JPH0418029A - Liposome capable of killing hiv-infected cell - Google Patents
Liposome capable of killing hiv-infected cellInfo
- Publication number
- JPH0418029A JPH0418029A JP2241591A JP24159190A JPH0418029A JP H0418029 A JPH0418029 A JP H0418029A JP 2241591 A JP2241591 A JP 2241591A JP 24159190 A JP24159190 A JP 24159190A JP H0418029 A JPH0418029 A JP H0418029A
- Authority
- JP
- Japan
- Prior art keywords
- hiv
- peptide
- liposome
- bound
- phospholipid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明は後天性免疫不全症候群(AIDS)の治療剤ま
たは発症予防剤に関する。さらに詳しくは、本発明はヒ
ト免疫不全ウィルス(HIV)に感染した細胞を選択的
に殺傷するリポソームに関する。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a therapeutic agent or agent for preventing the onset of acquired immunodeficiency syndrome (AIDS). More specifically, the present invention relates to liposomes that selectively kill cells infected with human immunodeficiency virus (HIV).
従来の技術および問題点
後天性免疫不全症候群(AIDS)はヒト免疫不全ウィ
ルス(HIV)の感染により引き起こされるウィルス感
染症てあり、日和見感染症や日和見腫瘍などを伴うこと
を特徴とする免疫不全症である。Conventional techniques and problems Acquired immunodeficiency syndrome (AIDS) is a viral infection caused by infection with the human immunodeficiency virus (HIV), and is an immunodeficiency disease characterized by being accompanied by opportunistic infections and opportunistic tumors. It is.
AIDSの原因ウィルスであるHIVはRNAを遺伝子
とするレトロウィルスの一種であり、ヒトヘルパーTリ
ンパ球なと表面にヒトのCD4抗原を持つ細胞に感染す
る。ヒ1−CD4はヒト白血球の分化抗原の一種てあり
、HIVの外被糖蛋白質gp120に対し親和性を有し
ている。HIVはこのCD4を受容体として細胞内へ侵
入する。HIV, the virus that causes AIDS, is a type of retrovirus whose gene is RNA, and it infects cells that have the human CD4 antigen on their surface, such as human helper T lymphocytes. Human 1-CD4 is a type of differentiation antigen for human leukocytes and has an affinity for the coat glycoprotein gp120 of HIV. HIV uses this CD4 as a receptor to enter cells.
宿主細胞内に侵入したHIVは自身の逆転写酵素を用い
て遺伝子RNAを鋳型として対応するDNAを合成する
。合成されたDNAは宿主細胞の染色体中に組み込まれ
プロウィルスとなり感染か成立する。プロウィルスの状
態から、ひとたびHIVの複製か開始されると感染細胞
はHIVの産生細胞となり、複製されたHIVによる感
染の拡大か繰り返される。HIV that has invaded the host cell uses its own reverse transcriptase to synthesize the corresponding DNA using the gene RNA as a template. The synthesized DNA is integrated into the chromosome of the host cell and becomes a provirus, causing infection. Once HIV replication starts from the provirus state, the infected cell becomes an HIV producing cell, and the infection by the replicated HIV continues to expand.
AIDSの治療剤としては、これまでにアシドチミジン
(AZT)のみか実用化されているにすぎない。AZT
は抗HIV作用を持つヌクレオントアナログである。そ
の作用は、HIVの遺伝子RNAか逆転写によりDNA
に複製される過程を阻害することによる感染阻止作用で
ある( IJi tsuya、 1.et al、、R
roc、 Natl、 Acad、 Sci、 USA
、84゜2033〜2037.1987)。従ってAZ
Tは、HTVの宿主細胞への感染の最初の段階を阻止す
ることはできても、ひとたび感染か成立して、プロウィ
ルスの形でHIVゲノムを持つようになった持続感染細
胞におけるHIVの複製は阻止することかできない。(
Nakashima、 H,et al、、 Anti
microb。As a therapeutic agent for AIDS, only acidothymidine (AZT) has so far been put into practical use. AZT
is a nucleontoanalogue with anti-HIV activity. Its action is to convert HIV gene RNA or DNA through reverse transcription.
It has an anti-infection effect by inhibiting the process of replication in
roc, Natl, Acad, Sci, USA
, 84°2033-2037.1987). Therefore AZ
Although T can block the initial stage of HTV infection of host cells, once infection is established, HIV replication in persistently infected cells that carry the HIV genome in the form of a provirus. can only be prevented. (
Nakashima, H. et al., Anti
microb.
AgentsChemother、、 30.933〜
937.1986) 、またAZTには骨髄(造血組織
)に対する副作用か強いという問題かある。このように
、AZTは抗HIV作用を持ち、臨床における効果も認
められているものの、その効果は十分でなく、また副作
用の問題かある。従ってより効果的なAIDSの治療及
び発症予防には、HIVの複製の場である持続感染細胞
を殺傷、破壊して、HIVか複製されないようにするこ
とか必要である。Agents Chemother,, 30.933~
937.1986), AZT also has the problem of strong side effects on bone marrow (hematopoietic tissue). As described above, although AZT has an anti-HIV effect and has been shown to be effective in clinical practice, the effect is not sufficient and there are problems with side effects. Therefore, for more effective treatment and prevention of AIDS, it is necessary to kill and destroy persistently infected cells, which are the sites of HIV replication, to prevent HIV from replicating.
HIV感染細胞を選択的に傷害する薬剤または組成物と
しては、ジフテリア毒素フラグメントAを封入したリポ
ソーム製剤か知られている( rkuta、 K、 e
t al、、 Jpn、J、Cancer Res、、
78.1159〜1163、1987および特開昭63
−290824)。また、gp120に親和性を有する
分子に毒素あるいは毒素フラグメン]・を直接させた薬
剤も知られている。As a drug or composition that selectively damages HIV-infected cells, a liposome preparation encapsulating diphtheria toxin fragment A is known (r.
tal,, Jpn, J, Cancer Res,,
78.1159-1163, 1987 and JP-A-63
-290824). Furthermore, drugs are also known in which a toxin or a toxin fragment] is directly attached to a molecule that has affinity for gp120.
このようなものとしては、可溶性のCD4分子にヒマ種
子毒素のA鎖を結合させたCD4−ヒマ種子毒素A鎖結
合物(Ti11.M、A、 et al、、 5cie
nce。As such, a CD4-castor seed toxin A chain conjugate (Ti11.M, A, et al., 5cie
nce.
242、1166〜1168.1988)、 CD 4
とンユートモナス外毒素へとの組換え融合蛋白質(Ch
audhary、 V、に、 eIal、、 Natu
re、335.369〜372.1988)およびgp
120に対するモノクローナル抗体にヒマ種子毒素A鎖
を結合させたイムノトキシン(Matsush目a、
S、 et al、、 Anti−gp120 i
mmunotoxin against HI V
1nfected Ce1ls、、 Four
th International Confere
nece on A I D S、 Stockhol
m、 Bo。242, 1166-1168.1988), CD 4
Recombinant fusion protein (Ch
audhary, V., eIal,, Natu
re, 335.369-372.1988) and gp
An immunotoxin (Matsulacea order A,
S. et al., Anti-gp120 i
mmunotoxin against HIV
1nfected Ce1ls,, Four
th International Conference
nece on AIDS, Stockhol
m, Bo.
k 1. p、234.+988)などがあげられる。k1. p, 234. +988).
HIV感染細胞の細胞股上にはHIVの外被糖蛋白質g
p】20か発現されており、CD4あるいは抗gp12
0モノクローナル抗体に毒素または毒素フラグメントを
結合させたこれらの薬剤は、HIV感染細胞に選択的に
結合して殺傷するものと考えられている。HIV coat glycoprotein g is present on the cell crotch of HIV-infected cells.
p]20 is expressed, CD4 or anti-gp12
These drugs, which are conjugated toxins or toxin fragments to 0 monoclonal antibodies, are thought to selectively bind to and kill HIV-infected cells.
以上、上記したようにHIV感染細胞を選択的に殺傷す
る方法かいくつか明らかにされているものの、いずれも
安全性および生体内での安定性の点から、必ずしも満足
てきるものではない。As mentioned above, although several methods for selectively killing HIV-infected cells have been revealed, none of them are necessarily satisfactory in terms of safety and in-vivo stability.
一方、最近、脂質成分のみから成るある組成のリポソー
ムかウィルス感染細胞を選択的に殺傷することか明らか
にされた(特願平1−322217)。On the other hand, it has recently been revealed that liposomes with a certain composition consisting only of lipid components selectively kill virus-infected cells (Japanese Patent Application No. 1-322217).
該発明のリポソームは、相転移温度か37℃未満である
フォスファチジルコリンと酸性リン脂質とコレステロー
ルとを最も好ましくは5二l:2(モル比)の組成とす
るリポソームであって、HIV感染細胞を選択的に殺傷
することか示されている。しかしなから、選択性をさら
に高めることかできればより効果的である。The liposome of the present invention is a liposome having a composition of phosphatidylcholine whose phase transition temperature is less than 37°C, acidic phospholipid, and cholesterol, most preferably in a molar ratio of 52 l:2, and which is suitable for HIV infection. It has been shown to selectively kill cells. However, it would be more effective if the selectivity could be further increased.
