JPH04356421A - Fat spherule composition containing prostaglandins - Google Patents
Fat spherule composition containing prostaglandinsInfo
- Publication number
- JPH04356421A JPH04356421A JP3250166A JP25016691A JPH04356421A JP H04356421 A JPH04356421 A JP H04356421A JP 3250166 A JP3250166 A JP 3250166A JP 25016691 A JP25016691 A JP 25016691A JP H04356421 A JPH04356421 A JP H04356421A
- Authority
- JP
- Japan
- Prior art keywords
- derived
- lipid
- fat
- pge1
- saturated
- 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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- 150000003180 prostaglandins Chemical class 0.000 title abstract description 7
- 229940094443 oxytocics prostaglandins Drugs 0.000 title abstract description 5
- 150000002632 lipids Chemical class 0.000 claims abstract description 46
- 125000002252 acyl group Chemical group 0.000 claims abstract description 32
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 28
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- 210000002468 fat body Anatomy 0.000 claims description 85
- 125000005257 alkyl acyl group Chemical group 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- 125000004432 carbon atom Chemical group C* 0.000 claims description 4
- 235000021122 unsaturated fatty acids Nutrition 0.000 claims description 3
- 150000004670 unsaturated fatty acids Chemical class 0.000 claims description 3
- 229930195735 unsaturated hydrocarbon Natural products 0.000 claims description 3
- -1 lipid compound Chemical class 0.000 claims 4
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- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
Abstract
Description
【０００１】0001
【産業上の利用分野】本発明は、プロスタグランジン類
（以下、ＰＧ類ともいう）の放出速度の抑制等を目的と
したＰＧ類を含有してなる脂肪小体組成物に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fat body composition containing PGs for the purpose of suppressing the release rate of prostaglandins (hereinafter also referred to as PGs).
【０００２】0002
【従来技術】ＰＧ類は、血管拡張、末梢循環改善、降圧
、抗脂肪分解、ナトリウム利尿など種々多彩な生理作用
を有することから、医薬品への適用がなされている。
この有用なＰＧ類を医薬品として適用する際には、生体
内で容易に不活性物質へ代謝されることと、病巣選択性
が問題となる。そのため、一般にＰＧ類製剤は瀕回投与
が必要となり、患者の苦痛を増すばかりでなく、頻回投
与に基づく目的以外の組織に対する作用が副作用となる
恐れがあり、これらの問題点の解決の手段としてＰＧ類
を脂肪小体に含有させた製剤が提案されている。BACKGROUND OF THE INVENTION PGs have various physiological effects such as vasodilation, improvement of peripheral circulation, lowering of blood pressure, anti-lipolysis, and natriuresis, and are therefore being applied to pharmaceutical products. When applying these useful PGs as pharmaceuticals, problems arise that they are easily metabolized into inactive substances in vivo and that they are selective in lesions. Therefore, PG preparations generally require multiple administrations, which not only increases the patient's pain, but also poses a risk of side effects due to effects on tissues other than the intended ones due to frequent administration. A preparation containing PGs in fat bodies has been proposed.
【０００３】0003
【発明が解決しようとする課題】しかし、ＰＧ類含有脂
肪小体製剤においても、組成物としての安定性が悪く、
一旦、脂肪小体に含有させたＰＧ類が遊離し、ＰＧ類の
活性の持続性に劣るなどの改善すべき問題点があり、様
々な検討が行われているが、未だ満足すべきＰＧ類含有
脂肪小体組成物は提案されていないのが実情である。従
って、本発明者らは脂肪小体からのＰＧ類の遊離が抑制
された脂肪小体組成物、即ち持続性を有する脂肪小体組
成物を提供することを目的とする。[Problems to be Solved by the Invention] However, even PG-containing fat body preparations have poor stability as a composition;
There are problems that need to be improved, such as PGs contained in fat bodies being released and poor sustainability of PG activity, and various studies have been carried out, but there is still no satisfactory level of PGs. The reality is that no composition containing fat bodies has been proposed. Therefore, the present inventors aim to provide a fat body composition in which release of PGs from fat bodies is suppressed, that is, a long-lasting fat body composition.
