JP2011026221A - Composition for increasing accumulation of protoporphyrin ix in cell - Google Patents
Composition for increasing accumulation of protoporphyrin ix in cell Download PDFInfo
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- JP2011026221A JP2011026221A JP2009171599A JP2009171599A JP2011026221A JP 2011026221 A JP2011026221 A JP 2011026221A JP 2009171599 A JP2009171599 A JP 2009171599A JP 2009171599 A JP2009171599 A JP 2009171599A JP 2011026221 A JP2011026221 A JP 2011026221A
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Abstract
Description
本発明はプロトポルフィリンＩＸ（以下「ＰｐＩＸ」ともいう）細胞内集積増強組成物に関し、より詳しくは、５−アミノレブリン酸（以下「ＡＬＡ」ともいう）類と、１８−クラウン−６又はその誘導体とを含有するＰｐＩＸ細胞内集積増強組成物や、該組成物を有効成分として含有するＡＬＡ−ＰＤＴ用薬剤やＡＬＡ−ＰＤＤ用診断剤等に関する。 The present invention relates to a composition for enhancing intracellular accumulation of protoporphyrin IX (hereinafter also referred to as “PpIX”), and more specifically, 5-aminolevulinic acid (hereinafter also referred to as “ALA”), 18-crown-6 or a derivative thereof. The present invention relates to a composition for enhancing intracellular PpIX intracellular accumulation, a drug for ALA-PDT, a diagnostic agent for ALA-PDD, and the like containing the composition as an active ingredient.
近年、がん、イボ、ニキビのような疾患組織の診断及び治療方法として光に反応する化合物を投与し、光を照射することにより標的箇所の特定や治療を行う光線力学的診断（Photodynamic diagnosis、以下ＰＤＤと略す）や、光線力学的治療（Photodynamic therapy、以下ＰＤＴと略す）が注目されている。ＰＤＤは、例えば、ポルフィリン系又はクロリン系光増感剤を患者に投与し、これを疾患組織に集積させ、波長４００ｎｍ付近の光を照射することによって蛍光発光させ、これを観察することにより、疾病の有無及び患部を特定する診断法であり、またＰＤＴは、集積した光増感剤に波長６００〜７００ｎｍの光を照射することによって活性酸素種を生成させ、これにより疾患組織のみを選択的に壊死させる治療法である。これらのＰＤＤ及びＰＤＴは、これまでの診断法及び治療法と比較して、低侵襲で副作用が少ないため、患者に対する負担が少ないという利点がある。 In recent years, photodynamic diagnosis (Photodynamic diagnosis, which identifies and treats a target site by administering a compound that reacts with light as a method for diagnosing and treating diseased tissues such as cancer, warts, and acne, and irradiating light. In the following, attention is focused on PDD) and photodynamic therapy (hereinafter abbreviated as PDT). PDD, for example, administers a porphyrin-type or chlorin-type photosensitizer to a patient, accumulates it in a diseased tissue, emits light by irradiating light with a wavelength of around 400 nm, and observes this to cause disease. PDT is a diagnostic method for identifying the presence or absence and the affected area, and PDT generates active oxygen species by irradiating the accumulated photosensitizer with light having a wavelength of 600 to 700 nm, thereby selectively selecting only the diseased tissue. Necrotic treatment. Since these PDDs and PDTs are less invasive and have fewer side effects than conventional diagnostic and therapeutic methods, there is an advantage that the burden on the patient is small.
このＰＤＤ及びＰＤＴで用いる光増感剤として、ＡＬＡを用いる光線力学的診断（以下「ＡＬＡ−ＰＤＤ」ともいう）や、ＡＬＡを用いる光線力学的療法（以下「ＡＬＡ−ＰＤＴ」ともいう）の開発がすすんでおり、ＡＬＡは、細胞内でヘム生合成経路の一連の酵素群によりＰｐＩＸに代謝活性化される（特許文献１参照）が、ＰｐＩＸはガン組織に対する集積性が非常に高いとは言えず、十分な治療効果を得るためには、多量のＡＬＡを投与する必要がある。しかしながら、ＡＬＡは、２０ｍｇ／ｋｇＢ．Ｗ．以上投与すると、嘔吐、日光過敏症あるいは一時的な肝機能障害を惹起することが知られているので、その投与量にはおのずから制限がある。 Development of photodynamic diagnosis using ALA (hereinafter also referred to as “ALA-PDD”) and photodynamic therapy using ALA (hereinafter also referred to as “ALA-PDT”) as photosensitizers used in PDD and PDT. ALA is metabolically activated to PpIX by a series of enzymes in the heme biosynthetic pathway in cells (see Patent Document 1), but it can be said that PpIX has very high accumulation in cancer tissues. In order to obtain a sufficient therapeutic effect, it is necessary to administer a large amount of ALA. However, ALA is 20 mg / kg B.I. W. Since it is known that vomiting, sunlight hypersensitivity, or temporary liver dysfunction is caused by the above administration, the dosage is naturally limited.
したがって、ＡＬＡの投与量を増加させることなく、ＰｐＩＸの疾患組織への集積性を増大させるために、粘着付着性剤、例えば水膨潤性ポリマーを添加したもの（特許文献２参照）、キレート化剤、例えばアミノポリカルボン酸キレート化剤を併用したもの（特許文献３参照）、５−アミノレブリン酸アルキルエステルを前駆体としたもの（特許文献４参照）、担体物質としてリポソームのような脂質を併用したもの（特許文献５参照）などが提案されている。 Therefore, in order to increase the accumulation of PpIX in diseased tissue without increasing the dose of ALA, an adhesive agent, for example, a water-swellable polymer added (see Patent Document 2), chelating agent For example, those using aminopolycarboxylic acid chelating agents in combination (see Patent Document 3), those using 5-aminolevulinic acid alkyl ester as a precursor (see Patent Document 4), and lipids such as liposomes as carrier materials A thing (refer patent document 5) etc. are proposed.