問題解決の手段
本発明者らは、安全にHIV感染細胞を選択的に殺傷す
る方法を種々検討して、転移温度か37℃未満のフォス
ファチジルコリンと酸性リン脂質とコレステロールとか
ら成るリポソームに、ヒトCD4の68番目から94番
目までのアミノ酸配列と同じアミノ酸配列よりなり、抗
HIV活性をイfするペプチドまたはその修飾ペプチド
を導入したリポソームか)(IV感染細胞に対し強い殺
傷活性と高い選択性を示すことを見出し、さらに検討を
重ねて本発明を完成するに至った。以下本発明について
詳細に説明する。Means of Solving the Problem The present inventors investigated various ways to safely and selectively kill HIV-infected cells, and developed a liposome consisting of phosphatidylcholine, acidic phospholipid, and cholesterol at a transition temperature of less than 37°C. , a liposome containing a peptide or its modified peptide that has the same amino acid sequence as the 68th to 94th amino acid sequence of human CD4 and exhibits anti-HIV activity) (strong killing activity and high selectivity against IV-infected cells) After further study, the present invention was completed.The present invention will be described in detail below.
ヒトCD 4 (Asn−Phe−Pro−Leu−
11e−11e−Lys−Asn−Leu−Lys−1
1e−G I u−Asp−Ser−As p−Thr
−Tyr−r l e−Cys−Glu−Val−Gl
u−Asp−Gln−Lys−Glu−Glu以下、単
にCD4と略す)は分子量約6万ダルトンの糖蛋白質で
、その1次構造は公知である( Maddon、 P、
J 。Human CD4 (Asn-Phe-Pro-Leu-
11e-11e-Lys-Asn-Leu-Lys-1
1e-G I u-Asp-Ser-As p-Thr
-Tyr-r l e-Cys-Glu-Val-Gl
u-Asp-Gln-Lys-Glu-Glu (hereinafter simply abbreviated as CD4) is a glycoprotein with a molecular weight of approximately 60,000 Daltons, and its primary structure is known (Maddon, P.
J.
et al、、 Ce11.42.93〜104.19
85) 。CD 4分子のうちHIV外被蛋白質を相互
作用を示す領域の特定が合成ペプチドを用いた検討て行
われ、N末端より68番目のアミノ酸から94番目のア
ミノ酸までの27個のアミノ酸から成る領域か関与して
いる可能性か示唆されている(Hayashi、Y、
etal、、Arch、 Virol、、 105.1
29〜135.1989;大本浩司ら、エイズ研究会第
3回学術集会(松江)、抄録集p、79.1989;お
よび大木洗用ら、第37回日本ウィルス学会総会((大
阪)演説抄録p、 83.1989)。et al, Ce11.42.93-104.19
85). A synthetic peptide was used to identify the region of the CD4 molecule that interacts with the HIV coat protein, and a region consisting of 27 amino acids from the 68th amino acid to the 94th amino acid from the N-terminus was identified. It has been suggested that they may be involved (Hayashi, Y.
etal, Arch, Virol, 105.1
29-135.1989; Koji Omoto et al., 3rd Academic Meeting of the AIDS Research Society (Matsue), Abstracts p. p, 83.1989).
また、この68番目から94番目までのアミノ酸鎖と同
し配列を有するベプチ1−化合物(以下、CD4 (6
8〜94)、!:略す)か、HIvの感染を阻止する活
性を有していることか明らかにされている(特願平1−
172053 ’)。CD4(68〜94)ペプチド
は、N末端より19番目のアミノ酸残基としてシスティ
ン残基を有するか、このシスティン残基をヘンシル基(
Bzl)で修飾したもの(以下、CD4 (68〜94
’) Bzl と略す)、あるいはアセタミドメチル
基(Acm)で修飾したもの(以下、CD4 (68〜
94) Acmと略す)についてもHIV感染を阻止す
る活性を有していることが明らかにされている(特願平
1−172053)。本発明において1よ、ヒトCD4
の68番目から94番目までのアミノ酸配列と同じアミ
ノ酸配列より成り、抗HIV活性を有するペプチドまた
はその修飾ペプチドとして、上記したペプチドのいずれ
も用いることかできるが、特に好適なものとしてはCD
4(68〜94)ペプチド、CD4(68〜94)Bz
lペプチド、CD4 (68〜94) Acmペプチド
をあげることかてきる。 CD4(68〜94)ペプチ
ド、CD4 (68〜94) Bzlペプチド、および
CD4 (68〜94) Acmペプチドは特願平1−
172053に記載されている方法で調製することかで
きる。該調製方法は、CD4分子をコードしているDN
A塩基配列(Maddon、 P、 J、 et al
、、 Ce1l、42.93〜104.1985)によ
り決定されているアミノ酸配列に基づき、CD4分子の
部分ペプチドの合成を行うものである。合成にはFIT
locアミノ酸を使用した固相合成法(Sheppar
d、 R,C。In addition, Vepti-1-compounds (hereinafter referred to as CD4 (6
8-94),! (abbreviated)), and has been shown to have the activity of inhibiting HIV infection (Patent Application No.
172053'). CD4 (68-94) peptide has a cysteine residue as the 19th amino acid residue from the N-terminus, or this cysteine residue is replaced with a Hensyl group (
Bzl) (hereinafter referred to as CD4 (68-94
') Bzl), or those modified with an acetamidomethyl group (Acm) (hereinafter referred to as CD4 (68-
94) Acm) has also been shown to have the activity of inhibiting HIV infection (Japanese Patent Application No. 1-172053). In the present invention, 1, human CD4
Although any of the above-mentioned peptides can be used as the peptide or its modified peptide, which has the same amino acid sequence as the 68th to 94th amino acid sequence and has anti-HIV activity, CD
4(68-94) peptide, CD4(68-94)Bz
1 peptide, CD4 (68-94) Acm peptide. CD4 (68-94) peptide, CD4 (68-94) Bzl peptide, and CD4 (68-94) Acm peptide are
172053. The preparation method involves the preparation of a DN encoding the CD4 molecule.
A base sequence (Maddon, P, J, et al
, Ce1l, 42.93-104.1985) to synthesize a partial peptide of the CD4 molecule. FIT for synthesis
Solid-phase synthesis method using loc amino acids (Sheppar
d, R, C.
et at、、 J、 Chem、 Soc、
、 Chem、 Commun、、 165〜1
66、1985)を用いることかできる。即ち、まずそ
れぞれのペプチドのC末端に相当する9−フルオレニル
メチルオキシカルボニル(Fmoc)アミノ酸ペンタフ
ルオロフェニル(Pfp)エステルをジメチルホルムア
ミド中で4−ジメチルアミノピリジン存在下、p−アル
コキシベンジルアルコール樹脂に結合させ、次いで結合
すべき別のFmoc−アミノ酸Pfpエステルを縮合反
応により結合させる。各アミノ酸のカップリング反応終
了後、ペプチドの結合した樹脂を20%ピペリジン・ジ
メチルホルムアミド混液て処理してN末端Fmoc基を
除去し、次いてn−クレゾール存在下、室温にてトリフ
ルオロ酢酸(T F A)−チオアニソールを作用させ
て脱保護をすると同時に、目的とするペプチドを樹脂よ
り回収する。et at,, J, Chem, Soc,
, Chem, Commun,, 165-1
66, 1985). That is, first, 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid pentafluorophenyl (Pfp) ester corresponding to the C-terminus of each peptide was added to p-alkoxybenzyl alcohol resin in dimethylformamide in the presence of 4-dimethylaminopyridine. and then another Fmoc-amino acid Pfp ester to be bound is bound by a condensation reaction. After the coupling reaction of each amino acid is completed, the resin bound to the peptide is treated with a 20% piperidine/dimethylformamide mixture to remove the N-terminal Fmoc group, and then trifluoroacetic acid (T FA)-Thioanisole is applied to perform deprotection, and at the same time, the target peptide is recovered from the resin.
また更に、合成の第2の方法として、第三ブチルオキシ
力ルホニル(Boc)−アミノ酸を使用した固相合成法
(Merrifield、 R,B、、 J、 Am、
Chem、 Soc、、85.2149〜2154.
1963)を用いることもてきる。Furthermore, as a second method of synthesis, a solid phase synthesis method using tert-butyloxysulfonyl (Boc)-amino acids (Merrifield, R.B., J. Am.
Chem, Soc, 85.2149-2154.
1963) can also be used.
これらの方法において、アミノ酸側鎖を修飾する場合は
修飾アミノ酸の形で、脱保護条件に安定なものをペプチ
ド中に導入する。In these methods, when modifying an amino acid side chain, a modified amino acid that is stable under deprotection conditions is introduced into the peptide.
また、他の化学合成法や、当該ペプチドに対応するDN
Aを得て、これを適当なベクターに挿入し、動物細胞や
微生物で発現させて目的とするペプチドを得ても良く、
また、でき上ったペプチドを得た後に、適当な化学修飾
を加えても良い。In addition, other chemical synthesis methods and DN corresponding to the peptide may also be used.
A may be obtained, inserted into an appropriate vector, and expressed in animal cells or microorganisms to obtain the desired peptide.
Further, after obtaining the completed peptide, appropriate chemical modification may be added.