【０００４】0004
【課題を解決するための手段】さて、脂肪小体二分子膜
は温度によってその流動性が大きく変化することが知ら
れている（ゲル−液晶相転位）。膜の流動性を変化させ
ることのできる因子には、温度の他脂質組成などが知ら
れているが、中でも、脂肪小体の形成に用いるジアシル
グリセロリン脂質やジアシルグリセロ糖脂質の脂肪酸残
基の鎖長や不飽和度を変えることにより、脂肪小体の膜
の流動性およびそれと密接に関連する膜のバリヤー能を
調節できることは興味深い。一般に、疏水性脂肪酸残基
の鎖長の短いものや、不飽和度の高い脂質で形成された
脂肪小体は膜流動性が高く、他方飽和で鎖長の長いもの
では膜流動性が低く、比較的相転移温度も高いことが知
られている（野島庄七、砂本順三、井上圭三編集「リポ
ソーム」（南江堂、１９８８年発行）。[Means for Solving the Problems] It is known that the fluidity of a fat body bilayer membrane changes greatly depending on the temperature (gel-liquid crystal phase transition). Factors that can change membrane fluidity include temperature and lipid composition, among others, chains of fatty acid residues in diacylglycerophospholipids and diacylglyceroglycolipids, which are used to form fat bodies. It is interesting that by changing the length and degree of unsaturation, the fluidity of the lipid body membrane and its closely related membrane barrier capacity can be controlled. In general, lipid bodies formed from hydrophobic fatty acid residues with short chain lengths or highly unsaturated lipids have high membrane fluidity, whereas saturated fatty acid residues with long chain lengths have low membrane fluidity. It is known that the phase transition temperature is relatively high (Liposome, edited by Shoshichi Nojima, Junzo Sunamoto, and Keizo Inoue (Nankodo, published in 1988).
【０００５】かかる技術水準の下に、本発明者らは種々
研究を重ねてきた結果、脂肪小体を形成するための脂質
をジアシルグリセロリン脂質、ジアシルグリセロ糖脂質
、アルキルグリセロリン脂質、アルキルグリセロ糖脂質
、リゾリン脂質の群から選び、該脂質がジアシルグリセ
ロリン脂質又はジアシルグリセロ糖脂質の際にはそのア
シル基が飽和脂肪酸由来であり、アルキルグリセロリン
脂質又はアルキルグリセロ糖脂質の際には、ジアルキル
型の場合、その両方のアルキル基が飽和炭化水素由来で
あり、アルキルアシル型の場合、アルキル基が飽和炭化
水素由来であり且つアシル基が飽和脂肪酸由来であり、
リゾリン脂質の際にはアシル型の場合、そのアシル基は
飽和脂肪酸由来であり、アルキル型の場合、そのアルキ
ル基は飽和炭化水素由来であるものを用いることにより
、上記課題を解決できることを見出し、本発明を完成す
るに至った。Based on this technical level, the present inventors have conducted various studies and found that the lipids for forming fat bodies are diacylglycerophospholipids, diacylglyceroglycolipids, alkylglycerophospholipids, and alkylglyceroglycolipids. , lysophospholipids, when the lipid is a diacylglycerophospholipid or a diacylglyceroglycolipid, the acyl group is derived from a saturated fatty acid, and when the lipid is an alkylglycerophospholipid or an alkylglyceroglycolipid, it is of the dialkyl type. , both alkyl groups are derived from saturated hydrocarbons, and in the case of an alkylacyl type, the alkyl group is derived from a saturated hydrocarbon and the acyl group is derived from a saturated fatty acid,
We have discovered that the above problem can be solved by using lysophospholipids whose acyl groups are derived from saturated fatty acids in the case of acyl-type lysophospholipids, and whose alkyl groups are derived from saturated hydrocarbons in the case of alkyl-type lysophospholipids, The present invention has now been completed.