しかしながら、５−アミノレブリン酸アルキルエステルは、疾患組織内へのＰｐＩＸの集積性は増加するものの、細胞に対する毒性も増加するため、安全性の点で問題があるし、キレート剤は、安全性は高いものの非経口投与製剤として用いなければならないため、患者に対する侵襲性が高くなるという欠点がある。また、リポソームによる送達は安全性の点では問題はないが、調製工程が煩雑であり、コスト高になるのを免れない。 However, although 5-aminolevulinic acid alkyl ester increases the accumulation of PpIX in diseased tissue, it also increases the toxicity to cells, so there is a problem in terms of safety, and chelating agents are highly safe. However, since it must be used as a preparation for parenteral administration, there is a drawback in that it is highly invasive to patients. In addition, delivery by liposome is not problematic in terms of safety, but the preparation process is complicated and inevitably costly.
本発明の課題は、ＡＬＡの投与量を増加させることなく、安全かつ簡単に疾患組織中へのＰｐＩＸの集積量を増加させることができるＰｐＩＸ細胞内集積増強組成物を提供することにある。 An object of the present invention is to provide a PpIX intracellular accumulation enhancing composition that can increase the amount of PpIX accumulated in a diseased tissue safely and easily without increasing the dose of ALA.
本発明者らは、ＰｐＩＸの前駆物質であるＡＬＡを投与した場合、体内においてＡＬＡから誘導されるＰｐＩＸの疾患組織中への集積量を増大させるため、ＡＬＡの投与方法の他、ＡＬＡ誘導体や併用化合物について鋭意検討してきたが、クラウンエーテルの一種である１８−クラウン−６又はその誘導体が、ＡＬＡと併用することでＰｐＩＸの疾患組織中への集積量を増大させる作用を有することを見い出した。また、他のクラウンエーテルの添加を検討する過程において、１５−クラウン−５を添加した場合には、同様の効果を得られないことを確認し、さらに、１８−クラウン−６又はその誘導体をＡＬＡ類と併用した場合、ＰｐＩＸの細胞内蓄積量の増加効果では説明しきれないほどのＰＤＴの増強効果も確認し、本発明を完成した。 When the present inventors administer ALA which is a precursor of PpIX, in order to increase the accumulation amount of PpIX derived from ALA in the diseased tissue in the body, in addition to the administration method of ALA, ALA derivatives and combinations Although compounds have been intensively studied, it has been found that 18-crown-6, which is a kind of crown ether, or a derivative thereof has an action of increasing the amount of PpIX accumulated in diseased tissues when used in combination with ALA. In addition, in the process of examining the addition of other crown ethers, it was confirmed that the same effect could not be obtained when 15-crown-5 was added. Further, 18-crown-6 or a derivative thereof was converted to ALA. When used in combination with the other species, the PDT enhancement effect that cannot be explained by the effect of increasing the intracellular accumulation of PpIX was confirmed, and the present invention was completed.
すなわち本発明は、（１）１８−クラウン−６又はその誘導体と、５−アミノレブリン酸類とを含有することを特徴とするプロトポルフィリンＩＸ細胞内集積増強組成物や、（２）１８−クラウン−６又はその誘導体と、５−アミノレブリン酸類とを、１８−クラウン−６又はその誘導体と、５−アミノレブリン酸類との複合体として、含有することを特徴とする上記（１）記載のプロトポルフィリンＩＸ細胞内集積増強組成物や、（３）１８−クラウン−６の誘導体が、ベンゾ−１８−クラウン−６、ジベンゾ−１８−クラウン−６、シクロヘキサノ−１８−クラウン−６、ジシクロヘキサノ−１８−クラウン−６、２−アミノメチル−１８−クラウン−６、２−ヒドロキシメチル−１８−クラウン−６であることを特徴とする上記（１）又は（２）記載のプロトポルフィリンＩＸ細胞内集積増強組成物や、（４）ＡＬＡ類が、５−アミノレブリン酸塩酸塩、臭化水素酸塩、ヨウ化水素酸塩、リン酸塩、硝酸塩、硫酸塩、酢酸塩、プロピオン酸塩、トルエンスルホン酸塩、コハク酸塩、シュウ酸塩、乳酸塩、酒石酸塩、グリコール酸塩、メタンスルホン酸塩、酪酸塩、吉草酸塩、クエン酸塩、フマル酸塩、マレイン酸塩、又はリンゴ酸塩であることを特徴とする上記（１）〜（３）のいずれか記載のプロトポルフィリンＩＸ細胞内集積増強組成物に関する。 That is, the present invention provides (1) 18-crown-6 or a derivative thereof and 5-aminolevulinic acid, and a composition for enhancing intracellular accumulation of protoporphyrin IX, (2) 18-crown-6 Or a derivative thereof and a 5-aminolevulinic acid compound as a complex of 18-crown-6 or a derivative thereof and a 5-aminolevulinic acid compound, The accumulation enhancing composition and (3) derivatives of 18-crown-6 are benzo-18-crown-6, dibenzo-18-crown-6, cyclohexano-18-crown-6, dicyclohexano-18-crown. -6, 2-aminomethyl-18-crown-6, 2-hydroxymethyl-18-crown-6 above (1) And (4) A composition for enhancing intracellular accumulation of protoporphyrin IX according to (2), or (4) ALA is 5-aminolevulinic acid hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfuric acid Salt, acetate, propionate, toluenesulfonate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumaric acid The composition for enhancing protoporphyrin IX intracellular accumulation according to any one of (1) to (3) above, which is a salt, maleate or malate.