CD4(68〜94)ペプチド、CD4 (68〜9
4 ) Bzlペプチド、あるいはCD4(68〜94
) Acmペプチドをリポソームに結合させる方法と
しては、抗体結合リポソームの製造で公知となった方法
(たとえばMethods in Enzymolog
y、 149.111〜119.1987に記載されて
いる方法)を用いることかてきる。該製造においては、
チオール基を導入されたペプチドと、チオール基と反応
し得る結合基を有するリン脂質とを反応させることによ
り、ペプチド−リン脂質結合物を調製し、このペプチド
−リン脂質結合物にリポソーム膜成分として必要な残り
の脂質(相転移温度か37℃未満のフォスファチジルコ
リンと酸性リン脂質とコレステロール)を加えてリポソ
ームを構成させる。CD4 (68-94) peptide, CD4 (68-9
4) Bzl peptide or CD4 (68-94
) Methods for binding Acm peptides to liposomes include methods known for producing antibody-bound liposomes (for example, Methods in Enzymolog).
y, 149.111 to 119.1987). In the production,
A peptide-phospholipid conjugate is prepared by reacting a peptide into which a thiol group has been introduced with a phospholipid having a bonding group that can react with the thiol group, and the peptide-phospholipid conjugate is added to the peptide-phospholipid conjugate as a liposome membrane component. The remaining necessary lipids (phosphatidylcholine, acidic phospholipids, and cholesterol with a phase transition temperature below 37°C) are added to form liposomes.
該製造の別の方法としては、チオール基と反応し得る結
合基を有するリン脂質と相転移温度か37℃未満のフォ
スファチジルコリンと酸性リン脂質とコレステロールと
を膜成分としてリポソームを調製しておき、このものと
、チオール基を導入されたペプチドとを反応、結合させ
ることにより、本発明のリポソームを製造することもて
きる。Another method for producing the liposome is to prepare a liposome using a phospholipid having a bonding group capable of reacting with a thiol group, a phosphatidylcholine having a phase transition temperature of less than 37°C, an acidic phospholipid, and cholesterol as membrane components. The liposome of the present invention can also be produced by reacting and bonding this with a peptide into which a thiol group has been introduced.
ペプチドにチオール基を導入する方法としてはN−サク
シニミンルビリジルシチオプロビオネート(N−suc
cinimidyl pyridyldithiop
ropionate)(SPDP)等の二官能性の蛋白
質修飾剤をペプチドに結合させ、これを還元する方法か
あげられる。別の方法としては、CD4 (68−94
)ペプチドの場合、ペプチド中のシスティン残基由来の
チオール基を利用することも可能である。As a method for introducing a thiol group into a peptide, N-succiniminerubilidylcythioprobionate (N-suc
cinimidyl pyridyl dithiop
One example is a method in which a bifunctional protein modifier such as ropionate (SPDP) is bonded to a peptide and this is reduced. Alternatively, CD4 (68-94
) In the case of peptides, it is also possible to utilize thiol groups derived from cysteine residues in the peptides.
一方、チオール基と反応し得る結合基を有するリン脂質
としては、フオスファチシルエタノールアミン(PE)
と5PDPまたはN−サクソニミジル 4−(パラマレ
イミドフェニル)ブチレート (N−succinim
idyl 4−(p−maleimidopheny
l)butyrate)等とを反応させて得られるピリ
シルジチオプロビオニルフォスファチジルエタノールア
ミン(pyr idy Id i th 1oprop
1ony Iphospha t idy le t
hano lamine (PDP−PE))または
4−(パラマレイミドフェニル)プチリルフオスファチ
ジルエタノールアミン(4−(p−maleimido
phenyl)butyrylphosphatidy
lethanolamine) (M P B −P
E ) )等をあげることかできる。上記で用いるフ
オスファチンルエタノールアミンとしては、動物起源の
フオスファチンルエタノールアミン、およびアンル基を
ラウロイル基、ミリストイル基、バルミトイル基、ステ
アロイル基、オレオイル基なとに置換しtこフオスファ
チシルエタノールアミンかあげられるか、特に好ましく
は動物起源のフオスファチソルエタノールアミンかあげ
られる。On the other hand, as a phospholipid having a bonding group that can react with a thiol group, phosphaticylethanolamine (PE)
and 5PDP or N-saxonimidyl 4-(paramaleimidophenyl)butyrate (N-succinim
idyl 4-(p-maleimidopheny
pyridy Id i th 1oprop
1ony Iphospha t idy le t
monolamine (PDP-PE)) or 4-(p-maleimidophenyl)butyrylphosphatidylethanolamine (4-(p-maleimido
phenyl)butyrylphosphotidy
lethanolamine) (M P B -P
E)) etc. The phosphatin ethanolamine used above includes animal-derived phosphatin ethanolamine, and phosphatin ethanolamine in which the anlu group is replaced with a lauroyl group, myristoyl group, valmitoyl group, stearoyl group, oleoyl group, etc. Mention may be made of silethanolamine, particularly preferably phosphatisolethanolamine of animal origin.
リポソーム膜に結合させるペプチドの数は、リポソーム
1個に理論的には、ペプチドか約8000個まで可能で
あるか、HIV感染細胞段傷剤としては、40〜120
個か望ましい。The number of peptides that can be bound to the liposome membrane is theoretically up to about 8,000 per liposome, or 40 to 120 as a wounding agent for HIV-infected cells.
Individuals or desirable.
本発明のリポソームは、上記のようなヒトCD4の68
番目から94番目までのアミノ察配列とと同しアミノ酸
配列より成り、抗HIV活性を有するペプチドまたはそ
の修飾ペプチドを結合したリン脂質と相転移温度か37
℃未満のフォスファチンルコリンと酸性リン脂質とコレ
ステロールとを膜成分とするリポソームである。相転移
温度はゲル−液晶相転移温度のことをさす。リン脂質の
二重膜は相転移温度以下では固い膜となるゲル状態て、
相転移温度以上ではやわらかい膜となる液晶状態をとる
。リン脂質の脂肪酸の種類により異なった相転移温度を
示す。The liposome of the present invention has the above-mentioned human CD4 68
It consists of the same amino acid sequence as the amino acid sequence from position to 94, and has a phase transition temperature of 37
It is a liposome whose membrane components are phosphatine lucholine, acidic phospholipid, and cholesterol at a temperature of less than ℃. The phase transition temperature refers to the gel-liquid crystal phase transition temperature. The phospholipid bilayer becomes a solid gel below the phase transition temperature.
Above the phase transition temperature, it assumes a liquid crystal state with a soft film. Phospholipids exhibit different phase transition temperatures depending on the type of fatty acid.
リポソームは内部に水層を有する脂質2重層から成る閉
鎖小胞体であり、その脂質2分子膜構造は生体膜に極め
て近似していることか知られている。リポソームは通常
の細胞膜中に存在する脂質なと天然の材料から調製する
ことかできるため、生体との適合性か高く、毒性や抗原
性をほとんと示さないという利点を有している。リポソ
ームは形態的にはマルチラメラベシクル(以下、MLV
と略する)とユニラメラベシクル(以下、ULVと略す
る)に分類され、ULVはさらにラージユニラメラベシ
クル(以下、LUVと略する)とスモールユニラメラベ
シクル(以下、SUVと略する)に分類されるか、本発
明のリポソームはこれらのいずれの形態でも用いること
かてきる。Liposomes are closed vesicles consisting of a lipid bilayer with an internal water layer, and it is known that the lipid bilayer membrane structure is extremely similar to biological membranes. Since liposomes can be prepared from natural materials such as lipids that are present in normal cell membranes, they have the advantage of being highly compatible with living organisms and showing almost no toxicity or antigenicity. Liposomes are morphologically multilamellar vesicles (hereinafter referred to as MLVs).
ULVs are further classified into large unilamellar vesicles (hereinafter abbreviated as LUV) and small unilamellar vesicles (hereinafter abbreviated as SUV). Alternatively, the liposomes of the present invention can be used in any of these forms.
本発明のリポソームの製造に用いられる相転移温度か3
7℃未満のフすスファチジルコリンとしては、動植物起
源のレシチン(たとえば卵黄レシチン、大豆レシチン)
や、アシル基をラウロイル基、ミリストイル基、オレオ
イル基などに置換しtこフォスファチジルコリンかあげ
られるか、特に好ましくはシミリストイルフォスファチ
ジルコリン(DMPC,相転移温度23℃)かあげられ
る。The phase transition temperature used in the production of the liposomes of the present invention is 3.
Fusphatidylcholine with a temperature below 7°C includes lecithins of animal and plant origin (e.g. egg yolk lecithin, soybean lecithin).
or phosphatidylcholine by substituting the acyl group with a lauroyl group, myristoyl group, oleoyl group, etc., and particularly preferably simiristoylphosphatidylcholine (DMPC, phase transition temperature 23°C). .
また酸性リン脂質としては、動物起源のフォスファチジ
ルセリン、フォスファチジルグリセロール、フォスファ
チジルイノシトール、フォスファチシン酸、および半合
成によりアシル基をラウロイル基、ミリストイル基、バ
ルミトイル基、ステアロイル基、オレオイル基などとし
たフォスファチンルセリン、フォスファチジルグリセロ
ール、フオスファチジルイノシトール、フォスファチジ
ン酸なとかあけられるか、特に好ましくは動物起源のフ
オスファチジルセリンがあげられる。In addition, acidic phospholipids include phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphaticic acid derived from animal sources, as well as semisynthetic acyl groups such as lauroyl, myristoyl, valmitoyl, stearoyl, and oleyl groups. Examples include oil-based phosphatine lucerine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid, and particularly preferred are phosphatidylserine of animal origin.
