JP2004238374A - Anti-chlamydia agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for markedly inhibiting the propagation of chlamydia by applying it by injection or through a cutaneous, mucosal, nasal or oral route. <P>SOLUTION: This anti-chlamydia agent contains interferon-α as an active ingredient, wherein the content of a subtype α8 is >10 mass % based on the amount of the interferon α. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００２０】
表１の結果は、同様にインターフェロンαに分類されるインターフェロンであっても、サブタイプによってクラミジア・トラコマティス及びクラミジア・ニューモニエの増殖を抑制する効果、すなわち、抗クラミジア活性が著しく異なることを示している。実験に供したサブタイプのうちでは、サブタイプα８の抗クラミジア活性が最も高く、他のサブタイプの２８倍以上にも達した。従来公知の医薬品に頻用されているサブタイプα２の抗クラミジア活性は著しく低く、サブタイプα８の１／４０以下であった。これらの結果は、インターフェロンαにおけるサブタイプのうちでも、サブタイプα８が呼吸器経疾患や循環器経疾患の発症原因であるクラミジアの増殖抑制に抜きんでて有効であることを物語っている。
【００２１】
【実験例４】
〈サブタイプα８と他のサブタイプとの併用〉
実験例２の方法により得た人のインターフェロンαにおけるサブタイプα８と、実験例２の方法により得た他のサブタイプとを表２に示す割合で併用した以外は実験例３におけると同様に試験した。結果を表２に併記する。
【００２２】
【表２】

【００２３】
表２の結果は、サブタイプの組合わせ方によって、発揮される抗クラミジア活性に著しい違いのあることを示している。サブタイプα８とサブタイプα２との組合わせは抗クラミジア活性が格別高く、他の組合わせの２倍以上にも達した。
実験例３において、サブタイプα２はサブタイプα８と比較して明らかに低い抗クラミジア活性しか示さなかったことから、本実験例において、サブタイプα８とサブタイプα２との組合わせが斯くも顕著な抗クラミジア活性を示したことは、両者の組合わせが、その余の組合わせでは決して達成されない、いわゆる、相乗効果をもたらしたものと推定される。なお、インターフェロンα混合物においても、その抗クラミジア効果はサブタイプα２の６倍程度にすぎず、このことも、クラミジアが発病原因となる疾患の治療・予防において、サブタイプα８とサブタイプα２の人為的な組合わせに意義があることを物語っている。
【００２４】
【実験例５】
〈サブタイプα８とサブタイプα２との配合比〉
実験例２の方法により得た人のインターフェロンαのサブタイプα８及びサブタイプα２につき、それらを表３に示す割合で併用した以外は実験例３におけると同様にして抗クラミジア活性を試験した。結果を表３に併記する。
【００２５】
【表３】

【００２６】
表３の結果は、サブタイプα８とサブタイプα２とを併用する場合には、インターフェロンαにおける前者の含量が１０質量％を越え、好ましくは、２０質量％以上にするのが望ましいことを物語っている。また、サブタイプα８とサブタイプα２との組合わせであっても、インターフェロンαにおける前者の割合が１０質量％以下になると実質的な相乗効果が認められず、また、４０質量％以上、いいかえると後者の割合が６０質量％以下になると、効果が頭打ちになることを示している。これらの結果は、人のインターフェロンαにおけるサブタイプα８とサブタイプα２とを併用する場合には、前者を１０質量％を越える含量、好ましくは、２０質量％以上、４０質量％未満の範囲で加減するのが望ましいことを物語っている。
【００２７】
【実験例６】
〈生体内試験〉
複数の成人男性から咽頭の上皮細胞を綿棒で採取し、ＳＰＧ緩衝液に浮遊させた後、超音波処理し、これを実験例３に記載の方法に準じて、ＨｅＬａ細胞に感染させ、３７℃インキュベーター（５％ＣＯ_２）中で４８時間培養した。これを実験例３に記載の蛍光標識法を適用してクラミジアの存在が確認された男性（年齢４６乃至７５歳、３６名）を本生体内試験の被験者とした。
【００２８】
被験者を無作為に選別して４群（９名／群）に分け、その３群に対して、実験例２の方法により得た人のインターフェロンαのサブタイプα８とサブタイプα２とを質量比１：０、１：２、１：３又は１：９のいずれかの割合で含んでなる、後記実施例５の方法により調製した口腔清浄剤を、１日３回、毎食後、２０日間に亙って適用させた。残る１群の被験者には、インターフェロンαを省略した以外は同様にして調製した対照の口腔清浄剤を適用した。２１日目にすべての被験者から、再度、咽頭の上皮細胞を採取してクラミジアによる封入体細胞数を計数し、試験開始前の封入体細胞数と比較した。結果を表４に示す。
【００２９】
【表４】

【００３０】
表４の結果は、インターフェロンαを含んでなり、そのインターフェロンαにおけるサブタイプα８の含量が１０質量％を越え、好ましくは、２０質量％以上である抗クラミジア用剤は、生体においても、クラミジアの増殖を顕著に抑制することを物語っている。本試験においても、サブタイプα８だけを配合した口腔清浄剤は、抗クラミジア活性においてサブタイプα８とサブタイプα２とを併用するものに劣り、また、サブタイプα８とサブタイプα２とを併用する場合でも、サブタイプα８の割合が小さいと所期の抗クラミジア効果が得られず、反対に、サブタイプα８の割合が大きすぎると、却って、抗クラミジア活性が低下するという、実験例５における生体外実験の結果が裏付けられた。