また、本発明は、（５）上記（１）〜（４）いずれか記載のプロトポルフィリンＩＸ細胞内集積増強組成物を有効成分として含有する、５−アミノレブリン酸−光線力学的診断剤や、（６）上記（１）〜（４）いずれか記載のプロトポルフィリンＩＸ細胞内集積増強組成物を有効成分として含有する、５−アミノレブリン酸−光線力学的治療剤に関する。 The present invention also includes (5) a 5-aminolevulinic acid-photodynamic diagnostic agent comprising, as an active ingredient, the protoporphyrin IX intracellular accumulation enhancing composition according to any one of (1) to (4) above, 6) A 5-aminolevulinic acid-photodynamic therapeutic agent comprising the protoporphyrin IX intracellular accumulation enhancing composition according to any one of (1) to (4) above as an active ingredient.
本発明のＰｐＩＸ細胞内集積増強組成物を用いることにより、ＡＬＡの投与量を増加させることなく、安全かつ簡単に疾患組織中へのＰｐＩＸの集積量を増加させることができ、ＡＬＡ−ＰＤＤやＡＬＡ−ＰＤＴの効果を増大させることができる。特に、ＡＬＡ−ＰＤＴについては相乗効果が期待できる。 By using the PpIX intracellular accumulation enhancing composition of the present invention, the amount of PpIX accumulated in a diseased tissue can be increased safely and easily without increasing the dose of ALA, and ALA-PDD and ALA -The effect of PDT can be increased. In particular, a synergistic effect can be expected for ALA-PDT.
本発明のＰｐＩＸ細胞内集積増強組成物としては、１８−クラウン−６（エーテル）又はその誘導体から構成される１８−クラウン−６類と、ＡＬＡ類とを含有する組成物であれば特に制限されず、上記１８−クラウン−６（１，４，７，１０，１３，１６−ヘキサオキサシクロオクタデカン）の誘導体として、具体的には、ベンゾ−１８−クラウン−６、ジベンゾ−１８−クラウン−６、シクロヘキサノ−１８−クラウン−６、ジシクロヘキサノ−１８−クラウン−６、２−アミノメチル−１８−クラウン−６、２−ヒドロキシメチル−１８−クラウン−６等を挙げることができる。さらに、Ｏが一箇所以上Ｓに置き換わった１８−チアクラウン−６エーテル又はその誘導体や、Ｏが一箇所以上ＮＨに置き換わった１８−アザクラウン−６エーテル又はその誘導体や、Ｏがそれぞれ一箇所以上Ｓ及びＮＨに置き換わった１８−アザチアクラウン−６エーテル又はその誘導体などを挙げることができる。 The PpIX intracellular accumulation enhancing composition of the present invention is not particularly limited as long as it is a composition containing 18-crown-6 composed of 18-crown-6 (ether) or a derivative thereof and ALAs. As derivatives of the above 18-crown-6 (1,4,7,10,13,16-hexaoxacyclooctadecane), specifically, benzo-18-crown-6, dibenzo-18-crown-6 Cyclohexano-18-crown-6, dicyclohexano-18-crown-6, 2-aminomethyl-18-crown-6, 2-hydroxymethyl-18-crown-6, and the like. Further, 18-thiacrown-6 ether or a derivative thereof in which O is replaced with one or more S, 18-azacrown-6 ether or a derivative thereof in which O is replaced with one or more NH, or O is one or more in each place Examples thereof include 18-azathiacrown-6 ether substituted with S and NH, or a derivative thereof.
上記ＡＬＡ類としては、ＡＬＡ又はＡＬＡの塩が含まれ、ＡＬＡは、式ＨＯＯＣ−（ＣＨ２）２−（ＣＯ）−ＣＨ２−ＮＨ２で表されるアミノ酸の一種であり、例えば特開平４−９３６０号公報等に記載された化学合成法や、微生物による生産、酵素による生産等のいずれの公知の方法によっても製造することができる。 The ALA includes ALA or an ALA salt, and ALA is a kind of amino acid represented by the formula HOOC- (CH 2 ) 2 — (CO) —CH 2 —NH 2. It can be produced by any known method such as the chemical synthesis method described in JP-9360, etc., production by microorganisms, production by enzymes, or the like.
ＡＬＡ類のうち、ＡＬＡの塩としては、ＡＬＡは両性電解質であり、ｐＨを変えることにより、酸性塩、中性塩又は塩基性塩のいずれの塩も形成可能であるが、安定性がよいという点でＡＬＡ酸性塩が好ましく、具体的には、塩酸塩、臭化水素酸塩、ヨウ化水素酸塩、リン酸塩、硝酸塩、硫酸塩、酢酸塩、プロピオン酸塩、トルエンスルホン酸塩、コハク酸塩、シュウ酸塩、乳酸塩、酒石酸塩、グリコール酸塩、メタンスルホン酸塩、酪酸塩、吉草酸塩、クエン酸塩、フマル酸塩、マレイン酸塩、リンゴ酸塩等を挙げることができ、なかでも塩酸塩が好ましい。なお、これらの塩は使用時において、溶液として用いることもでき、その作用は、ＡＬＡの場合と同効なものが好ましい。 Among the ALAs, ALA is an ampholyte as a salt of ALA. By changing pH, any salt of acid salt, neutral salt or basic salt can be formed, but it is said to have good stability. ALA acid salts are preferable in terms of points, and specifically, hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, propionate, toluenesulfonate, succinate Acid, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate, etc. Of these, hydrochloride is preferable. These salts can also be used as a solution at the time of use, and the action is preferably the same as that of ALA.
