JP6306825B2 - Saccharification product formation inhibitor - Google Patents
Saccharification product formation inhibitor Download PDFInfo
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- JP6306825B2 JP6306825B2 JP2013091428A JP2013091428A JP6306825B2 JP 6306825 B2 JP6306825 B2 JP 6306825B2 JP 2013091428 A JP2013091428 A JP 2013091428A JP 2013091428 A JP2013091428 A JP 2013091428A JP 6306825 B2 JP6306825 B2 JP 6306825B2
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Description
本発明は、ソルビトールを有効成分とする終末糖化産物生成抑制剤、ならびにソルビトールを有効成分として含む、糖尿病もしくは糖尿病合併症の予防および／または治療のための医薬組成物に関する。 The present invention relates to a glycation end product production inhibitor containing sorbitol as an active ingredient, and a pharmaceutical composition for the prevention and / or treatment of diabetes or diabetic complications containing sorbitol as an active ingredient.
アミノ酸と還元糖の混合物を加熱すると褐変する現象は、一般にメイラード反応と呼ばれ、食品分野では、食品の加熱処理や貯蔵中に生じる現象として知られている。メイラード反応は、生体内においても発生しており、１９６８年にはグリコシルヘモグロビン（ＨｂＡ１ｃ）が生体内で同定されたことにより、糖尿病や老化の進行に伴い蛋白質の糖化反応が進行することが明らかにされた。そして、近年では、蛋白質の糖化反応における終末糖化産物（Ａｄｖａｎｃｅｄ ｇｌｙｃａｔｉｏｎ ｅｎｄ ｐｒｏｄｕｃｔｓ、以下「ＡＧＥｓ」とも称する）が糖尿病合併症や動脈硬化といった生活習慣病の発症や老化の進行に関与することが報告されている（非特許文献１〜３）。ＡＧＥｓは特定の物質を指すものではなく、その全容は未だ解明されていないが、蛍光性や架橋構造の有無によって、ペントシジン、ピロピリジン、ピラリン等、様々な物質の存在が確認されている。 The phenomenon of browning when a mixture of amino acid and reducing sugar is heated is generally called the Maillard reaction, and is known in the food field as a phenomenon that occurs during heat treatment and storage of food. The Maillard reaction also occurs in vivo, and it was clear that glycosylation hemoglobin (HbA1c) was identified in vivo in 1968, leading to the progress of protein glycation with the progress of diabetes and aging. It was done. In recent years, advanced glycation end products (hereinafter also referred to as “AGEs”) in protein glycation reactions have been reported to be involved in the onset of lifestyle-related diseases such as diabetic complications and arteriosclerosis and the progression of aging. (Non-Patent Documents 1 to 3). AGEs do not refer to specific substances, and the whole of them has not yet been elucidated, but the presence of various substances such as pentosidine, pyropyridine, and pyralin has been confirmed depending on the presence of fluorescence and cross-linking structure.
生体内における蛋白質糖化反応の詳細は明らかとなっていないが、蛋白質に存在するアミノ基と還元糖に存在するアルデヒド基が反応し、シッフ塩基を形成した後、安定なアマドリ化合物が生成され、さらに長期の反応を経て、該アマドリ化合物から種々のＡＧＥｓが生成されると考えられている。アマドリ化合物からのＡＧＥｓの生成過程において、３−デオキシグルコソン（以下、「３−ＤＧ」とも称する）、グリオキサール、メチルグリオキサール等のジカルボニル化合物が中間体として生成することが知られている。これら中間体の中でも３−ＤＧはアミノ基との反応性が極めて高く、且つ最も生成量が多いことから、重要な中間体と考えられている。また、糖尿病患者においては、血清３−ＤＧ値が高いほど合併症の進行が速いとの報告もある（非特許文献４）。したがって、ＡＧＥｓの生成抑制のために３−ＤＧの生成を抑制することが特に注目されている。 Details of the protein saccharification reaction in vivo have not been clarified, but after the amino group present in the protein reacts with the aldehyde group present in the reducing sugar to form a Schiff base, a stable Amadori compound is generated, It is considered that various AGEs are generated from the Amadori compound through a long-term reaction. It is known that dicarbonyl compounds such as 3-deoxyglucosone (hereinafter also referred to as “3-DG”), glyoxal, and methylglyoxal are generated as intermediates in the process of generating AGEs from Amadori compounds. Among these intermediates, 3-DG is considered to be an important intermediate because it has extremely high reactivity with amino groups and has the largest production amount. There is also a report that in diabetic patients, the higher the serum 3-DG level, the faster the complications progress (Non-Patent Document 4). Therefore, it has been particularly noticed to suppress the generation of 3-DG in order to suppress the generation of AGEs.
３−ＤＧの生成抑制に着目した技術としては、３−ＤＧ等の初期グリコシル化産物のカルボニル基と反応して標的蛋白質の後期グリコシル化を抑制する、アミノグアニジン等の化合物を含むメイラード反応阻害剤が開示されている（特許文献１）。アミノグアニジンは当該技術分野における医薬品の開発において最も研究されている物質であるが、肝障害等の副作用を有することが確認されており、そのため、実用化には至っていない。 As a technique focused on the suppression of 3-DG production, a Maillard reaction inhibitor containing a compound such as aminoguanidine that reacts with a carbonyl group of an early glycosylation product such as 3-DG to suppress late glycosylation of a target protein Is disclosed (Patent Document 1). Aminoguanidine is the most studied substance in the development of pharmaceuticals in this technical field, but it has been confirmed that it has side effects such as liver damage and has not been put into practical use.
その他にも、ガンビールノキやシラカバ等の植物抽出物により３−ＤＧの生成反応を阻害するメイラード反応阻害剤（特許文献２）、グリオキサラーゼＩ活性を備える酵素およびカルボニル化合物還元剤を有効成分とする３−ＤＧの消去によるカルボニルストレス改善剤（特許文献３）、パラバン酸誘導体を有効成分とする３−ＤＧ生成阻害剤（特許文献４）、スルフィド化合物を有効成分とするデオキシグルコソン生成抑制剤（特許文献５）などが知られている。しかし、これらはいずれも有効性や安全性の点に課題を残すものであった。 In addition, a Maillard reaction inhibitor (Patent Document 2) that inhibits the 3-DG production reaction by plant extracts such as Gambirium and birch, and an enzyme having glyoxalase I activity and a carbonyl compound reducing agent as active ingredients Carbonyl stress ameliorating agent by elimination of 3-DG (Patent Document 3), 3-DG production inhibitor having a parabanic acid derivative as an active ingredient (Patent Document 4), Deoxyglucosone production inhibitor having a sulfide compound as an active ingredient ( Patent Document 5) is known. However, these all left problems in terms of effectiveness and safety.
そこで、種々の疾患の発症や悪化に関与するＡＧＥｓの生成抑制のために利用することができ、かつ、重篤な副作用を有さない安全性の高い薬剤が求められている。 Therefore, a highly safe drug that can be used for suppressing the production of AGEs involved in the onset and worsening of various diseases and that has no serious side effects is desired.
本発明は、種々の生活習慣病の要因物質と考えられているＡＧＥｓの生成を抑制することができる物質を含み、生活習慣病、特に糖尿病もしくは糖尿病合併症の予防および／または治療に有用であり且つ副作用の少ないＡＧＥｓ生成抑制剤を提供することを目的とする。 The present invention includes substances that can suppress the generation of AGEs that are considered to be a causative substance of various lifestyle-related diseases, and is useful for the prevention and / or treatment of lifestyle-related diseases, particularly diabetes or diabetic complications. And it aims at providing the AGE production | generation inhibitor with few side effects.
本発明者らは、鋭意検討の結果、ソルビトールがＡＧＥｓの生成およびＡＧＥｓ生成反応の中間体である３−ＤＧの生成を抑制する作用を有することを見出し、本発明を完成させた。 As a result of intensive studies, the present inventors have found that sorbitol has an action of suppressing the production of AGEs and the production of 3-DG, which is an intermediate of the AGEs production reaction, and completed the present invention.
すなわち本発明は、ソルビトールを有効成分として含有する終末糖化産物生成抑制剤（以下、「本発明のＡＧＥｓ生成抑制剤」とも称する）を提供する。 That is, the present invention provides a terminal glycation product production inhibitor (hereinafter, also referred to as “AGEs production inhibitor of the present invention”) containing sorbitol as an active ingredient.
本発明はまた、ソルビトールを有効成分として含有する３−デオキシグルコソン生成抑制剤（以下、「本発明の３−ＤＧ生成抑制剤」とも称する）を提供する。 The present invention also provides a 3-deoxyglucosone production inhibitor (hereinafter, also referred to as “the 3-DG production inhibitor of the present invention”) containing sorbitol as an active ingredient.
本発明はさらに、ソルビトールを有効成分として含む、糖尿病もしくは糖尿病合併症の予防および／または治療のための医薬組成物（以下、「本発明の糖尿病予防／治療用医薬組成物」とも称する）を提供する。 The present invention further provides a pharmaceutical composition for preventing and / or treating diabetes or diabetic complications (hereinafter also referred to as “the pharmaceutical composition for preventing / treating diabetes” of the present invention) comprising sorbitol as an active ingredient. To do.