相転移温度か37℃未満のフォスファチジルコリンと酸
性リン脂質とコレステロールとを膜成分とするリポソー
ムがウィルス感染細胞を選択的に12411することが
知られている(特願平1−322217)か、このリポ
ソーム膜組成に、ヒトCD4の688番目ら94番目ま
でのアミノ酸配列と同しアミノ酸配列より成り、抗HI
V活性を有するペプチドまたはその修飾ペプチドを結合
したリンIl’f’f質を0.1〜10モル%加えた本
発明のリポソームは、HIV感染細胞を選択的に殺傷す
る活性か非常に優れたものである。It is known that liposomes whose membrane components are phosphatidylcholine, acidic phospholipid, and cholesterol with a phase transition temperature of less than 37°C selectively target virus-infected cells (Patent Application No. 322217/1999). This liposome membrane composition consists of the same amino acid sequence as the amino acid sequence from 688th to 94th of human CD4, and anti-HI
The liposome of the present invention to which 0.1 to 10 mol% of phosphoIl'f'f substance bound to a peptide having V activity or a modified peptide thereof is added has an extremely excellent activity of selectively killing HIV-infected cells. It is something.
細胞にリポソームか接触した場合に起こる相互作用とし
て、細胞とリポソームとの間て膜構成成分の交換、移行
などが起こることか知られている(Mashino、に
、et at、、 J、Biochem、、 94.8
21〜831゜]983およびHuestjs、W、H
,et al、、 J、Biol、Chem、。It is known that interactions that occur when liposomes come into contact with cells include exchange and transfer of membrane components between the cells and liposomes (Mashino et al., J. Biochem. 94.8
21-831°] 983 and Huestjs, W, H
, et al., J. Biol.Chem.
261、16274〜16278.1986)。261, 16274-16278.1986).
一方、HTVか持続感染した細胞では、細胞膜の組成か
ウィルスの外被組成に対応して感染前とは異なっている
ことか知られている。最近の報告によれば、感染細胞の
細胞膜表面にはウィルスの外被糖蛋白質(gp120.
gp41)か産生されているだけでなく、細胞膜の脂質
組成そのものもHIVの外被組成に対応してリン脂質の
割合が減少し、コレステロールの比率が増加するなとの
変化を生していることか示されている( Lynn、
W、 S。On the other hand, it is known that cells persistently infected with HTV differ from those before infection, depending on the composition of the cell membrane or the composition of the viral coat. According to recent reports, the viral coat glycoprotein (gp120.
gp41) is produced, but also the lipid composition of the cell membrane itself changes in response to the HIV envelope composition, with the proportion of phospholipids decreasing and the proportion of cholesterol increasing. (Lynn,
W,S.
et al、、 Virology、+63.43〜5
1.1988)。従って、ヒl−CD 4の688番目
ら94番目までのアミノ酸配列と同しアミノ酸配列より
成り、抗HIV活性を有するペプチドまたはその修飾ペ
プチドを導入した本発明のリポソームは、HIV感染細
胞に選択的に接触し、膜構成成分の交換、移行などを介
して細胞に損傷を与え、細胞の正常な機能を損わせ死滅
に至らしめるものと推測される。et al., Virology, +63.43~5
1.1988). Therefore, the liposome of the present invention, which has the same amino acid sequence as the 688th to 94th amino acid sequence of human CD4 and into which a peptide having anti-HIV activity or a modified peptide thereof is introduced, is selective for HIV-infected cells. It is presumed that these substances damage cells through the exchange and migration of membrane components, impairing their normal functions and leading to their death.
上記した本発明のリポソームの製造に必要なリポソーム
膜構成成分から、リポソームを製造する方法自体は公知
の技術が用いられる。該製造法としては、
(1) リポソーム膜構成成分を溶媒中で混合した後
、溶媒を蒸発除去して薄膜とし、これに緩衝液を加えて
十分に水和させ、MLVのリポソームを得る方法、
(2) MLVをメンブランフィルタ−により濾過する
ことにより粒径をそろえたリポソームを得る方法、
(31MLVを超音波処理あるいは高圧噴射乳化機によ
る処理によりSUVのリポソームを得る方法、なとかあ
げられる。本発明のリポソームの製造にはこれらのいず
れの方法も用いることかできる。A known technique can be used to manufacture liposomes from the liposome membrane components necessary for manufacturing the liposomes of the present invention described above. The manufacturing method includes: (1) After mixing the liposome membrane components in a solvent, the solvent is removed by evaporation to form a thin film, and a buffer is added to the thin film to sufficiently hydrate it to obtain MLV liposomes; (2) A method for obtaining liposomes with uniform particle size by filtering MLV with a membrane filter; (a method for obtaining liposomes of SUV by processing 31 MLV with ultrasonication or a high-pressure injection emulsifier). Any of these methods can be used to produce the liposomes of the invention.
上記(11〜(3)項で用いられる緩衝液としては、リ
ポソームの製造に用いることのできるものであればいず
れでもよく、その例としてはリン酸緩衝液、クエン酸緩
衝液、乳酸緩衝液、酢酸緩衝液なとかあけられる。緩衝
液のpHとしては6〜8の中性付近のpHが好ましく、
また緩衝液の濃度としては5〜50mM程度か好ましい
。上記緩衝液はまた食塩、ショ糖なとの添加により等張
出したものを用いてもよい。また、緩衝液に代えて生理
食塩水を用いてもよい。The buffer used in the above (11 to (3)) may be any buffer as long as it can be used for producing liposomes, examples of which include phosphate buffer, citrate buffer, lactic acid buffer, An acetate buffer solution can be used.The pH of the buffer solution is preferably around neutral pH of 6 to 8.
Further, the concentration of the buffer solution is preferably about 5 to 50 mM. The above buffer may also be made isotonic by adding sodium chloride, sucrose, etc. Furthermore, physiological saline may be used instead of the buffer solution.
本発明のリポソームは、内部水層に薬物を保持させるこ
ともできる。このような薬物としては、細胞の蛋白合成
を阻害する毒素(たとえばジフテリア毒素フラグメント
A)や、AZTなとの抗HIV剤があげられる。HIV
感染細胞のあるものは貧食作用を有する(たとえばHI
Vに感染したマクロファージ)ので、本発明のリポソー
ムの内部水層に細胞の蛋白合成を阻害する毒素を保持さ
せたリポソームは、そのような細胞の殺傷に効果的と考
えられる。The liposome of the present invention can also retain a drug in its internal aqueous layer. Such drugs include toxins that inhibit cellular protein synthesis (eg, diphtheria toxin fragment A) and anti-HIV agents such as AZT. HIV
Some infected cells are phagocytic (e.g. HI
Therefore, the liposome of the present invention in which a toxin that inhibits cellular protein synthesis is retained in the internal aqueous layer of the liposome is considered to be effective in killing such cells.
本発明のリポソームはHIV感染細胞を選択的に殺傷、
破壊することかできるのでAIDSの治療剤または発症
予防剤として用いられる。本発明のリポソームは脂質成
分およびヒトのCD4と同しアミノ酸配列のCD4ペプ
チドから成るもので、安全に投与することかできる。A
IDS患者、AIDS関連症候群(ARC)患者、ある
いは無症候性のHIV感染者への本発明のリポソームの
投与は、適宜の量を注射または点滴により静脈内投与と
して行われる。HIV感染細胞は主として血中に存在す
るので、これらの細胞に本発明のリポソームを作用させ
ることは容易であると考えられる。このように本発明の
リポソームは、AIDSの治療あるいは発症予防におい
て優れた効果を発揮する。The liposome of the present invention selectively kills HIV-infected cells,
Since it can be destroyed, it is used as a therapeutic agent or preventive agent for AIDS. The liposome of the present invention consists of a lipid component and a CD4 peptide having the same amino acid sequence as human CD4, and can be safely administered. A
The liposome of the present invention is administered to IDS patients, AIDS-related syndrome (ARC) patients, or asymptomatic HIV-infected individuals by intravenous administration of an appropriate amount by injection or infusion. Since HIV-infected cells mainly exist in the blood, it is considered easy to cause the liposomes of the present invention to act on these cells. As described above, the liposome of the present invention exhibits excellent effects in treating or preventing the onset of AIDS.
発明の効果
本発明のリポソームはHIV感染細胞を選択的に殺傷す
ることにより、AIDSの治療あるいは発症予防におい
て優れた効果を発揮する。また非感染正常細胞に対して
は毒性を示さないので長期に渡って安全に投与しうる優
れたAIDS治療剤および発症予防剤である。Effects of the Invention The liposome of the present invention exhibits excellent effects in treating or preventing the onset of AIDS by selectively killing HIV-infected cells. In addition, it is not toxic to non-infected normal cells, so it is an excellent therapeutic agent and preventive agent for AIDS that can be safely administered over a long period of time.
次に実施例をあげて本発明をさらに詳細に説明する。な
お本発明は以下の実施例に限定されるものではない。Next, the present invention will be explained in more detail with reference to Examples. Note that the present invention is not limited to the following examples.
