JP2001064199A - Medicinal composition containing superoxide dismutase formed into lecithin - Google Patents
Medicinal composition containing superoxide dismutase formed into lecithinInfo
- Publication number
- JP2001064199A JP2001064199A JP2000190834A JP2000190834A JP2001064199A JP 2001064199 A JP2001064199 A JP 2001064199A JP 2000190834 A JP2000190834 A JP 2000190834A JP 2000190834 A JP2000190834 A JP 2000190834A JP 2001064199 A JP2001064199 A JP 2001064199A
- Authority
- JP
- Japan
- Prior art keywords
- sod
- superoxide dismutase
- pharmaceutical composition
- freeze
- composition according
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- 108010012715 Superoxide dismutase Proteins 0.000 title claims abstract description 35
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- 239000000787 lecithin Substances 0.000 title claims abstract description 13
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、レシチン化スーパ
ーオキシドジスムターゼ（以下、単にＰＣ−ＳＯＤとも
いう）及びシュークロースを含有する医薬組成物、およ
びこれからなる疾患の処置剤に関する。また本発明は、
シュークロースを有効成分とするＰＣ−ＳＯＤの活性低
下等を抑制するための剤及び方法に関する。TECHNICAL FIELD The present invention relates to a pharmaceutical composition containing lecithinated superoxide dismutase (hereinafter, also simply referred to as PC-SOD) and sucrose, and an agent for treating a disease comprising the same. The present invention also provides
The present invention relates to an agent and a method for suppressing a decrease in the activity of PC-SOD containing sucrose as an active ingredient.
【０００２】[0002]
【従来の技術】本発明に最も近い先行技術について説明
する。 （ａ）特開平９−１１７２７９号公報には、本発明にお
いて用いることができるＰＣ−ＳＯＤおよびこれを有効
成分として含む医薬について記載されている。 （ｂ）特開平３−１７０４３８号公報には、レシチン化
生物活性蛋白を含む経口及び局所投与用生物活性蛋白組
成物が記載されている。また生物活性蛋白の例示として
スーパーオキシドジスムターゼ（以下、単にＳＯＤとも
いう）が記載されており、また添加物として砂糖が例示
されている。2. Description of the Related Art The prior art closest to the present invention will be described. (A) JP-A-9-117279 describes PC-SOD which can be used in the present invention and a medicine containing the same as an active ingredient. (B) JP-A-3-170438 describes a bioactive protein composition for oral and topical administration containing a lecithinated bioactive protein. Further, superoxide dismutase (hereinafter, also simply referred to as SOD) is described as an example of the biologically active protein, and sugar is illustrated as an additive.
【０００３】しかしながらこれらの先行技術には、ＰＣ
−ＳＯＤ及びシュークロースを含有する医薬組成物の特
定の組み合わせについては記載されていない。またＰＣ
−ＳＯＤの長期間保存による活性の低下、凍結乾燥した
場合の性状、カラムクロマトグラフィーによる分析の際
の類縁物質ピークの出現の問題については解決されてい
なかった。[0003] However, these prior arts include PCs.
-No specific combinations of pharmaceutical compositions containing SOD and sucrose are described. Also PC
The problems of reduced activity due to long-term storage of -SOD, properties when freeze-dried, and appearance of related substance peaks during analysis by column chromatography have not been solved.
【０００４】[0004]
【発明が解決しようとする課題】本発明は、長期間保存
によるＰＣ−ＳＯＤの活性低下が少なく（すなわち安定
性が高く）、凍結乾燥した場合の性状が良好で、かつカ
ラムクロマトグラフィーによる分析の際の類縁物質ピー
クの出現が抑制されたＰＣ−ＳＯＤを含有する医薬組成
物を提供することを課題とする。DISCLOSURE OF THE INVENTION The present invention provides a PC-SOD having a reduced activity (that is, high stability) due to long-term storage, good properties when freeze-dried, and an analytical method using column chromatography. An object of the present invention is to provide a pharmaceutical composition containing PC-SOD in which the appearance of an analog peak at the time is suppressed.
【０００５】[0005]
【課題を解決するための手段】本発明者らは上記課題を
解決するために、ＰＣ−ＳＯＤを有効成分とする医薬組
成物について鋭意検討を行った結果、シュークロース
が、長期間保存によるＰＣ−ＳＯＤの活性低下を抑制し
（安定性を保持させ）、凍結乾燥した場合の性状を良好
に保ち、かつカラムクロマトグラフィーによる分析の際
の類縁物質ピークの出現を抑制する作用を有することを
見い出し、これにより上記課題を解決しうる医薬組成物
が提供できることを見い出し、本発明を完成した。Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have conducted intensive studies on pharmaceutical compositions containing PC-SOD as an active ingredient. -It has been found that the compound has an effect of suppressing a decrease in SOD activity (preserving stability), maintaining good properties when freeze-dried, and suppressing the appearance of peaks of related substances during analysis by column chromatography. Thus, they have found that a pharmaceutical composition that can solve the above-mentioned problems can be provided, and the present invention has been completed.
【０００６】すなわち本発明は、下記一般式（Ｉ）で表
されるＰＣ−ＳＯＤ及びシュークロースを含有すること
を特徴とする医薬組成物（以下、本発明医薬組成物とい
う）を提供する。 ＳＯＤ’（Ｑ−Ｂ）m （Ｉ） （式中、ＳＯＤ’はスーパーオキシドジスムターゼの残
基を表し、Ｑは化学的架橋を表し、Ｂはグリセロールの
２位に水酸基を有するリゾレシチンのその水酸基の水素
原子を除いた残基を表し、ｍはスーパーオキシドジスム
ターゼ１分子に対するリゾレシチンの平均結合数であっ
て、１以上の正数を表す）また本発明は、本発明医薬組
成物からなる、疾患の処置剤（以下、本発明処置剤とも
いう）を提供する。That is, the present invention provides a pharmaceutical composition comprising PC-SOD represented by the following general formula (I) and sucrose (hereinafter, referred to as the pharmaceutical composition of the present invention). SOD ′ (QB) m (I) (where SOD ′ represents a residue of superoxide dismutase, Q represents a chemical crosslink, and B represents the hydroxyl group of lysolecithin having a hydroxyl group at the 2-position of glycerol. M represents the average number of lysolecithin bonds to one molecule of superoxide dismutase, and represents a positive number of 1 or more.) The present invention also relates to a disease-related disease comprising the pharmaceutical composition of the present invention. A therapeutic agent (hereinafter, also referred to as the therapeutic agent of the present invention) is provided.
【０００７】本発明医薬組成物は、好ましくは下記の性
質を有する。 (a)性状：この医薬組成物を凍結乾燥したものに注射用
水（注射用蒸留水）を添加すると溶解し、不溶性異物は
認められない。 (b)安定性：この医薬組成物を凍結乾燥した直後におけ
る単位重量あたりのスーパーオキシドジスムターゼ活性
を１００とした場合、凍結乾燥した組成物を８℃で１２
ヶ月、２５℃で１２ヶ月、又は４０℃で６ヶ月間保存し
た時点における該活性の相対値が、いずれも９７％以上
である。[0007] The pharmaceutical composition of the present invention preferably has the following properties. (a) Properties: This pharmaceutical composition was freeze-dried and dissolved when water for injection (distilled water for injection) was added, and no insoluble foreign matter was observed. (b) Stability: Assuming that the superoxide dismutase activity per unit weight immediately after freeze-drying the pharmaceutical composition is 100, the freeze-dried composition is 12 at 8 ° C.
The relative value of the activity at the time of storage at 25 ° C. for 12 months or at 40 ° C. for 6 months is 97% or more.
【０００８】(c)ゲル濾過クロマトグラフィーにおける
類縁物質ピーク：凍結乾燥した組成物を再溶解し、ゲル
濾過クロマトグラフィーにかけ、溶出液の２２０ｎｍの
吸光度を測定した場合、当該吸光度の検出チャートにお
けるレシチン化スーパーオキシドジスムターゼのピーク
の形状と、凍結乾燥前のレシチン化スーパーオキシドジ
スムターゼの当該ピークの形状との間に、実質的な差が
認められない。(C) Related substance peak in gel filtration chromatography: When the freeze-dried composition is redissolved and subjected to gel filtration chromatography, and the absorbance at 220 nm of the eluate is measured, the lecithinization in the absorbance detection chart is determined. No substantial difference is observed between the shape of the superoxide dismutase peak and the shape of the lecithinated superoxide dismutase before lyophilization.
【０００９】(d)逆相クロマトグラフィーにおける類縁
物質ピーク：凍結乾燥した組成物を８℃で１２ヶ月、２
５℃で１２ヶ月、又は４０℃で６ヶ月間保存した時点で
再溶解し、逆相クロマトグラフィーにかけ、溶出液の２
２０ｎｍ及び２７０ｎｍの吸光度を測定した場合、検出
される類縁物質の量が、いずれも凍結乾燥した直後にお
けるものと実質的に変化しない。(D) Related substance peak in reverse phase chromatography: freeze-dried composition at 8 ° C. for 12 months,
Upon storage at 5 ° C. for 12 months or at 40 ° C. for 6 months, redissolved and subjected to reverse phase chromatography.
When the absorbances at 20 nm and 270 nm are measured, the amounts of the analogous substances detected do not substantially change from those immediately after freeze-drying.
【００１０】本明細書で「溶解」とは、溶質（医薬組成
物を凍結乾燥したもの）と溶媒（注射用水）が混合し
て、肉眼で観察して澄明な溶液が生じることをいう。
「溶解」するか否かは、室温（１℃〜３０℃）下におい
て、溶質に溶媒（注射用水）を添加して緩やかに振とう
し、１０秒以内に溶解するか否かで判断することが好ま
しい。As used herein, the term "dissolve" means that a solute (a lyophilized pharmaceutical composition) and a solvent (water for injection) are mixed to form a clear solution that is visually observed.
To determine whether or not to “dissolve”, add a solvent (water for injection) to the solute at room temperature (1 ° C. to 30 ° C.), shake gently, and determine whether or not it dissolves within 10 seconds. Is preferred.
【００１１】さらに本発明医薬組成物は、その凍結乾燥
物を、８℃で３６ヶ月、２５℃で３６ヶ月、又は４０℃
で６ヶ月間保存したいずれの時点においても上記(a)〜
(d)に記載の全性質を維持しているものであることが好
ましい。Further, the lyophilized product of the pharmaceutical composition of the present invention can be prepared at 8 ° C. for 36 months, at 25 ° C. for 36 months, or at 40 ° C.
(A) ~ at any time stored for 6 months at
It is preferable that all properties described in (d) be maintained.
【００１２】また本発明は、シュークロースを有効成分
とする剤であって、上記一般式（Ｉ）で表されるＰＣ−
ＳＯＤと共存させることにより該ＰＣ−ＳＯＤの活性低
下を抑制し、または、該ＰＣ−ＳＯＤのカラムクロマト
グラフィーによる分析の際の類縁物質ピークの出現を抑
制するための剤（以下、本発明抑制剤ともいう）を提供
する。Further, the present invention relates to an agent comprising sucrose as an active ingredient, wherein the agent comprises a PC-formula represented by the above general formula (I).
An agent for suppressing a decrease in the activity of the PC-SOD by coexisting with SOD, or an agent for suppressing the appearance of an analogous peak at the time of column chromatography analysis of the PC-SOD (hereinafter referred to as the inhibitor of the present invention) Also called).
【００１３】さらに本発明は、上記一般式（Ｉ）で表さ
れるＰＣ−ＳＯＤにシュークロースを共存させることに
より該ＰＣ−ＳＯＤの活性低下を抑制し、または、該Ｐ
Ｃ−ＳＯＤのカラムクロマトグラフィーによる分析の際
の類縁物質ピークの出現を抑制する方法（以下、本発明
抑制方法ともいう）を提供する。Further, the present invention suppresses a decrease in the activity of PC-SOD by allowing sucrose to coexist with PC-SOD represented by the above general formula (I),
Provided is a method for suppressing the appearance of a peak of an analogous substance at the time of analysis by column chromatography of C-SOD (hereinafter, also referred to as the suppression method of the present invention).