【００３１】
以下、本発明の実施の形態につき、実施例に基づいて説明する。
【００３２】
【実施例１】
〈有効成分としてのインターフェロンαの調製〉
常法にしたがって、市販されている人のインターフェロンα標品（商品名『インターフェロン−α』、コスモバイオ株式会社販売）へ抗ヒトインターフェロンα抗体カラムクロマトグラフィーを適用してウシ血清アルブミンを除去した後、限外濾過膜及びゲル濾過カラムクロマトグラフィーをそれぞれ適用することによって、人のインターフェロンαにおける活性なサブタイプα８ｂ及びサブタイプα２を質量比１：３の割合で含有するインターフェロンαを得た。
【００３３】
【実施例２】
〈有効成分としてのインターフェロンαの調製〉
常法にしたがって、ＢＡＬＬ−１細胞（ＪＣＲＢ００７１）からゲノムＤＮＡを調製し、制限酵素ＮｄｅＩ又はＢａｍＨＩによる切断部位を有する、配列表における配列番号４及び配列番号５に示す塩基配列のプライマーの存在下でＰＣＲ反応させ、配列表における配列番号２に併記されたアミノ酸配列を有する、人のインターフェロンαにおけるサブタイプα８ｂをコードするＤＮＡを得た。
【００３４】
常法により、このＤＮＡを制限酵素ＮｄｅＩ及びＢａｍＨＩにより切断した後、あらかじめ同様の制限酵素で切断しておいたプラスミド発現ベクターｐＥＴ−３ａへ連結した。斯くして得られた、Ｔ７プロモーター領域、サブタイプα８ｂをコードするＤＮＡ領域、Ｔ７ターミネーター領域、アンピシリン耐性遺伝子領域及びＣｏｌＥ１ Ｏｒｉ領域を含んでなる発現ベクターを電気パルス法により大腸菌ＢＬ−２１（ＤＥ３）コンピテントセルへ導入した。得られた形質転換体から、常法により、配列表における配列番号２に併記されるアミノ酸配列のＮ末端にメチオニン残基が付加してなる、人のインターフェロンαにおけるサブタイプα８ｂを発現するクローンを選別した。
【００３５】
斯くして得られた形質転換体を常法にしたがってアンピシリンを含むＬＢ培地で培養した後、培地へイソプロピル−１−チオ−β−Ｄ−ガラクトピラノシドを適量添加してサブタイプα８ｂの発現を誘導した。その後、さらに３時間培養した後、常法にしたがって菌体を採取し、尿素の存在下で超音波処理し、遠心分離により得られた上清を採取し、これに透析、疎水クロマトグラフィー、抗ヒトインターフェロンα抗体クロマトグラフィー、限外濾過膜、ゲル濾過クロマトグラフィーなどを適用することによって、人のインターフェロンα８における活性サブタイプα８ｂを得た。
【００３６】
【実施例３】
〈有効成分としてのインターフェロンαの調製〉
国際特許出願公開ＷＯ９５／１３０９０号明細書に記載された方法に準じて、活性化ポリエチレングリコール−Ｎ−スクシンイミドカーボネートを用い、実施例２の方法により得た人のインターフェロンαにおけるサブタイプα８ｂへ平均分子量１２，０００ダルトンのポリエチレングリコールを結合させた。反応混合物を実施例２における精製方法に準じて精製したところ、１分子当たりポリエチレングリコールが１．３分子結合してなる活性なサブタイプα８ｂが得られた。
【００３７】
【実施例４】
〈練歯磨〉
常法にしたがって、下記に示す練歯磨の基本処方に対して、実施例１乃至実施例３の方法により得た人のインターフェロンαのいずれかを濃度１×１０^４ＩＵ／ｇになるように配合した後、アルミニウムラミネートチューブへ１００ｇずつ充填して３種類のペースト状抗クラミジア口腔用剤を得た。
【００３８】
第二燐酸カルシウム ４２質量部
グリセリン １８質量部
カラギーナン ０．９質量部
ラウリル硫酸ナトリウム １．１質量部
香料 適量
サッカリン 適量
水 ３８質量部
【００３９】
本例の抗クラミジア口腔用剤は、通常の練歯磨と同様に常用することによって、口腔におけるクラミジアの増殖を抑制し、クラミジアが発症原因になる呼吸器系疾患や循環器系疾患を治療・予防することができる。
【００４０】
【実施例５】
〈口腔清浄剤〉
常法にしたがって、下記に示す口腔清浄剤の基本処方に対して、実施例１乃至実施例３の方法により得たインターフェロンαのいずれかを濃度５×１０^４ＩＵ／ｇになるように配合して３種類の液状抗クラミジア口腔用剤を得た。
【００４１】
エタノール ４０質量部
グリセリン １５質量部
ポリオキシエチレン硬化ヒマシ油 １質量部
サッカリン 適量
香料 適量
クロルヘキシジン 適量
水 ４４重量部
【００４２】
本例の抗クラミジア口腔用剤は、通常のうがい薬などと同様に常用することによって、口腔におけるクラミジアの増殖を抑制し、クラミジアが発症原因になる呼吸器系疾患や循環器系疾患を治療・予防することができる。
【００４３】
【実施例６】
〈トローチ剤〉
常法にしたがって、下記に示すトローチ剤の基本処方に対して、実施例１乃至実施例３の方法により得たインターフェロンαのいずれかを濃度５×１０^５ＩＵ／ｇになるように配合し、成形して３種類の固状抗クラミジア口腔用剤を得た。
【００４４】
マルトース ３５質量部
澱粉 ３４質量部
結晶セルロース １０質量部
ヒドロキシプロピルメチルセルロース １０質量部
ステアリン酸マグネシウム １質量部
【００４５】
本例の抗クラミジア口腔用剤は、通常のトローチ剤と同様に常用することによって、口腔におけるクラミジアの増殖を抑制し、クラミジアが発症原因になる呼吸器系疾患や循環器系疾患を治療・予防することができる。
【００４６】
【実施例７】
〈液剤〉