【０００６】即ち本発明は、脂肪小体を形成するための
脂質として、ジアシルグリセロリン脂質、ジアシルグリ
セロ糖脂質、アルキルグリセロリン脂質、アルキルグリ
セロ糖脂質、リゾリン脂質の群から選ばれた少なくとも
一種の脂質を含有し、該脂質がジアシルグリセロリン脂
質又はジアシルグリセロ糖脂質の際には、そのアシル基
が飽和脂肪酸由来であり、アルキルグリセロリン脂質又
はアルキルグリセロ糖脂質の際には、ジアルキル型の場
合、その両方のアルキル基が飽和炭化水素由来であり、
アルキルアシル型の場合、アルキル基が飽和炭化水素由
来であり且つアシル基が飽和脂肪酸由来であり、リゾリ
ン脂質の際にはアシル型の場合、そのアシル基は飽和脂
肪酸由来であり、アルキル型の場合、そのアルキル型は
飽和炭化水素由来であることを特徴とするプロスタグラ
ンジン類含有脂肪小体組成物に関するものである。That is, the present invention uses at least one lipid selected from the group of diacylglycerophospholipids, diacylglyceroglycolipids, alkylglycerophospholipids, alkylglyceroglycolipids, and lysophospholipids as a lipid for forming fat bodies. When the lipid is a diacylglycerophospholipid or a diacylglyceroglycolipid, the acyl group is derived from a saturated fatty acid, and when the lipid is an alkylglycerophospholipid or an alkylglyceroglycolipid, when it is a dialkyl type, both of the acyl groups are derived from a saturated fatty acid. the alkyl group is derived from a saturated hydrocarbon,
In the case of an alkylacyl type, the alkyl group is derived from a saturated hydrocarbon and the acyl group is derived from a saturated fatty acid; in the case of a lysophospholipid, in the case of an acyl type, the acyl group is derived from a saturated fatty acid; , relates to a prostaglandin-containing fat body composition characterized in that the alkyl type thereof is derived from a saturated hydrocarbon.
【０００７】本発明において、脂肪小体（リポソーム）
は、多重同心二重層、単一層あるいは多重層の構造をな
し、各種蛋白質その他の生理活性物質をその間隙に取り
込む能力を有するものである。当該脂肪小体を形成する
ためのジアシルグリセロリン脂質とは、グリセロリン酸
を骨格として持つリン脂質のうち、１位及び２位に飽和
脂肪酸由来のアシル基が結合したものである。ホスファ
チジルコリン、ホスファチジルセリン、ホスファチジン
酸、ホスファチジルグリセリン、ホスファチジルエタノ
ールアミン、ホスファチジルイノシートルなどが挙げら
れる。ジアシルグリセロ糖脂質とは、親水性部分が糖質
である糖脂質のうち、１位及び２位に飽和脂肪酸由来の
アシル基が結合したものである。[0007] In the present invention, fat bodies (liposomes)
has a structure of multiple concentric bilayers, a single layer, or a multilayer, and has the ability to incorporate various proteins and other physiologically active substances into the interstices. The diacylglycerophospholipid for forming the fat body is a phospholipid having glycerophosphoric acid as a skeleton, with acyl groups derived from saturated fatty acids bonded to the 1st and 2nd positions. Examples include phosphatidylcholine, phosphatidylserine, phosphatidic acid, phosphatidylglycerin, phosphatidylethanolamine, phosphatidylinositl, and the like. A diacylglyceroglycolipid is a glycolipid whose hydrophilic portion is a carbohydrate, with acyl groups derived from saturated fatty acids bonded to the 1st and 2nd positions.
【０００８】アルキルグリセロリン脂質においてジアル
キル型とは、グリセロリン酸を骨格として持つリン脂質
のうち、１位及び２位に飽和炭化水素由来のアルキル基
がエーテル結合したものである。アルキルアシル型とは
、１位又は２位の一方に飽和炭化水素由来のアルキル基
がエーテル結合し、他方に飽和脂肪酸由来のアシル基が
結合したものである。アルキルグリセロ糖脂質において
ジアルキル型とは、親水性部分が糖質である糖脂質のう
ち、１位及び２位に飽和炭化水素由来のアルキル基がエ
ーテル結合したものである。アルキルアシル型とは、１
位又は２位の一方に飽和炭化水素由来のアルキル基がエ
ーテル結合し、一方に飽和脂肪酸由来のアシル基が結合
したものである。[0008] The dialkyl type of alkylglycerophospholipids refers to phospholipids having glycerophosphoric acid as a backbone, in which alkyl groups derived from saturated hydrocarbons are bonded to ether at the 1st and 2nd positions. The alkylacyl type is one in which an alkyl group derived from a saturated hydrocarbon is bonded to one of the 1st or 2nd positions with an ether bond, and an acyl group derived from a saturated fatty acid is bonded to the other. The dialkyl-type alkylglyceroglycolipid refers to a glycolipid whose hydrophilic portion is a carbohydrate, in which alkyl groups derived from saturated hydrocarbons are bonded to the 1st and 2nd positions with ether. The alkyl acyl type is 1
An alkyl group derived from a saturated hydrocarbon is bonded to one of the 2-position and 2-position with an ether bond, and an acyl group derived from a saturated fatty acid is bonded to the other.