【００１４】[0014]
【発明の実施の形態】以下に、本発明の実施の形態を説
明する。Embodiments of the present invention will be described below.
【００１５】＜１＞本発明医薬組成物 (1)本発明医薬組成物に用いるＰＣ−ＳＯＤ 本明細書における「レシチン」とは、フォスファチジル
コリンを意味する通常のレシチンをいい、「リゾレシチ
ン」とは、レシチンのグリセロールの２位に結合した脂
肪酸１分子がとれて、２位の炭素原子に水酸基が結合し
た化合物をいう。<1> Pharmaceutical composition of the present invention (1) PC-SOD used in the pharmaceutical composition of the present invention "Lecithin" in the present specification means ordinary lecithin meaning phosphatidylcholine, and "lysolecithin" The term “lecithin” refers to a compound in which one molecule of fatty acid bonded to the 2-position of glycerol of lecithin is removed, and a hydroxyl group is bonded to the 2-position carbon atom.
【００１６】本発明医薬組成物に含有されるＰＣ−ＳＯ
Ｄは、通常、リゾレシチンの２位の水酸基に化学的架橋
剤を結合させたレシチン誘導体を、ＳＯＤに１個以上結
合させて得ることができる。このＰＣ−ＳＯＤは次式
（Ｉ）で示される。 ＳＯＤ’（Ｑ−Ｂ）m （Ｉ） 上記一般式（Ｉ）中、ＳＯＤ’はスーパーオキシドジス
ムターゼの残基を表す。PC-SO contained in the pharmaceutical composition of the present invention
D can be usually obtained by binding one or more lecithin derivatives in which a chemical crosslinking agent is bound to the hydroxyl group at position 2 of lysolecithin to SOD. This PC-SOD is represented by the following equation (I). SOD ′ (QB) m (I) In the above general formula (I), SOD ′ represents a residue of superoxide dismutase.
【００１７】前記式（Ｉ）中のＳＯＤ’は、生体内の活
性酸素Ｏ2 -の分解というその本来の機能を発揮し得る限
りにおいて、その起源等は特に限定されるものではな
く、各種の動植物又は微生物に由来するＳＯＤの残基を
広く用いることが可能である。しかしながら、医薬品用
途のＳＯＤとしては、生体内での抗原性を可能な限り減
じることが好ましいことを考慮すれば、本発明医薬組成
物を投与する動物種に応じて、適宜適切なＳＯＤを選択
することが好ましい。例えば、最も本発明医薬組成物が
必要とされると考えられるヒトを対象とする場合には、
ＳＯＤとしてヒト体内における抗原性を可能な限り減ず
るべく、ヒト由来のＳＯＤを用いることが好ましい。そ
してその中でも、ヒト由来のＣｕ／Ｚｎ ＳＯＤ（活性
中心に銅と亜鉛を含むヒト由来のＳＯＤ；以下、ヒトＣ
ｕ／Ｚｎ ＳＯＤと略記することもある）が、細胞内に
おける発現量が多く、また遺伝子工学的手法による生産
技術が確立しており、大量に調製することが可能である
ため、特に好ましい。[0017] SOD in the formula (I) ', the active oxygen O 2 in vivo - as long as capable of exhibiting its inherent function of decomposition, its origin and the like are not particularly limited, various It is possible to widely use residues of SOD derived from animals and plants or microorganisms. However, considering that it is preferable to reduce as much as possible the in vivo antigenicity of the SOD for pharmaceutical use, an appropriate SOD is appropriately selected according to the animal species to which the pharmaceutical composition of the present invention is administered. Is preferred. For example, when targeting a human who is most likely to need the pharmaceutical composition of the present invention,
It is preferable to use human-derived SOD in order to reduce antigenicity in the human body as much as possible. Among them, human-derived Cu / Zn SOD (human-derived SOD containing copper and zinc in the active center; hereinafter, human C
u / Zn SOD) is particularly preferable because it has a large amount of expression in cells, a production technology using a genetic engineering technique has been established, and it can be prepared in large quantities.
【００１８】このヒトＣｕ／Ｚｎ ＳＯＤには、ヒト組
織から製造される天然のヒトＣｕ／Ｚｎ ＳＯＤ；遺伝
子工学的手法により製造されるヒトＣｕ／Ｚｎ ＳＯ
Ｄ；天然のヒトＣｕ／Ｚｎ ＳＯＤと実質上同一のアミ
ノ酸配列を有する組換えヒトＣｕ／Ｚｎ ＳＯＤ、これ
らのヒトＣｕ／Ｚｎ ＳＯＤのアミノ酸配列中の一部の
アミノ酸を化学的に修飾もしくは改変したＳＯＤ等があ
り、いずれのヒトＣｕ／ＺｎＳＯＤであってもよい。The human Cu / Zn SOD includes natural human Cu / Zn SOD produced from human tissue; human Cu / Zn SOD produced by genetic engineering techniques.
D: Recombinant human Cu / Zn SOD having an amino acid sequence substantially identical to natural human Cu / Zn SOD, and some amino acids in the amino acid sequence of these human Cu / Zn SOD were chemically modified or modified. SOD and the like, and any human Cu / Zn SOD may be used.
【００１９】参考のため、この天然のヒトＣｕ／Ｚｎ
ＳＯＤのタンパク質のアミノ酸配列を配列番号１に示
す。なお、実際のヒトＣｕ／Ｚｎ ＳＯＤは、このアミ
ノ酸配列を有するタンパク質が二量体（ダイマー）とな
っている。For reference, this natural human Cu / Zn
The amino acid sequence of the SOD protein is shown in SEQ ID NO: 1. In the actual human Cu / Zn SOD, a protein having this amino acid sequence is a dimer.
【００２０】このタンパク質におけるアミノ酸配列に示
すごとく、天然のヒトＣｕ／ＺｎＳＯＤの１１１位のア
ミノ酸はシステインであるが、蛋白工学的手法、例えば
部位特異的変異法により、この１１１位をセリンに変換
したヒトＣｕ／Ｚｎ ＳＯＤ（特開昭62-130684号公報）
や、化学的にこの１１１位のシステインを修飾したヒト
Ｃｕ／Ｚｎ ＳＯＤ（特開平6-199895号公報）も報告さ
れており、これらのヒトＣｕ／Ｚｎ ＳＯＤを素材とし
て、本発明におけるＰＣ−ＳＯＤを得ることができる。
そして、これらのヒトＣｕ／Ｚｎ ＳＯＤのうちでも、
電荷的及び分子量的に均一であり、かつＳＯＤ活性が安
定している、１１１位のシステインを化学的に修飾し
た、例えばこのシステインをＳ−（２−ヒドロキシエチ
ルチオシステイン）とした、ヒトＣｕ／Ｚｎ ＳＯＤを
素材として、本発明におけるＰＣ−ＳＯＤを得るのが好
ましい。As shown in the amino acid sequence of this protein, the amino acid at position 111 of natural human Cu / ZnSOD is cysteine. This position was converted to serine by protein engineering techniques, for example, site-directed mutagenesis. Human Cu / Zn SOD (JP-A-62-130684)
Also, human Cu / Zn SOD in which the cysteine at position 111 is chemically modified (JP-A-6-199895) has been reported, and the PC-SOD in the present invention was prepared using these human Cu / Zn SOD as a material. Can be obtained.
And among these human Cu / Zn SODs,
Human Cu / which is homogeneous in charge and molecular weight and has a stable SOD activity, in which cysteine at position 111 is chemically modified, for example, this cysteine is S- (2-hydroxyethylthiocysteine) It is preferable to obtain PC-SOD in the present invention using Zn SOD as a material.
【００２１】なお本明細書においては、このようにヒト
Ｃｕ／Ｚｎ ＳＯＤにおいて部位特異的変異法等により
一部アミノ酸を変換したものや、ヒトＣｕ／Ｚｎ ＳＯ
Ｄの一部のアミノ酸を化学的に修飾して得られるものも
含めて、単にヒトＣｕ／ＺｎＳＯＤという。In this specification, human Cu / Zn SOD obtained by partially converting amino acids by site-directed mutagenesis or the like, or human Cu / Zn SO
It is simply called human Cu / ZnSOD, including those obtained by chemically modifying some amino acids of D.
【００２２】また、前記式（Ｉ）中のＢで示される「グ
リセロールの２位に水酸基を有するリゾレシチンの、そ
の水酸基の水素原子を除いた残基」は、次式(II)で表さ
れる。The “residue of lysolecithin having a hydroxyl group at the 2-position of glycerol except for the hydrogen atom of the hydroxyl group” represented by B in the above formula (I) is represented by the following formula (II): .
【００２３】 -O-CH(CH2OR)[CH2OP(O)(O-)(OCH2CH2N+(CH3)3)] （II） （式中、Ｒは脂肪酸残基（アシル基）である） Ｒは、炭素数１０〜２８の飽和又は不飽和の脂肪酸残基
が好ましく、より好ましくはミリストイル基、パルミト
イル基、ステアロイル基、イコサノイル基、ドコサノイ
ル基、その他炭素数が１４〜２２の飽和脂肪酸残基であ
り、特に好ましくは炭素数１６の飽和脂肪酸残基である
パルミトイル基である。[0023] -O-CH (CH 2 OR) [CH 2 OP (O) (O -) (OCH 2 CH 2 N + (CH 3) 3)] (II) ( wherein, R fatty acid residue ( R is preferably a saturated or unsaturated fatty acid residue having 10 to 28 carbon atoms, more preferably a myristoyl group, a palmitoyl group, a stearoyl group, an icosanoyl group, a docosanoyl group, or a group having 14 to 14 carbon atoms. And a palmitoyl group which is a saturated fatty acid residue having 16 carbon atoms.
【００２４】また前記式（Ｉ）中のＱは化学的架橋を表
す。化学的架橋は、ＳＯＤとレシチンとを架橋して化学
的に結合（共有結合）させ得るものであれば特に限定さ
れない。化学的架橋としては、残基−Ｃ（Ｏ）−（ＣＨ
2）n−Ｃ（Ｏ）−が例示され、かつ好ましい。この残基
は、 式ＨＯ−Ｃ（Ｏ）−（ＣＨ2）n−Ｃ（Ｏ）−ＯＨ
で表される直鎖状ジカルボン酸の両端の水酸基を除いた
残基、もしくは、このジカルボン酸の無水物、このジカ
ルボン酸のエステル、このジカルボン酸のハロゲン化
物、又はその他のこのジカルボン酸の反応性誘導体等の
両端における水酸基に対応する部分を除いた残基である
ものが好ましい。前記式（Ｉ）中のＱが上記直鎖状ジカ
ルボン酸の残基である場合、Ｑの一端とＢとは、エステ
ル結合で連結している。また、この場合、このＱの他端
は、ＳＯＤのアミノ基とアミド結合などにより直接結合
していると考えられる。この場合、ＳＯＤ’は、ＳＯＤ
の結合に関与するアミノ基から水素原子を１つ除いた残
基を示す。なお前記の化学的架橋において、基−（ＣＨ
2）n−におけるｎは２以上の整数であり、好ましくは２
〜１０の整数であり、特に好ましくは３である。Q in the formula (I) represents chemical crosslinking. Chemical cross-linking is not particularly limited as long as it can cross-link SOD and lecithin and chemically bond (covalently bond). As a chemical crosslink, the residue —C (O) — (CH
2 ) n- C (O)-is exemplified and preferred. This residue has the formula HO—C (O) — (CH 2 ) n —C (O) —OH
The residue obtained by removing the hydroxyl groups at both ends of the linear dicarboxylic acid represented by, or the anhydride of the dicarboxylic acid, the ester of the dicarboxylic acid, the halide of the dicarboxylic acid, or the reactivity of the dicarboxylic acid or other Preferably, the residue is a residue obtained by removing a portion corresponding to a hydroxyl group at both ends of a derivative or the like. When Q in the formula (I) is a residue of the above-mentioned linear dicarboxylic acid, one end of Q and B are connected by an ester bond. In this case, it is considered that the other end of Q is directly bonded to the amino group of SOD by an amide bond or the like. In this case, SOD 'is SOD
Represents a residue in which one hydrogen atom has been removed from the amino group involved in the bonding of. In the above chemical crosslinking, the group-(CH
2 ) n in n- is an integer of 2 or more, preferably 2
And an integer of 10 to 10, particularly preferably 3.