実施例
実施例1.CD4 (68〜94)ペプチドの調製Fm
oc−Glu(OBzl)−0Pfp(Immol)を
、触媒としてDMAP (4−ジメチルアミノピリジン
)(0,2mmoりを用い、ジメチルホルムアミド(D
MF)中、p−alkoxy benzyl alco
hol resin (0,2mmol)(0,35
meq OH/g、ポリスチレン−1%ジヒニルベンゼ
ンコボリマー (Polystyrene I%Div
inyl benzene Copolymer) 、
国産化学制)上にエステル結合により導入した。Examples Example 1. Preparation of CD4 (68-94) peptide Fm
oc-Glu(OBzl)-0Pfp (Immol) was reacted with dimethylformamide (D
MF), p-alkoxy benzyl alco
hol resin (0,2 mmol) (0,35
meq OH/g, Polystyrene I%Div
inyl benzene Copolymer),
(Domestic chemical system) was introduced through an ester bond.
使用したFmoc−アミノ酸誘導体(ミリジエン製)の
側鎖の保護は、Asp (OBu’ )、Glu(OB
u’ )、Thr(Bu’ ) 、5er(Bu’
) 、Tyr(Bu’ ) 、Lys(Bo
c)、Cys(Acm)で、ペプチド鎖伸長時の各縮合
反応はすへてprp(ペンタフロオルフェニル)活性エ
ステル体(2,5eq)を用い、触媒としてHOBT(
+−ハイトロキンベンゾトリアゾール) 0.2 mm
olの存在下DMF中で行った。それぞれのpmoc基
の除去は、20%ピペリジンのDMF溶液を用いた。The side chain of the Fmoc-amino acid derivative (manufactured by Myridien) used was protected by Asp (OBu') and Glu (OBu').
u'), Thr(Bu'), 5er(Bu'
), Tyr(Bu'), Lys(Bo
c), Cys (Acm), each condensation reaction during peptide chain elongation uses prp (pentafluorophenyl) active ester (2,5 eq), and HOBT (
+-hytroquine benzotriazole) 0.2 mm
The test was carried out in DMF in the presence of ol. Removal of each pmoc group used a 20% piperidine solution in DMF.
更にそれぞれのカップリング反応は、ニンヒドリンでモ
ニターした。Furthermore, each coupling reaction was monitored with ninhydrin.
すへてのカップリング反応終了後、N末端に残ったFm
oc基を20%ピペリジン/DMFにより除去し、N末
端アミノ基をフリーとし、その後、すへての他の保護基
の除去と、樹脂がらペブチ1〜を切り出すために、TF
A−チオアニソールでm−クレゾール存在下で室温3時
間処理し、グラスフィルターで濾過し、樹脂を除き、濾
液を室温で濃縮し、これにエーテルを加え粉末を得た。After the end of the coupling reaction, the Fm remaining at the N-terminus
The oc group was removed with 20% piperidine/DMF to free the N-terminal amino group, and then TF
The mixture was treated with A-thioanisole in the presence of m-cresol for 3 hours at room temperature, filtered through a glass filter, the resin was removed, the filtrate was concentrated at room temperature, and ether was added to obtain a powder.
それを、集め、適当な緩衝液に溶解し、5ephade
x G−25(ファルマシア社製)にかけ、0.5
N酢酸て溶出させ、脱塩、精製を行った。ゲル濾過後の
ペプチドは更にHPLCで精製した。It was collected, dissolved in a suitable buffer, and diluted with 5ephades.
x G-25 (manufactured by Pharmacia), 0.5
Desalting and purification were performed by elution with N acetic acid. The peptide after gel filtration was further purified by HPLC.
HPLCによる精製分取は、0.1%TFA中、アセト
ニトリル10−60%の濃度勾配てNucle。Purification by HPLC was carried out using a gradient of 10-60% acetonitrile in 0.1% TFA.
sil 100−5C18(4,OX150mm)カ
ラム(M ナーゲル社製)を流速1mt’/分て流し、
210.260nmにおける紫外吸収でモニターした。sil 100-5C18 (4, OX150mm) column (manufactured by M Nagel) at a flow rate of 1 mt'/min,
Monitored by ultraviolet absorption at 210.260 nm.
目的とするCD4(68〜94)ペプチドは保持時間1
6.8分て溶出した。得られたCD4 (68〜94)
ペプチドのアミノ酸分析の結果は、理論値と一致した。The target CD4 (68-94) peptide has a retention time of 1
It eluted after 6.8 minutes. Obtained CD4 (68-94)
The results of amino acid analysis of the peptide were consistent with the theoretical values.
実施例2.CD4 (68〜94)Bzlペプチドの調
製
Boc−Glu(OBzl)−フェニルアセタミドメチ
ル樹脂(0、5mmol 5cale、アプライド バ
イオ システムズ社製)をペプチドC末端アミノ酸であ
るグルタミン酸の出発原料として使用した。樹脂上にお
けるペプチド鎖の逐次延長合成には、アプライドバイオ
システムズ社製Model 430A(Program
versionl、 40)ペプチド自動合成機を使
用した。ペプチド鎖延長に使用したBocアミノ酸誘導
体(ペプチド研究断裂)の側鎖の保護は、Asp(Oc
Hex)、Glu(OcHex)、Thr(Bzl)、
5er(Bzl)、Tyr(Dr−Z) 、Lys (
CI −Z) 、 Cys(Bzl)で、ペプチド鎖の
延長時における各縮合反応はすへてDCC(ジシクロへ
キシルカルボジイミド)を用いてジクロロメタン−ジメ
チルホルムアミド混合液中で相当するBoc−アミノ酸
の対称無水物(2,0eq)を合成し、ジメチルホルム
アミド中で行った。それぞれのBoc基の除去は、50
%TFA()リフルオロ酢a!>−ジクロロメタン溶液
を用いた。Example 2. Preparation of CD4(68-94)Bzl peptide Boc-Glu(OBzl)-phenylacetamidomethyl resin (0.5 mmol 5cale, manufactured by Applied Biosystems) was used as a starting material for glutamic acid, the peptide C-terminal amino acid. Applied Biosystems Model 430A (Program
version 1, 40) An automatic peptide synthesizer was used. Protection of the side chain of the Boc amino acid derivative (peptide research cleavage) used for peptide chain elongation was performed using Asp(Oc
Hex), Glu (OcHex), Thr (Bzl),
5er (Bzl), Tyr (Dr-Z), Lys (
CI-Z), Cys(Bzl), each condensation reaction during the elongation of the peptide chain is performed using DCC (dicyclohexylcarbodiimide) in a dichloromethane-dimethylformamide mixture to form a symmetrical anhydride of the corresponding Boc-amino acid. (2.0 eq) was synthesized and carried out in dimethylformamide. Removal of each Boc group is 50
%TFA() Refluoro Vinegar a! >-dichloromethane solution was used.
全てのアミノ酸縮合反応終了後、システィン残基のBZ
Iを除く、N末端のBoc基並びにすべての保護基を氷
冷下10%アニソール/無水HF(フッ化水素)(−5
℃,30分)処理により除去すると共に、樹脂からペプ
チドの切り出しを行い、HFを減圧下に留去した後、エ
ーテルにて残渣を洗浄し、TFAにてペプチドを溶解し
、グラスフィルターで樹脂を除き、濾液を減圧濃縮し、
残渣に乾燥エーテルを加えて粉末を得た。得られた粗生
成物ペプチドは、分取用逆相HPLCにて実施例1と同
様に精製された。After completion of all amino acid condensation reactions, BZ of cysteine residue
The Boc group at the N-terminus and all protecting groups except I were removed with 10% anisole/anhydrous HF (hydrogen fluoride) (-5
℃, 30 minutes), the peptide was excised from the resin, the HF was distilled off under reduced pressure, the residue was washed with ether, the peptide was dissolved with TFA, and the resin was filtered with a glass filter. and concentrate the filtrate under reduced pressure.
Dry ether was added to the residue to obtain a powder. The obtained crude product peptide was purified in the same manner as in Example 1 using preparative reverse phase HPLC.
実施例3. CD4 (68〜94) Acmペプ
チドの調製
実施例2において、Cys(Bzl)の代わりにCys
(Acm)を用いて、その他の方法は実施例2と同し方
法でペプチドの合成と精製を行うことにより、CD4(
68〜94 ) Acmペプチドを得た。Example 3. Preparation of CD4 (68-94) Acm peptide In Example 2, Cys(Bzl) was replaced with Cys(Bzl).
(Acm) and the other methods were the same as in Example 2 to synthesize and purify the peptide.
68-94) Acm peptide was obtained.
CD4 (68〜94)ペプチド8■を2 mlの50
mMリン酸緩衝液(pH7)に溶解した。これに、エタ
ノールに溶解した60mM5PDPを172μl添加し
、密栓後室温にて2時間ゆるやかに攪拌を行った。次に
、この溶液にジチオスライトール16.7■を加え、再
び密栓して室温にて1.5時間ゆるやかに攪拌を行った
。次にこの溶液を、あらかじめ50mMリン酸緩衝液(
pH7)で平衡化した5ephadex G −15
(ファルマシア社製)16mlを充填したカラムの上端
に重層し、同じ緩衝液て溶出させることによりゲル濾過
Fit製を行った。ゲル濾過溶出液を逆相HPLCて分
析し、チオール基の導入されたCD4(68〜94)ペ
プチドのゲル濾過溶出画分を集め、7.2■の精製品を
得た。得られた精製品のチオール基の含有量を2.2′
−ジチオピリジンを用いる方法(Grassetti、
D、R,& Murray、J、F、Jr、、 Ar
ch、Biochem、Biophys 、+19.旧
〜49.1967)で定量した。CD4 (68〜9
4)ペプチド1分子当たり3.0個のチオール基を保持
していた。CD4 (68-94) Peptide 8■ 2 ml of 50
Dissolved in mM phosphate buffer (pH 7). To this, 172 μl of 60 mM 5PDP dissolved in ethanol was added, and after being tightly stoppered, the mixture was gently stirred at room temperature for 2 hours. Next, 16.7 μm of dithiothreitol was added to this solution, the solution was sealed again, and the solution was gently stirred at room temperature for 1.5 hours. Next, this solution was mixed in advance with 50mM phosphate buffer (
5ephadex G-15 equilibrated at pH 7)
(manufactured by Pharmacia) was layered on the top of a column filled with 16 ml, and gel filtration (Fit) was performed by eluating with the same buffer solution. The gel filtration eluate was analyzed by reverse phase HPLC, and the gel filtration eluate fraction of CD4 (68-94) peptide into which a thiol group had been introduced was collected to obtain a purified product of 7.2 μm. The content of thiol groups in the purified product obtained was 2.2'
-Method using dithiopyridine (Grassetti,
D. R. & Murray, J. F. Jr., Ar.
ch, Biochem, Biophys, +19. (old ~ 49.1967). CD4 (68~9
4) 3.0 thiol groups were retained per peptide molecule.