【０００９】リゾリン脂質においてアシル型とは、グロ
セロリン酸を骨格として持つリン脂質のうち、１位又は
２位の一方に飽和脂肪酸由来のアシル基が結合し、他方
には何も結合していないものであり、リゾホスファチジ
ルコリンなどが挙げられる。アルキル型とは、１位又は
２位の一方に飽和炭化水素由来のアルキル基がエーテル
結合し、他方には何も結合していないものである。[0009] The acyl type of lysophospholipid refers to a phospholipid that has glucerophosphoric acid as its backbone, and has an acyl group derived from a saturated fatty acid bound to either the 1st or 2nd position, and nothing is bound to the other. Examples include lysophosphatidylcholine. The alkyl type is one in which an alkyl group derived from a saturated hydrocarbon is bonded to one of the 1st and 2nd positions with an ether bond, and nothing is bonded to the other.
【００１０】ジアシルグリセロリン脂質及びジアシルグ
リセロ糖脂質のアシル基、アルキルグリセロリン脂質及
びアルキルグリセロ糖脂質のアルキルアシル型のアシル
基、リゾリン脂質のアシル型のアシル基は飽和脂肪酸由
来のものであれば直鎖状、分岐鎖状のいずれであっても
よい。また、ジアシルグリセロリン脂質及びジアシルグ
リセロ糖脂質の場合、２つのアシル基は同一の飽和脂肪
酸由来のものでもよく、また異なった飽和脂肪酸由来の
ものでもよい。The acyl groups of diacylglycerophospholipids and diacylglyceroglycolipids, the alkylacyl type acyl groups of alkylglycerophospholipids and alkylglyceroglycolipids, and the acyl type acyl groups of lysophospholipids are linear if they are derived from saturated fatty acids. It may be either chain-like or branched-chain. Furthermore, in the case of diacylglycerophospholipids and diacylglyceroglycolipids, the two acyl groups may be derived from the same saturated fatty acid or may be derived from different saturated fatty acids.
【００１１】アルキルグリセロリン脂質及びアルキルグ
リセロ糖脂質のジアルキル型のアルキル基、アルキルア
シル型のアルキル基、リゾリン脂質のアルキル型のアル
キル基は飽和炭化水素由来のものであれば、直鎖状、分
岐鎖状のいずれであってもよい。また、アルキルグリセ
ロリン脂質及びアルキルグリセロ糖脂質のジアルキル型
の場合、２つのアルキル基は同一の飽和炭化水素由来の
ものでもよく、また異なった飽和炭化水素由来のもので
もよい。[0011] The dialkyl-type alkyl group of the alkylglycerophospholipid and the alkylglyceroglycolipid, the alkylacyl-type alkyl group, and the alkyl-type alkyl group of the lysophospholipid may be linear or branched if they are derived from a saturated hydrocarbon. It may be in any form. Furthermore, in the case of dialkyl type alkylglycerophospholipids and alkylglyceroglycolipids, the two alkyl groups may be derived from the same saturated hydrocarbon, or may be derived from different saturated hydrocarbons.
【００１２】上記のアシル基としては、好ましくは炭素
数１４〜２２のアルカノイル基が、またアルキル基とし
ては、好ましくは炭素数１４〜２２のアルキル基が例示
される。これらの脂質は生理的に許容され、そして代謝
されうる無毒のものであればいずれも本発明に用いられ
、それらは天然由来の脂質であってもよいし、公知の方
法にて人工的に合成したものでもよい。また、単一の脂
質を用いてもよいし、複数の脂質を組み合わせて使用し
てもよい。The above acyl group is preferably an alkanoyl group having 14 to 22 carbon atoms, and the alkyl group is preferably an alkyl group having 14 to 22 carbon atoms. Any of these lipids can be used in the present invention as long as they are physiologically acceptable and metabolizable and non-toxic, and they may be naturally derived lipids or artificially synthesized lipids using known methods. It may be something you have done. Moreover, a single lipid may be used, or a plurality of lipids may be used in combination.