【００２５】また前記式（Ｉ）中のｍは、ＳＯＤ１分子
に対するリゾレシチンの平均結合数を表している。ｍは
１以上の正数であり、１〜１２の正数が好ましく、４が
特に好ましい。In the above formula (I), m represents the average number of lysolecithins bound to one SOD molecule. m is a positive number of 1 or more, preferably a positive number of 1 to 12, and particularly preferably 4.
【００２６】ＰＣ−ＳＯＤの製造において、レシチン誘
導体のＳＯＤへの結合は、公知の方法、例えば特開平６
−５４６８１号公報に記載された方法に従って行うこと
が可能である。このＰＣ−ＳＯＤの製造過程は、実施例
中に記載する。In the production of PC-SOD, the binding of a lecithin derivative to SOD is carried out by a known method,
The method can be performed according to the method described in JP-A-54681. The manufacturing process of this PC-SOD will be described in Examples.
【００２７】本発明医薬組成物に用いるＰＣ−ＳＯＤ
は、医薬として使用できる程度に精製され、医薬として
混入が許されない物質を実質的に含まないものであるこ
とが好ましい。PC-SOD used in the pharmaceutical composition of the present invention
Is preferably purified to such an extent that it can be used as a medicament, and is substantially free from substances that are not allowed to be contaminated as medicament.
【００２８】例えばＰＣ−ＳＯＤは、2,500 U/mg(ＰＣ
−ＳＯＤ)以上の比活性を有する精製されたものを用い
るのが好ましく、3,000 U/mg(ＰＣ−ＳＯＤ)以上の比活
性を有する精製されたものがより好ましい。なお、本明
細書において１U(ユニット)とは、pH7.8、30℃の条件下
でＮＢＴ(ニトロブルーテトラゾリウム)を用いてJ.Bio
l.Chem. Vol.244, No.22, 6049-6055(1969)に準じた方
法により測定し、ＮＢＴの還元速度を５０％阻害するＰ
Ｃ−ＳＯＤの酵素量を表す。For example, PC-SOD is 2,500 U / mg (PC
It is preferable to use a purified product having a specific activity of not less than 3,000 U / mg (PC-SOD). In this specification, 1 U (unit) refers to J. Bio using NBT (nitro blue tetrazolium) under conditions of pH 7.8 and 30 ° C.
Measured by a method according to l. Chem. Vol. 244, No. 22, 6049-6055 (1969), and P which inhibits the reduction rate of NBT by 50%
Indicates the amount of C-SOD enzyme.
【００２９】(2)本発明医薬組成物に用いるシュークロ
ース 本発明医薬組成物に用いるシュークロースは、医薬とし
て使用できる程度に精製され、医薬として混入が許され
ない物質を実質的に含まないものであり、活性炭で処理
されたシュークロースが好ましい。活性炭による処理
は、シュークロースを水溶液とし、これと活性炭とを接
触させることにより行うことができる。このような活性
炭処理により、たとえばシュークロース中のエンドトキ
シン濃度を低減させることができ、類縁物質ピークの出
現をさらに抑制でき、かつ安定性をさらに向上させう
る。(2) Sucrose used in the pharmaceutical composition of the present invention The sucrose used in the pharmaceutical composition of the present invention is purified to such an extent that it can be used as a medicament, and is substantially free from substances that cannot be mixed as a medicament. Yes, sucrose treated with activated carbon is preferred. The treatment with activated carbon can be performed by converting sucrose into an aqueous solution and bringing this into contact with activated carbon. By such activated carbon treatment, for example, the endotoxin concentration in sucrose can be reduced, the appearance of an analog peak can be further suppressed, and the stability can be further improved.
【００３０】本発明医薬組成物に用いるシュークロース
中のエンドトキシン濃度は、0.25 EU/mg以下が好まし
く、検出限界(0.006 EU/ml)以下がより好ましい。The concentration of endotoxin in sucrose used in the pharmaceutical composition of the present invention is preferably 0.25 EU / mg or less, more preferably the detection limit (0.006 EU / ml) or less.
【００３１】なおシュークロース中のエンドトキシン濃
度は、公知のエンドトキシンの測定法を用いて測定する
ことができるが、カブトガニ・アメボサイト・ライセー
ト成分を用いるリムルス試験法が好ましい。リムルス試
験法に用いるリムルス試薬としては、エンドトキシン特
異的リムルス試薬を用いることが好ましい。リムルス試
薬としては、例えば次に挙げる市販のリムルス試薬を用
いることができる；トキシカラーシステムＬＳ−６，Ｌ
Ｓ−２０，ＬＳ−５０Ｍ、エンドスペシーＥＳ−６，エ
ンドスペシーＥＳ−２００（生化学工業株式会社）。The endotoxin concentration in sucrose can be measured using a known method for measuring endotoxin, but the Limulus test using a horseshoe crab, amebosite and lysate component is preferred. As the Limulus reagent used in the Limulus test, it is preferable to use an endotoxin-specific Limulus reagent. As the Limulus reagent, for example, the following commercially available Limulus reagents can be used; Toxicolor System LS-6, L
S-20, LS-50M, Endspecy ES-6, Endspecy ES-200 (Seikagaku Corporation).
【００３２】(3)本発明医薬組成物 本発明の医薬組成物は、ＰＣ−ＳＯＤとシュークロース
を含む。該組成物は(1)で説明したＰＣ−ＳＯＤと、(2)
で説明したシュークロースを配合することにより得るの
が好ましい。これにより、長期間保存によるＰＣ−ＳＯ
Ｄの活性低下が少なく（安定性が高く）、凍結乾燥した
場合の性状が良好で、かつカラムクロマトグラフィーに
よる分析の時に類縁物質ピークの出現が抑制された本発
明医薬組成物を得ることができる。(3) Pharmaceutical composition of the present invention The pharmaceutical composition of the present invention contains PC-SOD and sucrose. The composition comprises PC-SOD described in (1) and (2)
It is preferably obtained by blending the sucrose described in the above. Thereby, PC-SO by long-term storage
It is possible to obtain the pharmaceutical composition of the present invention in which the activity of D is small (high in stability), the properties when freeze-dried are good, and the appearance of related substance peaks is suppressed during analysis by column chromatography. .
【００３３】本発明医薬組成物中のＰＣ−ＳＯＤとシュ
ークロースの配合比率は、投与量、本発明医薬組成物の
形態等に応じて当業者が適宜決定でき、特に限定されな
いが、「ＰＣ−ＳＯＤ／シュークロース」の重量比とし
て0.1/100〜80/100程度が好ましく、0.4/100〜60/100程
度がより好ましい。The mixing ratio of PC-SOD and sucrose in the pharmaceutical composition of the present invention can be appropriately determined by those skilled in the art according to the dose, the form of the pharmaceutical composition of the present invention, and the like. The weight ratio of "SOD / sucrose" is preferably about 0.1 / 100 to 80/100, more preferably about 0.4 / 100 to 60/100.
【００３４】また本発明医薬組成物は、その脂肪酸含量
が、０．１３〜０．１５μｍｏｌ／ｍｇ蛋白であること
が好ましい。脂肪酸含量は、例えば（株）ワイエムシイ
社製「長鎖、短鎖脂肪酸分析キット」を用いて測定する
ことができる。The pharmaceutical composition of the present invention preferably has a fatty acid content of 0.13 to 0.15 μmol / mg protein. The fatty acid content can be measured using, for example, “Long and short chain fatty acid analysis kit” manufactured by YMC.
【００３５】なお本発明医薬組成物には、ＰＣ−ＳＯＤ
の酵素活性に悪影響を与えず、かつ本発明の効果に影響
を与えない限りにおいて、他の医薬活性成分や、慣用の
賦形剤、結合剤、滑沢剤、着色剤、崩壊剤、緩衝剤、等
張化剤、保存剤、無痛化剤等、通常医薬に用いられる成
分を添加することができる。このような成分としては、
生理学上許容され、医薬として使用できる程度の純度で
あり、かつ医薬として混入が許されない物質を実質的に
含まないものを使用できる。The pharmaceutical composition of the present invention contains PC-SOD
Other pharmaceutically active ingredients, conventional excipients, binders, lubricants, coloring agents, disintegrants, buffering agents, as long as they do not adversely affect the enzyme activity of the present invention and the effects of the present invention. Ingredients usually used in medicine, such as a tonicity agent, a preservative, and a soothing agent, can be added. Such components include:
A substance which is physiologically acceptable and has a purity that can be used as a medicament and substantially does not contain a substance which is not allowed to be mixed as a medicament can be used.
【００３６】本発明医薬組成物の製造は、ＰＣ−ＳＯＤ
及びシュークロースを用い、製剤学的に公知の方法を用
いて行なうことができる。なお本発明医薬組成物は、溶
液状、凍結状、または凍結乾燥状の形態が好ましい。な
お溶液状の形態としては、以下の(1)および(2)の態様も
包含される。 (1)凍結状態とした場合の本発明医薬組成物における、
凍結前および融解後における溶液状態。 (2)凍結乾燥状態とした場合の本発明医薬組成物におけ
る、凍結乾燥前および溶媒添加による再溶解後の溶液状
態。The preparation of the pharmaceutical composition of the present invention is carried out by PC-SOD
And sucrose, and can be carried out by a method known in pharmaceutics. The pharmaceutical composition of the present invention is preferably in the form of a solution, a frozen form, or a lyophilized form. The form in the form of a solution also includes the following embodiments (1) and (2). (1) in the present pharmaceutical composition when in a frozen state,
Solution state before freezing and after thawing. (2) Solution state before freeze-drying and after re-dissolution by addition of a solvent in the pharmaceutical composition of the present invention in a freeze-dried state.
【００３７】これらの中で、本発明医薬組成物の形態は
凍結乾燥状の形態とするのが好ましい。すなわち本発明
医薬組成物は凍結乾燥医薬組成物であることが好まし
い。Of these, the pharmaceutical composition of the present invention is preferably in a lyophilized form. That is, the pharmaceutical composition of the present invention is preferably a freeze-dried pharmaceutical composition.
【００３８】凍結乾燥は、公知の凍結乾燥手法に従っ
て、当業者が適宜条件等を設定して行うことができる。
例えば市販の凍結乾燥機を用い、以下のステップ(1)〜
(5)の順に実行されるよう設定して行うことができる。
なお（ ）内は、各段階に要する時間を示した。The freeze-drying can be carried out by a person skilled in the art according to a known freeze-drying technique, by appropriately setting conditions and the like.
For example, using a commercially available freeze dryer, the following steps (1) ~
It can be set to be executed in the order of (5).
The times in parentheses indicate the time required for each step.
【００３９】(1)冷却する。例えば、室温から−４０℃
〜−５０℃まで冷却する（0.3〜1.5時間程度）。(1) Cool. For example, from room temperature to -40 ° C
Cool to ~ -50 ° C (about 0.3-1.5 hours).
【００４０】(2)真空状態を維持しながら冷却状態を維
持する。例えば、−４０℃〜−５０℃を維持する（１２
時間以上）。(2) Maintain a cooling state while maintaining a vacuum state. For example, a temperature of −40 ° C. to −50 ° C. is maintained (12
Hours).
【００４１】(3)真空状態を維持しながら冷却状態から
室温まで戻す。例えば、−４０℃〜−５０℃から室温ま
で戻す（３〜15時間程度）。(3) Returning from the cooling state to room temperature while maintaining the vacuum state. For example, the temperature is returned from −40 ° C. to −50 ° C. to room temperature (about 3 to 15 hours).
【００４２】(4)真空状態を維持しながら室温を維持す
る（例えば３〜20時間程度）。(4) Maintain the room temperature while maintaining the vacuum state (for example, about 3 to 20 hours).