(逆相HPLCの条件)
カラム:YMC−Pack AM−303(5μ、
100人)、4.6x250s+m(山村化学制)溶
出液、A液、トリフルオロ酢酸(TFA)を0゜1%含
有する水
B液: TFAを0.1%含有するアセトニトリルグラ
ジェント:0%B→7o%B、35分間のリニアグラジ
ェント
流速:1−7分
温度・室温
検出+2101mにおける紫外吸収
このHPLC条件て、CD4(68〜94)ペプチドは
、23.6分に溶出し、チオール基の導入されたCD4
(68〜94)ペプチドは約28分に溶出した。(Reverse phase HPLC conditions) Column: YMC-Pack AM-303 (5μ,
100 people), 4.6 x 250s + m (Yamamura Chemical System) Eluent, A solution, water containing 0.1% trifluoroacetic acid (TFA) B solution: Acetonitrile gradient containing 0.1% TFA: 0% B → 7o% B, linear gradient flow rate for 35 minutes: 1-7 minutes Temperature/room temperature detection + UV absorption at 2101 m Under these HPLC conditions, the CD4 (68-94) peptide was eluted at 23.6 minutes, and the thiol group CD4 introduced
(68-94) The peptide eluted at approximately 28 min.
実施例4において、CD4(68〜94)ペプチド8■
の代わりにCD4(68〜94)Bzlペプチド8■を
用いて、その他の方法は実施例4と同じ方法で操作を行
うことにより、1分子当たり2゜0個のチオール基が導
入されたCD4(68〜94)Bzlペプチドを7.0
■得た。In Example 4, CD4(68-94) peptide 8■
By using CD4 (68-94) Bzl peptide 8■ instead of CD4 ( 68-94) Bzl peptide 7.0
■I got it.
実施例4において、CD4(68〜94)ペプチド8■
の代わりにCD4 (68〜94)Acmペプチド8■
を用いて、その他の方法は実施例4と同じ方法で操作を
行うことにより、1分子当たり21個のチオール基か導
入されたCD4(68〜94)Acmペプチドを7.1
■得た。In Example 4, CD4(68-94) peptide 8■
CD4 (68-94) Acm peptide 8 instead of
By using the same procedure as in Example 4, the CD4 (68-94) Acm peptide into which 21 thiol groups were introduced per molecule was converted to 7.1
■I got it.
実施例7
マーチンら(Marjin、F、J、 et al、、
Biochemistry、翻、 4229〜4238
.1981)の方法に従い、牛脳由来フオスファチジル
エタノールアミン(PE、 シグマ社製)50■とN
−サクシニミシルビリシルンチオプロビオネート(SP
DP、ピアス社製)31.3■から46.2■のビリジ
ルジチオブロピオニルフオスファチジルエタノールアミ
ン(FDP−PE)を調製した。このFDP’−PEの
1.9■をバイヤルにとり、実施例4て得られたチオー
ル基の導入されたCD4(68〜94)ペプチド0.9
■を含む50mM’Jン酸緩衝液(p H7,5)
l m(!を加え、さらにアセトニトリルI mlを加
えて全量を溶解させた。バイヤル中の空気を窒素で置換
した後密栓し、4℃て16時間ゆるやかに攪拌を行い、
CD4(68〜94)ペプチドをリン脂質に結合させた
。次に、この溶液をなすフラスコに移し、ロータリーエ
バポレーターを用いて液を蒸発留去した。これに、ジミ
リストイルフオスファチジルコリン(DMPC,日本油
脂製)17.7■と牛脳由来フォスファチジルセリン(
PS、 シグマ社製)4.0■とコレステロール(C
H,和光紬薬製)4.0■、およびクロロホルム−メタ
ノール(9:l、容量比)5mj’を加え、溶解混合し
た。これをロータリーエバポレーターを用いて溶媒を減
圧上蒸発させ、フラスコの内側に脂質の薄膜を形成させ
た。これに300mMショ糖含WIOmMリン酸緩衝液
(pH7,2)3.5 mlを加え、フラスコ内の空気
を窒素で置換した後、30℃に1時間保って十分に水和
させた後ポルテックスミキサーにより10分間攪拌して
白濁液を得た。得られた白濁液を孔径1μmのポリカー
ボネートのメンブランフィルタ−にュクリポアー社製)
に10回通した後、さらに孔径200nmのポリカーボ
ネートのメンブランフィルタ−にュクリポアー社製)に
10回通した。この液をBio −Gel A 50m
(バイオラッド社製)を充填したカラムを用いてゲル濾
過精製を行い、CD4(68〜94)ペプチドの結合し
た本発明のリポソームを得た。Example 7 Marjin et al.
Biochemistry, translated, 4229-4238
.. 1981), bovine brain-derived phosphatidylethanolamine (PE, manufactured by Sigma) 50■ and N
-Succinimicylbilicylunthioprobionate (SP
DP (manufactured by Pierce, Inc.) 31.3 to 46.2 ■ biridyldithiopropionylphosphatidylethanolamine (FDP-PE) was prepared. Take 1.9 μ of this FDP'-PE into a vial, and add 0.9 μl of the thiol group-introduced CD4 (68-94) peptide obtained in Example 4.
50mM'J acid buffer (pH 7.5) containing ■
1 ml of acetonitrile was added to dissolve the entire amount. After replacing the air in the vial with nitrogen, the vial was tightly capped, and gently stirred at 4°C for 16 hours.
CD4(68-94) peptide was conjugated to phospholipids. Next, this solution was transferred to a hollow flask, and the liquid was evaporated using a rotary evaporator. In addition to this, dimyristoylphosphatidylcholine (DMPC, manufactured by NOF) 17.7■ and bovine brain-derived phosphatidylserine (
PS, manufactured by Sigma) 4.0■ and cholesterol (C
4.0 ml of chloroform-methanol (9:l, volume ratio) were added, and dissolved and mixed. The solvent was evaporated under reduced pressure using a rotary evaporator to form a thin lipid film inside the flask. Add 3.5 ml of WIOmM phosphate buffer containing 300mM sucrose (pH 7.2) to this, replace the air in the flask with nitrogen, keep it at 30°C for 1 hour to fully hydrate, and then remove the portex. The mixture was stirred with a mixer for 10 minutes to obtain a cloudy white liquid. The resulting cloudy liquid was passed through a polycarbonate membrane filter with a pore size of 1 μm (manufactured by Ucrypore).
After passing through it 10 times, it was further passed through a polycarbonate membrane filter with a pore size of 200 nm (manufactured by Ucrypore) 10 times. This liquid was added to Bio-Gel A 50m
Purification by gel filtration was performed using a column packed with 100% peptide (manufactured by Bio-Rad) to obtain a liposome of the present invention bound to CD4 (68-94) peptide.
得られたリポソームの540nmの波長における濁度(
ODs+a)は0.8+、平均粒径は1730m(光散
乱法による測定)であった。また、リポソームに結合し
たCD4(68〜94)ペブチ1〜の量を逆相HPLC
により定量した。逆相HPLCの条件は実施例4に記載
した条件と同一である。Turbidity of the obtained liposome at a wavelength of 540 nm (
ODs+a) was 0.8+, and the average particle size was 1730 m (measured by light scattering method). In addition, the amount of CD4 (68-94) peptide bound to liposomes was determined by reverse-phase HPLC.
It was quantified by The conditions for reverse phase HPLC are the same as those described in Example 4.
リポソームサンプルは、オクチルグルコシ1〜を終濃度
50mMとなるように加えて可溶化した後、ノチオスラ
イトールを終濃度50mMとなるように加え室温に2時
間保って、リン脂質−CD4 (68〜94)ペブチl
”間のジスルフィド結合を還元して、チオール基か結合
したCD4(68〜94)ペプチドを遊離させた。この
液の100μlをHPLCに注入し、得られたチオール
基の結合したCD4(68〜94)ペプチドのピーク面
積からリポソーム液中の該ペプチド量を求めた。Liposome samples were solubilized by adding octylglucosi 1~ to a final concentration of 50mM, then adding notiothreitol to a final concentration of 50mM and keeping at room temperature for 2 hours. 94) Pebuti l
By reducing the disulfide bonds between ``CD4 (68-94) peptides bound with thiol groups, 100 μl of this solution was injected into HPLC, and the resulting peptide CD4 (68-94) bound with thiol groups was released. ) The amount of the peptide in the liposome solution was determined from the peak area of the peptide.