本発明はさらに、非ヒト動物における糖尿病もしくは糖尿病合併症の予防および／または治療のための、ソルビトールを含む非ヒト動物用医薬組成物、該医薬組成物または本発明のＡＧＥｓ生成抑制剤もしくは３−ＤＧ生成抑制剤を非ヒト動物に投与することを含む、非ヒト動物における糖尿病もしくは糖尿病合併症を予防および／または治療する方法、本発明のＡＧＥｓ生成抑制剤または３−ＤＧ生成抑制剤を飲食品に配合または添加することを特徴とする機能性飲食品の製造方法、ならびにサプリメントの製造のための本発明のＡＧＥｓ生成抑制剤または３−ＤＧ生成抑制剤の使用を提供する。 The present invention further relates to a pharmaceutical composition for non-human animals containing sorbitol for the prevention and / or treatment of diabetes or diabetic complications in a non-human animal, the pharmaceutical composition or the AGEs production inhibitor of the present invention or 3- A method for preventing and / or treating diabetes or diabetic complications in a non-human animal, comprising administering the DG production inhibitor to a non-human animal, and a food or drink with the AGEs production inhibitor or the 3-DG production inhibitor of the present invention The present invention provides a method for producing a functional food or drink characterized by being added to or added to the above, and the use of the AGEs production inhibitor or 3-DG production inhibitor of the present invention for the production of supplements.
ＡＧＥｓ、およびＡＧＥｓの生成反応における主要な中間体である３−ＤＧは、種々の生活習慣病との関連が示唆されている物質であり、特に、糖尿病およびその合併症の発症および／または進行に深く関与するものである。したがって、本発明者らによりＡＧＥｓおよび３−ＤＧの生成を抑制する作用が見出されたソルビトールは、糖尿病もしくは糖尿病合併症の予防および／または治療のための、副作用の少ない医薬組成物の有効成分として有用である。 AGEs and 3-DG, which is the main intermediate in the AGEs production reaction, are substances that have been suggested to be associated with various lifestyle-related diseases, particularly in the onset and / or progression of diabetes and its complications. It is deeply involved. Therefore, sorbitol, which has been found by the present inventors to suppress the production of AGEs and 3-DG, is an active ingredient of a pharmaceutical composition with few side effects for the prevention and / or treatment of diabetes or diabetic complications. Useful as.
本発明のＡＧＥｓ生成抑制剤、３−ＤＧ生成抑制剤および糖尿病予防／治療用医薬組成物の有効成分であるソルビトールは、公知の製造方法により製造されたものであってもよく、市販のソルビトール製品であってもよい。ソルビトールの製造方法としては、例えば、ぶどう糖液を触媒存在下で高圧接触還元することで水素添加し、精製、濃縮する方法が例示される。市販のソルビトール製品としては、上野製薬株式会社製のソルパート、ソルビトールウエノ、粉末ソルビトールウエノを例示することができる。 Sorbitol, which is an active ingredient of the AGEs production inhibitor, 3-DG production inhibitor and diabetes prevention / treatment pharmaceutical composition of the present invention, may be produced by a known production method, and is a commercially available sorbitol product. It may be. Examples of the method for producing sorbitol include a method in which a glucose solution is hydrogenated by high-pressure catalytic reduction in the presence of a catalyst, and purified and concentrated. Examples of commercially available sorbitol products include sorbet, sorbitol ueno and powder sorbitol ueno manufactured by Ueno Pharmaceutical Co., Ltd.
本発明において使用するソルビトールの性状は、液体状または固体状のいずれであってもよく、例えば、液体状ソルビトールから公知の粉末化方法または結晶化方法により製造された、粉末状ソルビトールまたは結晶ソルビトールであってもよい。使用するソルビトールの性状は、目的とする剤形に応じて適宜選択することができる。 The property of sorbitol used in the present invention may be either liquid or solid, for example, powdered sorbitol or crystalline sorbitol produced from liquid sorbitol by a known powdering method or crystallization method. There may be. The properties of the sorbitol to be used can be appropriately selected according to the intended dosage form.
本発明において使用するソルビトールの純度としては、液体状ソルビトールであれば純度４５％以上のものが好ましい。また、粉末状ソルビトールであれば純度８０％以上のものが好ましく、純度８５％以上のものがより好ましく、純度８８％以上のものがさらに好ましい。 The purity of sorbitol used in the present invention is preferably 45% or more if it is liquid sorbitol. Moreover, if it is powdered sorbitol, a thing with a purity of 80% or more is preferable, a thing with a purity of 85% or more is more preferable, and a thing with a purity of 88% or more is further more preferable.
本発明のＡＧＥｓ生成抑制剤および３−ＤＧ生成抑制剤はいずれも、有効成分としてソルビトールを含有するものであればよく、ソルビトールによるＡＧＥｓ生成抑制効果または３−ＤＧ生成抑制効果を妨げない限り、さらに賦形剤等を含むものであってもよい。したがって、本発明のＡＧＥｓ生成抑制剤または３−ＤＧ生成抑制剤中のソルビトールの割合は特に限定されない。例えば、本発明のＡＧＥｓ生成抑制剤または３−ＤＧ生成抑制剤は、ソルビトールを４０重量％以上、４５重量％以上、５０重量％以上、６０重量％以上、７０重量％以上、８０重量％以上、８５重量％以上、９０重量％以上、９５重量％以上または９８重量％以上含むものであり得る。あるいは、本発明のＡＧＥｓ生成抑制剤または３−ＤＧ生成抑制剤は、ソルビトールのみからなるものであってもよい。一つの態様において、ソルビトールは、本発明のＡＧＥｓ生成抑制剤または３−ＤＧ生成抑制剤における単独の有効成分である。 Any of the AGEs production inhibitor and the 3-DG production inhibitor of the present invention may be those containing sorbitol as an active ingredient, and unless the AGEs production inhibitory effect or the 3-DG production inhibitory effect by sorbitol is hindered. It may contain an excipient or the like. Therefore, the ratio of sorbitol in the AGEs production | generation inhibitor or 3-DG production | generation inhibitor of this invention is not specifically limited. For example, the AGEs production inhibitor or 3-DG production inhibitor of the present invention comprises sorbitol at 40% by weight, 45% by weight, 50% by weight, 60% by weight, 70% by weight, 80% by weight, It may contain 85% or more, 90% or more, 95% or more, or 98% or more. Or the AGEs production | generation inhibitor or 3-DG production | generation inhibitor of this invention may consist only of sorbitol. In one embodiment, sorbitol is the sole active ingredient in the AGEs production inhibitor or 3-DG production inhibitor of the present invention.
本発明のＡＧＥｓ生成抑制剤は、ペントシジン、クロスリン、ピロピリジン、グリオキサール由来リジンダイマー（ＧＯＬＤ）、メチルグリオキサール由来リジンダイマー（ＭＯＬＤ）等の蛍光性ＡＧＥｓおよびピラリン、カルボキシメチルリジン（ＣＭＬ）、３−デオキシグルコソン由来リジンダイマー（ＤＯＬＤ）、イミダゾロン化合物等の非蛍光性ＡＧＥｓを含む種々のＡＧＥｓの生成を抑制することができる。一つの態様において、本発明のＡＧＥｓ生成抑制剤は、蛍光性ＡＧＥｓであるペントシジンの生成抑制のために用いられるものである。別の態様において、本発明のＡＧＥｓ生成抑制剤は、非蛍光性ＡＧＥｓであるカルボキシメチルリジン（ＣＭＬ）の生成抑制のために用いられるものである。 The AGEs production inhibitor of the present invention includes fluorescent AGEs such as pentosidine, croslin, pyropyridine, glyoxal-derived lysine dimer (GOLD), methylglyoxal-derived lysine dimer (MOLD), pyralin, carboxymethyllysine (CML), 3-deoxyglucose. Generation of various AGEs including non-fluorescent AGEs such as son-derived lysine dimer (DOLD) and imidazolone compounds can be suppressed. In one embodiment, the AGEs production inhibitor of the present invention is used for inhibiting the production of pentosidine, which is a fluorescent AGE. In another embodiment, the AGEs production inhibitor of the present invention is used for inhibiting the production of carboxymethyllysine (CML), which is a non-fluorescent AGE.
本発明の３−ＤＧ生成抑制剤は、３−ＤＧの生成抑制を介して、ＡＧＥｓのうち主として３−ＤＧから生じるピロピリジン、ピラリン、ＤＯＬＤ、イミダゾロン、３−ＤＧイミダゾロン等の生成を特に効果的に抑制することができる。 The 3-DG production inhibitor of the present invention particularly effectively produces pyropyridine, pyralin, DOLD, imidazolone, 3-DG imidazolone, and the like mainly produced from 3-DG among AGEs through the inhibition of 3-DG production. Can be suppressed.