【００１３】好ましい脂質としては、ジアシルグリセロ
リン脂質が挙げられ、より好ましい脂質はホスファチジ
ルコリンである。また、その際のアシル基は炭素数１４
〜２２の飽和脂肪酸由来のもの、例えばミリスチン酸、
パルミチン酸、ステアリン酸、エイコサン酸、ドコサン
酸由来のものが好ましい。脂質はＰＧ類１重量部当たり
、通常１０〜３０００重量部、好ましくは１５０〜２０
０重量部配合される。Preferred lipids include diacylglycerophospholipids, and a more preferred lipid is phosphatidylcholine. In addition, the acyl group at that time has 14 carbon atoms.
~22 saturated fatty acids, such as myristic acid,
Those derived from palmitic acid, stearic acid, eicosanoic acid, and docosanoic acid are preferred. The amount of lipid is usually 10 to 3000 parts by weight, preferably 150 to 20 parts by weight per 1 part by weight of PGs.
0 parts by weight is blended.
【００１４】当該脂質の簡単な合成法としては、例えば
不飽和脂肪酸由来のアシル基や不飽和炭化水素由来のア
ルキル基を有する脂質に、公知の方法にて水素添加反応
を行うことにより、対応する飽和脂肪酸由来のアシル基
や飽和炭化水素由来のアルキル基を有する化合物に交換
する方法が挙げられる。なお、不飽和脂肪酸由来のアシ
ル基や不飽和炭化水素由来のアルキル基を有する脂質と
、本発明に用いる脂質とを、適宜の量比で組み合わせる
とＰＧＥ１の放出速度に変化を持たせることができる。
即ち、不飽和由来のものをより多くすれば、放出速度を
早める方向の製剤が提供でき、逆に不飽和由来のものを
より少なくすれば、放出遅延させる方向の製剤が提供で
きる。[0014] As a simple method for synthesizing the lipid, for example, a lipid having an acyl group derived from an unsaturated fatty acid or an alkyl group derived from an unsaturated hydrocarbon is subjected to a hydrogenation reaction using a known method. Examples include a method of replacing it with a compound having an acyl group derived from a saturated fatty acid or an alkyl group derived from a saturated hydrocarbon. In addition, by combining a lipid having an acyl group derived from an unsaturated fatty acid or an alkyl group derived from an unsaturated hydrocarbon with the lipid used in the present invention in an appropriate quantitative ratio, the release rate of PGE1 can be varied. . That is, by increasing the unsaturated content, a formulation can be provided that accelerates the release rate, and conversely, by decreasing the unsaturated content, a formulation can be provided that has a delayed release rate.
【００１５】本発明で用いられるＰＧ類は、アラキドン
酸代謝物又はそれらの構造類似化合物であればよい。ア
ラキドン酸代謝物としては、例えばプロスタグランジン
、プロスタサイクリン、トロンボキサン、ロイコトリエ
ンなどが挙げられる。また、アラキドン酸代謝産物構造
類似化合物としては、例えばアラキドン酸代謝産物を基
本骨格とし構造修飾された６−ケト−ＰＧＥ１誘導体、
カルバサイクリン誘導体、ＰＧＤ２誘導体などが挙げら
れる。The PGs used in the present invention may be arachidonic acid metabolites or structurally similar compounds thereof. Examples of arachidonic acid metabolites include prostaglandins, prostacyclin, thromboxanes, and leukotrienes. In addition, examples of compounds similar to the structure of arachidonic acid metabolites include 6-keto-PGE1 derivatives that have a basic skeleton of arachidonic acid metabolites and are structurally modified;
Examples include carbacycline derivatives and PGD2 derivatives.