【００４３】(5)復圧する。(5) The pressure is restored.
【００４４】ステップ(1)〜(5)からなる凍結乾燥医薬組
成物の製造においては、上記のステップ(2)に要する時
間を工夫するのが好ましく、該時間が、１２時間以上で
あることが好ましい。In the production of the lyophilized pharmaceutical composition comprising the steps (1) to (5), it is preferable to devise the time required for the step (2), and the time is preferably 12 hours or more. preferable.
【００４５】凍結乾燥医薬組成物としては、その性質が
下記(a)〜(d)の全ての条件を満たすものが特に好まし
い。As the lyophilized pharmaceutical composition, those having properties satisfying all the following conditions (a) to (d) are particularly preferable.
【００４６】(a)性状：凍結乾燥医薬組成物を肉眼で観
察した場合、凍結乾燥組成物のケーキの形状が錠剤状で
あり、かつ凍結乾燥組成物に注射用水を添加した場合、
速やかに溶解し、かつ不溶性異物を認めない（凍結乾燥
医薬組成物の性状が良好に保持されている）。通常、注
射用水に１０秒以内に溶解する。(A) Properties: When the freeze-dried pharmaceutical composition is visually observed, when the cake of the freeze-dried composition is tablet-shaped and water for injection is added to the freeze-dried composition,
It dissolves quickly and does not show any insoluble foreign substances (the properties of the lyophilized pharmaceutical composition are well maintained). Usually, it is dissolved in water for injection within 10 seconds.
【００４７】(b)安定性：凍結乾燥直後の単位重量あた
りの酵素活性を１００(％)とした場合、凍結乾燥組成物
を８℃で１２ヶ月、２５℃で１２ヶ月、又は４０℃で６
ヶ月間保存した時の酵素活性の相対値(％)が９７％以
上、好ましくは９９％以上である。この安定性は、後述
する実施例１中の「(1-2-2)安定性試験」に記載された
方法で測定できる。(B) Stability: Assuming that the enzyme activity per unit weight immediately after freeze-drying is 100 (%), the freeze-dried composition is treated at 8 ° C. for 12 months, at 25 ° C. for 12 months, or at 40 ° C. for 6 months.
The relative value (%) of the enzyme activity after storage for months is 97% or more, preferably 99% or more. This stability can be measured by the method described in “(1-2-2) Stability test” in Example 1 described later.
【００４８】(c)ゲル濾過クロマトグラフィーによる類
縁物質ピーク：２２０ｎｍの吸光度を測定した場合、当
該吸光度の検出チャートにおけるＰＣ−ＳＯＤ(凍結乾
燥医薬組成物)のピークの形状と、凍結乾燥前のＰＣ−
ＳＯＤの当該ピークの形状との間に、実質的な差が認め
られない。該クロマトグラフィーは、後述する実施例１
中の「(1-2-3-1)ゲル濾過クロマトグラフィーによる類
縁物質ピークの検出試験」に記載された方法で測定する
のが好ましい。(C) Related substance peak by gel filtration chromatography: When the absorbance at 220 nm was measured, the shape of the PC-SOD (lyophilized pharmaceutical composition) peak in the absorbance detection chart and the PC before lyophilization were measured. −
No substantial difference is observed between the shape of the SOD and the peak. The chromatography was performed according to Example 1 described below.
It is preferably measured by the method described in “(1-2-3-1) Detection test of analogous substance peak by gel filtration chromatography” therein.
【００４９】(d)逆相クロマトグラフィーによる類縁物
質ピーク：凍結乾燥組成物を８℃で１２ヶ月、２５℃で
１２ヶ月、又は４０℃で６ヶ月間保存した時における、
２２０ｎｍ及び２７０ｎｍの吸光度の測定により検出さ
れる類縁物質の量が、凍結乾燥直後と実質的に変化しな
い。該クロマトグラフィーは、後述する実施例１中の
「(1-2-3-2)逆相クロマトグラフィーによる類縁物質ピ
ークの検出試験」に記載された方法で測定するのが好ま
しい。(D) Related substance peak by reverse phase chromatography: The lyophilized composition was stored for 12 months at 8 ° C., 12 months at 25 ° C., or 6 months at 40 ° C.
The amount of related substances detected by measuring the absorbance at 220 nm and 270 nm does not substantially change from that immediately after freeze-drying. The chromatography is preferably measured by the method described in “(1-2-3-2) Detection test of analogous substance peak by reversed phase chromatography” in Example 1 described later.
【００５０】さらに本発明医薬組成物は、その凍結乾燥
物を、８℃で３６ヶ月、２５℃で３６ヶ月、又は４０℃
で６ヶ月間保存したいずれの時点においても上記(a)〜
(d)に記載の全性質を維持しているものであることが好
ましい。Further, the pharmaceutical composition of the present invention may be prepared by lyophilizing the lyophilized product at 8 ° C. for 36 months, 25 ° C. for 36 months, or 40 ° C.
(A) ~ at any time stored for 6 months at
It is preferable that all properties described in (d) be maintained.
【００５１】本発明にいう類縁物質は、主としてレシチ
ンが種々の部分で切断されて生成した種々の物質からな
り、遊離脂肪酸等が含まれる。これらの類縁物質はＰＣ
−ＳＯＤに結合していたレシチンが切断されて生成する
ものと考えられ、ＰＣ−ＳＯＤに結合していたレシチン
が多量に切断されると、ＰＣ−ＳＯＤとしての機能が減
じられ、またそのような類縁物質が多量に含まれるとそ
の医薬品としての品質、規格等の管理上好ましくない。The analogous substances referred to in the present invention mainly consist of various substances formed by cutting lecithin at various parts, and include free fatty acids and the like. These related substances are PC
It is considered that lecithin bound to -SOD is generated by cleavage. When a large amount of lecithin bound to PC-SOD is cleaved, the function as PC-SOD is reduced, and such If a large amount of analogous substances are contained, it is not preferable in terms of control of the quality, specifications and the like as pharmaceuticals.
【００５２】本発明医薬組成物、さらには該組成物を種
々の形態にしたものは、そのまま医薬品として投与する
ための最終剤形として用いうるが、他の剤形の医薬品、
例えば、液剤、凍結乾燥剤等の原料として使用すること
もできる。The pharmaceutical composition of the present invention and the various forms of the composition can be used as a final dosage form for administration as a medicament as they are.
For example, it can be used as a raw material for liquid preparations, freeze-dried agents and the like.
【００５３】本発明医薬組成物は、ＰＣ−ＳＯＤを有効
成分とする注射用製剤として使用するのが好ましい。例
えば、本発明医薬組成物を溶液状態の注射用製剤として
提供する場合、前記の方法で製造される溶液状態の本発
明医薬組成物を、アンプル、バイアル、注射用シリンジ
等の適当な容器に充填・密封し、そのまま流通させある
いは保存し、注射剤として投与に供することができる。The pharmaceutical composition of the present invention is preferably used as an injectable preparation containing PC-SOD as an active ingredient. For example, when the pharmaceutical composition of the present invention is provided as an injectable preparation in the form of a solution, the pharmaceutical composition of the present invention in the form of a solution prepared by the above method is filled in an appropriate container such as an ampoule, a vial, or a syringe for injection. -It can be sealed, distributed or stored as it is, and used as an injection for administration.
【００５４】本発明医薬組成物を凍結状態の注射用製剤
として提供する場合、アンプル、バイアル、注射用シリ
ンジ等の適当な容器中に、凍結状態の本発明医薬組成物
を密封状態で保持させて、流通させあるいは保存し、投
与前に融解させて注射剤として投与に供することができ
る。When the pharmaceutical composition of the present invention is provided as a frozen preparation for injection, the frozen pharmaceutical composition of the present invention is held in a sealed container in an appropriate container such as an ampoule, a vial, or a syringe for injection. It can be distributed or stored, melted before administration, and provided as an injection.
【００５５】また、本発明医薬組成物を凍結乾燥状態の
注射用製剤として提供する場合、アンプル、バイアル、
注射用シリンジ等の適当な容器中に前記の方法で製造さ
れる凍結乾燥状態の本発明医薬組成物を密封状態で保持
させて、流通させあるいは保存し、投与前に注射用蒸留
水で溶解し、注射剤として投与に供することができる。
凍結乾燥状態の本発明医薬組成物は、溶解用の溶媒とセ
ットで提供してもよい。When the pharmaceutical composition of the present invention is provided as a lyophilized preparation for injection, an ampoule, a vial,
The lyophilized pharmaceutical composition of the present invention produced by the above-mentioned method is kept in a sealed state in a suitable container such as an injection syringe or the like, and is circulated or stored, and dissolved in distilled water for injection before administration. And can be administered as an injection.
The pharmaceutical composition of the present invention in a lyophilized state may be provided together with a solvent for dissolution.
【００５６】注射用製剤としては、凍結乾燥状態のもの
が好ましい。すなわち、本発明医薬組成物は注射用凍結
乾燥組成物の形態であることが特に好ましい。 ＜２＞本発明処置剤 本発明処置剤は、本発明医薬組成物からなる疾患の処置
剤である。また本発明処置剤は、各種疾患の治療剤、予
防剤、悪化防止剤、改善剤等の概念をも包含する。本発
明処置剤の構成成分として用いるＰＣ−ＳＯＤ、シュー
クロース、これらの配合量、含有させてもよい他の成
分、剤型等は、上記の本発明医薬組成物と全く同じ態様
が採用できる。The preparation for injection is preferably in a lyophilized state. That is, it is particularly preferable that the pharmaceutical composition of the present invention is in the form of a freeze-dried composition for injection. <2> Therapeutic agent of the present invention The therapeutic agent of the present invention is a therapeutic agent for diseases comprising the pharmaceutical composition of the present invention. The treatment agent of the present invention also includes the concept of a therapeutic agent, a preventive agent, a deterioration inhibitor, an ameliorating agent, and the like for various diseases. As the PC-SOD and sucrose used as the constituents of the treatment agent of the present invention, the amounts thereof, the other components that may be contained, the dosage form, and the like, the exactly same embodiment as the above-mentioned pharmaceutical composition of the present invention can be adopted.
【００５７】本発明処置剤を適用できる疾患は、ＰＣ−
ＳＯＤの作用によって処置（治療、予防、維持(悪化防
止)、軽減(症状の改善)等）が可能な疾患である限りに
おいて特に限定されない。The diseases to which the treatment agent of the present invention can be applied include PC-
There is no particular limitation as long as the disease can be treated (treatment, prevention, maintenance (prevention of deterioration), reduction (improvement of symptoms), etc.) by the action of SOD.