本発明の糖尿病予防／治療用医薬組成物は、本発明のＡＧＥｓ生成抑制剤および３−ＤＧ生成抑制剤と同様、有効成分としてソルビトールを含有するものである。 The pharmaceutical composition for preventing / treating diabetes of the present invention contains sorbitol as an active ingredient, like the AGEs production inhibitor and 3-DG production inhibitor of the present invention.
本発明の糖尿病予防／治療用医薬組成物中に有効成分として含有させるソルビトールの割合は、目的とする剤形等に応じて適宜設定することができるが、通常、医薬組成物全量に対し１〜９８重量％程度であればよく、２〜９５重量％程度であることが好ましく、３〜９０重量％程度であることがより好ましい。一つの態様において、ソルビトールは、本発明の糖尿病予防／治療用医薬組成物における単独の有効成分である。 The proportion of sorbitol contained as an active ingredient in the pharmaceutical composition for prevention / treatment of diabetes of the present invention can be appropriately set according to the intended dosage form and the like, but is usually 1 to the total amount of the pharmaceutical composition. It may be about 98% by weight, preferably about 2 to 95% by weight, and more preferably about 3 to 90% by weight. In one embodiment, sorbitol is the sole active ingredient in the pharmaceutical composition for preventing / treating diabetes according to the present invention.
本発明の好ましい態様において、糖尿病もしくは糖尿病合併症の予防および／または治療は、３−デオキシグルコソンまたは終末糖化産物の生成抑制を介するものである。 In a preferred embodiment of the present invention, the prevention and / or treatment of diabetes or diabetic complications is via suppression of the production of 3-deoxyglucosone or terminal glycation products.
本明細書において、糖尿病もしくは糖尿病合併症の「治療」には、既に糖尿病もしくは糖尿病合併症に罹患している患者における症状の進行を阻止することも含まれる。 As used herein, “treatment” of diabetes or diabetic complications includes preventing the progression of symptoms in a patient already suffering from diabetes or diabetic complications.
本発明の糖尿病予防／治療用医薬組成物の投与方法は、経口投与または非経口投与のいずれであってもよい。また、該組成物の投与時期は特に限定されず、食前、食中、食後、食間のいずれであってもよい。例えば、該組成物は、糖類含有量が５ｇ以上である食事、またはアミノ酸および／または蛋白質含有量が２０ｇ以上である食事の食前、食中、食後および食間のいずれかに投与するのが好ましく、糖類ならびにアミノ酸および／または蛋白質を前記含有量以上含有する食事の食前、食中、食後および食間のいずれかに投与するのがより好ましい。食事に含まれるアミノ酸としては、リジン、アルギニン等が挙げられ、その中でもＡＧＥｓ化し易いリジンおよび／またはアルギニンを前記含有量以上含有する食事の食前、食中、食後および食間のいずれかに該組成物を投与するのが好ましい。また、食事に含まれる蛋白質としては、卵白アルブミン、乳アルブミン、グリアジン、乳カゼイン、大豆カゼイン等が挙げられ、その中でもＡＧＥｓ化し易い卵白アルブミン、乳アルブミンおよび乳カゼインから選ばれる１種以上を前記含有量以上含有する食事の食前、食中、食後および食間のいずれかに該組成物を投与するのが好ましい。経口投与の場合には、多量のソルビトールによる下痢の副作用回避の点で、該組成物を食後に投与するか、または一日に複数回に分けて少量ずつ投与するのが特に好ましい。ここで、一般的に「食事」とは、生存に必要な栄養分をとるために毎日の習慣として物を食べること（飲食行為）あるいはその飲食物を意味するが、本明細書において用いる「食事」の用語は、一回の飲食行為において摂取する一まとまりの飲食物を意味するものとする。 The method for administering the pharmaceutical composition for preventing / treating diabetes of the present invention may be either oral administration or parenteral administration. Moreover, the administration time of this composition is not specifically limited, Any of before a meal, during a meal, after a meal, and between meals may be sufficient. For example, the composition is preferably administered before, during, after, and between meals of a meal having a saccharide content of 5 g or more, or a meal having an amino acid and / or protein content of 20 g or more, It is more preferable to administer sugars and amino acids and / or proteins before, during, after, and between meals of meals containing the above content or more. Examples of amino acids contained in the meal include lysine, arginine, etc. Among them, the composition may be any of before, during, after, and between meals of a meal containing lysine and / or arginine that is easily converted to AGEs. Is preferably administered. Examples of the protein contained in the meal include ovalbumin, milk albumin, gliadin, milk casein, soybean casein, and the like. Among these, one or more selected from ovalbumin, milk albumin, and milk casein that are easily converted to AGEs are contained. It is preferable to administer the composition either before, during, after, or between meals. In the case of oral administration, it is particularly preferable to administer the composition after meals, or in small portions divided into a plurality of times a day, in order to avoid side effects of diarrhea caused by a large amount of sorbitol. Here, “meal” generally means eating (drinking action) or eating or drinking food as a daily habit for taking the nutrients necessary for survival, but “meal” as used herein. The term "" means a set of food and drink taken in a single eating and drinking act.
本明細書において用いる場合、「糖類」とは、単糖および／または二糖を意味するものとする。これに対し「糖質」とは、炭水化物のうち食物繊維以外のものをいい、糖類の他に、オリゴ糖、多糖、糖アルコール等が含まれる。 As used herein, “saccharides” shall mean monosaccharides and / or disaccharides. On the other hand, “sugar” refers to carbohydrates other than dietary fiber, and includes oligosaccharides, polysaccharides, sugar alcohols and the like in addition to sugars.
本発明の糖尿病予防／治療用医薬組成物の投与量は、糖尿病またはその合併症の程度、その他の疾病の程度、年齢、性別等の条件に応じて適宜選択される。該組成物は、ＡＧＥｓの生成を抑制することができる量（以下、「ＡＧＥｓ生成抑制有効量」とも称する）あるいはＡＧＥｓの前駆体である３−ＤＧの生成を抑制することができる量（以下、「３−ＤＧ生成抑制有効量」とも称する）を投与すればよい。ＡＧＥｓ生成抑制有効量および３−ＤＧ生成抑制有効量は、当業者に周知の方法（各種の非臨床および／または臨床試験を含む）を用いて適宜決定することができる。 The dosage of the pharmaceutical composition for preventing / treating diabetes according to the present invention is appropriately selected according to conditions such as the degree of diabetes or its complications, the degree of other diseases, age, and sex. The composition can suppress the generation of AGEs (hereinafter also referred to as “AGEs generation effective effective amount”) or an amount capable of suppressing the generation of 3-DG which is a precursor of AGEs (hereinafter referred to as “AGEs”). (Also referred to as “3-DG production suppression effective amount”). The effective amount for inhibiting AGEs production and the effective amount for inhibiting 3-DG production can be appropriately determined using methods well known to those skilled in the art (including various non-clinical and / or clinical tests).
本発明の糖尿病予防／治療用医薬組成物は、ＡＧＥｓ生成抑制有効量または３−ＤＧ生成抑制有効量のソルビトールを一度に投与するものであっても良く、間隔を置いて複数回に分けて投与するものであっても良い。経口投与の場合は、緩下性の点から複数回に分けて投与するのが好ましい。複数回に分けて投与する場合は、一日に投与されるソルビトールの合計量がＡＧＥｓ生成抑制有効量または３−ＤＧ生成抑制有効量となればよく、食事の回数に合わせて投与するのが好ましい。 The pharmaceutical composition for preventing / treating diabetes according to the present invention may be one in which an AGEs production-inhibiting effective amount or a 3-DG production-inhibiting effective amount of sorbitol may be administered at once, and it may be administered in multiple doses at intervals. It may be what you do. In the case of oral administration, it is preferable to administer in multiple doses from the viewpoint of laxity. In the case of administration in multiple doses, the total amount of sorbitol administered per day may be an effective amount for inhibiting AGEs production or an effective amount for inhibiting 3-DG production, and is preferably administered according to the number of meals. .
本発明の糖尿病予防／治療用医薬組成物は、糖尿病もしくは糖尿病合併症に罹患した者、または健常者のいずれに対しても投与することができる。特に糖尿病はＡＧＥｓおよびその前駆体である３−ＤＧとの関連性が高いため、該組成物は糖尿病に罹患した者に対して投与するのが好ましい。糖尿病に罹患した者とは、ヘモグロビンＡ１ｃが６．１％（JDS値）以上で、且つ、以下の(1)〜(3)のいずれかに該当する者を指す：(1)空腹時血糖値が１２６ｍｇ／ｄＬ以上、(2)随時血糖値（空腹か食後かにかかわらず）が２００ｍｇ／ｄＬ以上、および(3)ブドウ糖負荷後２時間値が２００ｍｇ／ｄＬ以上。糖尿病診断基準の詳細は、日本糖尿病学会による「糖尿病の分類と診断基準に関する委員会報告」（同学会のホームページ等から入手可能）に記載されている。 The pharmaceutical composition for preventing / treating diabetes of the present invention can be administered to any person suffering from diabetes or diabetic complications, or a healthy person. In particular, since diabetes is highly related to AGEs and its precursor 3-DG, the composition is preferably administered to those suffering from diabetes. A person suffering from diabetes refers to a person whose hemoglobin A1c is 6.1% (JDS value) or more and falls under any of the following (1) to (3): (1) Fasting blood glucose level 126 mg / dL or higher, (2) Adequate blood glucose level (whether fasting or after eating) is 200 mg / dL or higher, and (3) 2 hours after glucose loading is 200 mg / dL or higher. Details of the diagnostic criteria for diabetes are described in the “Report of the Committee on Diabetes Classification and Diagnostic Criteria” by the Japanese Diabetes Association (available from the association's website).