一方、リポソームサンプルを上記の方法で可溶化後、ジ
チオスライトールによる還元は行わずにHPLCに注入
した。これにより、リポソーム内部水層に遊離の形で封
入されている該ペプチドの量を求めた。これらの分析結
果から、リポソームに結合している該ペプチドの量を次
式により求めた。On the other hand, after solubilizing the liposome sample by the above method, it was injected into HPLC without reduction with dithiothreitol. Thereby, the amount of the peptide encapsulated in a free form in the liposome internal aqueous layer was determined. From these analysis results, the amount of the peptide bound to the liposome was determined using the following formula.
リポソームに結合したCD4(68〜94)ペプチドの
定量値は26.1μg / meてあった(リポソーム
1個当たりのCD4(68〜94)ペプチドの結合数は
!14)。The quantitative value of CD4 (68-94) peptide bound to liposomes was 26.1 μg/me (the number of bound CD4 (68-94) peptides per liposome was !14).
次に、得られたリポソームのHIV感染細胞に対する殺
傷活性をin VitrOて調へた。HIVに持続感染
したヒトT細胞株(MOLT−4/HTLV−IIIs
) (2,5x I O’cells)を0.67
mlの培地(10%牛脂児血清含有RPM l−16
40)に懸濁して細胞浮遊液を調製した。この細胞浮遊
液と、300mMショ糖含有10mMリン酸緩衝液(p
H7,2)で適宜希釈することにより所定のOD、、。Next, the killing activity of the obtained liposomes against HIV-infected cells was examined in vitro. Human T cell line persistently infected with HIV (MOLT-4/HTLV-IIIs
) (2,5x I O'cells) to 0.67
ml of medium (RPM l-16 containing 10% tallow serum)
40) to prepare a cell suspension. This cell suspension and 10mM phosphate buffer containing 300mM sucrose (p
By appropriately diluting with H7,2), the predetermined OD is obtained.
とじたリポソーム液0.33 mlとを混合し、37℃
て1時間ゆっくり振とうして細胞とリポソームを接触さ
せた。その後4 mlの培地(10%牛脂児血清含有R
PMI−1640)を加え炭酸ガスインキュヘータ−(
37℃,5%C02)中にて3日間培養した。Mix with 0.33 ml of the closed liposome solution and heat at 37°C.
The cells were shaken slowly for 1 hour to bring the cells and liposomes into contact. Then add 4 ml of medium (R containing 10% tallow serum)
PMI-1640) and carbon dioxide incubator (
The cells were cultured for 3 days at 37°C and 5% CO2.
38後細胞をトリパンブルー染色法により染色して生細
胞数を測定した。リポソーム液の代わりに生理緩衝食塩
水(PBS)を用いて同様に行った実験を対照実験とし
て、次の式により増殖阻止率を求めた。After 38 days, the cells were stained by trypan blue staining method to measure the number of viable cells. Using a similar experiment using physiological buffered saline (PBS) instead of the liposome solution as a control experiment, the growth inhibition rate was determined using the following formula.
増殖阻止率(%)=
PBS処理(対照)での3日後の生細胞数×100
HIVを感染させていない非感染のMOLT−4細胞に
ついても同様の実験を行い、HIV感染細胞と非感染細
胞に対する選択性を見た。結果を第1図に示す。図中、
・□・はHIV感染細胞(MOLT−4/HI LV−
IIl、)1m対する増殖阻止率を、○−Oは非感染細
胞(MOLT−4)に対する増殖阻止率を示す。本発明
のリポソームはHTV感染細胞に対し濃度依存的に強い
増殖抑制活性を示した。感染細胞の増殖を50%抑制す
るリポソームOD、、。は0. lであり、ペプチドを
結合させていない場合(比較例)と比へ、7分の1の低
濃度て同等の増殖抑制効果を示すことか明らかとなった
。一方、非感染細胞に対しては、最も濃いリポソーム濃
度でも全く増殖抑制を示さなかった。このことから本発
明のリポソームはHIV感染細胞に対し特異的に強い傷
害作用をおよほすことか明らかとなった。Growth inhibition rate (%) = Number of viable cells after 3 days with PBS treatment (control) x 100 A similar experiment was performed on uninfected MOLT-4 cells that were not infected with HIV, and We looked at the selectivity for The results are shown in Figure 1. In the figure,
・□・ is HIV-infected cells (MOLT-4/HI LV-
IIl,) shows the growth inhibition rate against 1m, and ○-O shows the growth inhibition rate against uninfected cells (MOLT-4). The liposome of the present invention showed strong concentration-dependent growth-inhibiting activity against HTV-infected cells. Liposome OD suppresses the proliferation of infected cells by 50%. is 0. 1, and it became clear that the same growth-inhibiting effect was exhibited at a concentration one-seventh lower than that in the case where no peptide was bound (comparative example). On the other hand, no growth inhibition was observed in uninfected cells even at the highest liposome concentration. This revealed that the liposome of the present invention exerts a strong damaging effect specifically on HIV-infected cells.
実施例8゜
実施例7において、チオール基の導入されたCD4(6
8〜94)ペプチド0.9■を用いる代わりに、実施例
5て得られたチオール基の導入されたCD4 (68〜
94)Bzlペプチド0.9■を用いて、その他の方法
は実施例7と同じ方法でリポソームの調製を行うことに
より、CD4(68〜94)Bzlペプチドの結合した
本発明のリポソームを得た。得られたリポソームのOD
、4゜は0.82、平均粒径は172nmてあった。ま
たリポソームに結合したCD4(68〜94)Bzlペ
プチドの定量値は9.3μg / meてあった(リポ
ソーム1個当たりのCD4 (68〜94) Bzl
ペプチドの結合数は41)。このリポソームのHIV感
染細胞に対する傷害効果を実施例7と同様の方法で調べ
た。結果を第2図に示す。CD4 (68〜94)Bz
lペプチドを結合した本発明のリポソームはOD 、、
。が0.1以上てほぼ完全にHIV感染細胞の増殖を抑
制した。また感染細胞の増殖を50%抑制するリポソー
ムOD、、。は0.04で、ペプチドを結合させていな
い場合(比較例)と比へ、17分の1の低濃度で同等の
増殖抑制効果を示すことか明らかとなった。一方、非感
染細胞に対しては、最も濃いリポソーム濃度でも全く増
殖を阻害しなかった。このことから、CD4(68〜9
4)BZIペプチドを結合した本発明のリポソームは、
HIV感染細胞に対し選択的に極めて強い殺傷活性を有
することか明らかとなった。Example 8 In Example 7, CD4 (6
8-94) Instead of using peptide 0.9■, CD4 with a thiol group introduced in Example 5 (68-
94) A liposome of the present invention bound to CD4(68-94)Bzl peptide was obtained by preparing a liposome in the same manner as in Example 7 except for using 0.9■Bzl peptide. OD of the obtained liposome
, 4° was 0.82, and the average particle size was 172 nm. In addition, the quantitative value of CD4 (68-94) Bzl peptide bound to liposomes was 9.3 μg/me (CD4 (68-94) Bzl per liposome).
The number of peptide bonds is 41). The damaging effect of this liposome on HIV-infected cells was examined in the same manner as in Example 7. The results are shown in Figure 2. CD4 (68-94)Bz
The liposome of the present invention bound to l peptide has an OD of
. was 0.1 or more, and the proliferation of HIV-infected cells was almost completely suppressed. Liposome OD also suppresses the proliferation of infected cells by 50%. was 0.04, which revealed that the same growth-inhibiting effect was exhibited at a concentration 1/17 as low as in the case where no peptide was bound (comparative example). On the other hand, even the highest concentration of liposomes did not inhibit the proliferation of uninfected cells at all. From this, CD4 (68-9
4) The liposome of the present invention bound to BZI peptide is
It has become clear that this drug has extremely strong selective killing activity against HIV-infected cells.
実施例9
実施例7において、チオール基の導入されたCD4(6
8〜94)ペプチド0.9■を用いる代わりに、実施例
6て得られたチオール基の導入されたCD4 (68〜
94)Acmペプチド0.9■を用いて、その他の方法
は実施例7と同じ方法でリポソームの調製を行うことに
より、CD4(68〜94 ) Acmペプチドの結合
した本発明のリポソームを得た。得られたリポソームの
OD、、。は0.94、平均粒径は176nmであった
。またリポソームに結合したCD4 (68〜94)A
cmペプチドの定量値は12.8μg / mlであっ
た(リポソーム1個当たりのCD4 (68〜94)
Acmペプチドの結合数は49)。このリポソームのH
IV感染細胞に対する傷害効果を実施例7と同様の方法
て調へた。結果を第3図に示す。CD4 (68〜94
)Acmペプチドを結合した本発明のリポソームはHI
V感染細胞に対し濃度依存的に強い増殖抑制活性を示し
た。感染細胞の増殖を50%抑制するリポソームOD、
4゜は0.055で、ペプチドを結合させていない場合
(比較例)と比べ、12分の1の低濃度で同等の増殖抑
制効果を示すことか明らかとなった。一方、非感染細胞
に対しては、最も濃いリポソーム濃度でもほとんど増殖
を抑制しなかった。このことから、CD4(68〜94
) Acmペプチドを結合した本発明のリポソームは
、HIV感染細胞に対し選択的に強い駁傷活性を示すこ
とか明らかとなった。Example 9 In Example 7, CD4 (6
8-94) Instead of using peptide 0.9■, CD4 with a thiol group introduced in Example 6 (68-
94) A liposome was prepared in the same manner as in Example 7 except for using 0.9 μ of Acm peptide, thereby obtaining a liposome of the present invention bound to CD4(68-94) Acm peptide. OD of the obtained liposome. was 0.94, and the average particle size was 176 nm. Also, CD4 (68-94)A bound to liposomes
The quantitative value of cm peptide was 12.8 μg/ml (CD4 (68-94) per liposome).
