ブチルエステルなどの炭素数1〜4のアルキル基とエステル化されていてもよく、アミド化(CONH2)されていてもよい。 The peptides of the formula (I) or (Ia) of the present invention may be produced by solid-phase synthesis, and the DNA encoding these peptides is introduced into an appropriate host such as Escherichia coli and inherited by an engineering method. It may be manufactured. Further, the N-terminal of the peptide of the formula (I) or (Ia) may be methylated or acetylated, and the C-terminal may have 1 to 4 carbon atoms such as methyl ester, ethyl ester, propyl ester and butyl ester. It may be esterified with an alkyl group or may be amidated (CONH2).

また、本発明のペプチドは、塩酸、硫酸、硝酸、メタンスルホン酸、トルエンスルホン酸などの有機酸もしくは無機酸と酸付加塩を形成してもよく、ナトリウム、カリウム、リチウムなどのアルカリ金属、カルシウム、マグネシウムなどのアルカリ土類金属と塩基塩を形成してもよい。 Further, the peptide of the present invention may form an acid addition salt with an organic acid or an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, methanesulfonic acid and toluenesulfonic acid, and an alkali metal such as sodium, potassium and lithium, and calcium. , May form a base salt with an alkaline earth metal such as magnesium.

本発明のペプチドは、粘膜細胞の増殖を促進することで炎症性腸疾患の粘膜創傷治癒を促進することができ、潰瘍性大腸炎、クローン病などの炎症性腸疾患の予防又は治療に有効である。 The peptide of the present invention can promote mucosal wound healing of inflammatory bowel disease by promoting the proliferation of mucosal cells, and is effective for prevention or treatment of inflammatory bowel disease such as ulcerative colitis and Crohn's disease. is there.

また、本発明のペプチドは、大腸がんの増殖を抑制できるので、大腸がんの治療に有効である。 Moreover, since the peptide of the present invention can suppress the growth of colorectal cancer, it is effective for the treatment of colorectal cancer.

本発明のペプチドは、錠剤、カプセル剤、顆粒剤、液剤などの経口剤により投与した場合、腸管粘膜から吸収されることにより大腸に作用してもよく、吸収されなかったペプチドは大腸表面の粘膜もしくはがんに直接作用して効果を発現してもよい。或いは、注射剤、坐剤などの非経口剤により投与してもよい。本発明のペプチドを大腸に直接作用させる場合、腸溶性コーティングした錠剤、顆粒剤などの経口剤として投与すると、腸内で分解されずに大腸まで到達するので、好ましい。 When the peptide of the present invention is administered as an oral preparation such as tablets, capsules, granules, and liquids, it may act on the large intestine by being absorbed from the intestinal mucosa, and the unabsorbed peptide may act on the large intestine. Alternatively, it may act directly on the cancer to exert its effect. Alternatively, it may be administered by a parenteral agent such as an injection or a suppository. When the peptide of the present invention is allowed to act directly on the large intestine, it is preferable to administer it as an oral preparation such as an enteric coated tablet or granule because it reaches the large intestine without being decomposed in the intestine.

本発明のペプチドの潰瘍性大腸炎、大腸がんに対する有効投与量は、いずれも成人1日当たり0.01mg〜10g程度である。 The effective dose of the peptide of the present invention for ulcerative colitis and colorectal cancer is about 0.01 mg to 10 g per day for adults.

以下に実施例を示すが、本発明はこの実施例だけに限定されるものではない。 Examples are shown below, but the present invention is not limited to these examples.

本実施例で使用する配列番号2の36アミノ酸のペプチドの製造は、Invitragenに依頼し、質量分析の結果、理論値3906.53、実測値3906.40であった。
配列番号2:GVSGSCSLKTCWLQLADFRKVGDALKEKYDSAAAMR
The 36-amino acid peptide of SEQ ID NO: 2 used in this example was produced by Invitragen, and as a result of mass spectrometry, the theoretical value was 3906.53 and the measured value was 3906.40.
SEQ ID NO: 2: GVSGSCSLKTCWLQLADFRKVGDALKEKYDSAAAMR

実施例1:マウス大腸癌皮下移植モデル
6週齢BALB-Cマウスの背側に培養したColon26(マウス大腸癌細胞株)を皮下移植(皮下注)し、同日より投与を開始した。PBS(200μl)連日腹腔内投与群をコントロール群、PBSに溶解した36アミノ酸の配列番号1のWnt5a ペプチド(20μg/200μl)の連日腹腔内投与群を治療群とした(各群5匹)。その後経時的に両群の腫瘍径を測定し、比較検討した。結果を図1に示す。
Example 1: Mouse colorectal cancer subcutaneous transplant model
Colon26 (mouse colon cancer cell line) cultured on the dorsal side of 6-week-old BALB-C mice was subcutaneously transplanted (subcutaneous injection), and administration was started on the same day. The daily intraperitoneal administration group of PBS (200 μl) was used as the control group, and the daily intraperitoneal administration group of Wnt5a peptide (20 μg / 200 μl) of SEQ ID NO: 1 of 36 amino acids dissolved in PBS was used as the treatment group (5 animals in each group). After that, the tumor diameters of both groups were measured over time and compared. The results are shown in FIG.

実施例2:Wnt5a 36AA peptideの創傷治癒効果
YAMC細胞(Young Adult Mouse Colonic epithelial cell)におけるwound healing assay(YAMC細胞を用いたwound healing assayに関する参考論文:Uchiyama K, et al., BBRC 391(2010) 1122-1126)において、配列番号1の36アミノ酸のペプチドの濃度が10ng/mlでYAMC細胞の創傷治癒を亢進した。
Example 2: Wound healing effect of Wnt5a 36AA peptide
In the wound healing assay in YAMC cells (Young Adult Mouse Colonic epithelial cells) (reference paper on wound healing assay using YAMC cells: Uchiyama K, et al., BBRC 391 (2010) 1122-1126), 36 of SEQ ID NO: 1. The amino acid peptide concentration of 10 ng / ml enhanced wound healing of YAMC cells.

Claims (3)

アミノ酸配列GVSGSCSLKTCWLQLADFRKVGDALKEKYDSAAAMRで表される、ペプチド Represented by the amino acid sequence JibuiesujiesushiesuerukeitishiWLQLADFRKVGDALKEKYDSAAAMR, peptide 請求項1に記載のペプチドを有効成分として含む炎症性腸疾患の予防又は治療剤。 Prophylactic or therapeutic agent for inflammatory bowel disease comprising as an active ingredient the peptide of claim 1. 請求項1に記載のペプチドを有効成分として含む大腸がん治療剤。
Colorectal cancer therapeutic agent comprising as an active ingredient the peptide of claim 1.
JP2016163465A 2016-08-24 2016-08-24 peptide Active JP6841404B2 (en)

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