また、本発明の糖尿病予防／治療用医薬組成物は、糖尿病に伴って発症する糖尿病網膜症、糖尿病腎症、糖尿病神経障害、糖尿病血管合併症、動脈硬化症、腎不全、アルツハイマー病、神経変性疾患、がん等の糖尿病合併症の予防および／または治療のために用いることができる。これらの糖尿病合併症の中でも、発症率の高い糖尿病網膜症、糖尿病腎症および／または糖尿病神経障害の予防および／または治療のために該組成物を用いることが好ましい。 Further, the pharmaceutical composition for preventing / treating diabetes of the present invention comprises diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, diabetic vascular complications, arteriosclerosis, renal failure, Alzheimer's disease, neurodegeneration, which develops with diabetes. It can be used for the prevention and / or treatment of diabetic complications such as diseases and cancer. Among these diabetic complications, it is preferable to use the composition for the prevention and / or treatment of diabetic retinopathy, diabetic nephropathy and / or diabetic neuropathy with a high incidence.
さらに、本発明の糖尿病予防／治療用医薬組成物は、健常者に投与することにより、糖尿病およびその合併症を効果的に予防することができる。 Furthermore, diabetes and its complications can be effectively prevented by administering the pharmaceutical composition for preventing / treating diabetes of the present invention to healthy individuals.
本発明の糖尿病予防／治療用医薬組成物は、ソルビトールによる糖尿病もしくは糖尿病合併症の予防および／または治療効果を妨げない範囲であれば、ソルビトールの他にさらに賦形剤、安定剤、保存剤、緩衝剤、矯味剤、懸濁化剤、乳化剤、着香剤、溶解補助剤、着色剤、粘稠剤等の成分を添加して各種の剤形とすることができる。 The pharmaceutical composition for preventing / treating diabetes according to the present invention has an excipient, a stabilizer, a preservative, in addition to sorbitol, as long as it does not interfere with the prevention and / or treatment effects of diabetes or diabetic complications due to sorbitol. Various dosage forms can be obtained by adding components such as a buffer, a corrigent, a suspending agent, an emulsifier, a flavoring agent, a solubilizing agent, a coloring agent, and a thickening agent.
本発明の糖尿病予防／治療用医薬組成物の剤形としては、錠剤（口腔内崩壊錠、チュアブル錠、発泡錠、分散錠、溶解錠）、カプセル剤、顆粒剤（発泡顆粒剤）、散剤、経口液剤（エリキシル剤、懸濁剤、乳剤、リモナーデ剤）、シロップ剤（シロップ用剤）、経口ゼリー剤、口腔用錠剤（トローチ剤、舌下錠、バッカル錠、付着錠、ガム剤）、口腔用スプレー剤、口腔用半固形剤、含嗽剤、注射剤（輸液剤、埋め込み注射剤、持続性注射剤）、透析用剤（腹膜透析用剤、血液透析用剤）、吸入剤（吸入粉末剤、吸入液剤、吸入エアゾール剤）、坐剤、直腸用半固形剤、注腸剤、点眼剤、眼軟膏剤、点耳剤、点鼻剤（点鼻粉末剤、点鼻液剤）、膣錠、膣用坐剤、外用固形剤（外用散剤）、外用液剤（リニメント剤、ローション剤）、スプレー剤（外用エアゾール剤、ポンプスプレー剤）、軟膏剤、クリーム剤、ゲル剤、貼付剤（テープ剤、パップ剤）等が挙げられる。これらのの剤形中でも経口投与のし易さの点で、錠剤、カプセル剤、顆粒剤、散剤、経口液剤、シロップ剤、経口ゼリー剤、口腔用錠剤、口腔用スプレー剤、口腔用半固形剤および含嗽剤が好ましく、生体内に吸収され易い点で、錠剤、カプセル剤、顆粒剤、散剤がより好ましい。 Examples of the dosage form of the pharmaceutical composition for prevention / treatment of diabetes of the present invention include tablets (orally disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, dissolving tablets), capsules, granules (expanded granules), powders, Oral solution (elixir, suspension, emulsion, limonade), syrup (syrup), oral jelly, oral tablet (troche, sublingual, buccal, adhesive tablet, gum), oral Spray, oral semi-solid preparation, mouthwash, injection (infusion solution, implantable injection, continuous injection), dialysis agent (peritoneal dialysis agent, hemodialysis agent), inhalant (inhalation powder) , Inhalants, aerosols), suppositories, rectal semisolids, enemas, eye drops, eye ointments, ear drops, nasal drops (nasal powders, nasal drops), vaginal tablets, Vaginal suppository, external solid preparation (external powder), external liquid (liniment, lotion), spray (External aerosols, pump sprays), ointments, creams, gels, patches (tape, cataplasms), and the like. Even in these dosage forms, tablets, capsules, granules, powders, oral solutions, syrups, oral jelly, oral tablets, oral sprays, semi-solid oral preparations because of their ease of oral administration A gargle is preferable, and tablets, capsules, granules, and powders are more preferable in that they are easily absorbed in vivo.
また、本発明の糖尿病予防／治療用医薬組成物は、エキス剤、丸剤、酒精剤、浸剤・煎剤、茶剤、チンキ剤、芳香水剤、流エキス剤等の生薬関連製剤に本発明のＡＧＥｓ生成抑制剤または３−ＤＧ生成抑制剤を添加した剤形で使用することもできる。これら剤形は、糖尿病またはその合併症の程度、その他の疾病の程度、年齢、性別等の条件に応じて適宜選択される。 Further, the pharmaceutical composition for preventing / treating diabetes of the present invention can be applied to herbal medicine-related preparations such as extracts, pills, spirits, soaking agents, decoction, teas, tinctures, fragrances, and flow extracts. It can also be used in a dosage form to which an AGEs production inhibitor or a 3-DG production inhibitor is added. These dosage forms are appropriately selected according to conditions such as the degree of diabetes or its complications, the degree of other diseases, age, and sex.
更に、本発明の糖尿病予防／治療用医薬組成物は、ＡＧＥｓ生成抑制効果を有する他の薬剤をさらに含むものとすることができる。これらの薬剤としては、例えば、インスリン抵抗性改善薬、脂質異常症治療薬、アンジオテンシンII１型受容体拮抗薬、アンジオテンシン変換酵素阻害剤、メトホルミン等が例示される。 Furthermore, the pharmaceutical composition for preventing / treating diabetes according to the present invention may further contain other drugs having an AGEs production inhibitory effect. Examples of these drugs include insulin resistance improving drugs, dyslipidemic drugs, angiotensin II type 1 receptor antagonists, angiotensin converting enzyme inhibitors, metformin, and the like.
また、本発明の糖尿病予防／治療用医薬組成物は、ＡＧＥｓ生成抑制効果を有する天然物または天然由来物質をさらに含むものとすることもできる。これらの天然物または天然由来物質としては、例えば、ドクダミ、セイヨウサンザシ、カモミール、ブドウ葉等のハーブ類、桜の花、トウモロコシの花柱および柱頭、セイヨウオオバコ種子、マロニエ、シャクヤク、バラの花、梅果実、山ぶどう、紫菊花、小麦胚芽、小麦胚芽由来のポリアミン、マンゴスチン等が例示される。 In addition, the pharmaceutical composition for preventing / treating diabetes of the present invention may further contain a natural product or a naturally-derived material having an AGEs production inhibitory effect. Examples of these natural products or naturally-derived substances include herbs such as dokudami, hawthorn, chamomile, grape leaves, cherry blossoms, corn stigma and stigmas, plantain seeds, maronier, peonies, rose flowers, plum fruit , Mountain grape, purple chrysanthemum flower, wheat germ, wheat germ-derived polyamine, mangosteen and the like.