The number of Acm peptide bonds is 49). The H of this liposome
The damaging effect on IV-infected cells was investigated in the same manner as in Example 7. The results are shown in Figure 3. CD4 (68-94
) The liposome of the present invention bound to Acm peptide is HI
It showed strong concentration-dependent growth-inhibiting activity against V-infected cells. Liposome OD inhibits the proliferation of infected cells by 50%,
4° was 0.055, which revealed that the same growth-inhibiting effect was exhibited at a concentration 1/12 as low as in the case where no peptide was bound (comparative example). On the other hand, in uninfected cells, even the highest liposome concentration hardly inhibited proliferation. From this, CD4 (68-94
) It has become clear that the liposome of the present invention bound to the Acm peptide exhibits strong selective killing activity against HIV-infected cells.
比較例
実施例7において、CD4(68〜94)ペプチドは用
いずに、F D P −P E 3.8■とシミリスト
イルフォスファチジルコリン(DMPC)35.4■と
フォスファチジルセリン(PS)8.0■とコレステロ
ール(CH)8.0■とをリポソーム膜成分として、そ
の他の方法は実施例7と同じ方法でリポソームの調製を
行うことにより、膜脂質組成かDMPC−PS−CH−
PDP−PE (5: 1・2・0.42モル比)であ
って、ペプチドの結合しないリポソームを得た。得られ
たリポソームのOD 、、、は1.73、平均粒径は1
68nmであった。このリポソームのHIV感染細胞に
対する傷害効果を実施例7と同様の方法で調べた。結果
を第4図に示す。リポソームのOD s =。か0.5
以上の場合にHIV感染細胞に対する傷害効果か認めら
れた。感染細胞の増殖を50%抑制するリポソーム0D
s4゜は0,67てあった。なお、非感染細胞に対して
は、はとんと増殖抑制を示さなかった。Comparative Example In Example 7, CD4 (68-94) peptide was not used, but FDP-P E 3.8■, simyristoylphosphatidylcholine (DMPC) 35.4■ and phosphatidylserine (PS) were used. ) 8.0■ and cholesterol (CH) 8.0■ as liposome membrane components, and by otherwise preparing liposomes in the same manner as in Example 7, the membrane lipid composition was changed to DMPC-PS-CH-
A liposome of PDP-PE (5: 1.2.0.42 molar ratio) to which no peptide was bound was obtained. The obtained liposome had an OD of 1.73 and an average particle size of 1.
It was 68 nm. The damaging effect of this liposome on HIV-infected cells was examined in the same manner as in Example 7. The results are shown in Figure 4. OD s of liposomes =. or 0.5
In the above cases, a damaging effect on HIV-infected cells was observed. Liposome 0D suppresses the proliferation of infected cells by 50%
s4° was 0.67. It should be noted that no significant growth inhibition was observed in uninfected cells.
第1.2.3.4図はそれぞれ実施例7,8゜9および
比較例で得られたリポソームのHIV感染細胞および非
感染細胞に対する増殖阻止率を示す。縦軸は、増殖阻止
率(%)を表し、横軸はリポソームのOD、4.を表す
。各リポソームは図中に示されたODS、。に希釈調製
後、各細胞に作用させ増殖阻止率を測定した。なお各図
中、・−・はHIV感染細胞(MOLT−4/HTLV
−I[[、)に対する増殖阻止率、〇−〇は非感染細胞
(MOLT−4)に対する増殖阻止率を示す。
第1図
リポソーム 0D54゜
第2図
リポソーム 0D540
第3図
0.0050.01 α050.1 0.51リポ
ソーム OD540Figures 1.2.3.4 show the growth inhibition rates of the liposomes obtained in Examples 7, 8 and 9 and Comparative Example against HIV-infected cells and non-infected cells, respectively. The vertical axis represents the growth inhibition rate (%), and the horizontal axis represents the OD of liposomes. represents. Each liposome has an ODS indicated in the figure. After dilution preparation, it was allowed to act on each cell and the growth inhibition rate was measured. In each figure, ... indicates HIV-infected cells (MOLT-4/HTLV
-I [[, ) indicates the growth inhibition rate, 〇-〇 indicates the growth inhibition rate against uninfected cells (MOLT-4). Figure 1 Liposome 0D54° Figure 2 Liposome 0D540 Figure 3 0.0050.01 α050.1 0.51 Liposome OD540
Claims (8)
飾されていてもよい。)で示されるアミノ酸鎖よりなり
抗HIV活性を有するペプチドを結合したリン脂質と相
転移温度が37℃未満であるフォスファチジルコリンと
酸性リン脂質およびコレステロールを膜成分とするリポ
ソームからなるHIV感染細胞殺傷剤(1) [There is a gene sequence] (In the formula, the 19th cysteine group may be modified with a benzyl group or an acetamidomethyl group.) A peptide with anti-HIV activity consisting of an amino acid chain. An HIV-infected cell killer consisting of a liposome whose membrane components are bound phospholipids, phosphatidylcholine with a phase transition temperature of less than 37°C, acidic phospholipids, and cholesterol.
コリンが、ジミリストイルフォスファチジルコリンであ
ることを特徴とする特許請求の範囲第1項から第3項記
載のHIV感染細胞殺傷剤(2) The HIV-infected cell killing agent according to claims 1 to 3, wherein the phosphatidylcholine having a phase transition temperature of less than 37°C is dimyristoylphosphatidylcholine.
とを特徴とする特許請求の範囲第1項から第3項記載の
HIV感染細胞殺傷剤(3) The agent for killing HIV-infected cells according to claims 1 to 3, wherein the acidic phospholipid is phosphatidylserine.
0モル%、相転移温度が37℃未満であるフォスファチ
ジルコリンの割合が60〜85モル%、酸性リン脂質の
割合が5〜20モル%、コレステロールの割合が10〜
35モル%とする脂質組成であることを特徴とする特許
請求の範囲第1項から第3項記載のHIV感染細胞殺傷
剤(4) The ratio of peptide-bound phospholipids is 0.1 to 1
0 mol%, the proportion of phosphatidylcholine with a phase transition temperature of less than 37 ° C. is 60 to 85 mol%, the proportion of acidic phospholipid is 5 to 20 mol%, and the proportion of cholesterol is 10 to 85 mol%.
The HIV-infected cell killing agent according to claims 1 to 3, characterized in that the lipid composition is 35 mol%.
コリンと酸性リン脂質とコレステロールとの成分比が5
:1:2(モル比)であるリポソーム膜中に、ペプチド
を結合したリン脂質を0.1〜10モル%含むことを特
徴とする特許請求の範囲第1項から第3項記載のHIV
感染細胞殺傷剤(5) The component ratio of phosphatidylcholine with a phase transition temperature of less than 37°C, acidic phospholipid, and cholesterol is 5
HIV according to claims 1 to 3, characterized in that the liposome membrane contains 0.1 to 10 mol% of peptide-bound phospholipid in a liposome membrane having a molar ratio of : 1:2 (molar ratio).
Infected cell killer
合するペプチドの数が40〜120であることを特徴と
するHIV感染細胞殺傷剤(6) An agent for killing HIV-infected cells, which has the lipid composition according to claim 6 and binds 40 to 120 peptides.
〜120ペプチドが結合することを特徴とするHIV感
染細胞殺傷剤(7) In the liposome membrane according to claim 7,
An HIV-infected cell killer characterized by binding ~120 peptides
染細胞殺傷剤を用いることを特徴としたHIV感染細胞
の殺傷方法(8) A method for killing HIV-infected cells, characterized by using the HIV-infected cell killing agent according to claims 1 to 3.
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2241591A JPH0418029A (en) | 1990-04-27 | 1990-09-11 | Liposome capable of killing hiv-infected cell |
EP90123694A EP0432693A1 (en) | 1989-12-11 | 1990-12-10 | Liposomes cytotoxic to virus-infected cells |
CA002031973A CA2031973A1 (en) | 1989-12-11 | 1990-12-11 | Liposomes cytotoxic to virus-infected cells |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2-112424 | 1990-04-27 | ||
JP11242490 | 1990-04-27 | ||
JP2241591A JPH0418029A (en) | 1990-04-27 | 1990-09-11 | Liposome capable of killing hiv-infected cell |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH0418029A true JPH0418029A (en) | 1992-01-22 |
Family
ID=26451585
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2241591A Pending JPH0418029A (en) | 1989-12-11 | 1990-09-11 | Liposome capable of killing hiv-infected cell |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH0418029A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008279908A (en) * | 2007-05-10 | 2008-11-20 | Toei International:Kk | Trailer house |
-
1990
- 1990-09-11 JP JP2241591A patent/JPH0418029A/en active Pending
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008279908A (en) * | 2007-05-10 | 2008-11-20 | Toei International:Kk | Trailer house |
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