常法にしたがって、イソロイシンを４質量％含有する注射用精製水に、実施例１乃至３の方法により得た人のインターフェロンαのいずれかを濃度５×１０^５ＩＵ／ｍｌになるように溶解させた後、滅菌濾過して３種類の液状抗クラミジア用剤を得た。
【００４７】
本例の抗クラミジア用剤は、注射用剤、経粘皮用剤、経鼻腔用剤、経口用剤などとして、気道、鼻腔、口腔、視覚器、消化器、泌尿器、生殖器などにおけるクラミジアの増殖を抑制し、クラミジアが発症原因になる呼吸器系疾患や循環器系疾患を治療・予防することができる。
【００４８】
【実施例８】
〈軟膏〉
常法にしたがって、実施例１乃至実施例３の方法により得た人のインターフェロンαのいずれかを濃度１×１０^６ＩＵ／ｍｌになるように適量の燐酸緩衝生理食塩水（ｐＨ７．２）に溶解し、無水結晶マルトース４９ｇと均一に配合した後、さらに４５０ｇの白色ワセリンと練り合わせて３種類の液状抗クラミジア用剤を得た。
【００４９】
本例の抗クラミジア用剤は、皮膚外用剤、経粘皮用剤などとして、泌尿器、生殖器などにおけるクラミジアの増殖を抑制し、クラミジアが発症原因になる泌尿器系疾患や生殖器系疾患を治療・予防することができる。
【００５０】
【実施例９】
〈点眼剤〉
常法にしたがって、下記に示す点眼剤の基本処方に対して、実施例１乃至実施例３の方法により得たインターフェロンαのいずれかを濃度５×１０^６ＩＵ／ｇになるように配合して３種類の液状抗クラミジア点眼剤を得た。
【００５１】
トレハロース（２水和物） ２０質量部
塩化ナトリウム ０．０４質量部
塩化カリウム ０．０２質量部
燐酸２水素ナトリウム ０．０３質量部
硼砂 ０．０４質量部
塩化ベンザルコニウム ０．００４質量部
水 ８０質量部
【００５２】
本例の抗クラミジア用剤は、通常の点眼剤と同様に常用することによって、視覚器におけるクラミジアの増殖を抑制し、クラミジアが発症原因になる視覚器系疾患を治療・予防することができる。
【００５３】
【発明の効果】
以上説明したとおり、本発明は、生体内において、インターフェロンαにおけるサブタイプα８がクラミジアの増殖を著明に抑制するという独自の知見に基づくものである。本発明の抗クラミジア用剤は、注射又は経皮、経粘皮、経鼻腔若しくは経口経路で適用することによって、生体におけるクラミジアの増殖を著明に抑制することから、鼻腔、口腔や気道、さらには視覚器、消化器、泌尿器、生殖器などへ侵入したクラミジア・ニューモニエ、クラミジア・シッタシ、クラミジア・トラコマティスなどのクラミジアが発症原因となる、例えば、肺炎などの呼吸器系疾患や、動脈硬化、虚血性心疾患などの循環器系疾患、結膜炎などの視覚器系疾患、直腸炎などの消化器系疾患、尿道炎などの泌尿器系疾患、副睾丸炎、卵管炎、子宮頚管炎、切迫早産、卵管の炎症、癒着、閉塞などの生殖器系疾患の治療、予防に著効を発揮する。とりわけ、本発明の抗クラミジア口腔用剤は、抗生物質を有効成分とする経口用剤とは違って、耐性菌の出現を招来したり、長期間連用しても大腸における有用な菌叢を破壊することがないので、食欲不振、嘔吐、下痢、便秘、膨満感を生じたり、倦怠感、発湿などの副作用を惹起することがない。
【配列表】

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an anti-Chlamydia agent, and more particularly to an anti-Chlamydia agent containing interferon α as an active ingredient.
[0002]
[Prior art]
Chlamydia is known to include Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia spp., Among which Chlamydia pneumoniae and Chlamydia spp. It is said to cause cardiovascular diseases such as sclerosis and ischemic heart disease. Since these chlamydia are orally transmitted, it is thought that if the growth of chlamydia in the nasal cavity, oral cavity, respiratory tract, etc. can be suppressed, the incidence of diseases caused by chlamydia infection can be effectively suppressed. .
[0003]
Conventionally, tetracycline or macrolide antibiotics have been mainly used for the treatment of the above diseases. However, although these antibiotics have the advantage of being extremely effective when administered orally, common disadvantages of antibiotics generally include the emergence of resistant bacteria and the destruction of useful flora in the large intestine, resulting in anorexia. May cause vomiting, diarrhea, constipation, bloating, malaise, hydration.