本発明のＡＧＥｓ生成抑制剤もしくは３−ＤＧ生成抑制剤またはその有効成分であるソルビトールは、非ヒト動物における糖尿病もしくは糖尿病合併症を予防および／または治療するために用いることもできる。したがって、本発明の一態様において、非ヒト動物における糖尿病もしくは糖尿病合併症の予防および／または治療のための、ソルビトールを含む非ヒト動物用医薬組成物（以下、本発明の非ヒト動物用医薬組成物とも称する）が提供される。また、該非ヒト動物用医薬組成物または本発明のＡＧＥｓ生成抑制剤もしくは３−ＤＧ生成抑制剤を非ヒト動物に投与することを含む、非ヒト動物における糖尿病もしくは糖尿病合併症を予防および／または治療する方法も提供される。非ヒト動物としては、ウシ、ウマ、ブタ、ヒツジ、ヤギ、ニワトリ等の家畜類や、イヌ、ネコ等のペットとして飼育される動物等が例示される。 The AGEs production inhibitor or 3-DG production inhibitor of the present invention or sorbitol, which is an active ingredient thereof, can also be used to prevent and / or treat diabetes or diabetic complications in non-human animals. Therefore, in one aspect of the present invention, a pharmaceutical composition for non-human animals containing sorbitol for the prevention and / or treatment of diabetes or diabetic complications in a non-human animal (hereinafter, the pharmaceutical composition for non-human animals of the present invention). Also referred to as an object). In addition, prevention and / or treatment of diabetes or diabetic complications in a non-human animal, comprising administering the non-human animal pharmaceutical composition or the AGEs production inhibitor or 3-DG production inhibitor of the present invention to a non-human animal. A method is also provided. Examples of non-human animals include livestock such as cows, horses, pigs, sheep, goats and chickens, and animals raised as pets such as dogs and cats.
本発明の非ヒト動物用医薬組成物に含有させるソルビトールの量、該医薬組成物の投与方法、投与時期、投与量および剤形、該医薬組成物においてソルビトールと併用可能な他の薬剤、天然物および天然由来物質、ならびに本発明のＡＧＥｓ生成抑制剤または３−ＤＧ生成抑制剤を非ヒト動物に投与する場合における投与方法、投与時期、投与量等については、「本発明の糖尿病予防／治療用医薬組成物」について上述した内容と同様である。 The amount of sorbitol to be contained in the pharmaceutical composition for non-human animals of the present invention, the method of administering the pharmaceutical composition, the timing of administration, the dosage and the dosage form, other drugs that can be used in combination with sorbitol in the pharmaceutical composition, natural products For the administration method, administration time, dosage, etc. when administering the AGEs production inhibitor or 3-DG production inhibitor of the present invention to a non-human animal, The same as described above for the “pharmaceutical composition”.
さらに、本発明のＡＧＥｓ生成抑制剤または３−ＤＧ生成抑制剤は、食品添加剤として用いることもできる。したがって、本発明のＡＧＥｓ生成抑制剤または３−ＤＧ生成抑制剤を飲食品に配合または添加することにより、ＡＧＥｓまたは３−ＤＧの生成を抑制する機能性飲食品を提供することができる。また、本発明のＡＧＥｓ生成抑制剤または３−ＤＧ生成抑制剤を成分として使用することにより、ＡＧＥｓまたは３−ＤＧの生成を抑制するサプリメントを製造することもできる。この場合、製造するサプリメントの剤形に応じて適宜、賦形剤等を配合することができる。 Furthermore, the AGEs production | generation inhibitor or 3-DG production | generation inhibitor of this invention can also be used as a food additive. Therefore, the functional food / beverage products which suppress the production | generation of AGEs or 3-DG can be provided by mix | blending or adding the AGEs production | generation inhibitor or 3-DG production | generation inhibitor of this invention to food / beverage products. Moreover, the supplement which suppresses the production | generation of AGEs or 3-DG can also be manufactured by using the AGEs production | generation inhibitor or 3-DG production | generation inhibitor of this invention as a component. In this case, excipients and the like can be appropriately blended according to the dosage form of the supplement to be produced.
以下、実施例により本発明をさらに説明する。 Hereinafter, the present invention will be further described by examples.
試験例１
（１）試料の調製
アミノ酸、糖質およびリン酸緩衝液を表１に示す配合量で５０ｍＬのポリプロピレン製遠沈管に入れて密閉し、攪拌した後、３７℃で４２日間、インキュベーター内で反応させた。そして反応物を０．２μｍフィルターで濾過し、得られた濾液を試料とした。尚、使用したアミノ酸および糖質は下記の通りである。
［アミノ酸］
リジン（Ｎ−α−（ｔ−ブトキシカルボニル）−Ｌ−リジン、渡辺化学工業株式会社製）とアルギニン（Ｎ−α−（ｔ一ブトキシカルボニル）−Ｌ−アルギニン、渡辺化学工業株式会社製）の１：１混合物
［糖質］
グルコース（試薬特級、和光純薬工業株式会社製）
フルクトース（ＫＣ特級、片山化学工業株式会社製）
ソルビトール（シグマアルドリッチジャパン特級、シグマアルドリッチジャパン合同会社製）
Test example 1
(1) Preparation of sample Amino acid, saccharide and phosphate buffer solution were put in a 50 mL polypropylene centrifuge tube with the compounding amounts shown in Table 1, sealed, stirred, and reacted in an incubator at 37 ° C for 42 days. It was. The reaction product was filtered through a 0.2 μm filter, and the obtained filtrate was used as a sample. The amino acids and carbohydrates used are as follows.
[amino acid]
Of lysine (N-α- (t-butoxycarbonyl) -L-lysine, manufactured by Watanabe Chemical Co., Ltd.) and arginine (N-α- (t-butoxycarbonyl) -L-arginine, manufactured by Watanabe Chemical Co., Ltd.) 1: 1 mixture
[Sugar]
Glucose (special grade reagent, manufactured by Wako Pure Chemical Industries, Ltd.)
Fructose (KC special grade, manufactured by Katayama Chemical Co., Ltd.)
Sorbitol (Sigma Aldrich Japan Special Grade, Sigma Aldrich Japan GK)
（２）試験方法
上記のように調製した試料について、下記の方法によって３−ＤＧ、蛍光性ＡＧＥｓ、ペントシジン、ＣＭＬ、ＲＡＧＥアゴニスト活性およびＲＡＧＥアンタゴニスト活性を評価した。
(2) Test method The samples prepared as described above were evaluated for 3-DG, fluorescent AGEs, pentosidine, CML, RAGE agonist activity and RAGE antagonist activity by the following method.
（ａ）３−ＤＧの定量
試料２ｍＬに等量の６０％過塩素酸溶液を加えて除蛋白し、飽和炭酸水素ナトリウム水溶液４ｍＬで中和した後、２，３−ジアミノナフタレンの０．１％メタノール溶液０．８ｍＬを添加し、４℃で１６時間反応させた。次に反応物を４．４ｍＬの酢酸エチルで抽出し、エバポレーターで濃縮乾固後、メタノール５ｍＬに溶解させ、０．４５μｍフィルターで濾過した。濾過液を高速液体クロマトグラフィーに供試し、下記測定条件で３−ＤＧを定量した。
［高速液体クロマトグラフィー測定条件］
ＵＶ：２６８ｎｍ
注入量：２０μＬ
カラム：ＴＳＫ−ＧＥＬ，ＯＤＳ−８０Ｔｓ（４．６×１５０ｍｍ、東ソー株式会社製）
カラム温度：３０℃
移動相：５０ｍＭリン酸：メタノール：アセトニトリル＝７：１．５：１．５
流速：１ｍＬ／分
(A) Quantification of 3-DG After adding 2% sample of an equal amount of 60% perchloric acid solution to deproteinize and neutralizing with 4 mL of saturated aqueous sodium hydrogen carbonate solution, 0.1% of 2,3-diaminonaphthalene Methanol solution 0.8mL was added and it was made to react at 4 degreeC for 16 hours. Next, the reaction product was extracted with 4.4 mL of ethyl acetate, concentrated to dryness with an evaporator, dissolved in 5 mL of methanol, and filtered through a 0.45 μm filter. The filtrate was subjected to high performance liquid chromatography, and 3-DG was quantified under the following measurement conditions.
[High-performance liquid chromatography measurement conditions]
UV: 268 nm
Injection volume: 20 μL
Column: TSK-GEL, ODS-80Ts (4.6 × 150 mm, manufactured by Tosoh Corporation)
Column temperature: 30 ° C
Mobile phase: 50 mM phosphoric acid: methanol: acetonitrile = 7: 1.5: 1.5
Flow rate: 1 mL / min
（ｂ）蛍光性ＡＧＥｓの定量
標準物質であるペリレン２．５ｍｇを秤量し、脱気エタノール５０ｍＬを加えて十分に溶解させペリレン溶液を作製した。１０μＭの濃度まで希釈したペリレン溶液に波長３８６ｎｍの励起光を当て、４１０−６００ｎｍの範囲の蛍光強度を測定してエリアを算出した。一方で、試料には波長３８０ｎｍの励起光を当て、４００−６００ｎｍの範囲の蛍光強度を測定してエリアを算出した。試料のエリア測定値は１０μＭのペリレン溶液のエリアの値に対する相対値として蛍光性ＡＧＥｓを定量した。
(B) Quantification of fluorescent AGEs 2.5 mg of perylene as a standard substance was weighed, and 50 mL of degassed ethanol was added and sufficiently dissolved to prepare a perylene solution. The area was calculated by applying excitation light having a wavelength of 386 nm to a perylene solution diluted to a concentration of 10 μM and measuring the fluorescence intensity in the range of 410 to 600 nm. On the other hand, the area was calculated by applying excitation light having a wavelength of 380 nm to the sample and measuring the fluorescence intensity in the range of 400 to 600 nm. As the area measurement value of the sample, fluorescent AGEs were quantified as a relative value to the area value of the 10 μM perylene solution.
