JP3987562B2 - Peptides and their uses - Google Patents
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Description
この発明は、スギ花粉アレルゲンに特異的に反応するT細胞を不活性化するペプチド、及び、そのペプチドを有効成分として含んでなる免疫療法剤に関する。 The present invention relates to a peptide that inactivates a T cell that specifically reacts with a cedar pollen allergen, and an immunotherapeutic agent comprising the peptide as an active ingredient.
ここ数十年来、我国においては、春先になるとスギ花粉症による鼻炎や結膜炎を訴える人の数が増加し続けている。スギ花粉症はアルレギー症の一種であり、その主因はスギ花粉中の抗原性物質、すなわち、スギ花粉症アレルゲンであるといわれている。大気中に飛散したスギ花粉がヒトの体内に侵入すると、スギ花粉アレルゲンに対するイムノグロブリンE抗体が産生する。この状態で次にスギ花粉が侵入すると、その花粉中のアレルゲンとこのイムノグロブリンE抗体が免疫反応を起し、アレルギー症状を呈することとなる。 In recent decades, in Japan, the number of people complaining of rhinitis and conjunctivitis due to cedar pollinosis continues to increase in early spring. Cedar pollinosis is a type of arlegiosis, and the main cause is said to be an antigenic substance in cedar pollen, that is, cedar pollinosis allergen. When cedar pollen scattered in the atmosphere enters the human body, immunoglobulin E antibodies against cedar pollen allergen are produced. Next, when cedar pollen invades in this state, the allergen in the pollen and the immunoglobulin E antibody cause an immune reaction, and allergic symptoms are exhibited.
スギ花粉中に抗原性の相違する少なくとも二種類のアレルゲンの存在することが現在までに知られている。その一つは、ヤスエダ等が『ジャーナル・オブ・アレルギー・アンド・クリニカル・イムノロジー』、第71巻、第1号、第77〜86頁(1983年)に報告しているアレルゲンであり(非特許文献1)、今日、これは「Cryj1」と呼称されている。なお、Cryj1 はその全長アミノ酸配列が決定され、国際出願されている(WO 93/01213:特許文献1)。 It has been known to date that at least two types of allergens having different antigenicity are present in cedar pollen. One of them is an allergen reported by Yasuda et al. In “Journal of Allergy and Clinical Immunology”, Vol. 71, No. 1, pp. 77-86 (1983) (non-patent) Reference 1), today this is called “Cryj1”. Cryj1 has its full-length amino acid sequence determined and has been internationally filed (WO 93/01213: Patent Document 1).
もう一つは、タニアイ等『エフ・イー・ビー・エス・レターズ』、第239巻、第2号、第329〜332頁(1988年)(非特許文献2)やサカグチ等『アレルギー』、第45号、第309〜312頁(1990年)(非特許文献3)に報告されているアレルゲンであり、今日、これは「Cry j 2」と呼称されている。なお、Cry j 2はその全長アミノ酸配列が決定され、国際出願されている(WO 94/11512:特許文献2)。また、 Komiyama らも別個にCryj2の全長アミノ酸配列を決定しているがBiochem. Biophys. Res, Comm., vol.201, No.2, 1021-1028, (1994)(非特許文献4)、WO 94/11512(特許文献2)記載のアミノ酸配列とはアミノ酸残基が4か所異なっている。 The other is Taniyai et al. “EF SB Letters”, Vol. 239, No. 2, pp. 329-332 (1988) (Non-patent Document 2), Sakaguchi et al. No. 45, pp. 309-312 (1990) (Non-Patent Document 3), which is called “Cry j 2” today. The full-length amino acid sequence of Cry j 2 has been determined and has been internationally filed (WO 94/11512: Patent Document 2). Komiyama et al. Also separately determined the full-length amino acid sequence of Cryj2, but Biochem. Biophys. Res, Comm., Vol. 201, No. 2, 1021-1028, (1994) (non-patent document 4), WO It differs from the amino acid sequence described in 94/11512 (Patent Document 2) in 4 amino acid residues.
スギ花粉中には、通常、Cryj1とCryj2が約50:1乃至5:1の割合で存在し、花粉症患者から採取した血清の殆どがCryj1にもCryj2にも反応すると云われている。澤谷らは、『アレルギー』、第42巻、第6号、第738〜747頁(1993年)において、Cryj2は、皮内反応試験やRAST試験において、Cryj1と同程度の抗原性を発揮すると報告している(非特許文献5)。 In cedar pollen, Cryj1 and Cryj2 are usually present in a ratio of about 50: 1 to 5: 1, and it is said that most of the serum collected from hay fever responds to both Cryj1 and Cryj2. Sawatani et al., Allergy, Vol. 42, No. 6, pp. 738-747 (1993), reported that Cryj2 exhibits the same level of antigenicity as Cryj1 in intradermal reaction tests and RAST tests. (Non-Patent Document 5).
このように、スギ花粉アレルゲンが既に幾つか単離され、その性質、性状もある程度解明されたことから、精製スギ花粉アレルゲンをヒトに投与して減感作することにより、スギ花粉症を治療・予防できる見通しがついてきた。最近ではそのための減感作剤も幾つか考案されており、例えば、特開平1−156926号公報(特許文献3)や特開平3−93730号公報(特許文献4)には、N末端からのアミノ酸配列がAsp−Asn−Pro−Ile−Asp−Ser又はAla−Ile−Asn−Ile−Phe−Asnで表わされるスギ花粉アレルゲンに糖質を共有結合せしめ、生成した複合体を減感作剤としてヒトに投与する提案が為されている。 In this way, several cedar pollen allergens have already been isolated, and their properties and properties have been elucidated to some extent, so that purified cedar pollen allergen is administered to humans to desensitize it, thereby treating cedar pollinosis. The prospects that can be prevented have come. Recently, several desensitizers have been devised for this purpose. For example, JP-A-1-156926 (Patent Document 3) and JP-A-3-93730 (Patent Document 4) disclose the sensitizers from the N-terminal. A carbohydrate is covalently bound to a cedar pollen allergen whose amino acid sequence is represented by Asp-Asn-Pro-Ile-Asp-Ser or Ala-Ile-Asn-Ile-Phe-Asn, and the resulting complex is used as a desensitizing agent. Proposals for administration to humans have been made.
しかしながら、アレルギー症の診断や減感作療法には、通常、高純度のアレルゲンが大量に必要とされ、スギ花粉中のアレルゲンは僅少であるうえに安定性が低く、スギ花粉症の診断剤や減感作剤をスギ花粉だけで賄おうとすると、多大の困難が伴なう。このようなことから、最近のアレルギー疾患の治療・予防においては、これまでのように、患者にアレルゲン全体を投与するのではなく、アレルゲン中のT細胞が特異的に認識する最小領域、すなわち、本質的にT細胞エピトープのみからなる低分子のペプチドを投与する免疫療法が注目されている。 However, diagnosis and desensitization of allergies usually require large amounts of high-purity allergens, and allergens in cedar pollen are scarce and have low stability. It is very difficult to cover the desensitizer with cedar pollen alone. For this reason, in the recent treatment and prevention of allergic diseases, instead of administering the whole allergen to the patient as in the past, the minimum region that T cells in the allergen specifically recognize, that is, Attention has been focused on immunotherapy in which a small peptide consisting essentially of a T cell epitope is administered.
一般に、アレルゲンは、マクロファージなどの抗原提示細胞に取込まれると、そこで消化され、消化断片が免疫提示細胞表層のHLA(Human Leucocyte Antigen )蛋白質に結合し、抗原提示されることとなる。抗原提示される断片は、HLA蛋白質に対する親和性などにより、アレルゲンにおける一部の特定領域に限られ、斯かる領域のうち、T細胞が特異的に認識する領域は、通常、「T細胞エピトープ」と呼称される。実質的にT細胞エプトープのみからなるペプチドを投与する免疫療法には、
(i) ペプチドがB細胞エピトープを欠いている、すなわち、アレルゲンに特異的なイムノグロブリンE抗体が反応しないので、従来の粗製又は精製アレルゲンで頻発していたアナフィラキシーなどの副作用が起こり得ない。
(ii) 少量からスタートし、有効投与量に達するまでの期間が、従来の減感作剤に比較して、大幅に短縮できる。
(iii) 経口免疫寛容を誘導し、アレルゲンに対するアレルギー反応を減弱することができる。
などの利点がある。
In general, when allergens are taken into antigen-presenting cells such as macrophages, they are digested there, and the digested fragments bind to HLA (Human Leucocyte Antigen) protein on the surface of immune-presenting cells and presented as antigens. The antigen-presented fragment is limited to a specific region of the allergen due to its affinity for the HLA protein. Among these regions, the region specifically recognized by T cells is usually a “T cell epitope”. It is called. For immunotherapy in which a peptide consisting essentially of only a T cell eptop is administered,
(i) Since the peptide lacks the B cell epitope, that is, the immunoglobulin E antibody specific to the allergen does not react, side effects such as anaphylaxis that frequently occur with conventional crude or purified allergens cannot occur.
(ii) The period from starting with a small amount to reaching an effective dose can be significantly shortened compared to conventional desensitizers.
(iii) It can induce oral tolerance and attenuate allergic reactions to allergens.
There are advantages such as.
本発明者らは、上記T細胞エピトープを構成する最小単位のアミノ酸配列を見出し、本発明を完成した。この発明の第一の課題は、本質的にスギ花粉アレルゲンのT細胞エピトープのみからなるペプチドを提供することにある。この発明の第二の課題は、有効成分として上記ペプチドを含んでなる抗スギ花粉症剤を提供することにある。 The present inventors have found the amino acid sequence of the minimum unit constituting the T cell epitope and completed the present invention. The first object of the present invention is to provide a peptide consisting essentially of a T cell epitope of a cedar pollen allergen. The second object of the present invention is to provide an anti-cedar pollinosis agent comprising the above peptide as an active ingredient.