【００５８】ＰＣ−ＳＯＤの作用によって処置が可能な
疾患としては、過剰な活性酸素に起因する疾患が挙げら
れるが、より具体的には運動ニューロン疾患（例えば筋
萎縮性側索硬化症(ALS)、脊髄性進行性筋萎縮症、家族
性痙性麻痺、シャルコー・マリー足、進行性球麻痺、若
年性一側上肢筋萎縮症（平山病）等）、虚血再灌流障
害、急性腎不全（腎前性急性腎不全、腎性急性腎不全、
腎後性急性腎不全等）、ループス腎炎（正常糸球体にお
けるループス腎炎、メサンギウム増殖性変化を伴うルー
プス腎炎、巣状分節状糸球体腎炎、びまん性増殖性糸球
体腎炎、びまん性膜性糸球体腎炎、末期硬化性糸球体腎
炎等）、脳血管障害（脳出血、高血圧性脳内出血、脳梗
塞、脳血栓、脳塞栓、一過性脳虚血発作、高血圧性脳症
等）に伴う機能障害（発語障害、麻痺による運動機能障
害、視覚障害、聴覚障害、嗅覚障害、味覚障害等の感覚
障害、痴呆等）、間質性肺炎、線維症（肺線維症等）、
潰瘍性胃腸障害（潰瘍性大腸炎、クローン病等）、関節
炎、熱傷等が挙げられ、これらのなかでも運動ニューロ
ン疾患（特にＡＬＳ）や潰瘍性胃腸障害（特に潰瘍性大
腸炎やクローン病）が好ましい適用疾患である。また線
維症（特に肺線維症）、間質性肺炎も好ましい適用疾患
である。The diseases that can be treated by the action of PC-SOD include diseases caused by excessive active oxygen, and more specifically, motor neuron diseases (eg, amyotrophic lateral sclerosis (ALS) , Spinal progressive muscular atrophy, familial spastic palsy, Charcot-Marie foot, progressive bulbar palsy, juvenile unilateral upper limb muscular atrophy (Hirayama disease), ischemia-reperfusion injury, acute renal failure (kidney) Pre-acute renal failure, renal acute renal failure,
Postrenal acute renal failure, etc., lupus nephritis (lupus nephritis in normal glomeruli, lupus nephritis with mesangial proliferative changes, focal segmental glomerulonephritis, diffuse proliferative glomerulonephritis, diffuse membranous glomeruli Dysfunction due to nephritis, end-stage sclerotic glomerulonephritis, etc., cerebrovascular disorder (cerebral hemorrhage, hypertensive intracerebral hemorrhage, cerebral infarction, cerebral thrombosis, cerebral embolism, transient ischemic attack, hypertensive encephalopathy, etc.) Disorders, motor impairment due to paralysis, visual impairment, hearing impairment, olfactory impairment, sensory impairment such as taste disorder, dementia, etc.), interstitial pneumonia, fibrosis (pulmonary fibrosis, etc.),
Ulcerative gastrointestinal disorders (ulcerative colitis, Crohn's disease, etc.), arthritis, burns, etc., among which motor neuron disease (especially ALS) and ulcerative gastrointestinal disorders (especially ulcerative colitis, Crohn's disease) It is a preferred indication. Fibrosis (particularly pulmonary fibrosis) and interstitial pneumonia are also preferred indications.
【００５９】本発明処置剤は、含有するＰＣ−ＳＯＤの
働きで、これらの疾患並びにこれら疾患に伴う症状に対
して改善作用を有する。なお、ここに挙げた疾患名は例
示であり、かかる例示疾患に本発明処置剤の適用範囲は
限定されない。The treatment agent of the present invention has an effect of improving these diseases and symptoms associated with these diseases by the action of PC-SOD contained therein. In addition, the disease names listed here are examples, and the application range of the therapeutic agent of the present invention is not limited to these exemplified diseases.
【００６０】本発明においては、対象となる疾患の性質
や進行段階に応じて、後述する剤形を本発明処置剤の剤
形として適宜選択することが好ましい。In the present invention, it is preferable to appropriately select a dosage form to be described later as a dosage form of the treatment agent of the present invention according to the nature and progression stage of the target disease.
【００６１】本発明処置剤は、注射（筋肉内、皮下、皮
内、静脈内等）、経口、吸入等の投与方法によって、経
口あるいは非経口的に投与することができ、投与方法に
応じ適宜製剤化することができる。剤形としては、注射
剤（溶液、懸濁液、乳濁液、用時溶解用固形剤等）、錠
剤、カプセル剤、顆粒剤、散剤、液剤、リポ化剤、ゲル
剤、外用散剤、スプレー剤、吸入散剤、坐剤等が挙げら
れるが、注射剤が好ましく、静脈内投与用の注射剤とす
ることがより好ましい。The therapeutic agent of the present invention can be administered orally or parenterally, depending on the administration method such as injection (intramuscular, subcutaneous, intradermal, intravenous, etc.), oral, inhalation and the like. It can be formulated. Dosage forms include injections (solutions, suspensions, emulsions, solids for dissolution before use, etc.), tablets, capsules, granules, powders, solutions, lipoagents, gels, external powders, sprays Preparations, inhalation powders, suppositories, etc., but injections are preferable, and injections for intravenous administration are more preferable.
【００６２】本発明処置剤中の有効成分（ＰＣ−ＳＯ
Ｄ）の量および当該処置剤の投与量は、製剤調製の方
法、剤形、疾患の種類、当該処置剤の投与方法、投与形
態、使用目的、患者の具体的症状（疾患の程度）、患者
の体重等によって個別的であり特に限定されないが、例
えば、臨床量として成人１人１日当り 0.5〜100mg（150
0〜300000Ｕ）を例示することができる。また、上記製
剤の投与間隔は１日１回程度でも可能であり、３〜４回
に分けて投与することも可能である。The active ingredient (PC-SO) in the treatment agent of the present invention
The amount of D) and the dose of the therapeutic agent are determined by the method of preparation of the preparation, the dosage form, the type of disease, the method of administration of the therapeutic agent, the dosage form, the purpose of use, the specific symptoms of the patient (the degree of the disease), Although it is individual and not particularly limited depending on the body weight of the subject, for example, a clinical dose of 0.5 to 100 mg (150
0 to 300,000 U). In addition, the administration interval of the above-mentioned preparation can be about once a day, and the preparation can be divided into three to four times.
【００６３】また本発明処置剤は、前記の疾患を発症し
うる脊椎動物、特に哺乳動物に投与する処置剤とするこ
とができるが、ヒトの処置剤とすることが好ましい。The therapeutic agent of the present invention can be a therapeutic agent to be administered to vertebrates, particularly mammals, which can develop the above-mentioned diseases, but is preferably a human therapeutic agent.
【００６４】＜３＞本発明抑制剤及び本発明抑制方法 上記の通り、ＰＣ−ＳＯＤにシュークロースを共存させ
ることにより該ＰＣ−ＳＯＤの活性低下を抑制し、また
は、該ＰＣ−ＳＯＤのカラムクロマトグラフィーによる
分析の際の類縁物質ピークの出現を抑制することができ
る。従って本発明抑制剤は、シュークロースを有効成分
とする剤であって、前記一般式（Ｉ）で表されるＰＣ−
ＳＯＤと共存させることにより該ＰＣ−ＳＯＤの活性低
下を抑制し、または、該ＰＣ−ＳＯＤのカラムクロマト
グラフィーによる分析の際の類縁物質ピークの出現を抑
制するための剤である。好ましくは、前記ＰＣ−ＳＯＤ
の活性低下の抑制、および該ＰＣ−ＳＯＤのカラムクロ
マトグラフィーによる分析の際の類縁物質ピークの出現
の抑制の両方の目的のために使用される剤である。<3> The Inhibitor of the Present Invention and the Method of Inhibiting the Present Invention As described above, coexistence of sucrose in PC-SOD suppresses the decrease in the activity of PC-SOD, or the column chromatography of PC-SOD. Appearance of a peak of an analogous substance at the time of analysis by chromatography can be suppressed. Therefore, the inhibitor of the present invention is an agent containing sucrose as an active ingredient, and is a PC-based compound represented by the general formula (I).
It is an agent for suppressing a decrease in the activity of the PC-SOD by coexisting with SOD, or for suppressing the appearance of a peak of an analogous substance upon analysis of the PC-SOD by column chromatography. Preferably, the PC-SOD
Is an agent used for both the purpose of suppressing the decrease in the activity of, and the appearance of analogous substance peaks during the analysis of the PC-SOD by column chromatography.
【００６５】なお本発明抑制剤は、凍結乾燥医薬組成物
の調製の際にＰＣ−ＳＯＤと共存させると、上記の抑制
効果に加えてさらに凍結乾燥医薬組成物の性状を良好に
保持させるという効果を発揮する。従って本発明抑制剤
は、特に、前記ＰＣ−ＳＯＤを含有する凍結乾燥医薬組
成物を調製する際に前記ＰＣ−ＳＯＤと共存させて用い
ることが好ましい。When the inhibitor of the present invention is co-present with PC-SOD during the preparation of a lyophilized pharmaceutical composition, it has the effect of maintaining the properties of the lyophilized pharmaceutical composition in addition to the above inhibitory effects. Demonstrate. Therefore, it is preferable that the inhibitor of the present invention is used in combination with the PC-SOD when preparing the freeze-dried pharmaceutical composition containing the PC-SOD.
【００６６】また本発明抑制方法は、上記一般式（Ｉ）
で表されるＰＣ−ＳＯＤにシュークロースを共存させる
ことにより該ＰＣ−ＳＯＤの活性低下を抑制し、また
は、該ＰＣ−ＳＯＤのカラムクロマトグラフィーによる
分析の際の類縁物質ピークの出現を抑制する方法であ
る。好ましくは、本発明抑制方法は、前記ＰＣ−ＳＯＤ
の活性低下の抑制、および該ＰＣ−ＳＯＤのカラムクロ
マトグラフィーによる分析の際の類縁物質ピークの出現
の抑制の両方の目的のために使用される。Further, the method of the present invention is characterized in that the above general formula (I)
A method for suppressing a decrease in the activity of the PC-SOD by coexisting with sucrose in the PC-SOD represented by the following formula, or suppressing the appearance of a peak of an analogous substance during the analysis of the PC-SOD by column chromatography It is. Preferably, the method according to the present invention comprises the step of:
Is used for both the purpose of suppressing the decrease in the activity of PC-SOD and suppressing the appearance of analog peaks when the PC-SOD is analyzed by column chromatography.
【００６７】本発明抑制剤の有効成分として用いるシュ
ークロース、また本発明抑制方法に用いられるシューク
ロース、及びそのＰＣ−ＳＯＤへの添加量等は、前記＜
１＞に記載したものと同様である。The sucrose used as the active ingredient of the inhibitor of the present invention, the sucrose used in the method of the present invention, and the amount of the sucrose added to PC-SOD are as described above.
1>.
【００６８】またＰＣ−ＳＯＤの活性低下抑制、カラム
クロマトグラフィーによる分析の際の類縁物質ピークの
出現抑制、また凍結乾燥医薬組成物の調製に用いた場合
における凍結乾燥医薬組成物の性状の良好な保持は、後
述の実施例に記載された方法で確認することができる。In addition, suppression of the decrease in the activity of PC-SOD, suppression of the appearance of analogous substance peaks during column chromatography analysis, and excellent properties of the lyophilized pharmaceutical composition when used for the preparation of the lyophilized pharmaceutical composition. Retention can be confirmed by the method described in Examples described later.
【００６９】[0069]
【実施例】以下に、本発明を製造例及び実施例により具
体的に説明する。しかしながら、これらにより本発明の
技術的範囲が限定されるべきものではない。The present invention will be described below in more detail with reference to production examples and examples. However, these should not limit the technical scope of the present invention.
【００７０】〔製造例〕＜１＞ヒトＣｕ／Ｚｎ ＳＯＤ
の調製 該ＳＯＤは、特開平6-199895号公報に記載された方法で
調製したものを用いた。このヒトＣｕ／Ｚｎ ＳＯＤの1
11 位のアミノ酸はＳ−（２−ヒドロキシエチルチオ）
システインとなっている。[Production Example] <1> Human Cu / Zn SOD
Preparation of SOD The SOD used was prepared by the method described in JP-A-6-199895. This human Cu / Zn SOD
The amino acid at position 11 is S- (2-hydroxyethylthio)
Has become cysteine.
【００７１】＜２＞ＰＣ−ＳＯＤの調製 ＰＣ−ＳＯＤは、特開平6-54681号公報に記載された方
法によって、２−（４−ヒドロキシカルボニルブチロイ
ル）リゾレシチンの活性エステル体を調製し、これと上
記＜１＞のＳＯＤとを反応させることによって調製し
た。精製・濃縮後、蛋白質濃度をローリー法(Lowry, O.
h.ら、J. Biol. Chem., 193 巻, 265 頁 (1951年))、Ｓ
ＯＤの残存アミノ基をＴＮＢＳ法（トリニトロベンゼン
スルホン酸塩、Goodwin, J. F.ら、Clin. Chem., 16
巻, 24頁 (1970年))で分析することにより、ＳＯＤ１分
子あたりのレシチン誘導体の結合数を求めたところ、平
均４．０個であった。このＰＣ−ＳＯＤの水溶液は青緑
色〜緑色を呈し、ｐＨは７〜８であった。また、分子量
をＳＤＳ−ポリアクリルアミドゲル電気泳動法により調
べたところ、ＰＣ−ＳＯＤサブユニットのモノマー（Ｐ
Ｃ−ＳＯＤはサブユニットのホモダイマーである）あた
り約１８０００であった。<2> Preparation of PC-SOD PC-SOD was prepared by preparing an active ester of 2- (4-hydroxycarbonylbutyroyl) lysolecithin by the method described in JP-A-6-54681. And SOD of the above <1>. After purification and concentration, the protein concentration was determined by the Lowry method (Lowry, O.