（ｃ）ペントシジンの定量
ペントシジン測定キット「ＦＳＫペントシジン(登録商標)」（株式会社伏見製薬所）を用いて、以下の通りにペントシジン定量を行った： 使用する３０分以上前に試薬及びペントシジン固相化プレートを常温に戻しておいた。試料希釈後、キット添付のペントシジン固相化プレートに試料を５０μＬずつ分注し、試薬のブランク値測定用ウェル以外に第一抗体溶液５０μＬを分注した。プレートを３００ｒｐｍで１分間振盪することにより試料と抗体溶液を混合した後、３７℃で１時間反応させた。次に反応液を捨て、洗浄液２００μＬを分注してプレートを洗浄した。該洗浄は３回行った。プレートの水気を取り除いた後、試薬のブランク値測定用ウェル以外に第二抗体溶液１００μＬを分注し、３００ｒｐｍで１分間の振盪により混合した後、２５℃で１時間反応させた。そして、反応液を捨て、再び洗浄液２００μＬでプレートを３回洗浄した後、プレートの水気を取り、発色剤１００μＬを分注して遮光条件下で１０分間静置した。最後に反応停止液１００μＬを分注し、分注後１０分以内にマイクロプレートリーダーを用いて主波長４５０ｎｍ／参照波長６３０ｎｍで測定を行った。ペントシジン濃度は、試料の測定値に基づき、ペントシジン標準溶液の検量線を用いて決定した。
(C) Pentosidine Quantification Pentosidine quantification was performed as follows using a pentosidine measurement kit “FSK Pentosidine (registered trademark) ” (Fushimi Pharmaceutical Co., Ltd.): Reagent and pentosidine solid phase 30 minutes or more before use The conversion plate was returned to room temperature. After dilution of the sample, 50 μL of the sample was dispensed on a pentosidine solid phase plate attached to the kit, and 50 μL of the first antibody solution was dispensed in addition to the reagent blank measurement well. The sample and the antibody solution were mixed by shaking the plate at 300 rpm for 1 minute, and then reacted at 37 ° C. for 1 hour. Next, the reaction solution was discarded, and 200 μL of the washing solution was dispensed to wash the plate. The washing was performed 3 times. After removing the moisture from the plate, 100 μL of the second antibody solution was dispensed in addition to the well for measuring the blank value of the reagent, mixed by shaking at 300 rpm for 1 minute, and then reacted at 25 ° C. for 1 hour. Then, the reaction solution was discarded, and the plate was washed three times with 200 μL of the washing solution again. Then, the plate was drained, 100 μL of the color former was dispensed, and the plate was allowed to stand for 10 minutes under light-shielding conditions. Finally, 100 μL of the reaction stop solution was dispensed, and measurement was performed at a main wavelength of 450 nm / reference wavelength of 630 nm using a microplate reader within 10 minutes after dispensing. The pentosidine concentration was determined using a calibration curve of a pentosidine standard solution based on the measured value of the sample.
（ｄ）ＣＭＬの定量
ＣｉｒｃｕＬｅｘ ＣＭＬ／Ｎε−（ｃａｒｂｏｘｙｍｅｔｈｙｌ）ｌｙｓｉｎｅ ＥＬＩＳＡ ｋｉｔ（株式会社サイクレックス製）を用いて、以下の通りにＣＭＬ定量を行った： 使用する３０分以上前に試薬及びＣＭＬ固相化ウェルを常温に戻しておいた。またＣＭＬ−ＨＳＡスタンダード粉末品に超純水（ミリＱ水）１ｍＬを加えてマスタースタンダードを作製し、スタンダード希釈用バッファーで希釈を行いスタンダード１〜７を作製した。試料は必要に応じてサンプル希釈用バッファーで希釈した。キット付属品でない９６ウェルプレートにＣＭＬ−ＨＳＡスタンダードと試料６０μＬを分注した。次に、第一抗体粉末品に超純水（ミリＱ水）を加え、サンプル希釈用バッファーで希釈して第一抗体溶液を作成し、該抗体溶液６０μＬをプレートに分注して混合した。該混合液１００μＬをＣＭＬ固相化ウェルに添加し、３００ｒｐｍで振盪させながら２５℃で１時間反応させた。さらに反応液を捨て、洗浄液３５０μＬを分注してプレートを洗浄した。該洗浄は４回行った。プレートの水気を取り除いた後、第二抗体溶液１００μＬを分注し、３００ｒｐｍで振盪させながら２５℃で１時間反応させた。反応液を捨て、再び洗浄液３５０μＬで４回洗浄し、プレートの水気を取り、発色剤１００μＬを分注した。そして３００ｒｐｍで振盪させながら遮光条件下で１０分間静置した。最後に反応停止液１００μＬを分注し、分注後３０分以内にマイクロプレートリーダーを用いて主波長４５０ｎｍ／参照波長５９５ｎｍで測定を行った。ＣＭＬ濃度は、試料の測定値に基づき、ＣＭＬ標準溶液の検量線を用いて決定した。
(D) Quantification of CML CML quantification was performed as follows using CircuLex CML / Nε- (carbomethyl) lysine ELISA kit (manufactured by Cyclex Co., Ltd.): Reagent and CML solid phase 30 minutes or more before use The chemical well was returned to room temperature. Moreover, 1 mL of ultrapure water (Milli Q water) was added to the CML-HSA standard powder product to prepare a master standard, and diluted with a standard dilution buffer to prepare standards 1 to 7. Samples were diluted with sample dilution buffer as needed. A CML-HSA standard and a sample of 60 μL were dispensed into a 96-well plate not included in the kit. Next, ultrapure water (Milli Q water) was added to the first antibody powder product, diluted with a sample dilution buffer to prepare a first antibody solution, and 60 μL of the antibody solution was dispensed onto a plate and mixed. 100 μL of the mixed solution was added to the CML-immobilized well and reacted at 25 ° C. for 1 hour while shaking at 300 rpm. Further, the reaction solution was discarded, and 350 μL of the washing solution was dispensed to wash the plate. The washing was performed 4 times. After removing the moisture from the plate, 100 μL of the second antibody solution was dispensed and reacted at 25 ° C. for 1 hour while shaking at 300 rpm. The reaction solution was discarded, and the plate was again washed with 350 μL of washing solution four times, the plate was drained, and 100 μL of color former was dispensed. And it left still for 10 minutes under light-shielding conditions, shaking at 300 rpm. Finally, 100 μL of the reaction stop solution was dispensed, and measurement was performed at a main wavelength of 450 nm / reference wavelength of 595 nm using a microplate reader within 30 minutes after dispensing. The CML concentration was determined using a calibration curve of a CML standard solution based on the measured value of the sample.
（ｅ）ＲＡＧＥアゴニスト活性の評価
Ｌｕｃｉｆｅｒａｓｅ Ａｓｓａｙ Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ）を用いてＲＡＧＥアゴニスト活性の評価を行った：（１日目）Ｄｉａｂｅｔｅｓ， ２００６， ｖｏｌ．５５， ｐｐ．２５１０−２５２２に記載される方法に従い、ｐＮＦ−κＢ−Ｌｕｃプラスミド（ｓｔｒａｔａｇｅｎｅ社）とヒトＲＡＧＥプラスミド（全長ヒトＲＡＧＥ ｃＤＮＡを含む）を導入したＣ６グリオーマ細胞を９６ウェルプレートに１００μＬ／ウェルで播種し、細胞がコンフルエントになるまでＣＯ２インキュベーターで培養した。培養には１０％ＦＢＳ／ＤＭＥＭ培地を使用した。（２日目）培地を除去し、無血清ＤＭＥＭ培地３００μＬでウェル内を洗浄後、０．１％ＦＢＳ／ＤＭＥＭ培地１００μＬをゆっくり添加した。その後、ＣＯ２インキュベーターで一晩培養した。（３日目）培地を用いて試料を希釈し、各ウェルに希釈した試料５０μＬを添加した。また、対照のウェルにはグリセルアルデヒドでＡＧＥ化されたＡＧＥ−ＢＳＡ（５０μｇ／ｍＬ）を添加した。ＣＯ２インキュベーターで４時間培養後、培地を除去してＰＢＳ（−）３００μＬでウェル内を２回洗浄した。そして氷上で１×リシスバッファー２５μＬを添加した。プレートを１５秒間振盪した後、新しい白色プレートに可溶化液２０μＬを入れた。ルシフェラーゼアッセイリージェント１００μＬをさらに添加し、ルミノメーターで発光強度を測定した。また、可溶化液５μＬを使用してタンパク定量も行った。
(E) Evaluation of RAGE agonist activity The RAGE agonist activity was evaluated using Luciferase Assay System (Promega): (Day 1) Diabetes, 2006, vol. 55, pp. In accordance with the method described in 2510-2522, C6 glioma cells into which pNF-κB-Luc plasmid (Stratagene) and human RAGE plasmid (including full-length human RAGE cDNA) were introduced were seeded in a 96-well plate at 100 μL / well, The cells were cultured in a CO 2 incubator until the cells became confluent. For culture, 10% FBS / DMEM medium was used. (Day 2) After removing the medium and washing the well with 300 μL of serum-free DMEM medium, 100 μL of 0.1% FBS / DMEM medium was slowly added. Thereafter, the cells were cultured overnight in a CO 2 incubator. (Day 3) The sample was diluted with the medium, and 50 μL of the diluted sample was added to each well. In addition, AGE-BSA (50 μg / mL) AGE-modified with glyceraldehyde was added to the control well. After culturing for 4 hours in a CO 2 incubator, the medium was removed, and the wells were washed twice with 300 μL of PBS (−). Then 25 μL of 1 × lysis buffer was added on ice. After shaking the plate for 15 seconds, 20 μL of lysate was placed in a new white plate. 100 μL of luciferase assay reagent was further added, and the luminescence intensity was measured with a luminometer. Protein quantification was also performed using 5 μL of the solubilized solution.