本発明は、
(1)配列番号7のアミノ酸配列から成るペプチド、
(2)配列番号8のアミノ酸配列から成るペプチド、
(3)配列番号9のアミノ酸配列から成るペプチド、
(4)配列番号10のアミノ酸配列から成るペプチド、
(5)配列番号11のアミノ酸配列から成るペプチド、
(6)配列番号7のアミノ酸配列から成るペプチドを有効成分とする抗スギ花粉症剤、
(7)配列番号8のアミノ酸配列から成るペプチドを有効成分とする抗スギ花粉症剤、
(8)配列番号9のアミノ酸配列から成るペプチドを有効成分とする抗スギ花粉症剤、
(9)配列番号10のアミノ酸配列から成るペプチドを有効成分とする抗スギ花粉症剤、
(10)配列番号11のアミノ酸配列から成るペプチドを有効成分とする抗スギ花粉症剤、
(11)更に、ぶどう糖、果糖、しょ糖、麦芽糖、乳糖、トレハロース、ソルビトール、マンニトール、マルチトールおよび/またはラクチトールを含む、前記抗スギ花粉症剤、
に関する。
The present invention
(1) a peptide comprising the amino acid sequence of SEQ ID NO: 7,
(2) a peptide consisting of the amino acid sequence of SEQ ID NO: 8,
(3) a peptide consisting of the amino acid sequence of SEQ ID NO: 9,
(4) a peptide consisting of the amino acid sequence of SEQ ID NO: 10,
(5) a peptide consisting of the amino acid sequence of SEQ ID NO: 11,
(6) an anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 7 as an active ingredient,
(7) an anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 8 as an active ingredient,
(8) an anti-cedar pollinosis agent comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 9 as an active ingredient,
(9) an anti-cedar pollinosis agent comprising a peptide comprising the amino acid sequence of SEQ ID NO: 10 as an active ingredient,
(10) an anti-cedar pollinosis agent comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 11 as an active ingredient,
(11) The anti-cedar pollinosis agent further comprising glucose, fructose, sucrose, maltose, lactose, trehalose, sorbitol, mannitol, maltitol and / or lactitol,
About.
本発明により、本質的にスギ花粉アレルゲンに特異的なT細胞エピトープのみからなる新規ペプチド群、さらにはそれらのポリペプチドを有効成分として含んでなる抗スギ花粉症剤を提供することができた。そして、本発明のペプチドは、スギ花粉アレルゲンに特異的なイムノグロブリンE抗体に実質的に反応しないので、ヒトを含む哺乳類一般に投与すると、実質的にアナフィラキシーを引起こすことなく、スギ花粉アレルゲンに特異的なT細胞を不活性化することができる。 According to the present invention, a novel peptide group consisting essentially of only a T cell epitope specific to a cedar pollen allergen, and further an anti-cedar pollinosis agent comprising such a polypeptide as an active ingredient can be provided. The peptide of the present invention does not substantially react with the immunoglobulin E antibody specific for the cedar pollen allergen. Therefore, when administered to mammals including humans, it does not cause anaphylaxis substantially and is specific for the cedar pollen allergen. Normal T cells can be inactivated.
以下、本発明を詳しく説明する。本発明における好ましいペプチドの例は表1の通りである。 The present invention will be described in detail below. Examples of preferred peptides in the present invention are shown in Table 1.
なお、上記のペプチド1は、配列表の配列番号1のアミノ酸配列で示されるペプチド、上記のペプチド2は、配列表の配列番号2のアミノ酸配列で示されるペプチド、上記のペプチド3は、配列表の配列番号3のアミノ酸配列で示されるペプチド、上記のペプチド4は、配列表の配列番号4のアミノ酸配列で示されるペプチド、上記のペプチド5は、配列表の配列番号5のアミノ酸配列で示されるペプチド、上記のペプチド6は、配列表の配列番号6のアミノ酸配列で示されるペプチド、上記のペプチド7は、配列表の配列番号7のアミノ酸配列で示されるペプチド、上記のペプチド8は、配列表の配列番号8のアミノ酸配列で示されるペプチド、上記のペプチド9は、配列表の配列番号9のアミノ酸配列で示されるペプチド、上記のペプチド10は、配列表の配列番号10のアミノ酸配列で示されるペプチド、上記のペプチド11は、配列表の配列番号11のアミノ酸配列で示されるペプチド、をそれぞれ示す。 In addition, said peptide 1 is a peptide shown by the amino acid sequence of sequence number 1 of a sequence table, said peptide 2 is a peptide shown by the amino acid sequence of sequence number 2 of a sequence table, said peptide 3 is a sequence table. The peptide represented by the amino acid sequence of SEQ ID NO: 3, the above peptide 4 is the peptide represented by the amino acid sequence of SEQ ID NO: 4 in the sequence listing, and the above peptide 5 is represented by the amino acid sequence of SEQ ID NO: 5 of the sequence listing. Peptide, the above peptide 6 is a peptide represented by the amino acid sequence of SEQ ID NO: 6 of the sequence listing, the above peptide 7 is a peptide represented by the amino acid sequence of SEQ ID NO: 7 of the sequence listing, and the above peptide 8 is the sequence listing A peptide represented by the amino acid sequence of SEQ ID NO: 8, and the peptide 9 described above are a peptide represented by the amino acid sequence of SEQ ID NO: 9 10 is a peptide represented by the amino acid sequence of SEQ ID NO: 10 of the Sequence Listing, said peptide 11 indicates a peptide represented by the amino acid sequence of SEQ ID NO: 11 in Sequence Listing, respectively.
上記(1)乃至(11)に記載のペプチドは、「固相法」又は「液相法」として知られる斯界において慣用のペプチド合成法により、容易に調製することができる。例えば、社団法人日本生化学会編『新生化学実験講座』、第1巻、「タンパク質VI」、第3〜44頁、1992年、東京化学同人発行などにはペプチド合成の詳細が記載されている。また、該ペプチドは、マルチペプチドシンセサイザー SYMPHONY (プロティンテクノロジー社製)を用い、Fmoc (9-fluorenyl methyloxycarbonyl) 固相合成法にて同装置のプロトコールに従って合成することができる。すなわち、合成する各ペプチドのC末端に相当するアミノ酸が導入されている Fmoc-L-アミノ酸 Wang 樹脂を上記ペプチド合成装置の反応容器にセットし、デプロテクション溶液を用いて Fmoc を除く。さらにC末端から2番目のアミノ酸に相当するアミノ酸溶液とアクチベーター溶液を反応せしめ、反応後再び Fmoc 基のデプロテクションを行い、同様の操作を繰り返すことにより、目的とするペプチドを合成することができる。 The peptides described in the above (1) to (11) can be easily prepared by a peptide synthesis method commonly used in this field known as “solid phase method” or “liquid phase method”. For example, the details of peptide synthesis are described in the Japan Biochemical Society, “New Chemistry Laboratory”, Volume 1, “Protein VI”, pp. 3-44, 1992, published by Tokyo Kagaku Dojin. Further, the peptide can be synthesized according to the protocol of the same apparatus by Fmoc (9-fluorenyl methyloxycarbonyl) solid phase synthesis method using a multi-peptide synthesizer SYMPHONY (manufactured by Protein Technology). That is, Fmoc-L-amino acid Wang resin into which an amino acid corresponding to the C-terminus of each peptide to be synthesized has been introduced is set in the reaction vessel of the peptide synthesizer, and Fmoc is removed using a deprotection solution. Furthermore, the target peptide can be synthesized by reacting an amino acid solution corresponding to the second amino acid from the C-terminus with an activator solution, deprotecting the Fmoc group again after the reaction, and repeating the same operation. .
本発明のペプチドは化学合成により調製されたものに限定されず、例えば、スギの花粉又は雄花から採取するか、組換えDNA技術により調製したスギ花粉アレルゲンを適宜分解し、分解物から採取したものであってもよく、また、例えば、上記(1)乃至(11)に記載されたペプチドをコードするDNAを調製し、これを自律複製可能なベクターに挿入して組換えDNAとし、これを大腸菌、枯草菌、放線菌、酵母などの適宜宿主に導入して形質転換体とし、その培養物からこの発明のペプチドを採取してもよい。 The peptides of the present invention are not limited to those prepared by chemical synthesis, for example, those collected from cedar pollen or male flowers, or from cedar pollen allergens prepared by recombinant DNA technology, and collected from degradation products Alternatively, for example, a DNA encoding the peptide described in (1) to (11) above is prepared and inserted into a vector capable of autonomous replication to obtain a recombinant DNA. , Bacillus subtilis, actinomycetes, yeast or the like may be introduced into a suitable host to obtain a transformant, and the peptide of the present invention may be collected from the culture.
さらに、この発明のペプチドは、斯くして得られるペプチドに糖質やポリエチレングリコールを付加して得られる複合体としての形態、さらには、ペプチドをアセチル化、アミド化及び/又は多官能試験により架橋重合させて得られる誘導体又は重合体としての形態であってもよい。 Furthermore, the peptide of the present invention is in the form of a complex obtained by adding a carbohydrate or polyethylene glycol to the peptide thus obtained, and further, the peptide is crosslinked by acetylation, amidation and / or multifunctional test. It may be in the form of a derivative or polymer obtained by polymerization.