以上の１８−クラウン−６類やＡＬＡ類を含む組成物は、水和物又は溶媒和物を形成していてもよく、溶媒としては、水又は生理的食塩水が好ましいが、溶解性を高めるために、水溶性有機溶剤、例えばメタノール、エタノール、ｎ−プロパノール、アセトン、グリセリン、プロビレングリコール、酢酸、ジメチルスルホキシドを併用することもできる。また、本発明の組成物は、散剤、顆粒剤、錠剤、カプセル剤、シロップ剤、液剤、軟膏、巴布剤等の剤型で経口・経皮・静脈投与することができ、軟膏又は巴布剤として経皮的に施用する場合には、経皮吸収を促進するために経皮吸収促進剤、例えばｌ−メントールやｄ−リモネンなどのテルペン系化合物、Ｌ−α−ホスファチジルコリンなどのリン脂質、Ｔｗｅｅｎ系又はＳｐａｎ系界面活性剤、ラウリン酸やオレイン酸などの脂肪酸又はその塩などを適宜配合して使用してもよい。 The composition containing the above 18-crown-6 or ALA may form a hydrate or a solvate, and the solvent is preferably water or physiological saline, but increases the solubility. Therefore, a water-soluble organic solvent such as methanol, ethanol, n-propanol, acetone, glycerin, propylene glycol, acetic acid or dimethyl sulfoxide can be used in combination. The composition of the present invention can be administered orally, transdermally or intravenously in dosage forms such as powders, granules, tablets, capsules, syrups, liquids, ointments, and sachets. When applied transdermally as an agent, in order to promote percutaneous absorption, transdermal absorption enhancers, for example, terpene compounds such as l-menthol and d-limonene, phospholipids such as L-α-phosphatidylcholine, A Tween-based or Span-based surfactant, a fatty acid such as lauric acid or oleic acid, or a salt thereof may be appropriately blended and used.
また、ＡＬＡ類を経口・静脈投与する場合の、ＡＬＡ類の投与量は、ＡＬＡ類の合計が、ＡＬＡ換算で体重１ｋｇあたり、１ｍｇ〜１０００ｍｇ、好ましくは５ｍｇ〜１００ｍｇ、より好ましくは１０ｍｇ〜３０ｍｇ、さらに好ましくは１５ｍｇ〜２５ｍｇである。また、例えばＡＬＡ２０％含有クリームを用いて、ＡＬＡ類を経皮投与する場合の、ＡＬＡ類の投与量は、ＡＬＡ類の合計が、ＡＬＡ換算で、１ｍｇ〜９０ｍｇ、好ましくは２ｍｇ〜５０ｍｇ、より好ましくは５ｍｇ〜３０ｍｇである。 In addition, when ALAs are administered orally or intravenously, the total amount of ALAs is 1 mg to 1000 mg, preferably 5 mg to 100 mg, more preferably 10 mg to 30 mg per kg body weight in terms of ALA. More preferably, it is 15 mg-25 mg. For example, when ALA is transdermally administered using a cream containing ALA 20%, the total amount of ALAs is 1 mg to 90 mg, preferably 2 mg to 50 mg, more preferably ALA in terms of the total amount of ALAs. Is 5-30 mg.
ＡＬＡ類と１８−クラウン−６類との使用割合は、モル比で１：１〜１：２０、好ましく１：３〜１：１５であり、また、上記１８−クラウン−６類とＡＬＡ類とは、複合体を形成していてもよく、ＡＬＡ類のアミノ基と１８−クラウン−６類とが複合体を形成すると、ＡＬＡ類は疎水性が増加し、ＡＬＡ類が細胞膜を直接に通過する受動的取込が促進されるため、ＰｐＩＸの細胞内集積性が増加すると考えることもできるが、細胞内ＰｐＩＸ集積性の増加から類推される効果を超えたＡＬＡ−ＰＤＴによる殺細胞効果からすると、本発明におけるＡＬＡ−ＰＤＴ効果の作用機序は不明である。 The use ratio of ALAs to 18-crown-6 is 1: 1 to 1:20, preferably 1: 3 to 1:15 in molar ratio, and the 18-crown-6 and ALA May form a complex, and when the amino group of ALA and 18-crown-6 form a complex, the hydrophobicity of ALA increases and the ALA directly passes through the cell membrane. Since passive uptake is promoted, it can be considered that the intracellular accumulation of PpIX is increased, but from the cell killing effect by ALA-PDT that exceeds the effect estimated from the increase in intracellular PpIX accumulation, The mechanism of action of the ALA-PDT effect in the present invention is unknown.