H. et al., J. Biol. Chem., 193, 265 (1951)), S.
The remaining amino groups of the OD were determined by the TNBS method (trinitrobenzene sulfonate, Goodwin, JF et al., Clin. Chem., 16
Vol. 24, 1970 (1970)), the number of lecithin derivatives bound per SOD molecule was determined to be 4.0 on average. This aqueous solution of PC-SOD exhibited a bluish green to green color, and had a pH of 7 to 8. In addition, when the molecular weight was examined by SDS-polyacrylamide gel electrophoresis, the monomer of PC-SOD subunit (P
C-SOD is a homodimer of subunits).
【００７２】〔実施例１〕本実施例において「ＰＥＧ４
０００」とは、ポリエチレングリコール（平均分子量４
０００）をいう。[Embodiment 1] In this embodiment, "PEG4
000 ”means polyethylene glycol (average molecular weight 4
000).
【００７３】(1)以下、本実施例で用いた方法をまとめ
て説明する。 (1-1)凍結乾燥 凍結乾燥機（モデルNo.RL402BS；共和真空製）を用いて
溶液組成物を凍結乾燥した。なお凍結乾燥のプログラム
は、下記表１のステップ(1)〜(5)の順に実行されるよう
設定した。(1) Hereinafter, the methods used in this embodiment will be described together. (1-1) Freeze-drying The solution composition was freeze-dried using a freeze-dryer (Model No. RL402BS; manufactured by Kyowa Vacuum). The freeze-drying program was set to be executed in the order of steps (1) to (5) in Table 1 below.
【００７４】[0074]
【表１】 [Table 1]
【００７５】(1-2)各種試験方法 (1-2-1)性状試験 凍結乾燥された組成物について、以下の方法で組成物の
性状を調べた。(1-2) Various Test Methods (1-2-1) Property Test The composition of the freeze-dried composition was examined by the following method.
【００７６】肉眼で観察して、凍結乾燥組成物のケーキ
の形状が錠剤状であり、かつ凍結乾燥組成物に注射用水
を添加した際、１０秒以内に溶解し、かつ不溶性異物を
認めなかったものを「○」、凍結乾燥組成物のケーキの
形状が錠剤状でなかったもの、又は凍結乾燥組成物に注
射用水を添加した際、１０秒以内に溶解しなかったかも
しくは不溶性異物を認めたものを「×」として評価し
た。When visually observed, the freeze-dried composition cake was tablet-shaped, and when water for injection was added to the freeze-dried composition, it was dissolved within 10 seconds and no insoluble foreign matter was observed. The product was marked with “O”, the freeze-dried composition cake was not tablet-shaped, or when the water for injection was added to the freeze-dried composition, it did not dissolve within 10 seconds or found insoluble foreign matter Was evaluated as "x".
【００７７】(1-2-2)安定性試験 凍結乾燥された組成物について、以下の方法で組成物中
のＰＣ−ＳＯＤの酵素活性を測定した。(1-2-2) Stability test The freeze-dried composition was measured for PC-SOD enzyme activity in the composition by the following method.
【００７８】凍結乾燥組成物に注射用水を添加して再溶
解した溶液を用い、pH7.8、30℃の条件下でＮＢＴ(ニト
ロブルーテトラゾリウム)を用いてJ.Biol.Chem. Vol.24
4,No.22, 6049-6055(1969)に準じた方法により測定し、
ＮＢＴの還元速度を５０％阻害する酵素量を１Uとして
求めた。凍結乾燥直後の酵素活性を１００(％)とした時
の保存後の酵素活性の相対値(％)を求めた。 (1-2-3)カラムクロマトグラフィーによる類縁物質ピー
クの検出試験 (1-2-3-1)ゲル濾過クロマトグラフィーによる類縁物質
ピークの検出試験（以下、「ＧＰＣ」と略記することも
ある） 凍結乾燥組成物に注射用水を添加して再溶解した溶液を
試料とし、以下のカラム及び移動相を用いた高速液体ク
ロマトグラフィーに付した。Using a solution obtained by adding water for injection to the freeze-dried composition and redissolving it, using NBT (nitro blue tetrazolium) at pH 7.8 and 30 ° C., J. Biol. Chem. Vol.
4, Measured by the method according to No. 22, 6049-6055 (1969),
The amount of the enzyme that inhibits the reduction rate of NBT by 50% was determined as 1 U. The relative value (%) of the enzyme activity after storage, where the enzyme activity immediately after freeze-drying was defined as 100 (%), was determined. (1-2-3) Detection test of analogous substance peak by column chromatography (1-2-3-1) Detection test of analogous substance peak by gel filtration chromatography (hereinafter sometimes abbreviated as "GPC") A solution obtained by adding water for injection to the lyophilized composition and redissolving it was used as a sample, and subjected to high-performance liquid chromatography using the following column and mobile phase.
【００７９】カラム：YMC-Pack Diol-200（(株)ワイエ
ムシイ製） 移動相：45％アセトニトリル／0.01M リン酸緩衝液 溶出液の２２０ｎｍの吸光度を連続的に検出し、その検
出チャートを得た。検出チャートにおけるＰＣ−ＳＯＤ
のピークの形状が、凍結乾燥前のＰＣ−ＳＯＤのピーク
の形状と実質的な差が認められなかった場合を「○」、
凍結乾燥前のＰＣ−ＳＯＤのピークには認められないピ
ークが認められた場合を「×」として評価した。Column: YMC-Pack Diol-200 (manufactured by YMC) Mobile phase: 45% acetonitrile / 0.01M phosphate buffer The absorbance at 220 nm of the eluate was continuously detected, and a detection chart was obtained. . PC-SOD in detection chart
When the shape of the peak was not substantially different from the shape of the peak of PC-SOD before freeze-drying, "O"
The case where a peak not observed in the PC-SOD peak before freeze-drying was observed was evaluated as “x”.
【００８０】(1-2-3-2)逆相クロマトグラフィーによる
類縁物質ピークの検出試験（以下、「ＣＮ」と略記する
こともある） 上記(1-2-3-1)と同様に試料を調製し、以下のカラム及
び移動相を用いた高速液体クロマトグラフィーに付し
た。(1-2-3-2) Test for detecting peaks of related substances by reversed-phase chromatography (hereinafter sometimes abbreviated as “CN”) A sample was prepared in the same manner as in (1-2-3-1) above. Was subjected to high performance liquid chromatography using the following column and mobile phase.
【００８１】カラム：シアノプロピル化シリカゲル
（(株)ワイエムシイ製） 移動相：アセトニトリル／0.1％トリフルオロ酢酸（ア
セトニトリルの２０％〜９０％の濃度勾配により溶出）Column: cyanopropylated silica gel (manufactured by YMC) Mobile phase: acetonitrile / 0.1% trifluoroacetic acid (eluted with a concentration gradient of 20% to 90% of acetonitrile)
【００８２】溶出液の２２０ｎｍ及び２７０ｎｍの吸光
度を連続的に検出した。ＰＣ−ＳＯＤのピーク（主要な
ピーク）の前に出現するピーク群（類縁物質のピーク
群）の合計面積（ＳＩ）と、ＰＣ−ＳＯＤのピークとＳ
Ｉとの合計面積（ＳＴ）を測定し、各々のサンプルにつ
いて類縁物質の量（ＳＩ／ＳＴ；％）を算出した。The absorbance at 220 nm and 270 nm of the eluate was continuously detected. The total area (SI) of the peak group (peak group of related substances) appearing before the PC-SOD peak (main peak), and the PC-SOD peak and S
The total area (ST) with I was measured, and the amount of related substances (SI / ST;%) was calculated for each sample.
【００８３】なお、後述の表７と表８については、比較
を容易にするために、処方１における凍結乾燥直後の類
縁物質の量を１としたときの相対値で示した。In Tables 7 and 8, which will be described later, for ease of comparison, relative amounts are shown assuming that the amount of related substances immediately after freeze-drying in Formulation 1 is 1.
【００８４】(2)ＰＣ−ＳＯＤ含有医薬組成物について
の試験 (2-1)組成物中の成分の種類による影響１ 前記製造例で製造したＰＣ−ＳＯＤ(0.4mg/バイアル)及
び下記の表２に示した各種成分(表中のカッコ内の数値
は１バイアルあたりの重量を示す)を注射用水に溶解し
て溶液組成物を製造し、これを凍結乾燥した。(2) Test on Pharmaceutical Composition Containing PC-SOD (2-1) Influence by Type of Ingredients in Composition 1 PC-SOD (0.4 mg / vial) produced in the above Production Example and the following table The various components shown in Table 2 (the values in parentheses in the table indicate the weight per vial) were dissolved in water for injection to prepare a solution composition, which was freeze-dried.
【００８５】この凍結乾燥物を、前記の各種試験に付し
た。結果を表２に示す。評価項目中１つでも×があった
ものは「×」と判定し、評価項目中全く×がなかったも
のを「◎」と判定した。The lyophilized product was subjected to the various tests described above. Table 2 shows the results. If any one of the evaluation items had x, it was judged as "x", and if there was no x in the evaluation items, it was judged as "◎".
【００８６】[0086]
【表２】 [Table 2]
【００８７】表２の結果から、ＰＣ−ＳＯＤはシューク
ロースと共に配合することによって、極めて凍結乾燥医
薬組成物の性状が良好となり、またカラムクロマトグラ
フィー（ＧＰＣ）による分析の際の類縁物質ピークの出
現が抑制されることが明らかになった。From the results in Table 2, it can be seen that when PC-SOD is blended with sucrose, the properties of the lyophilized pharmaceutical composition are extremely good, and the appearance of analogous substance peaks during analysis by column chromatography (GPC). Was found to be suppressed.
【００８８】(2-2)成分の種類による影響２ 前記製造例で製造したＰＣ−ＳＯＤ及び下記の表３に示
した各種成分を注射用水に溶解して溶液組成物を製造
し、0.5 mLずつバイアルに分注し、前記の方法で凍結乾
燥した。(2-2) Influence 2 of the kind of the component The PC-SOD prepared in the above-mentioned preparation example and various components shown in the following Table 3 were dissolved in water for injection to prepare a solution composition, and 0.5 mL each was prepared. Dispensed into vials and lyophilized as described above.
【００８９】なお表中の重量は、１バイアル中に含有さ
れる重量を示し、「−」は配合していないことを示す。The weight in the table indicates the weight contained in one vial, and "-" indicates that no vial was blended.
【００９０】[0090]
【表３】 [Table 3]
【００９１】上記処方１〜４の凍結乾燥医薬組成物を、
以下の(a)〜(d)の時点において、性状試験、安定性試
験、ゲル濾過クロマトグラフィーによる類縁物質ピーク
の検出試験および逆相クロマトグラフィーによる類縁物
質ピークの検出試験に付した。The lyophilized pharmaceutical compositions of the above Formulations 1 to 4 were
At the following points (a) to (d), they were subjected to a property test, a stability test, a test for detecting a peak of a related substance by gel filtration chromatography, and a test for detecting a peak of a related substance by reverse phase chromatography.
【００９２】 (a)凍結乾燥直後（保存せず） (b)凍結乾燥後、４０℃で３ヶ月間保存した時点 (c)凍結乾燥後、２５℃で３ヶ月間保存した時点 (d)凍結乾燥後、８℃で３ヶ月間保存した時点（処方１
については６ヶ月間保存した時点） 各試験の結果を表４〜表７に示す。 (1)性状試験(A) Immediately after freeze-drying (not stored) (b) After freeze-drying and storing at 40 ° C. for 3 months (c) After freeze-drying and storing at 25 ° C. for 3 months (d) Freezing After drying, when stored at 8 ° C. for 3 months (formulation 1
At the time of storage for 6 months) The results of each test are shown in Tables 4 to 7. (1) Property test
【００９３】[0093]
【表４】 [Table 4]
【００９４】いずれの処方も、良好な性状を示した。All the formulations showed good properties.