この発明のペプチドは、比較的粗な形態で投与しても所期の治療・予防効果を発揮するが、通常は使用に先立って精製される。精製には、例えば、濾過、濃縮、遠心分離、ゲル濾過クロマトグラフィー、イオン交換クロマトグラフィー、高速液体クロマトグラフィー、アフィニティークロマトグラフィー、ゲル電気泳動、等電点電気泳動などのペプチド乃至蛋白質を精製するための斯界における慣用の方法が用いられ、必要に応じて、これら方法を適宜組合せればよい。そして、最終使用形態に応じて、精製したペプチドを濃縮、凍結乾燥して液状又は固状にすればよい。本発明のペプチドがT細胞エピトープとしての活性を有することは、スギ花粉アレルゲンに特異的なT細胞のトリチウム化チミジンの取込を計測することにより確認することができる。 The peptide of the present invention exerts the intended therapeutic / preventive effect even when administered in a relatively crude form, but is usually purified prior to use. For purification, for example, to purify peptides or proteins such as filtration, concentration, centrifugation, gel filtration chromatography, ion exchange chromatography, high performance liquid chromatography, affinity chromatography, gel electrophoresis, isoelectric focusing etc. Conventional methods in this field are used, and these methods may be appropriately combined as necessary. Then, the purified peptide may be concentrated and lyophilized to make it liquid or solid depending on the final use form. The activity of the peptide of the present invention as a T cell epitope can be confirmed by measuring the uptake of tritiated thymidine in T cells specific for the cedar pollen allergen.
すなわち、フィコール・ハイパック比重遠心法等により花粉症患者の末梢血単核細胞群を分離し、この細胞群をRPMI1640等の培地に浮遊させ、96穴マイクロプレート上に分注する。次に、被検物質であるペプチドを加え培養する。この培養の温度、時間等の条件は各実験毎に適宜調整することができるが、37℃、3日間が好適である。その後トリチウム化チミジンを培地に加え、さらに一定時間培養を続け、単核細胞群におけるトリチウム化チミジンの取込み量を測定することにより、本発明のペプチドのT細胞エピトープとしての活性を算定することができる。なお、本発明では、同時にペプチドを含まない系を設けてこれを陰性対照とし、トリチウム化チミジンの取込み量が陰性対照の2倍以上に達した系を「陽性」、達しなかった系を「陰性」とした。 That is, a peripheral blood mononuclear cell group of a hay fever patient is separated by Ficoll-Hipac specific gravity centrifugation or the like, and the cell group is suspended in a medium such as RPMI 1640 and dispensed onto a 96-well microplate. Next, a peptide as a test substance is added and cultured. Conditions such as the temperature and time of the culture can be appropriately adjusted for each experiment, but 37 ° C. and 3 days are preferable. Thereafter, tritiated thymidine is added to the medium, and the culture is further continued for a certain period of time. By measuring the amount of tritiated thymidine incorporated in the mononuclear cell group, the activity of the peptide of the present invention as a T cell epitope can be calculated. . In the present invention, a system not containing a peptide is provided at the same time as a negative control. A system in which the amount of tritiated thymidine taken up more than twice that of the negative control is “positive”, and a system that has not reached “negative” "
本発明のペプチドは、スギ花粉アレルゲンに特異的なイムノグロブリンE抗体に実質的に反応しないので、ヒトを含む哺乳類一般に投与すると、実質的にアナフィラキシーを引き起こすことなく、スギ花粉アレルゲンに特異的なT細胞を不活性化することができる。 Since the peptide of the present invention does not substantially react with an immunoglobulin E antibody specific for a cedar pollen allergen, when administered to mammals including humans, a peptide specific for a cedar pollen allergen is substantially produced without causing anaphylaxis. Cells can be inactivated.
有効成分としてかかるペプチドを含んでなる本発明の抗スギ花粉症剤は、ヒトを含む哺乳類一般に投与すると、実質的にアナフィラキシーを引起こすことなくスギ花粉症に対して顕著な治療・予防効果を発揮する。有効成分としてこの発明のペプチドを含んでなる抗スギ花粉症剤は、スギ花粉症に罹患したヒトを含む哺乳類一般に投与すると、アナフィラキシーなどの副作用を実質的に引起こすことなく、スギ花粉症を治療することができる。一方、この発明の抗スギ花粉症例を、スギ花粉が飛散し始める前に健常な個体や潜在的なスギ花粉症の個体に投与するときには、スギ花粉症に対して顕著な予防効果を発揮するとともに、発症時のアレルギー症状の緩解に著効を発揮する。 The anti-cedar pollinosis agent of the present invention comprising such a peptide as an active ingredient exerts a remarkable therapeutic / preventive effect on cedar pollinosis without substantially causing anaphylaxis when administered to mammals including humans in general. To do. An anti-cedar pollinosis agent comprising the peptide of the present invention as an active ingredient treats cedar pollinosis without substantially causing side effects such as anaphylaxis when administered to mammals including humans suffering from cedar pollinosis can do. On the other hand, when the anti-cedar pollen case of this invention is administered to a healthy individual or an individual with potential cedar pollinosis before the cedar pollen begins to scatter, it exhibits a remarkable preventive effect against cedar pollinosis Effective in relieving allergic symptoms at the time of onset.
この発明の抗スギ花粉症剤につきさらに詳しく説明すると、この発明の抗スギ花粉症剤は、通常、この発明によるペプチドの1種又は2種以上を0.01乃至100%(w/w) 、望ましくは、0.05乃至50%(w/v) 、さらに望ましくは、0.5乃至5.0%(w/w) 含んでなる。この発明の抗スギ花粉症剤は、当該ペプチド単独の形態はもとより、その以外の生理的に許容される、例えば、血清アルブミン、ゼラチン、ぶどう糖、果糖、しょ糖、麦芽糖、乳糖、トレハロース、ソルビトール、マンニトール、マルチトール、ラクチトール、りん酸緩衝液剤もしくはクエン酸緩衝液剤を主体とする担体、賦形剤、免疫助成剤および安定剤、さらには、必要に応じて、スギ花粉に特異的な公知のT細胞エピトープ、ステロイドホルモンやクリモグリク酸ナトリウムなどの抗炎症剤や抗ヒスタミン剤及び免疫抑制剤を含む1種又は2種以上の他の薬剤との組成物としての形態を包含する。さらに、この発明の抗スギ花粉症剤は、投薬単位形態の薬剤をも包含し、その投薬単位形態の薬剤とは、この発明のポリペプチドを、例えば、1日当たりの用量又はその整数倍(4倍まで)又はその約数(1/40まで)に相当する量を含有し、投与に適する物理的に分離した一体の剤形にある薬剤を意味する。このような投薬単位形態の薬剤としては、散剤、細粒剤、顆粒剤、丸剤、錠剤、カプセル剤、トローチ剤、シロップ剤、乳剤、軟こう剤、硬膏剤、パップ剤、坐剤、点眼剤、点鼻剤、噴霧剤、注射剤などが挙げられる。 The anti-cedar pollinosis agent of the present invention will be described in more detail. The anti-cedar pollinosis agent of the present invention usually contains 0.01 to 100% (w / w) of one or more of the peptides according to the present invention. Preferably, it comprises 0.05 to 50% (w / v), more preferably 0.5 to 5.0% (w / w). The anti-cedar pollinosis agent of the present invention is not only in the form of the peptide alone but also physiologically acceptable, for example, serum albumin, gelatin, glucose, fructose, sucrose, maltose, lactose, trehalose, sorbitol, mannitol , Carriers based on maltitol, lactitol, phosphate buffer or citrate buffer, excipients, immune aids and stabilizers, and, if necessary, known T cells specific for cedar pollen It includes forms as compositions with one or more other drugs including epitopes, anti-inflammatory drugs such as steroid hormones and sodium crimoglycate, antihistamines and immunosuppressants. Further, the anti-cedar pollinosis agent of the present invention includes a drug in a dosage unit form, and the drug in the dosage unit form includes, for example, a dose per day or an integral multiple thereof (4 Means an agent in a physically separate, unitary dosage form that contains an amount equivalent to (up to twice) or a fraction thereof (up to 1/40) and is suitable for administration. Such dosage unit forms include powders, fine granules, granules, pills, tablets, capsules, troches, syrups, emulsions, ointments, plasters, poultices, suppositories, eye drops. Nasal drops, sprays, injections and the like.
この発明の抗スギ花粉症剤の使用方法について説明すると、この発明の抗スギ花粉症剤は、スギ花粉症の治療・予防を目的に、ヒトを含む哺乳類一般に経皮、経口、点鼻、点眼又は注射投与される。ヒトにおける投与量は、投与の目的や症状に依っても変わるが、通常、対象者の症状や投与後の経過を観察しながら、成人1日当たり本発明のポリペプチドの量にして0.01乃至1.0g 、望ましくは、0.01乃至0.1g を目安に、毎週1回乃至毎月1回の頻度で、約1乃至6カ月間、通常、用量を増やしながら反復投与される。 The method of using the anti-cedar pollinosis agent of the present invention will be described. The anti-cedar pollinosis agent of the present invention is generally transdermally, orally, nasally, and instilled for mammals including humans for the purpose of treating and preventing cedar pollinosis. Or it is administered by injection. The dose in humans varies depending on the purpose and symptom of administration, but usually, the amount of the polypeptide of the present invention per day for an adult is 0.01 to observing the subject's symptom and the course after administration. The dose is usually repeatedly administered in increasing doses for about 1 to 6 months at a frequency of once a week to once a month with a standard of 1.0 g, preferably 0.01 to 0.1 g.