[0004]
[Problems to be solved by the invention]
In view of such circumstances, an object of the present invention is to provide a means for significantly suppressing the growth of Chlamydia by injection or transdermal, transmucosal, nasal or oral routes.
[0005]
[Means for Solving the Problems]
The present inventor focused on interferon, which is a kind of cytokine, and conducted extensive research. As a result of the search, interferon α having a subtype α8 content of more than 10% by mass was found to be respiratory disease, It has been found that the growth of Chlamydia, which causes circulatory, digestive, urinary, reproductive and visual diseases, is significantly suppressed.
[0006]
That is, the present invention solves the above-mentioned problems by providing an anti-chlamydia agent comprising interferon α as an active ingredient, wherein the content of subtype α8 in the interferon α exceeds 10% by mass in interferon α. is there.
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
Interferon α in the present invention is, for example, interferon obtained by allowing an interferon-inducing agent such as Sendai virus to act on blood cells such as leukocytes or lymphoblastoid cells. As is well known, molecules of interferon α have polymorphisms. For example, in the case of human interferon α, molecules classified as interferon α are subtype α1, subtype α2, They are classified into type α5, subtype α6, subtype α7, subtype α8, subtype α14, subtype ω, and the like. The present invention relates to an anti-Chlamydia agent comprising interferon α as an active ingredient, wherein the content of subtype α8 in the interferon α exceeds 10% by mass.
[0008]
The subtype α8 of interferon α in the anti-Chlamydia agent of the present invention usually consists of 161 or 166 amino acid residues and has a nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in the sequence listing, respectively. Subtype α8a, subtype 8b, and subtype α8c of human interferon α having the amino acid sequence described in the above are preferable. Among them, subtype α8b is the most preferable in terms of economy and a large effect of suppressing the growth of Chlamydia. preferable.
[0009]
The anti-chlamydial agent of the present invention usually contains one or more components for constituting an injection, an external preparation for the skin, a transdermal agent, a nasal agent or an oral agent, and a subtype α8 content. Exceeds 10% by mass, preferably 20% by mass or more. Interferon α to be added to the anti-Chlamydia agent of the present invention has a subtype α8 content in the interferon α within the above range, and is applied by injection or transdermal, transdermal, transnasal or oral route. As long as the desired anti-Chlamydia effect is exhibited, it may be a natural type or a recombinant type, regardless of its origin and preparation method. When the content of the subtype α8 is below this range, no anti-chlamydia effect is obtained or the level is extremely low. Therefore, it is desirable to adjust the content within the above range.
[0010]
Examples of the natural interferon α to be added to the anti-Chlamydia agent of the present invention include, for example, the Sendai virus acting on BALL-1 cells (JCRB0071), which is a kind of human lymphoblastoid cells, to the interferon produced. The content of subtype α2 obtained by applying antibody chromatography is more than 60% by mass, preferably 70 to 80% by mass, and the content of subtype α8b is more than 15% by mass, preferably 20% by mass. And 30% by mass of human interferon α. When the anti-chlamydia agent containing such interferon α is applied to a living body, the balance between subtype α2 and subtype α8b is exquisite, and since they synergistically cooperate with each other, it is never possible to use only one of them. An extremely high anti-Chlamydia effect, which is not achieved, is obtained.
[0011]
Examples of the recombinant interferon α to be added to the anti-Chlamydia agent according to the present invention include, for example, human interferon having an amino acid sequence described in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in the sequence listing. A gene encoding any of subtype α8a, subtype α8b or subtype α8c in α is inserted into an appropriate vector, and a transformant obtained by transforming, for example, Escherichia coli or the like is used in a conventional manner. And obtained by collecting the interferon protein from the culture. Such interferon α is slightly inferior in the anti-chlamydia effect as compared to the natural type produced by blood cells such as BALL-1 cells as described above, but if necessary, for example, the dose applied to a living body is reduced. For example, polyethylene glycol may be used for the protein of subtype α8 by the method described in International Patent Application Publication No. WO95 / 13090 or WO96 / 10089, or according to those methods. Dextran, pullulan or other polysaccharides to increase the residence time in the living body, or use in combination with a recombinant subtype α2 prepared in the same manner to produce an antibody that does not hinder practical use. An agent for chlamydia can be obtained.