【００９５】(2)安定性試験(2) Stability test
【００９６】[0096]
【表５】 [Table 5]
【００９７】ソルビトールとポリエチレングリコールと
を含有する処方（処方４）は、他の処方に比してＰＣ−
ＳＯＤ活性の低下が大きかった。The formulation containing sorbitol and polyethylene glycol (formulation 4) has a PC-
The decrease in SOD activity was significant.
【００９８】(3)ゲル濾過クロマトグラフィーによる類
縁物質ピークの検出試験(3) Test for detecting peaks of related substances by gel filtration chromatography
【００９９】[0099]
【表６】 表６中、Ｎ．Ｔ．は試験していないことを示す。[Table 6] In Table 6, N.I. T. Indicates not tested.
【０１００】いずれの処方も、ゲル濾過クロマトグラフ
ィーによる類縁物質ピークは検出されなかった。In any of the formulations, no analog peak was detected by gel filtration chromatography.
【０１０１】(4)逆相クロマトグラフィーによる類縁物
質ピークの検出試験（２２０ｎｍ）(4) Detection test of analogous substance peak by reverse phase chromatography (220 nm)
【０１０２】[0102]
【表７】 [Table 7]
【０１０３】ソルビトールとマンニトールとを含有する
処方（処方３）及びソルビトールとポリエチレングリコ
ールとを含有する処方（処方４）では、逆相クロマトグ
ラフィーによる類縁物質ピークの面積が、シュークロー
スを含有する処方（処方１）の約１．４〜３．４倍であ
った。In the formulation containing sorbitol and mannitol (formulation 3) and the formulation containing sorbitol and polyethylene glycol (formulation 4), the area of the peak of a related substance determined by reverse phase chromatography was reduced in the formulation containing sucrose (formulation 3). It was about 1.4 to 3.4 times that of the prescription 1).
【０１０４】また上記処方において、ＰＣ−ＳＯＤの含
量を0.4mg、あるいは10mgとしたものについても同様に
実験をした結果、同様の結果が得られた。Further, the same experiment was carried out with the above formulation in which the content of PC-SOD was 0.4 mg or 10 mg, and the same result was obtained.
【０１０５】以上の結果から、ＰＣ−ＳＯＤ及びシュー
クロースを含有する凍結乾燥組成物は、他の処方に比し
て、(1)性状が良好であり、かつ(2)長期間の保存による
酵素活性の低下が非常に少なく（極めて安定である）、
かつ(3)ゲル濾過クロマトグラフィー及び逆相クロマト
グラフィーによる類縁物質ピークが極めて少ないという
極めて有利な効果を有することが見いだされた。From the above results, the freeze-dried composition containing PC-SOD and sucrose has (1) better properties and (2) an enzyme obtained by long-term storage as compared with other formulations. Very little loss of activity (very stable),
And (3) it has been found to have a very advantageous effect that the peaks of related substances by gel filtration chromatography and reverse phase chromatography are extremely small.
【０１０６】〔実施例２〕ＰＣ−ＳＯＤ及びシュークロ
ースを含有する凍結乾燥組成物が、より長期間の保存に
耐えうるか否かを調べるために、以下の試験を行った。Example 2 The following test was conducted to examine whether or not a freeze-dried composition containing PC-SOD and sucrose can withstand longer-term storage.
【０１０７】上記の処方１によりＰＣ−ＳＯＤを含有す
る凍結乾燥組成物を製造し、以下の(e)〜(h)の時点にお
いて、実施例１と全く同様の方法で各種試験を行った。
本試験においては各処方においてそれぞれ３ロットを試
験に供した。A freeze-dried composition containing PC-SOD was prepared according to the above Formulation 1, and various tests were carried out at the following points (e) to (h) in exactly the same manner as in Example 1.
In this test, three lots of each formulation were subjected to the test.
【０１０８】 (e)凍結乾燥直後（保存せず） (f)凍結乾燥後、４０℃で６ヶ月間保存した時点 (g)凍結乾燥後、２５℃で１２ヶ月間(g-1)、２４ヶ月間
(g-2)、３６ヶ月間(g-3)保存した時点 (h)凍結乾燥後、８℃で１２ヶ月間(h-1)、２４ヶ月間(h
-2)、３６ヶ月間(h-3)保存した時点 試験の結果をまとめて表８に示す。(E) Immediately after freeze-drying (not stored) (f) After freeze-drying and storage at 40 ° C. for 6 months (g) After freeze-drying and 25 ° C. for 12 months (g−1), 24 Months
(g-2) at the time of storage for 36 months (g-3) (h) After freeze-drying, at 8 ° C for 12 months (h-1), 24 months (h
-2), when stored for 36 months (h-3) The results of the test are summarized in Table 8.
【０１０９】[0109]
【表８】 [Table 8]
【０１１０】またこの処方において、ＰＣ−ＳＯＤの含
量を０．４ｍｇ、あるいは１０ｍｇとしたものについて
も同様に実験した結果をそれぞれ表９及び表１０に示
す。Tables 9 and 10 also show the results of the same experiment conducted with this formulation having a PC-SOD content of 0.4 mg or 10 mg, respectively.
【０１１１】[0111]
【表９】 [Table 9]
【０１１２】[0112]
【表１０】 [Table 10]
【０１１３】［実施例３］前記製造例で製造したＰＣ−
ＳＯＤ ３０ｍｇ及びシュークロース５０ｍｇを注射用
水に溶解して溶液組成物を製造し、１．５ｍＬずつバイ
アルに分注し、前記の方法で凍結乾燥した。[Embodiment 3] The PC-
A solution composition was prepared by dissolving 30 mg of SOD and 50 mg of sucrose in water for injection, dispensed in 1.5 mL aliquots into vials, and lyophilized as described above.
【０１１４】上記組成物の凍結乾燥直後、８℃で９ヶ月
保存した時点において、比活性の測定方法を除き、実施
例１と全く同様の方法で各種試験を行った。比活性は、
凍結乾燥組成物に注射用水を添加して再溶解した溶液を
用い、ｐＨ７．８、 ２５℃の条件下でシトクロムＣを
用いて J. Biol. Chem., Vol. 244, No.22, 6049-6055
(1969) に準じた方法により測定し、シトクロムＣの還
元速度を５０％阻害する酵素量を１Ｕとして求めた。凍
結乾燥直後の酵素活性を１００（％）とした時の保存後
の酵素活性の相対値（％）を求めた。試験の結果を表１
１に示す。Immediately after the composition was freeze-dried and stored at 8 ° C. for 9 months, various tests were carried out in exactly the same manner as in Example 1, except for the method for measuring the specific activity. The specific activity is
Using a solution obtained by adding water for injection to the lyophilized composition and re-dissolving it, using cytochrome C under conditions of pH 7.8 and 25 ° C, J. Biol. Chem., Vol. 244, No. 22, 6049- 6055
The measurement was carried out according to the method according to (1969), and the amount of the enzyme which inhibits the reduction rate of cytochrome C by 50% was determined as 1 U. The relative value (%) of the enzyme activity after storage, where the enzyme activity immediately after freeze-drying was defined as 100 (%), was determined. Table 1 shows the test results.
It is shown in FIG.
【０１１５】[0115]
【表１１】 [Table 11]
【０１１６】以上の結果から、シュークロースは、従来
知られている医薬担体（結合剤）としての効果の他に、
ＰＣ−ＳＯＤに添加することによって長期間保存後でも
凍結乾燥医薬組成物の性状を良好に保持し、ＰＣ−ＳＯ
Ｄ活性の低下を抑制し（安定に保持し）、かつゲル濾過
クロマトグラフィー及び逆相クロマトグラフィーによる
分析の際の類縁物質ピークを抑制する等の効果を発揮す
ることが示された。From the above results, it can be seen that sucrose is not only effective as a conventionally known pharmaceutical carrier (binder),
By adding to the PC-SOD, the properties of the lyophilized pharmaceutical composition can be maintained well even after long-term storage,
It has been shown that the compound exhibits effects such as suppressing the decrease in D activity (keeping it stable) and suppressing peaks of related substances during analysis by gel filtration chromatography and reverse phase chromatography.
【０１１７】[0117]
【発明の効果】本発明医薬組成物は、組成物中のシュー
クロースの作用により、長期間保存によるＰＣ−ＳＯＤ
の活性低下が少なく（安定性が高く）、凍結乾燥した場
合の性状が良好で、かつカラムクロマトグラフィーによ
る分析の際の類縁物質ピークの出現が抑制されているの
で、医薬品、例えば本発明処置剤の成分として有用であ
る。EFFECT OF THE INVENTION The pharmaceutical composition of the present invention can be stored for a long period of time by the action of sucrose in the composition.
Activity (high stability), good properties when freeze-dried, and suppression of peaks of related substances upon analysis by column chromatography. It is useful as a component of
【０１１８】本発明処置剤は、長期間保存によるＰＣ−
ＳＯＤの活性低下が少なく（安定性が高く）、凍結乾燥
した場合の性状が良好で、かつカラムクロマトグラフィ
ーによる分析の際の類縁物質ピークの出現が少ない医薬
品として、例えば過剰な活性酸素に起因する疾患の処置
剤として有用である。The treatment agent of the present invention can be stored for a long time in PC-
Pharmaceuticals that have a small decrease in SOD activity (high stability), good properties when lyophilized, and little appearance of related substance peaks during analysis by column chromatography, for example, due to excess active oxygen It is useful as a therapeutic agent for diseases.
【０１１９】また本発明抑制剤は、ＰＣ−ＳＯＤの長期
間保存による活性低下を抑制し（安定性を保持させ）、
凍結乾燥した場合の性状を良好に保ち、かつカラムクロ
マトグラフィーによる分析の際の類縁物質ピークの出現
を抑制する作用を有するので、本発明医薬組成物や本発
明処置剤を調製する際に有用である。Further, the inhibitor of the present invention suppresses a decrease in activity of PC-SOD due to long-term storage (maintains stability),
It is useful for preparing the pharmaceutical composition of the present invention or the treatment agent of the present invention because it has an effect of maintaining good properties when lyophilized and suppressing the appearance of analogous substance peaks during analysis by column chromatography. is there.