本発明のポリペプチドの急性毒性
常法により、生後20日のマウスに後述の製剤例1乃至4の方法により得た免疫治療剤を経口又は腹腔内投与した。その結果、これら抗スギ花粉症剤は、いずれの投与経路によっても200mg/kg 以上のLD50であることが判明した。このことは、この発明のペプチドが、ヒトを含むほ乳類に対する抗スギ花粉症剤に安全に配合使用し得ることを示している。
According to the conventional acute toxicity of the polypeptide of the present invention, the immunotherapeutic agent obtained by the method of Preparation Examples 1 to 4 described later was orally or intraperitoneally administered to a 20-day-old mouse. As a result, these anti-cedar pollinosis agents were found to have an LD 50 of 200 mg / kg or more by any route of administration. This indicates that the peptide of the present invention can be safely used in an anti-cedar pollinosis agent for mammals including humans.
試験例1
スギ花粉症患者由来のT細胞を用い、本発明のペプチド1ないし11がスギ花粉症抗原T細胞エピトープ活性を有することを以下に記載する方法により確認した。
Test example 1
Using T cells derived from a cedar pollinosis patient, it was confirmed by the method described below that peptides 1 to 11 of the present invention have a cedar pollinosis antigen T cell epitope activity.
抗スギ花粉アレルゲンIgE反応に陽性を示す患者から40mlの末梢血を採取した。遠心分離後、バフィーコートを得て、さらにフィコール・ハイパック比重遠心法により、末梢血単核球(Peripheral Blood Mononuclear Cells; PBMC)を採取した。このPBMCを培地(5%のヒトAB型血清を含むRPMI)に懸濁した。 40 ml of peripheral blood was collected from a patient positive for anti-cedar pollen allergen IgE reaction. After centrifugation, a buffy coat was obtained, and peripheral blood mononuclear cells (PBMC) were collected by Ficoll-Hypac specific gravity centrifugation. This PBMC was suspended in a medium (RPMI containing 5% human type AB serum).
96穴の平底プレートに1ウェルあたり5×105個の細胞を分注し、各ウェル200ngのペプチド1ないし11を含む200μlの培地中で37℃、5%炭酸ガス下で72時間培養した。また別に、ペプチドを含まない培養群を設けてこれを陰性対照とした。その後、0.5μCiのトリチウム化チミジンを加え、さらに16時間培養した。セルハーベスターを用いて各ウェルの細胞をガラス繊維フィルター上に集め、それぞれ液体シンチレーションカウンターで細胞に取込まれたトリチウム化チミジン量を測定した。取込みチミジン量が陰性対照群の取込み量の2倍以上であった群をT細胞エピトープ活性「陽性」とし、達しなかった系を「陰性」とした。
この結果を以下の表2に示す。
5 × 10 5 cells per well were dispensed into a 96-well flat bottom plate and cultured in 200 μl of medium containing 200 ng of peptide 1 to 11 in each well at 37 ° C. under 5% carbon dioxide for 72 hours. Separately, a culture group containing no peptide was provided as a negative control. Thereafter, 0.5 μCi of tritiated thymidine was added and further cultured for 16 hours. Cells in each well were collected on a glass fiber filter using a cell harvester, and the amount of tritiated thymidine incorporated into the cells was measured with a liquid scintillation counter. The group in which the amount of thymidine incorporated was more than twice that of the negative control group was defined as T cell epitope activity “positive”, and the system that did not reach it was defined as “negative”.
The results are shown in Table 2 below.
以上の結果より、これらのペプチドは、Cryj1アレルゲンのT細胞エピトープを含有していることが示された。配列表における配列番号1乃至11に示すアミノ酸配列を有するT細胞エピトープは未だ知られておらず、新規T細胞エピトープと判断される。 From the above results, these peptides were shown to contain the T cell epitope of Cryj1 allergen. T cell epitopes having the amino acid sequences shown in SEQ ID NOs: 1 to 11 in the sequence listing are not yet known, and are judged to be novel T cell epitopes.
以上のように、本発明のペプチドは、ヒトを含む哺乳類一般に投与すると、実質的にアナフィラキシーを引起こすことなく、スギ花粉アレルゲンに特異的なT細胞を不活性化することができる。 As described above, the peptide of the present invention can inactivate T cells specific for the cedar pollen allergen without substantially causing anaphylaxis when administered to mammals including humans in general.
有効成分として斯かるペプチドを含んでなる本発明の抗スギ花粉症剤は、ヒトを含む哺乳類一般に投与すると、実質的にアナフィラキシーを引起こすことなくスギ花粉症に対して顕著な治療・予防効果を発揮する。有効成分としてこの発明のペプチドを含んでなる抗スギ花粉症剤は、スギ花粉症に罹患したヒトを含む哺乳類一般に投与すると、アナフィラキシーなどの副作用を実質的に引起こすことなく、スギ花粉症を治療することができる。一方、この発明の抗スギ花粉症剤を、スギ花粉が飛散し始める前に健常な個体や潜在的なスギ花粉症の個体に投与するときには、スギ花粉症に対して顕著な予防効果を発揮するとともに、発症時のアレルギー症状の緩解に著効を発揮する。 The anti-cedar pollinosis agent of the present invention comprising such a peptide as an active ingredient has a remarkable therapeutic / preventive effect on cedar pollinosis substantially without causing anaphylaxis when administered to mammals including humans. Demonstrate. An anti-cedar pollinosis agent comprising the peptide of the present invention as an active ingredient treats cedar pollinosis without substantially causing side effects such as anaphylaxis when administered to mammals including humans suffering from cedar pollinosis can do. On the other hand, when the anti-cedar pollinosis agent of this invention is administered to healthy individuals or individuals with potential cedar pollinosis before the cedar pollen begins to scatter, it exerts a remarkable preventive effect against cedar pollinosis. At the same time, it is effective in relieving allergic symptoms at the onset.
以下、実施例、製剤例により本発明をさらに詳細に説明するが、本発明はこれらによりその技術的範囲が限定されるものではない。
〔実施例1〕ペプチド1の合成
樹脂に固定したアミノ酸誘導体に、1個ずつアミノ酸をカルボキシル末端側から結合させていく方法(固相合成法)でペプチドを化学合成した。各サイクルで使用するアミノ酸はアミノ基および残基部分の反応基が保護基でブロックされた特殊なアミノ酸誘導体を用いた。ここで、それぞれのアミノ基がFmoc (9-フルオレニル・メチロキシカルボニル)によりブロックされているアミノ酸を用いた(Fmoc法)。またペプチド合成は樹脂に結合したアミノ酸のアミノ基のFmocを脱保護し、次にカルボキシル基が活性化したアミノ酸誘導体を結合させるという反応を順次繰り返して行なった。
EXAMPLES Hereinafter, although an Example and a formulation example demonstrate this invention further in detail, this invention does not limit the technical scope by these.
[Example 1] Synthesis of Peptide 1 A peptide was chemically synthesized by a method (solid phase synthesis method) in which amino acids were bound to the amino acid derivative fixed on the resin one by one from the carboxyl terminal side. The amino acid used in each cycle was a special amino acid derivative in which the reactive group of the amino group and the residue part was blocked with a protecting group. Here, an amino acid in which each amino group was blocked with Fmoc (9-fluorenylmethyloxycarbonyl) was used (Fmoc method). Peptide synthesis was performed by sequentially repeating the reaction of deprotecting the Fmoc of the amino group of the amino acid bonded to the resin, and then binding the amino acid derivative activated with the carboxyl group.
ペプチド1のぺプチドは、マルチペプチドシンセサイザー(シンフォニー;プロテインテクノロジー社製)を用い、上記のFmoc固相合成法にて同装置のプロトコールに従って合成した。 The peptide 1 peptide was synthesized using a multi-peptide synthesizer (Symphony; manufactured by Protein Technology) according to the protocol of the same apparatus by the Fmoc solid phase synthesis method described above.
すなわち、合成するペプチドのC末端残基に相当するアミノ酸(Gly)が導入されているFmoc-Gly-Wang樹脂(0.65mmol/g)の25μmol相当を上記ペプチド合成装置の反応容器にセットし、デプロテクション溶液(20%ピぺリジン/ジメチルホルムアミド)1.25mlを5分間2回反応させ、樹脂に結合しているアミノ酸のFmoc基を除いた。樹脂をジメチルホルムアミド液1.25mlで30秒間6回洗浄し、C末端側から2番目のアミノ酸に相当する100mMのFmoc-Pheジメチルホルムアミド溶液1.25 mlに、100mMのアクチベーター溶液(100mM 2−(1H−ベンゾトリアゾール−1−イル−)−1、1、3、3−テトラメチルウロニウム−ヘキサフルオロホスフェート/400mM N−メチルモルフォリン/ジメチルホルムアミド) 1.25mlを加え (結合アミノ酸に対して5倍等量)、20分間室温で反応させた。ここで生成した Fmoc- Phe-Gly-Wang 樹脂をジメチルホルムアミド1.25mlで30秒間6回洗浄後、再びFmoc基のデプロテクションを行い、ジメチルホルムアミド1.25mlで30秒間6回洗浄後、Fmoc-Gln(Trt) 溶液とアクチベーター溶液を加え反応させた。 That is, 25 μmol equivalent of Fmoc-Gly-Wang resin (0.65 mmol / g) into which an amino acid (Gly) corresponding to the C-terminal residue of the peptide to be synthesized was introduced was set in the reaction vessel of the peptide synthesizer, 1.25 ml of a deprotection solution (20% piperidine / dimethylformamide) was reacted twice for 5 minutes to remove the Fmoc group of the amino acid bound to the resin. The resin was washed 6 times with 1.25 ml of dimethylformamide solution for 30 seconds, and 1.25 ml of 100 mM Fmoc-Phe dimethylformamide solution corresponding to the second amino acid from the C-terminal side was added with 100 mM activator solution (100 mM 2- (1H-benzotriazol-1-yl-)-1,1,3,3-tetramethyluronium-hexafluorophosphate / 400 mM N-methylmorpholine / dimethylformamide) Add 1.25 ml (for bound amino acids) 5 times equivalent), and allowed to react at room temperature for 20 minutes. The Fmoc-Phe-Gly-Wang resin produced here was washed with 1.25 ml of dimethylformamide 6 times for 30 seconds, deprotected with Fmoc group again, washed with 1.25 ml of dimethylformamide 6 times for 30 seconds, and then Fmoc- Gln (Trt) solution and activator solution were added and reacted.