本発明のＰｐＩＸ細胞内集積増強組成物は、ＡＬＡ−ＰＤＤ用の診断剤として使用することができ、本発明のＡＬＡ−ＰＤＤ診断剤を用いると、皮膚又は他の上皮組織の種々の異常部位、疾患部位等の病変部の範囲を判断することができる。すなわち、病変部の治療（手術）において、病変部の範囲を判断する際に、それ自身は光増感作用を有さないＡＬＡ類を１８−クラウン−６類と共に投与し、ＡＬＡ類から色素生合成経路を経て誘導されたＰｐＩＸが、病変部組織内に特異的に蓄積し、紫色〜青色の励起光を照射すると、赤色の蛍光を放射することを利用して、赤色の波長を検出することで、がん等の病変部の範囲を判断することができる。具体的には、本発明の組成物を投与し、３〜６時間経過後、波長４００ｎｍ付近の光を照射して発光させ、生じる蛍光を観察する方法を例示することができる。このように、本発明の実施態様のひとつに、本発明のＰｐＩＸ細胞内集積増強組成物を用いたＡＬＡ−ＰＤＤ方法が含まれる。 The PpIX intracellular accumulation enhancing composition of the present invention can be used as a diagnostic agent for ALA-PDD, and when the ALA-PDD diagnostic agent of the present invention is used, various abnormal sites in the skin or other epithelial tissues, The extent of a lesion such as a disease site can be determined. That is, in the treatment (surgery) of a lesioned part, when determining the range of the lesioned part, ALAs that do not themselves have a photosensitizing action are administered together with 18-crown-6, and pigmentation from the ALAs occurs. Detecting the red wavelength by utilizing the fact that PpIX induced through the synthetic pathway accumulates specifically in the lesion tissue and emits red fluorescence when irradiated with purple to blue excitation light Thus, the range of a lesion such as cancer can be determined. Specifically, a method of observing the generated fluorescence by administering the composition of the present invention and emitting light by irradiating light having a wavelength of around 400 nm after 3 to 6 hours has elapsed can be exemplified. Thus, one of the embodiments of the present invention includes an ALA-PDD method using the PpIX intracellular accumulation enhancing composition of the present invention.
また、本発明のＰｐＩＸ細胞内集積増強組成物は、ＡＬＡ−ＰＤＴ用治療剤として使用することもでき、上記ＡＬＡ−ＰＤＴ治療剤を用いると、皮膚又は他の上皮組織の種々の異常部位、疾患部位等の病変部組織を壊死させることができる。すなわち、光に反応する化合物を投与し、光を照射することにより病変部組織を治療するＰＤＴを行う際に、それ自身は光増感作用を有さないＡＬＡ類を、１８−クラウン−６類と共に投与し、ＡＬＡ類から色素生合成経路を経て誘導されたＰｐＩＸが、病変部組織内に特異的に蓄積し、光照射されることにより周囲の酸素分子を光励起し、その結果生成する一重項酸素が、その強い酸化力による殺細胞効果を有することを利用する病変部組織を壊死させる方法・手段を挙げることができる。具体的には、本発明の組成物を投与して、３〜６時間経過後、患部に５０〜５００ｍＷ／ｃｍ２で波長６００〜７００ｎｍの光を全照射エネルギーが５０〜３００Ｊ／ｃｍ２になるように照射する方法を例示することができる。このように、本発明の実施態様のひとつに、本発明のＰｐＩＸ細胞内集積増強組成物を用いたＡＬＡ−ＰＤＴ方法が含まれる。 In addition, the PpIX intracellular accumulation enhancing composition of the present invention can also be used as a therapeutic agent for ALA-PDT. When the ALA-PDT therapeutic agent is used, various abnormal sites or diseases of the skin or other epithelial tissues are used. The lesioned tissue such as a site can be necrotized. That is, when PDT for treating a lesioned tissue by administering a compound that reacts with light and irradiating light is performed, ALAs that do not themselves have a photosensitizing action are converted to 18-crown-6s. PpIX, which is administered together with ALAs via the pigment biosynthetic pathway, specifically accumulates in the lesion tissue and is irradiated with light to photoexcite surrounding oxygen molecules, resulting in singlet Examples include methods and means for necroticizing lesion tissue utilizing the fact that oxygen has a cell-killing effect due to its strong oxidizing power. Specifically, 3 to 6 hours after administration of the composition of the present invention, the affected area is irradiated with light having a wavelength of 600 to 700 nm at 50 to 500 mW / cm 2 and a total irradiation energy of 50 to 300 J / cm 2 . The method of irradiating can be illustrated. Thus, one embodiment of the present invention includes an ALA-PDT method using the PpIX intracellular accumulation enhancing composition of the present invention.
（ＰｐＩＸ集積性に対する１８−クラウン−６添加効果）
５×１０５個／ｍＬに調整したヒト組織球性リンパ腫由来細胞株Ｕ９３７細胞（以下、Ｕ９３７細胞）５ｍＬにＡＬＡ塩酸塩１．０ｍＭと１８−クラウン−６エーテルとを所定濃度添加し、３７℃恒温下にて３時間インキュベーションした。インキュベーション後、ハンクス緩衝溶液を用いて２回洗浄し、蛍光分光光度計（ＦＰ-６５００、日本分光社製）を用いて細胞内ＰｐＩＸの蛍光強度を測定した。励起波長及び測定波長領域は、それぞれ４１０ｎｍ及び６２０〜６５０ｎｍとした。
(Effect of 18-crown-6 addition on PpIX accumulation)
ALA hydrochloride 1.0 mM and 18-crown-6 ether were added at predetermined concentrations to 5 mL of human histiocytic lymphoma-derived cell line U937 cells (hereinafter referred to as U937 cells) adjusted to 5 × 10 5 cells / mL, and 37 ° C. Incubated for 3 hours at constant temperature. After incubation, the plate was washed twice with Hanks buffer solution, and the fluorescence intensity of intracellular PpIX was measured using a fluorescence spectrophotometer (FP-6500, manufactured by JASCO Corporation). The excitation wavelength and measurement wavelength regions were 410 nm and 620 to 650 nm, respectively.