[0012]
The anti-chlamydia agent of the present invention may be, for example, in the form of a liquid, paste or solid injection, skin external preparation, transmucosal agent, nasal agent, oral agent, etc. Prepared and used, specifically, injections, eye drops, suppositories, ointments, and further, toothpastes, mouthwashes, mouthwashes such as lozenges, and oral disinfectants in the dental field. Oral forms are preferred. Therefore, the anti-Chlamydia agent of the present invention includes powders, fine granules, pills, tablets, capsules, troches, limonades, elixirs, syrups, fragrances, suspensions, emulsions, dips, and decoctions , Tinctures, extracts, fluid extracts, alcoholic beverages, liniments, lotions, ointments, cataplasms, plasters, suppositories, eye drops, eye ointments, injections, for example, abrasives , Foaming agents, wetting agents, binders, flavors, sweeteners, preservatives, fluorine compounds, enzyme agents, deodorants, as well as excipients, ointment bases, dissolving agents, flavoring agents, flavoring agents, It does not prevent use in combination with one or more preparation agents such as colorants, emulsifiers, and propellants. Further, within the scope not departing from the object of the present invention, commonly used in the drug of such a dosage form, for example, a hemostatic agent, an anti-inflammatory agent, a tissue activator, and further, a subtype other than the subtype α8 in interferon α, It does not prevent the combined use with one or more other active ingredients including interferon β and interferon γ. Injectable preparations and transmucosal preparations, nasal cavity, oral preparations, in terms of having a property to stabilize interferon α, for example, sucrose, glucose, maltose, trehalose, fructose, In addition to sugars or sugar alcohols such as mannitol, alanine, arginine, asparagine, aspartic acid, citrulline, cysteine, cystine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, phenylglycine, proline, serine, Threonine, tryptophan, tyrosine, valine, hydroxyproline, γ-carboxyglutamate, O-phosphoserine, β-alanine, α-aminobutyric acid, γ-aminobutyric acid, α-aminoisobutyric acid, 4- (4-aminophenyl Butyric acid, aminophenylbutyric acid, aminobenzoic acid, 4-aminohippuric acid, aminomethylbenzoic acid, ε-aminocaproic acid, 7-aminoheptanoic acid, β-aspartic acid, γ-glutamic acid, ε-lysine, methionine = sulfone, Amino acids such as norleucine, norvaline, ornithine, d-ornithine, p-nitro-phenylalanine, hydroxyproline, thioproline, and derivatives or oligopeptides of these amino acids are preferred, and these may be used in combination as necessary.
[0013]
The dose of interferon α8 to be added to the anti-Chlamydia agent of the present invention depends on the type of subtype α8, the indication of the anti-Chlamydia agent, the administration route, and the like. ³ ~ 5 × 10 ⁷ IU / day / adult, preferably 1 × 10 ⁴ ~ 1 × 10 ⁷ IU / day / adult. In this specification, the activity of interferon α is expressed in terms of international units (IU) unless otherwise specified.
[0014]
The anti-Chlamydia agent of the present invention can be used in the body at such doses by injection once or more frequently once a day or once a week, or by transdermal, transmucosal, nasal or oral route, to achieve Chlamydia in vivo. Chlamydia such as Chlamydia pneumoniae, Chlamydia shittashi and Chlamydia trachomatis invades the nasal cavity, oral cavity and respiratory tract, as well as the visual, digestive, urinary and reproductive organs. For example, respiratory diseases such as pneumonia, circulatory diseases such as arteriosclerosis, ischemic heart disease, visual diseases such as conjunctivitis, digestive diseases such as proctitis, urinary diseases such as urethritis It is extremely effective in treating and preventing reproductive diseases such as systemic diseases, epididymis, salpingitis, cervicitis, premature labor, inflammation of the fallopian tubes, adhesions and obstruction. In particular, the oral anti-Chlamydia agent of the present invention, unlike an oral agent containing an antibiotic as an active ingredient, causes the emergence of resistant bacteria and destroys a useful flora in the large intestine even after long-term continuous use. It does not cause anorexia, vomiting, diarrhea, constipation, bloating, and does not cause side effects such as malaise and humidification. Regarding the safety of the anti-Chlamydia agent of the present invention, as is well known, interferon α is a substance originally existing in a living body, and today, it is safe to administer at the dose as described above. It should be added that has been confirmed.
[0015]
Hereinafter, the anti-Chlamydia effect of the anti-Chlamydia agent of the present invention will be described based on experimental examples.
[0016]
[Experimental example 1]
<Preparation of Chlamydia stock solution>
A549 cells (ATCC CCL-185), a kind of human lung-derived fibroblast cell line, were placed in an Eagle minimum medium (pH 7.2) supplemented with 10% by volume of calf serum at 8.6 × as a host cell. 10 ⁵ The cells were suspended to a cell density of cells / ml, aliquoted into a culture plate, and incubated at 37 ° C. in an incubator (5% CO 2). ₂ ), And the medium was removed by centrifugation. Separately, either Chlamydia trachomatis UW-3 / Cx strain (ATCC VR-885) or Chlamydia pneumoniae TWARAR-39 strain (ATCC53592) of trachoma type D was added to an SPG buffer (75 g sucrose, 0.52 g dihydrogen phosphate). Potassium, 1.529 g disodium hydrogen phosphate dihydrate and 0.72 g glutamic acid were dissolved in 1000 ml of distilled water, and then diluted with a filter and sterilized by filtration, and added to A549 cells on a culture plate in an appropriate amount. Thereafter, the cells were centrifuged at 1,500 × g for 1 hour to infect the cells with Chlamydia. Then, the buffer was removed and added to Eagle's minimal medium (pH 7.2) supplemented with 8% by volume of fetal bovine serum at 5 ml / well, and the mixture was added at 37 ° C. in an incubator (5% CO 2). ₂ ) For 48 hours. Thereafter, the medium was removed from the culture, fresh SPG buffer was added at 3 ml / well, and the cells were collected in suspension using a cell scraper and diluted with fresh SPG buffer to obtain 1 ml / ml. 1 x 10 inclusion body forming units (IFU) ⁷ An IFU, Chlamydia stock solution was obtained.
[0017]
[Experimental example 2]
<Preparation of subtype in interferon α>
According to the method described in U.S. Pat. No. 6,350,589, buffy coat hesindai virus prepared from human blood is allowed to act on the resulting interferon-alpha, and hydroxyapatite chromatography, exclusion chromatography, ion exchange chromatography, By applying hydrophobic chromatography, etc., the active subtype α1, subtype α2, subtype α5, subtype α7 and subtype α8 of human interferon α and subtype of interferon α produced by buffy coat are produced. Each mixture of interferon α contained in the composition as it was (hereinafter, referred to as “interferon α mixture”) was isolated.