同様の操作を繰り返すことにより保護ペプチド樹脂(Fmoc- Met-Lys(Boc)-Val-Thr(tBu)-Val-Ala-Phe-Asn(Trt)-Gln(Trt)-Phe-Gly -Wang樹脂)を合成した。 Protected peptide resin (Fmoc-Met-Lys (Boc) -Val-Thr (tBu) -Val-Ala-Phe-Asn (Trt) -Gln (Trt) -Phe-Gly-Wang resin) by repeating similar operations Was synthesized.
本実施例以下のペプチド合成に使用したアミノ酸は以下の通りである:
Fmoc-Asp(OtBu) Fmoc-Asn(Trt) Fmoc-Ala Fmoc-Glu(OtBu) Fmoc-Gln(Trt) Fmoc-Gly Fmoc-His(Trt) Fmoc-Ile Fmoc-LeuFmoc-Lys(Boc) Fmoc-Met Fmoc-Phe Fmoc-Pro Fmoc-Ser(tBu)Fmoc-Thr(tBu) Fmoc-Trp(Boc) Fmoc-Tyr(tBu) Fmoc-Val
(( )内は残基部分の反応基を保護する保護基を表わす。;(株)パーキンエルマージャパンアプライドバイオシステムズ事業部 製)
The amino acids used in the peptide synthesis below this example are as follows:
Fmoc-Asp (OtBu) Fmoc-Asn (Trt) Fmoc-Ala Fmoc-Glu (OtBu) Fmoc-Gln (Trt) Fmoc-Gly Fmoc-His (Trt) Fmoc-Ile Fmoc-LeuFmoc-Lys (Boc) Fmoc-Met Fmoc-Phe Fmoc-Pro Fmoc-Ser (tBu) Fmoc-Thr (tBu) Fmoc-Trp (Boc) Fmoc-Tyr (tBu) Fmoc-Val
(() Represents a protecting group for protecting the reactive group of the residue part; manufactured by PerkinElmer Japan Applied Biosystems Division)
まず、上記のように合成し得られた保護ぺプチド樹脂 (Fmoc- Met-Lys(Boc)-Val-Thr(tBu)-Val-Ala-Phe-Asn(Trt)-Gln(Trt)-Phe-Gly -Wang樹脂)にデプロテクション溶液 1.25mlを5分間2回反応させてN末端 Fmoc基を脱保護した。次に1.25mlのDMFにて6回洗浄後、ジクロロメタンにて9回洗浄し、さらに窒素ガスを吹き付けることにより20分間乾燥させた。 First, the protected peptide resin synthesized as described above (Fmoc-Met-Lys (Boc) -Val-Thr (tBu) -Val-Ala-Phe-Asn (Trt) -Gln (Trt) -Phe- Gly-Wang resin) was reacted with 1.25 ml of the deprotection solution twice for 5 minutes to deprotect the N-terminal Fmoc group. Next, it was washed 6 times with 1.25 ml of DMF, then 9 times with dichloromethane, and further dried by blowing nitrogen gas for 20 minutes.
樹脂を取り出し、クリベージ溶液(トリフルオロ酢酸(以下「TFA」という):フェノール:水:チオアニソール:エタンジチオール=82.5:5:5:5:2.5)を4ml加え、室温で2時間反応させることにより樹脂からのペプチドの切断およびアミノ酸側鎖保護基の除去を行い、ペプチド溶液を得た[King,D.S.(1990)Int.J.peptide Protein Reg. 36,255]。このぺプチド溶液をフィルターを用いて濾過し、濾液を遠心管に回収した。これに、10mlの冷エーテルを加え、ペプチドを沈殿させた。しばらく冷却後、これを遠心して(3000rpm,10分間)沈殿物を集め、再び冷エーテルを加えて分散させては回収する操作を4回繰り返してペプチドを洗浄した。得られたペプチドを乾燥させ、粗ペプチドを得た。以下、上記の操作を「クリベージ反応」という。 By removing the resin, adding 4 ml of cleaved solution (trifluoroacetic acid (hereinafter referred to as “TFA”): phenol: water: thioanisole: ethanedithiol = 82.5: 5: 5: 5: 2.5) and reacting at room temperature for 2 hours The peptide was cleaved from the resin and the amino acid side chain protecting group was removed to obtain a peptide solution [King, DS (1990) Int. J. peptide Protein Reg. 36, 255]. The peptide solution was filtered using a filter, and the filtrate was collected in a centrifuge tube. To this, 10 ml of cold ether was added to precipitate the peptide. After cooling for a while, this was centrifuged (3000 rpm, 10 minutes), the precipitate was collected, and cold ether was added again to disperse and recover, and the peptide was washed 4 times. The obtained peptide was dried to obtain a crude peptide. Hereinafter, the above operation is referred to as “cribage reaction”.
得られた粗ペプチドのうち11.7mgを0.1%のTFAを含む30%アセトニトリル水溶液に溶解後、高速液体クロマトグラフィー(以下「HPLC」という)に供した。HPLCの条件は以下の通りであった。
カラム:ODSカラム(TSKガードカラムODS, 21.5mmx75mm;東ソー(株)社製)
移動相:25−28%アセトニトリル/0.1% TFA,15分
流速:5ml/min.
検出波長:220nm
11.7 mg of the obtained crude peptide was dissolved in 30% acetonitrile aqueous solution containing 0.1% TFA, and then subjected to high performance liquid chromatography (hereinafter referred to as “HPLC”). The HPLC conditions were as follows.
Column: ODS column (TSK guard column ODS, 21.5 mm x 75 mm; manufactured by Tosoh Corporation)
Mobile phase: 25-28% acetonitrile / 0.1% TFA, 15 minutes flow rate: 5 ml / min.
Detection wavelength: 220 nm
12〜14分に溶出された画分を分取し、濃縮後、凍結乾燥を行い目的とするペプチド 4.9mgを得た。この合成したペプチド100pmolについて、アミノ酸配列装置(PPSQ−10型;島津製作所(株)社製)を用いてアミノ酸配列分析を行なったところ、配列表の配列番号1に示されるアミノ酸配列が確認された。 The fraction eluted at 12 to 14 minutes was collected, concentrated, and lyophilized to obtain 4.9 mg of the desired peptide. When 100 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequencer (PPSQ-10 type; manufactured by Shimadzu Corporation), the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing was confirmed. .
[実施例2]ペプチド2の合成
C末端がProであるペプチドの合成にはFmoc-Pro-2-Chlorotrityl樹脂を用い実施例1と同様の操作により、保護ペプチド樹脂 (Fmoc- Ser(tBu)-Met-Lys(Boc)-Val-Thr(tBu)-Val-Ala-Phe-Asn(Trt)-Gln(Trt)-Phe-Gly-Pro-2-Chlorotrityl樹脂 )を合成しクリベージ反応を行なって、粗ペプチドを得た。
[Example 2] Synthesis of peptide 2
For the synthesis of a peptide having C-terminal Pro, Fmoc-Pro-2-Chlorotrityl resin was used and the same procedure as in Example 1 was followed to obtain a protected peptide resin (Fmoc-Ser (tBu) -Met-Lys (Boc) -Val- Thr (tBu) -Val-Ala-Phe-Asn (Trt) -Gln (Trt) -Phe-Gly-Pro-2-Chlorotrityl resin) was synthesized and subjected to a cleavage reaction to obtain a crude peptide.
得られた粗ペプチドのうち10mgを0.1%のTFAを含む20%アセトニトリル水溶液に溶解後、HPLCに供した。HPLCの条件は以下の通りであった。
カラム:ODSカラム(TSKgel ODS−80T, 7.8mmx300mm;東ソー(株)社製)
移動相:30%アセトニトリル/0.1% TFA,30分
流速:0.7ml/min.
検出波長:230nm
10 mg of the obtained crude peptide was dissolved in 20% acetonitrile aqueous solution containing 0.1% TFA, and then subjected to HPLC. The HPLC conditions were as follows.
Column: ODS column (TSKgel ODS-80T, 7.8 mm x 300 mm; manufactured by Tosoh Corporation)
Mobile phase: 30% acetonitrile / 0.1% TFA, 30 minutes flow rate: 0.7 ml / min.
Detection wavelength: 230 nm
21〜22分に溶出された画分を分取し、濃縮後、凍結乾燥を行い目的とするペプチド 1.3mgを得た。この合成したペプチド100pmolについて、アミノ酸配列装置(PPSQ−10型;島津製作所(株)社製)を用いてアミノ酸配列分析を行なったところ、配列表の配列番号2に示されるアミノ酸配列が確認された。 The fraction eluted at 21-22 minutes was collected, concentrated, and lyophilized to obtain 1.3 mg of the desired peptide. When 100 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequencer (PPSQ-10 type; manufactured by Shimadzu Corporation), the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing was confirmed. .
[実施例3]ペプチド3の合成
実施例1と同様の操作により、保護ペプチド樹脂(Fmoc-Tyr(tBu)-Gly-Leu-Val-His(Trt)-Val-Ala-Asn(Trt)-Asn(Trt)-Asn(Trt)-Tyr(tBu)-Asp(OtBu)-Pro-Trp(Boc)-Thr(tBu)-Ile Wang 樹脂 )を合成しクリベージ反応を行なって、粗ペプチドを得た。
Example 3 Synthesis of Peptide 3 According to the same procedure as in Example 1, the protected peptide resin (Fmoc-Tyr (tBu) -Gly-Leu-Val-His (Trt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp (OtBu) -Pro-Trp (Boc) -Thr (tBu) -Ile Wang resin) was synthesized and subjected to a cleave reaction to obtain a crude peptide.