（結果）
１．０ｍＭのＡＬＡ塩酸塩に１８−クラウン−６を、濃度を変えて添加した場合の、細胞内ＰｐＩＸの蛍光強度比を図１に示す。ただし蛍光強度比とは、ＡＬＡ塩酸塩１．０ｍＭのみを添加した際のＰｐＩＸに対する蛍光強度の百分率である。この結果、１８−クラウン−６の添加量が増すにつれて。細胞内ＰｐＩＸ集積性が向上し、７．５ｍＭ付近で蛍光強度比が極大値を示した。一方、１０ｍＭ以上添加すると細胞内ＰｐＩＸ集積性が減少するが、これは１８−クラウン−６の細胞毒性によるためと考えられる。このことは、ＡＬＡ塩酸塩無添加系において、１８−クラウン−６の細胞生存率に対する影響を検討した図２の結果からも示唆される。
(result)
FIG. 1 shows the fluorescence intensity ratio of intracellular PpIX when 18-crown-6 was added to 1.0 mM ALA hydrochloride at different concentrations. However, the fluorescence intensity ratio is the percentage of the fluorescence intensity with respect to PpIX when only 1.0 mM ALA hydrochloride is added. As a result, as the amount of 18-crown-6 added increases. Intracellular PpIX accumulation was improved, and the fluorescence intensity ratio showed a maximum at around 7.5 mM. On the other hand, addition of 10 mM or more decreases intracellular PpIX accumulation, which is considered to be due to cytotoxicity of 18-crown-6. This is also suggested from the results of FIG. 2 in which the effect of 18-crown-6 on the cell viability was examined in the ALA hydrochloride-free system.
［比較例１］
上記実施例１と同様に調整したＵ９３７細胞に、ＡＬＡ塩酸塩１．０ｍＭと１５−クラウン−５とを添加した際の細胞内ＰｐＩＸの蛍光強度比の変化を図３に示す。１８−クラウン−６と異なり、１５−クラウン−５を添加するとＰｐＩＸの細胞内蛍光強度は増加するどころか、逆に減少する傾向にあることがわかる。このことから、細胞内ＰｐＩＸ集積性に及ぼすクラウンエーテルの添加効果は、１８−クラウン−６にのみ見いだされる現象であることが示唆された。
[Comparative Example 1]
FIG. 3 shows changes in the fluorescence intensity ratio of intracellular PpIX when ALA hydrochloride 1.0 mM and 15-crown-5 were added to U937 cells prepared in the same manner as in Example 1 above. It can be seen that, unlike 18-crown-6, when 15-crown-5 is added, the intracellular fluorescence intensity of PpIX tends to decrease rather than increase. This suggests that the effect of adding crown ether on intracellular PpIX accumulation is a phenomenon found only in 18-crown-6.
（ＰｐＩＸ集積性に対する１８−クラウン−６の細胞接触時間の影響）
５×１０５個／ｍＬに調整したＵ９３７細胞５ｍＬにＡＬＡ塩酸塩１．０ｍＭを添加したものに、さらに１８−クラウン−６を７．５ｍＭ添加し、３７℃恒温下で所定時間インキュベーションした。ただし、ＡＬＡ塩酸塩添加後のインキュベーション時間は３時間で固定した。インキュベーション後、ハンクス緩衝溶液を用いて２回洗浄し、蛍光分光光度計を用いて上記実施例１と同様の方法で、細胞内ＰｐＩＸの蛍光強度を測定した。
(Effect of 18-crown-6 cell contact time on PpIX accumulation)
To 5 mL of U937 cells adjusted to 5 × 10 5 cells / mL, 1.0 mM of ALA hydrochloride was added to 7.5 mL of 18-crown-6, and incubated at 37 ° C. for a predetermined time. However, the incubation time after addition of ALA hydrochloride was fixed at 3 hours. After incubation, the plate was washed twice with Hanks buffer solution, and the fluorescence intensity of intracellular PpIX was measured using a fluorescence spectrophotometer in the same manner as in Example 1 above.
（結果）
図４に、１８−クラウン−６添加後のインキュベーション時間に対する細胞内ＰｐＩＸの蛍光強度比を示す。ただし蛍光強度比とは、ＡＬＡ塩酸塩１．０ｍＭのみを添加した際のＰｐＩＸに対する蛍光強度比の百分率である。１８−クラウン−６添加後のインキュベーション時間の増加に伴い細胞内ＰｐＩＸ集積性は増加し、３時間で極大値を示すことがわかった。
(result)
FIG. 4 shows the fluorescence intensity ratio of intracellular PpIX with respect to the incubation time after addition of 18-crown-6. However, the fluorescence intensity ratio is the percentage of the fluorescence intensity ratio with respect to PpIX when only 1.0 mM ALA hydrochloride is added. It was found that intracellular PpIX accumulation increased with an increase in incubation time after addition of 18-crown-6, and showed a maximum value at 3 hours.
（ＰＤＴに対するＡＬＡ及び／又は１８−クラウン−６の添加効果）
５×１０５個／ｍＬに調整したＵ９３７細胞５ｍＬに、ＡＬＡ塩酸塩を１．０ｍＭ及び／又は１８−クラウン−６を７．５ｍＭ添加し、３７℃恒温下で３時間インキュベーションした。インキュベーション後、ヨウ化ナトリウムとヨウ化リチウムとを封入したメタルハライドランプ（ウシオ電機社製）を用いて波長領域５５０〜７５０ｎｍの光（放射強度：４０ｍＷ／ｃｍ２）を６０分間照射し、生細胞数を測定して細胞生存率を算出した。ただし、コントロールには生理食塩水を用いた。
(Additional effect of ALA and / or 18-crown-6 on PDT)
To 5 mL of U937 cells adjusted to 5 × 10 5 cells / mL, 1.0 mM ALA hydrochloride and / or 7.5 mM 18-crown-6 was added, and incubated at 37 ° C. for 3 hours. After incubation, a metal halide lamp (USHIO INC.) Encapsulating sodium iodide and lithium iodide was used to irradiate light with a wavelength region of 550 to 750 nm (radiation intensity: 40 mW / cm 2 ) for 60 minutes, and the number of living cells Was measured to calculate the cell viability. However, physiological saline was used for control.