[0018]
[Experimental example 3]
<Subtype anti-Chlamydia activity>
HeLa cells (ATCC CCL-2), a type of fibroblast cell line derived from human uterus, were suspended in Eagle's minimal medium (pH 7.2) supplemented with 10% by volume of calf serum, and the cell density was 8 × 10 ⁴ Cells / ml, seeded in a 0.1 ml well in a culture plate, and then inoculated in a 37 ° C. incubator (5% CO 2). ₂ ) For 16 hours. Next, Eagle's minimum medium (pH 7.20) supplemented with 8% by volume of calf serum containing any of subtype α1, subtype α2, subtype α5, subtype α7 or subtype α8 of interferon α prepared by the method of Experimental Example 2. 2) was added to the concentration shown in Table 1, and then added to a 37 ° C. incubator (5% CO 2). ₂ ) For 24 hours. After removing the medium, the Chlamydia stock solution prepared by the method of Experimental Example 1 was sonicated, and 1 × 10 5 ⁴ After addition of IFU / well, the cells were centrifuged at 1,500 × g for 1 hour to adsorb and infected Chlamydia to Hela cells. After removing the excess chlamydia stock solution, fresh Eagle's minimal medium (pH 7.2) supplemented with 8% by volume calf serum containing the same interferon α subtype as above was added, and the cells were further cultured for 48 hours. At the same time, a system to which interferon α was not added was provided and treated in the same manner as above to serve as a control. Thereafter, the number of the inclusion body forming cells in the cultured cells is counted by the usual fluorescent labeling method, and the concentration of the subtype that reduces the number of the inclusion body forming cells in the control without interferon α to 10% is calculated by the serial dilution method. did. The results are also shown in Table 1.
[0019]
[Table 1]

[0020]
The results in Table 1 show that the effect of suppressing the growth of Chlamydia trachomatis and Chlamydia pneumoniae depending on the subtype, that is, the anti-Chlamydia activity is significantly different even for interferon similarly classified as interferon α. I have. Among the subtypes subjected to the experiment, the anti-Chlamydia activity of subtype α8 was the highest, and reached 28 times or more of the other subtypes. The anti-chlamydia activity of subtype α2, which has been frequently used in conventionally known pharmaceuticals, is remarkably low, being 1/40 or less of subtype α8. These results indicate that among the subtypes of interferon α, subtype α8 is most effective in suppressing the growth of Chlamydia, which is a cause of respiratory and cardiovascular diseases.
[0021]
[Experimental example 4]
<Use of subtype α8 with other subtypes>
A test was conducted in the same manner as in Experimental Example 3 except that subtype α8 in human interferon α obtained by the method of Experimental Example 2 and another subtype obtained by the method of Experimental Example 2 were used in the ratio shown in Table 2. did. The results are also shown in Table 2.
[0022]
[Table 2]

[0023]
The results in Table 2 show that there is a marked difference in the anti-Chlamydia activity exhibited by the combination of subtypes. The combination of subtype α8 and subtype α2 has remarkably high anti-Chlamydia activity, more than twice as high as the other combinations.
In Experimental Example 3, the combination of subtype α8 and subtype α2 was so remarkable in sub-type α2 because subtype α2 showed only clearly lower anti-chlamydia activity as compared with subtype α8. The fact that it exhibited anti-Chlamydia activity is presumed that the combination of the two resulted in a so-called synergistic effect which was never achieved with the other combinations. In addition, the anti-Chlamydia effect of the interferon α mixture is only about six times that of the subtype α2, which also indicates that in the treatment / prevention of diseases caused by chlamydia, the subtype α8 and the subtype α2 It tells the significance of a special combination.
[0024]
[Experimental example 5]
<Compounding ratio of subtype α8 and subtype α2>
Anti-Chlamydia activity was tested in the same manner as in Experimental Example 3 except that human interferon α subtype α8 and subtype α2 obtained by the method of Experimental Example 2 were used in combination at the ratios shown in Table 3. The results are also shown in Table 3.
[0025]
[Table 3]

[0026]
The results in Table 3 indicate that when subtype α8 and subtype α2 are used in combination, the content of the former in interferon α should be more than 10% by mass, preferably 20% by mass or more. I have. Also, even with the combination of subtype α8 and subtype α2, if the ratio of the former in interferon α is 10% by mass or less, a substantial synergistic effect is not recognized, and if it is 40% by mass or more. When the ratio of the latter is 60% by mass or less, the effect reaches a plateau. These results indicate that when subtype α8 and subtype α2 in human interferon α are used in combination, the former is adjusted to a content of more than 10% by mass, preferably 20% by mass or more and less than 40% by mass. Tells you what it is desirable to do.
[0027]
[Experimental example 6]
<In vivo test>
Pharyngeal epithelial cells were collected from a plurality of adult males with a cotton swab, suspended in an SPG buffer, sonicated, and infected with HeLa cells according to the method described in Experimental Example 3 at 37 ° C. Incubator (5% CO ₂ ) For 48 hours. The fluorescent labeling method described in Experimental Example 3 was applied thereto, and men (age: 46 to 75 years, 36 persons) whose presence of chlamydia was confirmed were taken as subjects in this in vivo test.