得られた粗ペプチドのうち8.9mgを0.1%のTFAを含む20%アセトニトリル水溶液に溶解後、HPLCに供した。HPLCの条件は以下の通りであった。
カラム:ODSカラム(TSKガードカラムODS, 21.5mmx75mm;東ソー(株)社製)
移動相:20−30%アセトニトリル/0.1% TFA,20分
流速:8ml/min.
検出波長:220nm
8.9 mg of the obtained crude peptide was dissolved in 20% acetonitrile aqueous solution containing 0.1% TFA, and then subjected to HPLC. The HPLC conditions were as follows.
Column: ODS column (TSK guard column ODS, 21.5 mm x 75 mm; manufactured by Tosoh Corporation)
Mobile phase: 20-30% acetonitrile / 0.1% TFA, 20 minutes flow rate: 8 ml / min.
Detection wavelength: 220 nm
17〜20分に溶出された画分を分取し、濃縮後、凍結乾燥を行い目的とするペプチド 5.0mgを得た。この合成したペプチド100pmolについて、アミノ酸配列装置(PPSQ−10型;島津製作所(株)社製)を用いてアミノ酸配列分析を行なったところ、配列表の配列番号3に示されるアミノ酸配列が確認された。 The fraction eluted at 17 to 20 minutes was collected, concentrated, and lyophilized to obtain 5.0 mg of the desired peptide. When 100 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequencer (PPSQ-10 type; manufactured by Shimadzu Corporation), the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing was confirmed. .
[実施例4]ペプチド4の合成
実施例1と同様の操作により、保護ペプチド樹脂(Fmoc-Tyr(tBu)-Gly-Leu-Val-His(Trt)-Val-Ala-Asn(Trt)-Asn(Trt)-Asn(Trt)-Tyr(tBu)-Asp(OtBu)-Pro-Trp(Boc)- Wang 樹脂 )を合成しクリベージ反応を行なって、粗ペプチドを得た。得られた粗ペプチドのうち 9mgを0.1%のTFAを含む20%アセトニトリル水溶液に溶解後、HPLCに供した。HPLCの条件は以下の通りであった。
カラム:ODSカラム(TSKガードカラムODS, 21.5mmx75mm;東ソー(株)社製)
移動相: 20−30%アセトニトリル/0.1% TFA,20分
流速:8ml/min.
検出波長:220nm
Example 4 Synthesis of Peptide 4 According to the same procedure as in Example 1, protected peptide resin (Fmoc-Tyr (tBu) -Gly-Leu-Val-His (Trt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp (OtBu) -Pro-Trp (Boc) -Wang resin) was synthesized and subjected to a cleavage reaction to obtain a crude peptide. Of the obtained crude peptide, 9 mg was dissolved in 20% acetonitrile aqueous solution containing 0.1% TFA and subjected to HPLC. The HPLC conditions were as follows.
Column: ODS column (TSK guard column ODS, 21.5 mm x 75 mm; manufactured by Tosoh Corporation)
Mobile phase: 20-30% acetonitrile / 0.1% TFA, 20 minutes flow rate: 8 ml / min.
Detection wavelength: 220 nm
13〜16分に溶出された画分を分取し、濃縮後、凍結乾燥を行い目的とするペプチド 6.4mgを得た。 The fraction eluted at 13 to 16 minutes was collected, concentrated, and lyophilized to obtain 6.4 mg of the desired peptide.
この合成したペプチド100pmolについて、アミノ酸配列装置(PPSQ−10型;島津製作所(株)社製)を用いてアミノ酸配列分析を行なったところ、配列表の配列番号4に示されるアミノ酸配列が確認された。 When 100 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequencer (PPSQ-10 type; manufactured by Shimadzu Corporation), the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing was confirmed. .
[実施例5]ペプチド5の合成
実施例1と同様の操作により、保護ペプチド樹脂(Fmoc-Gly-Leu-Val-His(Trt)-Val-Ala-Asn(Trt)-Asn(Trt)-Asn(Trt)-Tyr(tBu)-Asp(OtBu)-Pro-Trp(Boc)-Thr(tBu) Wang 樹脂 )を合成しクリベージ反応を行なって、粗ペプチドを得た。
Example 5 Synthesis of Peptide 5 According to the same procedure as in Example 1, protected peptide resin (Fmoc-Gly-Leu-Val-His (Trt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp (OtBu) -Pro-Trp (Boc) -Thr (tBu) Wang resin) was synthesized and subjected to a cleave reaction to obtain a crude peptide.
得られた粗ペプチドのうち8mgを0.1%のTFAを含む20%アセトニトリル水溶液に溶解後、HPLCに供した。HPLCの条件は以下の通りであった。
カラム:ODSカラム(TSKガードカラムODS, 21.5mmx75mm;東ソー(株)社製)
移動相: 20−30%アセトニトリル/0.1% TFA,20分
流速:8ml/min.
検出波長:220nm
8 mg of the obtained crude peptide was dissolved in 20% acetonitrile aqueous solution containing 0.1% TFA, and then subjected to HPLC. The HPLC conditions were as follows.
Column: ODS column (TSK guard column ODS, 21.5 mm x 75 mm; manufactured by Tosoh Corporation)
Mobile phase: 20-30% acetonitrile / 0.1% TFA, 20 minutes flow rate: 8 ml / min.
Detection wavelength: 220 nm
8〜11分に溶出された画分を分取し、濃縮後、凍結乾燥を行い目的とするペプチド 4.1mgを得た。 The fraction eluted at 8 to 11 minutes was collected, concentrated, and lyophilized to obtain 4.1 mg of the desired peptide.
この合成したペプチド100pmolについて、アミノ酸配列装置(PPSQ−10型;島津製作所(株)社製)を用いてアミノ酸配列分析を行なったところ、配列表の配列番号6に示されるアミノ酸配列が確認された。 When 100 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequencer (PPSQ-10 type; manufactured by Shimadzu Corporation), the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing was confirmed. .
[実施例6]ペプチド6の合成
C末端がProであるペプチドの合成にはFmoc-Pro-2-Chlorotrityl樹脂を用い、実施例1と同様の操作により、保護ペプチド樹脂 (Fmoc-Tyr(tBu)-Gly-Leu-Val-His(Trt)-Val-Ala-Asn(Trt)-Asn(Trt)-Asn(Trt)-Tyr(tBu)-Asp(OtBu)-Pro-2-Chlorotrityl樹脂 )を合成しクリベージ反応を行なって、粗ペプチドを得た。
[Example 6] Synthesis of peptide 6
Fmoc-Pro-2-Chlorotrityl resin was used for the synthesis of a peptide having C-terminal Pro, and a protected peptide resin (Fmoc-Tyr (tBu) -Gly-Leu-Val-His ( Trt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp (OtBu) -Pro-2-Chlorotrityl resin) Got.
得られた粗ペプチドのうち5.5mgを0.1%のTFAを含む20%アセトニトリル水溶液に溶解後、HPLCに供した。HPLCの条件は以下の通りであった。
カラム:ODSカラム(TSKガードカラムODS, 21.5mmx75mm;東ソー(株)社製)
移動相: 21−26%アセトニトリル/0.1% TFA,15分
流速:5ml/min.
検出波長:220nm
5.5 mg of the obtained crude peptide was dissolved in 20% acetonitrile aqueous solution containing 0.1% TFA, and then subjected to HPLC. The HPLC conditions were as follows.
Column: ODS column (TSK guard column ODS, 21.5 mm x 75 mm; manufactured by Tosoh Corporation)
Mobile phase: 21-26% acetonitrile / 0.1% TFA, 15 min flow rate: 5 ml / min.
Detection wavelength: 220 nm
9〜10.5分に溶出された画分を分取し、濃縮後、凍結乾燥を行い目的とするペプチド 2.8mgを得た。 The fraction eluted at 9 to 10.5 minutes was collected, concentrated, and lyophilized to obtain 2.8 mg of the desired peptide.
この合成したペプチド100pmolについて、アミノ酸配列装置(PPSQ−10型;島津製作所(株)社製)を用いてアミノ酸配列分析を行なったところ、配列表の配列番号6に示されるアミノ酸配列が確認された。 When 100 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequencer (PPSQ-10 type; manufactured by Shimadzu Corporation), the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing was confirmed. .
[実施例7]ペプチド7の合成
実施例1と同様の操作により、保護ペプチド樹脂(Fmoc- Ser(tBu)- Ser(tBu)-Gly-Lys(Boc)-Tyr(tBu)-Glu(OtBu)-Gly-Gly-Asn(Trt)-Ile-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Ala-Phe-Asn(Trt)-Val−Wang樹脂)を合成しクリベージ反応を行なって、粗ペプチドを得た。 得られた粗ペプチドのうち 11mgを0.1%のTFAを含む15%アセトニトリル水溶液に溶解後、HPLCに供した。HPLCの条件は以下の通りであった。
カラム:ODSカラム(TSKガードカラムODS, 21.5mmx75mm;東ソー(株)社製)
移動相:15−25%アセトニトリル/0.1% TFA,20分
流速:8ml/min.
検出波長:220nm
[Example 7] Synthesis of peptide 7 By the same operation as in Example 1, protected peptide resin (Fmoc-Ser (tBu) -Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu)) -Gly-Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Wang resin ) And a cleave reaction was performed to obtain a crude peptide. 11 mg of the obtained crude peptide was dissolved in a 15% acetonitrile aqueous solution containing 0.1% TFA, and then subjected to HPLC. The HPLC conditions were as follows.