（結果）
結果を図５に示す。細胞生存率はコントロールの細胞生存数を１００％として表した。細胞生存率が低いほどＰＤＴ効果が高いことを意味する。１８−クラウン−６のみを添加した系ではＰＤＴ効果はまったく認められなかった。また、ＡＬＡ塩酸塩のみを添加した系では細胞生存率は７５．２％であった。しかし、ＡＬＡ塩酸塩と１８−クラウン−６とを併用すると細胞生存率は２１．０％と急激に低下した。これはＡＬＡ塩酸塩と１８−クラウン−６共存下において、ＰｐＩＸの細胞内集積性が増加したという理由だけでは説明できない。
(result)
The results are shown in FIG. The cell viability was expressed with the cell viability of the control as 100%. The lower the cell viability, the higher the PDT effect. In the system to which only 18-crown-6 was added, no PDT effect was observed. In the system to which only ALA hydrochloride was added, the cell viability was 75.2%. However, when ALA hydrochloride and 18-crown-6 were used in combination, the cell viability decreased rapidly to 21.0%. This cannot be explained only by the fact that the intracellular accumulation of PpIX was increased in the presence of ALA hydrochloride and 18-crown-6.
（ＰｐＩＸ集積性に対する１８−クラウン−６の濃度依存性）
５×１０５個／ｍＬに調整したＵ９３７細胞５ｍＬに０．２５、０．５、又は１．０ｍＭのＡＬＡ塩酸塩を添加し、さらに１８−クラウン−６をそれぞれ０、２．５、５．０、７．５、又は１０ｍＭ添加し、３７℃恒温下で３時間インキュベーションした。インキュベーション後、ハンクス緩衝溶液を用いて２回洗浄し、蛍光分光光度計を用いて上記実施例１と同様の方法で、細胞内ＰｐＩＸの蛍光強度を測定した。図６に、ＡＬＡ塩酸塩濃度を０．２５、０．５及び１．０ｍＭとした時の１８−クラウン−６の添加濃度に対する細胞内ＰｐＩＸの蛍光強度比を示す。ただし蛍光強度比は、各濃度のＡＬＡのみを添加した際のＰｐＩＸの蛍光強度を１００％とした値を示した。また、図６の結果を基に、縦軸の蛍光強度比を１．０ｍＭのＡＬＡのみを添加した際のＰｐＩＸの蛍光強度を基準に整理しなおした図を図７に示す。
(Dependence of 18-crown-6 concentration on PpIX accumulation)
0.25, 0.5, or 1.0 mM ALA hydrochloride is added to 5 mL of U937 cells adjusted to 5 × 10 5 cells / mL, and 18-crown-6 is added to 0, 2.5, 5. 0, 7.5, or 10 mM was added and incubated at 37 ° C. for 3 hours. After incubation, the plate was washed twice with Hanks buffer solution, and the fluorescence intensity of intracellular PpIX was measured using a fluorescence spectrophotometer in the same manner as in Example 1 above. FIG. 6 shows the fluorescence intensity ratio of intracellular PpIX with respect to the added concentration of 18-crown-6 when the ALA hydrochloride concentration is 0.25, 0.5 and 1.0 mM. However, the fluorescence intensity ratio showed a value where the fluorescence intensity of PpIX when adding only ALA of each concentration was 100%. Moreover, based on the result of FIG. 6, the figure which rearranged the fluorescence intensity ratio of the vertical axis | shaft on the basis of the fluorescence intensity of PpIX at the time of adding only 1.0 mM ALA is shown in FIG.
（結果）
図６より、いずれのＡＬＡ濃度においてもＡＬＡ単独投与よりも、１８−クラウン−６を添加することで、細胞内ＰｐＩＸ集積性が増加し、その傾向はＡＬＡ濃度が高いほど顕著であることがわかった。また、図７より、ＡＬＡ濃度が０．５ｍＭの場合、１８−クラウン−６を２．５ｍＭ以上共存させると、１．０ｍＭのＡＬＡ単独投与の場合よりもＰｐＩＸの細胞内集積性が増加することがわかった。
(result)
From FIG. 6, it can be seen that by adding 18-crown-6 at any ALA concentration, intracellular PpIX accumulation is increased by adding 18-crown-6, and the tendency is more prominent as the ALA concentration is higher. It was. Further, from FIG. 7, when the ALA concentration is 0.5 mM, the presence of 18-crown-6 in the presence of 2.5 mM or more increases the intracellular accumulation of PpIX compared to the case of administering 1.0 mM ALA alone. I understood.
（ＰｐＩＸ集積性に対するベンゾ−１８−クラウン−６添加効果）
５×１０５個／ｍＬに調整したＵ９３７細胞５ｍＬにＡＬＡ塩酸塩１．０ｍＭとベンゾ−１８−クラウン−６とを所定濃度添加し、３７℃恒温下にて３時間インキュベーションした。インキュベーション後、ハンクス緩衝溶液を用いて２回洗浄し、蛍光分光光度計を用いて上記実施例１と同様の方法で、細胞内ＰｐＩＸの蛍光強度を測定した。ただし、ベンゾ−１８−クラウン−６を５ｍＭ以上投与すると細胞毒性が大きくなるので（図８参照）、ベンゾ−１８−クラウン−６の添加濃度は５ｍＭ以下とした。
(Effect of adding benzo-18-crown-6 to PpIX accumulation)
Predetermined concentrations of 1.0 mM ALA hydrochloride and benzo-18-crown-6 were added to 5 mL of U937 cells adjusted to 5 × 10 5 cells / mL, and incubated at 37 ° C. for 3 hours. After incubation, the plate was washed twice with Hanks buffer solution, and the fluorescence intensity of intracellular PpIX was measured using a fluorescence spectrophotometer in the same manner as in Example 1 above. However, since the cytotoxicity increases when benzo-18-crown-6 is administered at 5 mM or more (see FIG. 8), the concentration of benzo-18-crown-6 added is 5 mM or less.