[0028]
Subjects were randomly selected and divided into 4 groups (9 persons / group), and the mass ratio of human interferon α subtype α8 and subtype α2 obtained by the method of Experimental Example 2 to the three groups was determined. An oral cleanser prepared according to the method of Example 5 described below, comprising any ratio of 1: 0, 1: 2, 1: 3 or 1: 9, three times a day, after each meal, for 20 days Was applied over a period of time. The remaining group of subjects was applied with a control mouthwash prepared in the same manner except that interferon α was omitted. On the 21st day, pharyngeal epithelial cells were collected again from all subjects, and the number of Chlamydia inclusion bodies was counted, and compared with the number of inclusion bodies before the start of the test. Table 4 shows the results.
[0029]
[Table 4]

[0030]
The results in Table 4 show that the anti-Chlamydia agent comprising interferon α, wherein the content of subtype α8 in the interferon α exceeds 10% by mass, and preferably 20% by mass or more, shows that the anti-Chlamydia It indicates that growth is significantly suppressed. In this test, the mouthwash containing only subtype α8 is inferior to the one using subtype α8 and subtype α2 in anti-chlamydia activity, and the case where subtype α8 and subtype α2 are used together. However, if the proportion of subtype α8 is small, the intended anti-Chlamydia effect cannot be obtained, whereas if the proportion of subtype α8 is too large, the anti-Chlamydia activity is rather reduced. The results of the experiment were supported.
[0031]
Hereinafter, embodiments of the present invention will be described based on examples.
[0032]
Embodiment 1
<Preparation of interferon α as active ingredient>
After removing bovine serum albumin by applying anti-human interferon α antibody column chromatography to a commercially available human interferon α standard (trade name “Interferon-α”, sold by Cosmo Bio Inc.) in accordance with a conventional method. By applying an ultrafiltration membrane and gel filtration column chromatography, interferon α containing active subtype α8b and subtype α2 in human interferon α at a mass ratio of 1: 3 was obtained.
[0033]
Embodiment 2
<Preparation of interferon α as active ingredient>
Genomic DNA is prepared from BALL-1 cells (JCRB0071) according to a conventional method, and is prepared in the presence of a primer having the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 having a cleavage site with the restriction enzyme NdeI or BamHI. A PCR was carried out to obtain a DNA encoding the human subferon α8 subtype α8b having the amino acid sequence described in SEQ ID NO: 2 in the sequence listing.
[0034]
This DNA was cut with restriction enzymes NdeI and BamHI and ligated to a plasmid expression vector pET-3a which had been cut with the same restriction enzymes in the usual manner. The thus obtained expression vector containing the T7 promoter region, the DNA region encoding the subtype α8b, the T7 terminator region, the ampicillin resistance gene region, and the ColE1 Ori region was subjected to the electric pulse method by E. coli BL-21 (DE3). Introduced to competent cells. From the obtained transformant, a clone expressing a subtype α8b in human interferon α, which is obtained by adding a methionine residue to the N-terminus of the amino acid sequence described in SEQ ID NO: 2 in the sequence listing, by a conventional method. Sorted out.
[0035]
After culturing the thus obtained transformant in an LB medium containing ampicillin according to a conventional method, an appropriate amount of isopropyl-1-thio-β-D-galactopyranoside is added to the medium to express the subtype α8b. Was induced. Then, after further culturing for 3 hours, the cells were collected according to a conventional method, sonicated in the presence of urea, and the supernatant obtained by centrifugation was collected. The active subtype α8b in human interferon α8 was obtained by applying human interferon α antibody chromatography, ultrafiltration membrane, gel filtration chromatography and the like.
[0036]
Embodiment 3
<Preparation of interferon α as active ingredient>
Average molecular weight to subtype α8b in human interferon α obtained by the method of Example 2 using activated polyethylene glycol-N-succinimide carbonate according to the method described in International Patent Application Publication No. WO95 / 13090. 12,000 daltons of polyethylene glycol were bound. The reaction mixture was purified according to the purification method in Example 2 to obtain an active subtype α8b in which 1.3 molecules of polyethylene glycol were bonded per molecule.
[0037]
Embodiment 4
<Toothpaste>
According to a conventional method, any one of human interferon α obtained by the method of Examples 1 to 3 was added to a basic prescription of toothpaste shown below at a concentration of 1 × 10 ⁴ After blending to give IU / g, 100 g of each was filled into an aluminum laminate tube to obtain three kinds of paste-like anti-Chlamydia oral agents.
[0038]
42 parts by mass of dicalcium phosphate
Glycerin 18 parts by mass
Carrageenan 0.9 parts by mass
Sodium lauryl sulfate 1.1 parts by mass
Appropriate amount of fragrance
Saccharin qs
38 parts by mass of water
[0039]
The anti-Chlamydia oral preparation of this example is used in the same manner as ordinary toothpaste to suppress the growth of Chlamydia in the oral cavity and treat and prevent respiratory and cardiovascular diseases caused by Chlamydia. can do.
[0040]
Embodiment 5
<Mouthwash>
According to a conventional method, any one of the interferon αs obtained by the methods of Examples 1 to 3 was added to the basic formulation of the mouthwash shown below at a concentration of 5 × 10 ⁴ Three kinds of liquid anti-Chlamydia oral preparations were obtained by blending to give IU / g.