Column: ODS column (TSK guard column ODS, 21.5 mm x 75 mm; manufactured by Tosoh Corporation)
Mobile phase: 15-25% acetonitrile / 0.1% TFA, 20 minutes flow rate: 8 ml / min.
Detection wavelength: 220 nm
10〜12分に溶出された画分を分取し、濃縮後、凍結乾燥を行い目的とするペプチド 5.9mgを得た。 The fraction eluted at 10 to 12 minutes was collected, concentrated, and lyophilized to obtain 5.9 mg of the desired peptide.
この合成したペプチド100pmolについて、アミノ酸配列装置(PPSQ−10型;島津製作所(株)社製)を用いてアミノ酸配列分析を行なったところ、配列表の配列番号7に示されるアミノ酸配列が確認された。 When 100 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequencer (PPSQ-10 type; manufactured by Shimadzu Corporation), the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing was confirmed. .
[実施例8]ペプチド8の合成
実施例1と同様の操作により、保護ペプチド樹脂(Fmoc- Ser(tBu) -Gly-Lys(Boc)-Tyr(tBu)-Glu(OtBu)-Gly-Gly-Asn(Trt)-Ile-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Ala-Phe-Asn(Trt)-Val-Glu(OtBu)−Wang樹脂)を合成しクリベージ反応を行なって、粗ペプチドを得た。
[Example 8] Synthesis of peptide 8 By the same operation as in Example 1, protected peptide resin (Fmoc-Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly- Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Wang resin ) And a cleave reaction was performed to obtain a crude peptide.
得られた粗ペプチドのうち11mgを0.1%のTFAを含む15%アセトニトリル水溶液に溶解後、HPLCに供した。HPLCの条件は以下の通りであった。
カラム:ODSカラム(TSKガードカラムODS, 21.5mmx75mm;東ソー(株)社製)
移動相: 15−22%アセトニトリル/0.1% TFA,20分
流速:8ml/min.
検出波長:220nm
11 mg of the resulting crude peptide was dissolved in a 15% acetonitrile aqueous solution containing 0.1% TFA and then subjected to HPLC. The HPLC conditions were as follows.
Column: ODS column (TSK guard column ODS, 21.5 mm x 75 mm; manufactured by Tosoh Corporation)
Mobile phase: 15-22% acetonitrile / 0.1% TFA, 20 minutes flow rate: 8 ml / min.
Detection wavelength: 220 nm
10〜12分に溶出された画分を分取し、濃縮後、凍結乾燥を行い目的とするペプチド 6.4mgを得た。 The fraction eluted at 10 to 12 minutes was collected, concentrated, and lyophilized to obtain 6.4 mg of the desired peptide.
この合成したペプチド100pmolについて、アミノ酸配列装置(PPSQ−10型;島津製作所(株)社製)を用いてアミノ酸配列分析を行なったところ、配列表の配列番号8に示されるアミノ酸配列が確認された。 When 100 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequencer (PPSQ-10 type; manufactured by Shimadzu Corporation), the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing was confirmed. .
[実施例9]ペプチド9の合成
実施例1と同様の操作により、保護ペプチド樹脂(Fmoc- Ser(tBu)-Gly-Lys(Boc)-Tyr(tBu)-Glu(OtBu)-Gly-Gly-Asn(Trt)-Ile-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Ala-Phe-Asn(Trt)-Val −Wang樹脂)を合成しクリベージ反応を行なって、粗ペプチドを得た。
[Example 9] Synthesis of peptide 9 By the same operation as in Example 1, protected peptide resin (Fmoc-Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly- Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Wang resin) Reaction was performed to obtain a crude peptide.
得られた粗ペプチドのうち11mgを0.1%のTFAを含む15%アセトニトリル水溶液に溶解後、HPLCに供した。HPLCの条件は以下の通りであった。
カラム:ODSカラム(TSKガードカラムODS, 21.5mmx75mm;東ソー(株)社製)
移動相: 15−20%アセトニトリル/0.1% TFA,20分
流速:8ml/min.
検出波長:220nm
11 mg of the resulting crude peptide was dissolved in a 15% acetonitrile aqueous solution containing 0.1% TFA and then subjected to HPLC. The HPLC conditions were as follows.
Column: ODS column (TSK guard column ODS, 21.5 mm x 75 mm; manufactured by Tosoh Corporation)
Mobile phase: 15-20% acetonitrile / 0.1% TFA, 20 minutes flow rate: 8 ml / min.
Detection wavelength: 220 nm
16〜18分に溶出された画分を分取し、濃縮後、凍結乾燥を行い目的とするペプチド 4.8mgを得た。 The fraction eluted at 16 to 18 minutes was collected, concentrated, and lyophilized to obtain 4.8 mg of the desired peptide.
この合成したペプチド100pmolについて、アミノ酸配列装置(PPSQ−10型;島津製作所(株)社製)を用いてアミノ酸配列分析を行なったところ、配列表の配列番号9に示されるアミノ酸配列が確認された。 When 100 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequencer (PPSQ-10 type; manufactured by Shimadzu Corporation), the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing was confirmed. .
[実施例10]ペプチド10の合成
実施例1と同様の操作により、保護ペプチド樹脂(Fmoc-Gly-Lys(Boc)-Tyr(tBu)-Glu(OtBu)-Gly-Gly-Asn(Trt)-Ile-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Ala-Phe-Asn(Trt)-Val-Glu(OtBu)−Wang樹脂)を合成しクリベージ反応を行なって、粗ペプチドを得た。
[Example 10] Synthesis of peptide 10 By the same operation as in Example 1, protected peptide resin (Fmoc-Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly-Asn (Trt)- Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Wang resin) Reaction was performed to obtain a crude peptide.
得られた粗ペプチドのうち5.4mgを0.1%のTFAを含む20%アセトニトリル水溶液に溶解後、HPLCに供した。HPLCの条件は以下の通りであった。
カラム:ODSカラム(TSKガードカラムODS, 21.5mmx75mm;東ソー(株)社製)
移動相: 20−30%アセトニトリル/0.1% TFA,15分
流速:5ml/min.
検出波長:230nm
5.4 mg of the obtained crude peptide was dissolved in 20% acetonitrile aqueous solution containing 0.1% TFA, and then subjected to HPLC. The HPLC conditions were as follows.
Column: ODS column (TSK guard column ODS, 21.5 mm x 75 mm; manufactured by Tosoh Corporation)
Mobile phase: 20-30% acetonitrile / 0.1% TFA, 15 minutes flow rate: 5 ml / min.
Detection wavelength: 230 nm
9〜10分に溶出された画分を分取し、濃縮後、凍結乾燥を行い目的とするペプチド 3.6mgを得た。 The fraction eluted at 9 to 10 minutes was collected, concentrated, and lyophilized to obtain 3.6 mg of the desired peptide.
この合成したペプチド100pmolについて、アミノ酸配列装置(PPSQ−10型;島津製作所(株)社製)を用いてアミノ酸配列分析を行なったところ、配列表の配列番号10に示されるアミノ酸配列が確認された。 When 100 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequencer (PPSQ-10 type; manufactured by Shimadzu Corporation), the amino acid sequence shown in SEQ ID NO: 10 in the sequence listing was confirmed. .
[実施例11]ペプチド11の合成
実施例1と同様の操作により、保護ペプチド樹脂(Fmoc- Glu(OtBu)-Gly-Gly-Asn(Trt)-Ile-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Ala-Phe-Asn(Trt)-Val−Wang樹脂)を合成しクリベージ反応を行なって、粗ペプチドを得た。
[Example 11] Synthesis of peptide 11 According to the same procedure as in Example 1, a protected peptide resin (Fmoc-Glu (OtBu) -Gly-Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu)- Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Wang resin) was synthesized and subjected to a cleavage reaction to obtain a crude peptide.
得られた粗ペプチドのうち5mgを0.1%のTFAを含む20%アセトニトリル水溶液に溶解後、HPLCに供した。HPLCの条件は以下の通りであった。
カラム:ODSカラム(TSKガードカラムODS, 21.5mmx75mm;東ソー(株)社製)
移動相: 20−35%アセトニトリル/0.1% TFA,15分
流速:5ml/min.
検出波長:230nm
Of the obtained crude peptide, 5 mg was dissolved in 20% acetonitrile aqueous solution containing 0.1% TFA, and then subjected to HPLC. The HPLC conditions were as follows.
Column: ODS column (TSK guard column ODS, 21.5 mm x 75 mm; manufactured by Tosoh Corporation)
Mobile phase: 20-35% acetonitrile / 0.1% TFA, 15 min flow rate: 5 ml / min.
Detection wavelength: 230 nm
9.5〜10.5分に溶出された画分を分取し、濃縮後、凍結乾燥を行い目的とするペプチド 3.5mgを得た。 The fraction eluted at 9.5 to 10.5 minutes was collected, concentrated, and lyophilized to obtain 3.5 mg of the desired peptide.
この合成したペプチド100pmolについて、アミノ酸配列装置(PPSQ−10型;島津製作所(株)社製)を用いてアミノ酸配列分析を行なったところ、配列表の配列番号11に示されるアミノ酸配列が確認された。 When 100 pmol of the synthesized peptide was subjected to amino acid sequence analysis using an amino acid sequencer (PPSQ-10 type; manufactured by Shimadzu Corporation), the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing was confirmed. .
製剤例1.