（結果）
図９に、ベンゾ−１８−クラウン−６の添加濃度に対する細胞内ＰｐＩＸの蛍光強度比を示す。ただし、ＡＬＡ塩酸塩１．０ｍＭのみを添加した際のＰｐＩＸの蛍光強度を１００％とした。その結果、１８−クラウン−６と同様に、ＡＬＡ塩酸塩にベンゾ−１８−クラウン−６を添加することによって細胞内ＰｐＩＸ集積性が向上することがわかった。
(result)
FIG. 9 shows the fluorescence intensity ratio of intracellular PpIX with respect to the added concentration of benzo-18-crown-6. However, the fluorescence intensity of PpIX when only 1.0 mM ALA hydrochloride was added was taken as 100%. As a result, it was found that the intracellular PpIX accumulation property was improved by adding benzo-18-crown-6 to ALA hydrochloride as in the case of 18-crown-6.
（ＰＤＴに対するＡＬＡ及び／又は１８−クラウン−６の添加効果）
５×１０５個／ｍＬに調整したＵ９３７細胞５ｍＬに０．２５、０．５、又は１．０ｍＭのＡＬＡ塩酸塩を添加し、さらに１８−クラウン−６をそれぞれ７．５ｍＭ添加し、３７℃恒温下で３時間インキュベーションした。インキュベーション後、上記実施例３と同様な方法で光照射し、生細胞数を測定して細胞生存率を算出した。ただし、コントロールには生理食塩水を用いた。
(Additional effect of ALA and / or 18-crown-6 on PDT)
0.25, 0.5, or 1.0 mM ALA hydrochloride was added to 5 mL of U937 cells adjusted to 5 × 10 5 cells / mL, and 7.5 mM of 18-crown-6 was added to each of the cells. Incubated for 3 hours under constant temperature. After incubation, the cells were irradiated with light in the same manner as in Example 3 above, and the number of viable cells was measured to calculate the cell viability. However, physiological saline was used for control.
（結果）
結果を図１０に示す。細胞生存率はコントロールの細胞生存数を１００％として表した。細胞生存率が低いほどＰＤＴ効果が高いことを意味する。０．５、又は１．０ｍＭのＡＬＡ塩酸塩を添加した系では１８−クラウン−６を併用すると細胞生存率が急激に低下した。しかし、０．２５ｍＭのＡＬＡ塩酸塩を添加した系では顕著な変化は認められなかった。
(result)
The results are shown in FIG. The cell viability was expressed with the cell viability of the control as 100%. The lower the cell viability, the higher the PDT effect. In the system to which 0.5 or 1.0 mM ALA hydrochloride was added, cell viability decreased rapidly when 18-crown-6 was used in combination. However, no significant change was observed in the system to which 0.25 mM ALA hydrochloride was added.
（ＰＤＴに対するＡＬＡ及び／又はベンゾ−１８−クラウン−６の添加効果）
５×１０５個／ｍＬに調整したＵ９３７細胞５ｍＬに、ＡＬＡ塩酸塩を１．０ｍＭ及び／又はベンゾ−１８−クラウン−６を３．７５ｍＭ添加し、３７℃恒温下で３時間インキュベーションした。インキュベーション後、上記実施例３と同様な方法で光照射し、生細胞数を測定して細胞生存率を算出した。ただし、コントロールには生理食塩水を用いた。
(Additional effect of ALA and / or benzo-18-crown-6 on PDT)
To 5 mL of U937 cells adjusted to 5 × 10 5 cells / mL, 1.0 mM of ALA hydrochloride and / or 3.75 mM of benzo-18-crown-6 were added and incubated at 37 ° C. for 3 hours. After incubation, the cells were irradiated with light in the same manner as in Example 3 above, and the number of viable cells was measured to calculate the cell viability. However, physiological saline was used for control.
（結果）
結果を図１１に示す。細胞生存率はコントロールの細胞生存数を１００％として表した。細胞生存率が低いほどＰＤＴ効果が高いことを意味する。ベンゾ−１８−クラウン−６のみを添加した系ではＰＤＴ効果はまったく認められなかった。また、ＡＬＡ塩酸塩のみを添加した系では細胞生存率は８５．０％であった。しかし、ＡＬＡ塩酸塩と１８−クラウン−６とを併用すると細胞生存率は２０．６％と急激に低下した。
(result)
The results are shown in FIG. The cell viability was expressed with the cell viability of the control as 100%. The lower the cell viability, the higher the PDT effect. In the system to which only benzo-18-crown-6 was added, no PDT effect was observed. In the system to which only ALA hydrochloride was added, the cell viability was 85.0%. However, when ALA hydrochloride and 18-crown-6 were used in combination, the cell viability decreased rapidly to 20.6%.
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| US11633505B2 (en) | 2012-12-11 | 2023-04-25 | National University Corporation Kumamoto University | Nuclear magnetic resonance diagnostic agent, and method for detecting or diagnosing state of cell, tissue or organ in subject using same |
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