【０１２０】[0120]
【配列表】 <110> LTT Institute Co.,Ltd., Asahi Glass Co., Ltd., and Seikagaku Corpo ration <120> Pharmaceutical composition containing lecithinized-superoxide dism utase <130> <160> 1 <210> 1 <211> 153 <212> PRT <213> Homo sapiens <400> 1 Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly 1 5 10 15 Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp 20 25 30 Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val His 35 40 45 Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe 50 55 60 Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His 65 70 75 80 Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp 85 90 95 Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile 100 105 110 Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys 115 120 125 Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu 130 135 140 Ala Cys Gly Val Ile Gly Ile Ala Gln 145 150[Sequence Table] <110> LTT Institute Co., Ltd., Asahi Glass Co., Ltd., and Seikagaku Corporation <120> Pharmaceutical composition containing lecithinized-superoxide dism utase <130> <160> 1 <210> 1 < 211> 153 <212> PRT <213> Homo sapiens <400> 1 Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly 1 5 10 15 Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp 20 25 30 Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val His 35 40 45 Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe 50 55 60 Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His 65 70 75 80 Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp 85 90 95 Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile 100 105 110 Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys 115 120 125 Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu 130 135 140 Ala Cys Gly Val Ile Gly Ile Ala Gln 145 150
───────────────────────────────────────────────────── フロントページの続き (72)発明者 池田 義仁 東京都東大和市立野３−594−１ アルテ ミス立野305 (72)発明者 佐藤 裕美 東京都武蔵村山市大南４−23−２ クレー ル202 Ｆターム(参考） 4C076 AA29 CC01 CC16 DD67 FF65 GG06 4C084 AA02 AA03 BA01 BA08 BA22 BA33 BA36 BA37 CA18 CA59 DC24 MA05 MA44 NA03 NA14 ZA221 ZA681 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yoshihito Ikeda 3-594-1 Tateno, Higashiyamato-shi, Tokyo 305 Artemis Tateno 305 (72) Inventor Hiromi Sato 4-23-2 Onan, Musashimurayama-shi, Tokyo 202 F term (reference) 4C076 AA29 CC01 CC16 DD67 FF65 GG06 4C084 AA02 AA03 BA01 BA08 BA22 BA33 BA36 BA37 CA18 CA59 DC24 MA05 MA44 NA03 NA14 ZA221 ZA681
Claims (18)
スーパーオキシドジスムターゼ及びシュークロースを含
有することを特徴とする医薬組成物。 ＳＯＤ’（Ｑ−Ｂ）m （Ｉ） （式中、ＳＯＤ’はスーパーオキシドジスムターゼの残
基を表し、Ｑは化学的架橋を表し、Ｂはグリセロールの
２位に水酸基を有するリゾレシチンのその水酸基の水素
原子を除いた残基を表し、ｍはスーパーオキシドジスム
ターゼ１分子に対するリゾレシチンの平均結合数であっ
て、１以上の正数を表す）1. A pharmaceutical composition comprising a lecithinated superoxide dismutase represented by the following general formula (I) and sucrose. SOD ′ (QB) m (I) (where SOD ′ represents a residue of superoxide dismutase, Q represents a chemical crosslink, and B represents the hydroxyl group of lysolecithin having a hydroxyl group at the 2-position of glycerol. Represents a residue excluding a hydrogen atom, and m is the average number of lysolecithin bonds to one molecule of superoxide dismutase, and represents a positive number of 1 or more.
求項１に記載の医薬組成物。 (a)性状：この医薬組成物を凍結乾燥したものに注射用
水を添加すると溶解し、不溶性異物は認められない。 (b)安定性：この医薬組成物を凍結乾燥した直後におけ
る単位重量あたりのスーパーオキシドジスムターゼ活性
を１００とした場合、凍結乾燥した組成物を８℃で１２
ヶ月、２５℃で１２ヶ月、又は４０℃で６ヶ月間保存し
た時点における該活性の相対値が、いずれも９７％以上
である。 (c)ゲル濾過クロマトグラフィーにおける類縁物質ピー
ク：凍結乾燥した組成物を再溶解し、ゲル濾過クロマト
グラフィーにかけ、溶出液の２２０ｎｍの吸光度を測定
した場合、当該吸光度の検出チャートにおけるレシチン
化スーパーオキシドジスムターゼのピークの形状と、凍
結乾燥前のレシチン化スーパーオキシドジスムターゼの
当該ピークの形状との間に、実質的な差が認められな
い。 (d)逆相クロマトグラフィーにおける類縁物質ピーク：
凍結乾燥した組成物を８℃で１２ヶ月、２５℃で１２ヶ
月、又は４０℃で６ヶ月間保存した時点で再溶解し、逆
相クロマトグラフィーにかけ、溶出液の２２０ｎｍ及び
２７０ｎｍの吸光度を測定した場合、検出される類縁物
質の量が、いずれも凍結乾燥した直後におけるものと実
質的に変化しない。2. The pharmaceutical composition according to claim 1, which has the following properties. (a) Properties: This pharmaceutical composition is freeze-dried and dissolved when water for injection is added, and no insoluble foreign matter is observed. (b) Stability: Assuming that the superoxide dismutase activity per unit weight immediately after freeze-drying the pharmaceutical composition is 100, the freeze-dried composition is 12 at 8 ° C.
The relative value of the activity at the time of storage at 25 ° C. for 12 months or at 40 ° C. for 6 months is 97% or more. (c) Related substance peak in gel filtration chromatography: When the freeze-dried composition is redissolved and subjected to gel filtration chromatography, and the absorbance at 220 nm of the eluate is measured, the lecithinated superoxide dismutase in the absorbance detection chart is obtained. No substantial difference is observed between the shape of the peak of No. 1 and the shape of the peak of lecithinated superoxide dismutase before freeze-drying. (d) Related substance peak in reverse phase chromatography:
When the lyophilized composition was stored at 8 ° C. for 12 months, at 25 ° C. for 12 months, or at 40 ° C. for 6 months, it was redissolved, subjected to reverse phase chromatography, and the absorbance at 220 nm and 270 nm of the eluate was measured. In this case, the amount of the analogs detected does not substantially change from that immediately after freeze-drying.
月、２５℃で３６ヶ月、又は４０℃で６ヶ月間保存した
いずれの時点においても請求項２に記載の全性質を維持
していることを特徴とする、請求項１に記載の医薬組成
物。3. The lyophilized composition is stored at 8 ° C. for 36 months, at 25 ° C. for 36 months, or at 40 ° C. for 6 months, while maintaining all properties according to claim 2. The pharmaceutical composition according to claim 1, characterized in that:
シドジスムターゼのレシチン部分が切断されて生成した
物質であることを特徴とする請求項２又は３に記載の医
薬組成物。4. The pharmaceutical composition according to claim 2, wherein the analogous substance is a substance produced by cleavage of a lecithin portion of a lecithinated superoxide dismutase.
〜０．１５μｍｏｌ／ｍｇ蛋白である、請求項１〜４の
いずれか１項に記載の医薬組成物。5. The method according to claim 5, wherein the fatty acid content of the pharmaceutical composition is 0.13.
The pharmaceutical composition according to any one of claims 1 to 4, which has a protein content of 0.1 to 0.15 µmol / mg protein.
ある、請求項１〜５のいずれか１項に記載の医薬組成物
（ｎは２以上の整数を表す）。6. The pharmaceutical composition according to claim 1, wherein Q is —C (O) — (CH 2 ) n —C (O) —, wherein n is 2 or more. Represents an integer).
スムターゼの残基である、請求項１〜６いずれか１項に
記載の医薬組成物。7. The pharmaceutical composition according to claim 1, wherein SOD ′ is a residue of human superoxide dismutase.
スムターゼのアミノ酸配列１１１位のアミノ酸がＳ−
（２−ヒドロキシエチルチオ）システインとなったスー
パーオキシドジスムターゼ修飾体の残基である、請求項
１〜７のいずれか１項に記載の医薬組成物。8. The SOD ′, wherein the amino acid at position 111 in the amino acid sequence of human superoxide dismutase is S-amino acid.
The pharmaceutical composition according to any one of claims 1 to 7, which is a residue of a modified superoxide dismutase that has become (2-hydroxyethylthio) cysteine.
中心に銅と亜鉛を含むスーパーオキシドジスムターゼで
ある、請求項７又は８に記載の医薬組成物。9. The pharmaceutical composition according to claim 7, wherein the superoxide dismutase is a superoxide dismutase containing copper and zinc in an active center.
〜９のいずれか１項に記載の医薬組成物。10. The method according to claim 6, wherein n is an integer of 2 to 10.
The pharmaceutical composition according to any one of claims 9 to 9.
〜１０のいずれか１項に記載の医薬組成物。11. The method according to claim 1, wherein m is a positive number from 1 to 12.
The pharmaceutical composition according to any one of claims 10 to 10.
シュークロースである、請求項１〜１１のいずれか１項
に記載の医薬組成物。12. The pharmaceutical composition according to claim 1, wherein the sucrose is activated carbon-treated sucrose.
求項１〜１２のいずれか１項に記載の医薬組成物。13. The pharmaceutical composition according to claim 1, which is in the form of a lyophilized pharmaceutical composition.
ーゼとシュークロースの重量比が０．４／１００〜６０
／１００である請求項１〜１３のいずれか１項に記載の
医薬組成物。14. The weight ratio of lecithinated superoxide dismutase to sucrose is 0.4 / 100-60.
The pharmaceutical composition according to any one of claims 1 to 13, wherein the ratio is / 100.
の医薬組成物からなる疾患の処置剤。15. An agent for treating a disease, comprising the pharmaceutical composition according to any one of claims 1 to 14.
性胃腸障害である、請求項１５に記載の処置剤。16. The therapeutic agent according to claim 15, wherein the disease is a motor neuron disease or ulcerative gastrointestinal disorder.
あって、下記一般式（Ｉ）で表されるレシチン化スーパ
ーオキシドジスムターゼと共存させることにより該スー
パーオキシドジスムターゼの活性低下を抑制し、また
は、該スーパーオキシドジスムターゼのカラムクロマト
グラフィーによる分析の際の類縁物質ピークの出現を抑
制するための剤。 ＳＯＤ’（Ｑ−Ｂ）m （Ｉ） （式中、ＳＯＤ’はスーパーオキシドジスムターゼの残
基を表し、Ｑは化学的架橋を表し、Ｂはグリセロールの
２位に水酸基を有するリゾレシチンのその水酸基の水素
原子を除いた残基を表し、ｍはスーパーオキシドジスム
ターゼ１分子に対するリゾレシチンの平均結合数であっ
て、１以上の正数を表す）17. An agent comprising sucrose as an active ingredient, wherein the agent comprises co-existence with a lecithinated superoxide dismutase represented by the following general formula (I) to suppress a decrease in the activity of the superoxide dismutase; An agent for suppressing the appearance of a peak of an analogous substance upon analysis of the superoxide dismutase by column chromatography. SOD ′ (QB) m (I) (where SOD ′ represents a residue of superoxide dismutase, Q represents a chemical crosslink, and B represents the hydroxyl group of lysolecithin having a hydroxyl group at the 2-position of glycerol. Represents a residue excluding a hydrogen atom, and m is the average number of lysolecithin bonds to one molecule of superoxide dismutase, and represents a positive number of 1 or more.
化スーパーオキシドジスムターゼにシュークロースを共
存させることにより該スーパーオキシドジスムターゼの
活性低下を抑制し、または、該スーパーオキシドジスム
ターゼのカラムクロマトグラフィーによる分析の際の類
縁物質ピークの出現を抑制する方法。 ＳＯＤ’（Ｑ−Ｂ）m （Ｉ） （式中、ＳＯＤ’はスーパーオキシドジスムターゼの残
基を表し、Ｑは化学的架橋を表し、Ｂはグリセロールの
２位に水酸基を有するリゾレシチンのその水酸基の水素
原子を除いた残基を表し、ｍはスーパーオキシドジスム
ターゼ１分子に対するリゾレシチンの平均結合数であっ
て、１以上の正数を表す）18. A decrease in the activity of superoxide dismutase is suppressed by coexisting sucrose with lecithinated superoxide dismutase represented by the following general formula (I), or the superoxide dismutase is subjected to column chromatography. A method for suppressing the appearance of related substance peaks during analysis. SOD ′ (QB) m (I) (where SOD ′ represents a residue of superoxide dismutase, Q represents a chemical crosslink, and B represents the hydroxyl group of lysolecithin having a hydroxyl group at the 2-position of glycerol. Represents a residue excluding a hydrogen atom, and m is the average number of lysolecithin bonds to one molecule of superoxide dismutase, and represents a positive number of 1 or more.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006062976A (en) * | 2004-08-24 | 2006-03-09 | Nippon Medical School | Oral agent for reducing oxidation stress of muscle |
| WO2008075706A1 (en) | 2006-12-19 | 2008-06-26 | Ltt Bio-Pharma Co., Ltd. | Therapeutic agent for interstitial pneumonia |
| WO2010064522A1 (en) | 2008-12-03 | 2010-06-10 | 株式会社Ｌｔｔバイオファーマ | Inhalant comprising modified superoxide dismutase |
| WO2010103959A1 (en) | 2009-03-13 | 2010-09-16 | 株式会社Ｌｔｔバイオファーマ | Ameliorating agent for chronic obstructive pulmonary disease |
| WO2017073255A1 (en) * | 2015-10-29 | 2017-05-04 | 株式会社Ｌｔｔバイオファーマ | Acute respiratory distress syndrome therapeutic agent |
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