液剤
実施例1乃至11記載の方法により得た11種類のペプチドのいずれかを最終濃度0.1g/mlになるように安定剤として1%(w/v) 精製ゼラチンを含む蒸留水に溶解し、常法により滅菌濾過して11種類の液剤を得た。
Formulation Example 1
Solution 11 Any of the 11 peptides obtained by the method described in Examples 1 to 11 was dissolved in distilled water containing 1% (w / v) purified gelatin as a stabilizer to a final concentration of 0.1 g / ml. The solution was sterilized and filtered in a conventional manner to obtain 11 types of solutions.
本発明のペプチドに対する感受性は個体毎に変わるのが通例であるから、本品は個々の個体に最も適した組成になるよう、11種類の液剤を適宜配合して使用する。本品は安定性に優れているので、スギ花粉症を治療・予防するための点眼剤、点鼻剤、口腔内噴霧剤用の液剤として有用である。 Since susceptibility to the peptide of the present invention usually varies from individual to individual, this product is used by appropriately blending 11 kinds of liquids so that the composition is most suitable for each individual. Since this product is excellent in stability, it is useful as a solution for eye drops, nasal drops, and oral sprays for treating and preventing cedar pollinosis.
製剤例2.
注射剤
安定剤として1%(w/v) ヒト血清アルブミンを含む生理食塩水に実施例1乃至11記載の方法により得た24種類のペプチドをそれぞれ最終濃度0.01、0.1又は1mg/ml になるように溶解し、滅菌濾過した後、滅菌バイアル瓶に2mlずつ分注し、凍結乾燥し、密栓した。
Formulation Example 2
24 kinds of peptides obtained by the method described in Examples 1 to 11 in physiological saline containing 1% (w / v) human serum albumin as an injection stabilizer were respectively prepared at final concentrations of 0.01, 0.1 or 1 mg / The solution was dissolved to a volume of 1 ml, filtered sterilized, and dispensed in 2 ml sterilized vials, lyophilized, and sealed.
本品は投与に先立ち、まず、バイアル瓶内に注射用蒸留水等を1ml加え、次いで、内容物を均一に溶解して使用する。安定性に優れ、有効成分として本発明による11種類のポリペプチドを含んでなる本品は、スギ花粉症を治療・予防するための乾燥注射剤として有用である。 Prior to administration, first add 1 ml of distilled water for injection into a vial, and then dissolve the contents uniformly before use. The product having excellent stability and comprising 11 kinds of polypeptides according to the present invention as an active ingredient is useful as a dry injection for treating and preventing cedar pollinosis.
製剤例3.
錠剤
平均分子量約20,000ダルトンの精製プルラン2g を蒸留水100mlに均一に溶解し、溶液に塩化シアヌルの1.7%(w/v) アセトン溶液を2ml加え、5%(w/v)炭酸ナトリウム水溶液でpHを7付近に保ちつつ、攪拌下、5℃で2時間反応させた。その後、同様にして反応物のpHを7付近に保ちながら、4℃の冷水に対して一晩透析し、活性化プルランを含む水溶液20mlを得た。
Formulation Example 3
Tablets average molecular weight of about 20,000 daltons purified pullulan 2g uniformly dissolved in distilled water 100 ml, 1.7% solution in cyanuric chloride (w / v) acetone was added 2 ml, 5% (w / v) aqueous solution of sodium carbonate The mixture was reacted at 5 ° C. for 2 hours with stirring while maintaining the pH at around 7. Subsequently, the reaction product was dialyzed overnight against 4 ° C. cold water while maintaining the pH of the reaction at around 7 to obtain 20 ml of an aqueous solution containing activated pullulan.
実施例1乃至11記載の方法により得たペプチドをそれぞれ0.2mg加え、溶液のpHを7付近に保ちつつ、穏やかに攪拌しながら、37℃で12時間反応させた。反応後、反応物にグリシンを4g を加え、穏やかに攪拌しながら、37℃で5時間インキュベートし、未反応の活性基をブロックした。 0.2 mg of each peptide obtained by the method described in Examples 1 to 11 was added, and the mixture was allowed to react at 37 ° C. for 12 hours with gentle stirring while keeping the pH of the solution at around 7. After the reaction, 4 g of glycine was added to the reaction product, and incubated at 37 ° C. for 5 hours with gentle stirring to block unreacted active groups.
反応物を濃縮し、あらかじめ0.1Mリン酸緩衝液(pH 7.0) で平衡化させておいたセファデックス G-50 カラムに負荷し、カラムに新鮮な同一緩衝液を通液して、この発明のペプチドとプルランの複合体を含む画分を採取した。収量は、原料ペプチド固形分当たり、約30%であった。 The reaction was concentrated and loaded onto a Sephadex G-50 column that had been equilibrated with 0.1 M phosphate buffer (pH 7.0), and the same buffer was passed through the column. Fractions containing a complex of the peptide and pullulan were collected. The yield was about 30% per raw peptide solid content.
常法に従って、この画分を滅菌濾過し、濃縮し、凍結乾燥し、粉砕後、マンニトールを均一に混合し、混合物を打錠して製品1錠(200mg)当たり複合体を2、10又は50mg含む錠剤を得た。 This fraction is sterile filtered, concentrated, lyophilized, pulverized, mixed uniformly with mannitol, and the mixture is compressed to give 2, 10, or 50 mg of complex per tablet (200 mg) according to conventional methods. A tablet containing was obtained.
摂取性、安定性に優れた本品は、スギ花粉症を治療・予防するための舌下剤として有用である。 This product with excellent ingestion and stability is useful as a sublingual agent for treating and preventing cedar pollinosis.
製剤例4.
シロップ剤
大腸菌由来の精製リポ多糖1g を10mMリン酸カルシウム溶液100mlに溶解し、溶液に100mM過ヨウ素酸ナトリウムを6ml加え、室温下で20分間反応させてリポ多糖を活性化した。反応物を4℃の1Mグリシン−塩酸緩衝液(pH 4.4) に対して一晩透析して未反応の過ヨウ素酸を除去した後、0.1M 炭酸水素ナトリウム緩衝液によりpH 9.5付近に調整する一方、別途、実施例1乃至11記載の方法により得た11種類のペプチドを0.1M リン酸緩衝液(pH 7.0) 100mlにそれぞれ10mgずつ溶解し、活性化リポ多糖を含む上記反応物に加え、室温下で12時間静置して反応させた。
Formulation Example 4
1 g of purified lipopolysaccharide derived from syrup Escherichia coli was dissolved in 100 ml of 10 mM calcium phosphate solution, 6 ml of 100 mM sodium periodate was added to the solution and reacted at room temperature for 20 minutes to activate the lipopolysaccharide. The reaction product is dialyzed overnight against 4 ° C. 1M glycine-hydrochloric acid buffer (pH 4.4) to remove unreacted periodic acid, and then adjusted to about pH 9.5 with 0.1M sodium bicarbonate buffer. Separately, eleven types of peptides obtained by the methods described in Examples 1 to 11 were dissolved 10 mg each in 100 ml of 0.1M phosphate buffer (pH 7.0), and added to the reaction product containing activated lipopolysaccharide. The reaction was allowed to stand at room temperature for 12 hours.
その後、新たに得られた反応物を製剤例3の方法により精製し、得られた本発明のペプチドとリポ多糖の複合体を含む画分を濃縮し、凍結乾燥し、粉砕して固状物とした。収量は、原料ペプチド固形分当たり、約30%であった。 Thereafter, the newly obtained reaction product is purified by the method of Formulation Example 3, and the resulting fraction containing the complex of the peptide of the present invention and lipopolysaccharide is concentrated, lyophilized, pulverized and solidified. It was. The yield was about 30% per raw peptide solid content.
この固形物を蔗糖をそれぞれ最終濃度が0.1若しくは1mg/ml 又は50%(w/w) になるように安定剤として精製ゼラチンを1%(w/w) 含む蒸留水に溶解し、溶液を常法により滅菌濾過してシロップ状物を得た。このシロップ状物を2mlずつ滅菌バイアル瓶に分注し、密栓して製品とした。安定性に優れ、有効成分としてこの発明のペプチドとリポ多糖の複合体を含む本品は、スギ花粉症を治療・予防するためのシロップ剤として有用である。 This solid is dissolved in distilled water containing 1% (w / w) purified gelatin as a stabilizer so that the final concentration is 0.1 or 1 mg / ml or 50% (w / w). Was sterilized and filtered by a conventional method to obtain a syrup. Each 2 ml of this syrup was dispensed into sterile vials and sealed to give a product. The product having excellent stability and containing the complex of the peptide of the present invention and lipopolysaccharide as an active ingredient is useful as a syrup for treating and preventing cedar pollinosis.
本発明は、免疫療法剤、特にスギ花粉症治療剤として有用である。 The present invention is useful as an immunotherapeutic agent, particularly a cedar pollinosis therapeutic agent.
配列番号1:合成ペプチド
配列番号2:合成ペプチド
配列番号3:合成ペプチド
配列番号4:合成ペプチド
配列番号5:合成ペプチド
配列番号6:合成ペプチド
配列番号7:合成ペプチド
配列番号8:合成ペプチド
配列番号9:合成ペプチド
配列番号10:合成ペプチド
配列番号11:合成ペプチド
Sequence number 1: Synthetic peptide Sequence number 2: Synthetic peptide sequence number 3: Synthetic peptide sequence number 4: Synthetic peptide sequence number 5: Synthetic peptide sequence number 6: Synthetic peptide sequence number 7: Synthetic peptide sequence number 8: Synthetic peptide sequence number 9: Synthetic peptide SEQ ID NO: 10: Synthetic peptide SEQ ID NO: 11: Synthetic peptide
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JP3474898B2 (en) * | 1993-10-20 | 2003-12-08 | 明治乳業株式会社 | T cell epitope peptide of cedar pollen allergen and its analog peptide |
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