（ｆ）ＲＡＧＥアンタゴニスト活性の評価
Ｌｕｃｉｆｅｒａｓｅ Ａｓｓａｙ Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ）を用いてＲＡＧＥアンタゴニスト活性の評価を行った：（１日目）Ｄｉａｂｅｔｅｓ，２００６，ｖｏｌ．５５，ｐｐ，２５１０−２５２２に記載される方法に従い、ｐＮＦ−κＢ−Ｌｕｃプラスミド（ｓｔｒａｔａｇｅｎｅ社）とヒトＲＡＧＥプラスミド（全長ヒトＲＡＧＥ ｃＤＮＡを含む）を導入したＣ６グリオーマ細胞を９６ウェルプレートに１００μＬ／ウェルで播種し、細胞がコンフルエントになるまでＣＯ２インキュベーターで培養した。培養には１０％ＦＢＳ／ＤＭＥＭ培地を使用した。（２日目）培地を除去し、無血清ＤＭＥＭ培地３００μＬでウェル内を洗浄後、０．１％ＦＢＳ／ＤＭＥＭ培地１００μＬをゆっくり添加した。その後、ＣＯ２インキュベーターで一晩培養した。（３日目）培地を用いて試料を希釈し、各ウェルに希釈した試料５０μＬ、対照としてグリセルアルデヒドでＡＧＥ化されたＡＧＥ−ＢＳＡ（５０μｇ／ｍＬ）、さらに希釈した試料とＡＧＥ−ＢＳＡの混合液を添加した。ＣＯ２インキュベーターで４時間培養後、培地を除去してＰＢＳ（−）３００μＬでウェル内を２回洗浄した。そして氷上で１×リシスバッファー２５μＬを添加した。プレートを１５秒間振盪した後、新しい白色プレートに可溶化液２０μＬを入れた。ルシフェラーゼアッセイリージェント１００μＬをさらに添加し、ルミノメーターで発光強度を測定した。また、可溶化液５μＬを使用してタンパク定量も行った。
(F) Evaluation of RAGE antagonist activity The RAGE antagonist activity was evaluated using Luciferase Assay System (Promega): (Day 1) Diabetes, 2006, vol. 55, pp, 2510-2522, C6 glioma cells into which pNF-κB-Luc plasmid (Stratagene) and human RAGE plasmid (including full-length human RAGE cDNA) were introduced into a 96-well plate at 100 μL / well. And cultured in a CO 2 incubator until the cells were confluent. For culture, 10% FBS / DMEM medium was used. (Day 2) After removing the medium and washing the well with 300 μL of serum-free DMEM medium, 100 μL of 0.1% FBS / DMEM medium was slowly added. Thereafter, the cells were cultured overnight in a CO 2 incubator. (Day 3) Sample was diluted with medium, 50 μL of sample diluted in each well, AGE-BSA AGE-treated with glyceraldehyde (50 μg / mL) as a control, further diluted sample and AGE-BSA The mixture was added. After culturing for 4 hours in a CO 2 incubator, the medium was removed, and the wells were washed twice with 300 μL of PBS (−). Then 25 μL of 1 × lysis buffer was added on ice. After shaking the plate for 15 seconds, 20 μL of lysate was placed in a new white plate. 100 μL of luciferase assay reagent was further added, and the luminescence intensity was measured with a luminometer. Protein quantification was also performed using 5 μL of the solubilized solution.
（３）結果
糖質としてソルビトールを含有する試験区３では、糖質としてグルコースおよびフルクトースを含有する試験区１および２に比べ、３−ＤＧ生成量が大幅に減少し、かつ、蛍光性ＡＧＥｓ、ペントシジンおよびＣＭＬ生成量も減少していた。また、糖質としてソルビトールを含有する試験区３では、糖質としてフルクトースを含有する試験区２に比べてＲＡＧＥアゴニスト活性が低下し、ＲＡＧＥアンタゴニスト活性が低下していた。かかる結果から、アミノ酸存在下におけるソルビトールのＡＧＥｓおよび３−ＤＧ生成抑制効果が認められる。各種ＡＧＥｓおよび３−ＤＧの定量結果を表２に示す。
(3) Results In test group 3 containing sorbitol as a carbohydrate, the amount of 3-DG produced was significantly reduced compared to test groups 1 and 2 containing glucose and fructose as carbohydrates, and fluorescent AGEs, Pentosidine and CML production were also reduced. Moreover, in the test group 3 containing sorbitol as a carbohydrate, the RAGE agonist activity was reduced and the RAGE antagonist activity was reduced compared to the test group 2 containing fructose as a carbohydrate. From this result, the AGEs and 3-DG production inhibitory effect of sorbitol in the presence of amino acids is recognized. Table 2 shows quantitative results of various AGEs and 3-DG.
試験例２
（１）試料の調製
生体蛋白質、糖質およびリン酸緩衝液を表３に示す配合量で５０ｍＬのポリプロピレン製遠沈管に入れて密閉し、攪拌した後、３７℃で４２日間、インキュベーター内で反応させた。次に、反応物を限外濾過ユニット（ビバスピン２０、Ｓａｒｔｏｒｉｕｓ社製）を用いて、３０００ｒｐｍで３時間、遠心分離することにより分画し、低分子画分を３−ＤＧ定量用、高分子画分を蛍光性ＡＧＥｓ、ペントシジン、ＣＭＬ、ＲＡＧＥアゴニスト活性およびＲＡＧＥアンタゴニスト活性測定用の試料とした。尚、使用した生体蛋白質および糖質は下記の通りである。
［生体蛋白質］
ウシ血清アルブミン（脂肪酸無・低エンドトキシン、シグマアルドリッチジャパン合同会社製）
ヒト血清アルブミン（フラクションＶ、東京化成工業株式会社製）
γグロブリン（ウシ血液由来、シグマアルドリッチジャパン合同会社製）
［糖質］
グルコース（試薬特級、和光純薬工業株式会社製）
フルクトース（ＫＣ特級、片山化学工業株式会社製）
ソルビトール（シグマアルドリッチジャパン特級、シグマアルドリッチジャパン合同会社製）
Test example 2
(1) Sample preparation Biological protein, carbohydrate, and phosphate buffer were mixed in a 50 mL polypropylene centrifuge tube with the compounding amounts shown in Table 3, sealed, stirred, and reacted in an incubator at 37 ° C for 42 days. I let you. Next, the reaction product was fractionated by centrifuging at 3000 rpm for 3 hours using an ultrafiltration unit (Vivapin 20, manufactured by Sartorius), and the low molecular fraction was used for 3-DG quantification. The sample was used as a sample for measuring fluorescent AGEs, pentosidine, CML, RAGE agonist activity and RAGE antagonist activity. The biological proteins and carbohydrates used are as follows.
[Bioprotein]
Bovine serum albumin (no fatty acid, low endotoxin, manufactured by Sigma-Aldrich Japan LLC)
Human serum albumin (Fraction V, manufactured by Tokyo Chemical Industry Co., Ltd.)
γ globulin (derived from bovine blood, manufactured by Sigma Aldrich Japan LLC)
[Sugar]
Glucose (special grade reagent, manufactured by Wako Pure Chemical Industries, Ltd.)
Fructose (KC special grade, manufactured by Katayama Chemical Co., Ltd.)
Sorbitol (Sigma Aldrich Japan Special Grade, Sigma Aldrich Japan GK)
（２）試験方法
上記のように調製した低分子画分を試料として用いた以外は、試験例１と同様の方法によって３−ＤＧを定量した。高分子画分は０．２μｍフィルターで濾過を行い、濾液を試料として用いた以外は、試験例１と同様の方法によって蛍光性ＡＧＥｓ、ペントシジン、ＣＭＬ、ＲＡＧＥアゴニスト活性およびＲＡＧＥアンタゴニスト活性の評価を行った。
(2) Test method 3-DG was quantified by the same method as in Test Example 1 except that the low molecular fraction prepared as described above was used as a sample. The polymer fraction was filtered through a 0.2 μm filter, and the fluorescent AGEs, pentosidine, CML, RAGE agonist activity and RAGE antagonist activity were evaluated in the same manner as in Test Example 1 except that the filtrate was used as a sample. It was.