[0041]
40 parts by mass of ethanol
Glycerin 15 parts by mass
Polyoxyethylene hydrogenated castor oil 1 part by mass
Saccharin qs
Appropriate amount of fragrance
Chlorhexidine qs
44 parts by weight of water
[0042]
The anti-Chlamydia oral agent in this example is used in the same manner as ordinary mouthwashes to suppress the growth of Chlamydia in the oral cavity and treat respiratory and cardiovascular diseases caused by Chlamydia. Can be prevented.
[0043]
Embodiment 6
<Troche>
According to a conventional method, any one of the interferon αs obtained by the methods of Examples 1 to 3 was added to a basic formulation of a troche shown below at a concentration of 5 × 10 ⁵ IU / g was blended and molded to obtain three types of solid anti-Chlamydia oral agents.
[0044]
Maltose 35 parts by mass
Starch 34 parts by mass
Crystalline cellulose 10 parts by mass
Hydroxypropyl methylcellulose 10 parts by mass
Magnesium stearate 1 part by mass
[0045]
The oral anti-Chlamydia agent of this example is used in the same manner as ordinary lozenges to suppress the growth of Chlamydia in the oral cavity and treat and prevent respiratory and cardiovascular diseases caused by Chlamydia. can do.
[0046]
Embodiment 7
<Liquid>
According to a conventional method, any of human interferon α obtained by the method of Examples 1 to 3 was added to purified water for injection containing 4% by mass of isoleucine at a concentration of 5 × 10 5. ⁵ After dissolving to IU / ml, three types of liquid anti-Chlamydia agents were obtained by sterile filtration.
[0047]
The anti-Chlamydia agent of the present example may be used as an injection, transdermal, nasal, oral, etc., Chlamydia in the respiratory tract, nasal cavity, oral cavity, visual system, digestive system, urinary system, genitalia, etc. And prevent and treat respiratory and cardiovascular diseases caused by Chlamydia.
[0048]
Embodiment 8
<ointment>
According to a conventional method, any one of human interferon α obtained by the methods of Examples 1 to 3 was used at a concentration of 1 × 10 ⁶ After dissolving in an appropriate amount of phosphate buffered saline (pH 7.2) so as to have an IU / ml and uniformly blending with 49 g of anhydrous crystalline maltose, the mixture is further kneaded with 450 g of white petrolatum to obtain three kinds of liquid anti-Chlamydia agents. Got.
[0049]
The anti-Chlamydia agent of this example, as an external preparation for skin and transmucosal skin, suppresses the growth of Chlamydia in the urinary and genital organs, and treats and prevents urinary and reproductive diseases caused by Chlamydia. can do.
[0050]
Embodiment 9
<Eye drops>
According to a conventional method, any one of the interferon α obtained by the methods of Examples 1 to 3 was added to the basic ophthalmic solution shown below at a concentration of 5 × 10 5 ⁶ Three kinds of liquid anti-Chlamydia eye drops were obtained by blending to give IU / g.
[0051]
Trehalose (dihydrate) 20 parts by mass
Sodium chloride 0.04 parts by mass
Potassium chloride 0.02 parts by mass
Sodium dihydrogen phosphate 0.03 parts by mass
Borax 0.04 parts by mass
Benzalkonium chloride 0.004 parts by mass
80 parts by weight of water
[0052]
The anti-Chlamydia agent of this example is commonly used like ordinary eye drops, thereby suppressing the growth of Chlamydia in the visual organ and treating / preventing visual organ diseases caused by Chlamydia.
[0053]
【The invention's effect】
As described above, the present invention is based on the unique finding that subtype α8 in interferon α significantly suppresses the growth of Chlamydia in vivo. The anti-Chlamydia agent of the present invention, by injection or transdermal, transdermal, transnasal or oral route, markedly inhibits the growth of Chlamydia in the living body, so that the nasal cavity, oral cavity and respiratory tract, Chlamydia such as Chlamydia pneumoniae, Chlamydia shittashi, Chlamydia trachomatis, etc. invading the visual, digestive, urinary, and genital organs can cause respiratory diseases such as pneumonia, arteriosclerosis, and illness. Cardiovascular diseases such as bloody heart disease, visual diseases such as conjunctivitis, gastrointestinal diseases such as proctitis, urinary diseases such as urethritis, epididymitis, salpingitis, cervicitis, and premature labor It is extremely effective in treating and preventing genital diseases such as inflammation, adhesion and obstruction of the fallopian tubes. In particular, the oral anti-Chlamydia agent of the present invention, unlike an oral agent containing an antibiotic as an active ingredient, causes the emergence of resistant bacteria and destroys the useful flora in the large intestine even after long-term use. It does not cause anorexia, vomiting, diarrhea, constipation, bloating, and does not cause side effects such as malaise and humidification.
[Sequence list]

Claims (4)

An anti-Chlamydia agent comprising interferon α as an active ingredient, wherein the content of subtype α8 in interferon α exceeds 10% by mass in interferon α. The anti-Chlamydia agent according to claim 1, wherein the content of subtype α2 in interferon α exceeds 60% by mass in interferon α. The anti-chlamydia agent according to claim 1 or 2, which is in the form of an injection, an external preparation for the skin, a transdermal agent, a nasal agent or an oral agent. The anti-Chlamydia agent according to claim 3, wherein the oral agent is any one of a toothpaste, a mouthwash, and an oral fungicide.