（３）結果
上記いずれの生体蛋白質との組合せにおいても、糖質としてソルビトールを含有する試験区３、６および９では、糖質としてグルコースを含有する試験区１、４および７に比べて３−ＤＧ、蛍光性ＡＧＥｓ、ペントシジンおよびＣＭＬの生成量が減少し、ＲＡＧＥアゴニスト活性およびＲＡＧＥアンタゴニスト活性が低下していた。また、糖質としてフルクトースを含有する試験区２、５および８と比べても、試験区３、６および９では３−ＤＧ生成量が大幅に減少し、蛍光性ＡＧＥｓ、ペントシジンおよびＣＭＬ生成量も減少し、かつ、ＲＡＧＥアゴニスト活性が低下していた。試験区３および６ではＲＡＧＥアンタゴニスト活性が低下していた。かかる結果から、生体蛋白質存在下におけるソルビトールの各種ＡＧＥｓおよび３−ＤＧ生成抑制効果が認められる。各種ＡＧＥｓおよび３−ＤＧの定量結果を表４に示す。
(3) Results In the combination with any of the above biological proteins, the test groups 3, 6 and 9 containing sorbitol as a saccharide had a higher concentration than the test groups 1, 4 and 7 containing glucose as a saccharide. The production of DG, fluorescent AGEs, pentosidine and CML was decreased, and RAGE agonist activity and RAGE antagonist activity were decreased. In addition, compared to test groups 2, 5 and 8 containing fructose as a carbohydrate, the amount of 3-DG produced in test groups 3, 6 and 9 was significantly reduced, and the amounts of fluorescent AGEs, pentosidine and CML were also generated. The RAGE agonist activity was decreased. In test groups 3 and 6, the RAGE antagonist activity decreased. From these results, various AGEs and 3-DG production inhibitory effects of sorbitol in the presence of biological proteins are recognized. Table 4 shows quantitative results of various AGEs and 3-DG.
試験例３
（１）試料の調製
コラーゲン、糖質およびリン酸緩衝液を表５に示す配合量で５０ｍＬのポリプロピレン製遠沈管に入れて密閉し、攪拌した後、３７℃で４２日間、インキュベーター内で反応させた。次に、反応物を限外濾過ユニット（ビバスピン２０、Ｓａｒｔｏｒｉｕｓ社製）を用いて、３０００ｒｐｍで３時間、遠心分離することにより分画し、低分子画分を３−ＤＧ定量用試料とした。一方で、アンプル管に高分子画分０．４ｇおよび６Ｎ塩酸８ｍＬを入れ、管内を窒素置換した後、１１０℃で１６時間、ヒートブロックにて加水分解を行った。反応後、５Ｎ水酸化ナトリウム８ｍＬを加えた後、ｐＨ７．２〜７．６の範囲内に納まるよう１Ｎ塩酸および１Ｎ水酸化ナトリウムを用いてｐＨ調整を行い、０．２μｍフィルターで濾過した。得られた濾液を蛍光性ＡＧＥｓ、ペントシジン、ＣＭＬ、ＲＡＧＥアゴニスト活性およびＲＡＧＥアンタゴニスト活性測定用試料とした。尚、使用したコラーゲンおよび糖質は下記の通りである。
［生体蛋白質］
コラーゲン（コラーゲンタイプＩウシ真皮由来、ペプシン可溶化、株式会社ニッピ）
［糖質］
グルコース（試薬特級、和光純薬工業株式会社製）
フルクトース（ＫＣ特級、片山化学工業株式会社製）
ソルビトール（シグマアルドリッチジャパン特級、シグマアルドリッチジャパン合同会社製）
Test example 3
(1) Preparation of sample Collagen, saccharide and phosphate buffer solution were put in a 50 mL polypropylene centrifuge tube with the compounding amounts shown in Table 5, sealed, stirred, and reacted in an incubator at 37 ° C for 42 days. It was. Next, the reaction product was fractionated by centrifugation at 3000 rpm for 3 hours using an ultrafiltration unit (Vivapin 20, manufactured by Sartorius), and the low molecular fraction was used as a sample for 3-DG quantification. On the other hand, 0.4 g of the polymer fraction and 8 mL of 6N hydrochloric acid were placed in an ampule tube, and the inside of the tube was purged with nitrogen, followed by hydrolysis in a heat block at 110 ° C. for 16 hours. After the reaction, 8 mL of 5N sodium hydroxide was added, pH was adjusted with 1N hydrochloric acid and 1N sodium hydroxide so as to be within the range of pH 7.2 to 7.6, and filtered through a 0.2 μm filter. The obtained filtrate was used as a sample for measuring fluorescent AGEs, pentosidine, CML, RAGE agonist activity and RAGE antagonist activity. In addition, the used collagen and carbohydrate are as follows.
[Bioprotein]
Collagen (Collagen type I calf dermis, pepsin solubilized, Nippi Co., Ltd.)
[Sugar]
Glucose (special grade reagent, manufactured by Wako Pure Chemical Industries, Ltd.)
Fructose (KC special grade, manufactured by Katayama Chemical Co., Ltd.)
Sorbitol (Sigma Aldrich Japan Special Grade, Sigma Aldrich Japan GK)
（２）試験方法
上記のように調製した低分子画分を試料として用いた以外は、試験例１と同様の方法によって３−ＤＧを定量した。また、上記のように調製した高分子画分濾液を試料として用いた以外は、試験例１と同様の方法によって蛍光性ＡＧＥｓ、ペントシジン、ＣＭＬ、ＲＡＧＥアゴニスト活性およびＲＡＧＥアンタゴニスト活性の評価を行った。
(2) Test method 3-DG was quantified by the same method as in Test Example 1 except that the low molecular fraction prepared as described above was used as a sample. Moreover, fluorescent AGEs, pentosidine, CML, RAGE agonist activity and RAGE antagonist activity were evaluated by the same method as in Test Example 1 except that the polymer fraction filtrate prepared as described above was used as a sample.
（３）結果
糖質としてソルビトールを含有する試験区３では、糖質としてグルコースおよびフルクトースを含有する試験区１および２よりも３−ＤＧ生成量が大幅に減少し、蛍光性ＡＧＥｓ、ペントシジンおよびＣＭＬ生成量が減少し、ＲＡＧＥアゴニスト活性およびＲＡＧＥアンタゴニスト活性が低下していた。各種ＡＧＥｓおよび３−ＤＧの定量結果を表６に示す。
(3) Results In test group 3 containing sorbitol as a carbohydrate, 3-DG production was significantly reduced compared to test groups 1 and 2 containing glucose and fructose as carbohydrates, and fluorescent AGEs, pentosidine and CML The amount produced was reduced and the RAGE agonist activity and RAGE antagonist activity were reduced. Table 6 shows the results of quantification of various AGEs and 3-DG.
処方例１（散剤）
表７に示す原材料を混合し、散剤を製造した。該散剤は、本発明のＡＧＥｓ生成抑制剤もしくは３−ＤＧ生成抑制剤、または本発明の糖尿病予防／治療用医薬組成物の一形態である。
The raw materials shown in Table 7 were mixed to produce a powder. The powder is one form of the AGEs production inhibitor or 3-DG production inhibitor of the present invention, or the pharmaceutical composition for prevention / treatment of diabetes of the present invention.
処方例２（錠剤）
表８に示す原材料を混合した後、連続式打錠機（Ｐｉｃｃｏｌａ Ｂ−１０／ＲＩＶＡ社製）を用い、杵金型（φ８ｍｍ、Ｒ１２ｍｍ）、１錠あたりの重量１５０〜２００ｍｇ、回転盤の回転数１２ｒｐｍ、打錠圧４ｋＮの打錠条件で直接打錠し、錠剤を製造した。該錠剤は、本発明のＡＧＥｓ生成抑制剤もしくは３−ＤＧ生成抑制剤、または本発明の糖尿病予防／治療用医薬組成物の一形態である。
After mixing the raw materials shown in Table 8, using a continuous tableting machine (Piccola B-10 / RIVA), a die (φ8 mm, R12 mm), a weight of 150 to 200 mg per tablet, rotation of the rotating plate Tableting was performed directly under tableting conditions of several 12 rpm and a tableting pressure of 4 kN to produce tablets. The tablet is one form of the AGEs production inhibitor or 3-DG production inhibitor of the present invention, or the pharmaceutical composition for prevention / treatment of diabetes of the present invention.
処方例３（経口液剤）
表９に示す原材料を蒸留水５００ｍＬに溶解し、経口液剤を製造した。該経口液剤は、本発明の糖尿病予防／治療用医薬組成物の一形態である。
The raw materials shown in Table 9 were dissolved in 500 mL of distilled water to produce an oral solution. The oral solution is one form of the pharmaceutical composition for preventing / treating diabetes of the present invention.
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