【００１６】脂肪小体の製造の概略は、次の通りである
。即ち、適当な脂質を含有する溶液（当該溶液用の溶媒
としては、脂質を変成しない溶媒、例えばクロロホルム
等の溶媒を使用した溶液が例示される）から溶媒を留去
して脂質の薄膜を作り、この薄膜にＰＧ類を含有する溶
液を加えて激しく振とうし、好ましくは超音波処理を行
い、脂質を均一分散させて脂質薄膜懸濁液を調製する。
ここで、ＰＧ類を含有する溶液に使用される溶媒として
は脂肪小体を変成、分解せず、かつ生理的に許容される
ものであればよく、例えば水、生理食塩水、ｐＨ４〜７
の緩衝液などが挙げられる。勿論、ＰＧ類は脂肪小体調
製後に加えてもよい。The outline of the production of fat bodies is as follows. That is, the solvent is distilled off from a solution containing a suitable lipid (an example of a solvent for the solution is a solution using a solvent that does not denature lipids, such as chloroform) to form a thin film of lipid. A solution containing PGs is added to this thin film, followed by vigorous shaking and preferably ultrasonic treatment to uniformly disperse the lipids to prepare a lipid thin film suspension. Here, the solvent used in the solution containing PGs may be any solvent that does not denature or decompose fat bodies and is physiologically acceptable, such as water, physiological saline, pH 4 to 7.
buffer solutions and the like. Of course, PGs may be added after the preparation of fat bodies.
【００１７】本発明の脂肪小体の粒子径３μｍ以下とす
るためには、上記方法における超音波処理は、０℃〜７
０℃、好ましくは３５℃〜４５℃で１〜６０分間程度行
えばよい。また、均一なサイズ分布（２００ｎｍ）にま
でサイズをフィルター等を用いて減少させることができ
る。なお、脂質薄膜の調製に際し、トコフェロール（ビ
タミンＥ）、コレステロール、ホスファチジン酸、ジセ
チルホスフェート、バルミチン酸などの脂肪酸等を安定
化剤として添加してもよい。特にコレステロールは脂肪
小体の安定性に寄与するばかりでなく、後述の脂肪小体
にｐＨ勾配を付与する場合、その勾配の維持を容易にす
るためにも有利であり、好ましくはコレステロールを脂
質１モル当たり０．５〜１．０モル添加するのがよく、
更に好ましくは０．８モル程度添加するのがよい。[0017] In order to make the particle size of the fat bodies of the present invention 3 μm or less, the ultrasonic treatment in the above method is carried out at a temperature of 0°C to 7°C.
It may be carried out at 0°C, preferably 35°C to 45°C, for about 1 to 60 minutes. Further, the size can be reduced to a uniform size distribution (200 nm) using a filter or the like. In addition, in preparing the lipid thin film, tocopherol (vitamin E), cholesterol, fatty acids such as phosphatidic acid, dicetyl phosphate, and valmitic acid may be added as stabilizers. In particular, cholesterol not only contributes to the stability of fat bodies, but also has the advantage of facilitating the maintenance of a pH gradient when imparting a pH gradient to fat bodies, which will be described later. It is best to add 0.5 to 1.0 mol per mol,
More preferably, it is added in an amount of about 0.8 mol.
【００１８】上記の製造方法は超音波処理法による製造
法［Ｈｕａｎｇ，Ｃ．：Ｂｉｏｃｈｅｍｉｓｔｒｙ，
８，３４４（１９６９）］であるが、これに限らず、
凍結融解法［Ｐｉｃｋ，Ｕ．：Ａｒｃｈ．Ｂｉｏｃｈｅ
ｍ．Ｂｉｏｐｈｙｓ．，２１２，１８６（１９８１）］
、界面活性剤除去法［Ｂｒｕｎｎｅｒ，Ｊ．ｅｔ ａ
ｌ：Ｂｉｏｃｈｅｍ．Ｂｉｏｐｈｙｓ・Ａｃｔａ，４５
５，３２２（１９７６）］、逆相蒸発法 ［Ｓｚｏｋ
ａ，Ｆ．ｅｔ ａｌ：Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ
．Ｓｃｉ．ＵＳＡ，７５，４１９４（１９７８）］、乳
化機による方法［Ｔａｌｓｍａ，Ｈ．ｅｔ ａｌ．：
Ｄｒｕｇ Ｄｅｖ．ａｎｄＩｎｄ．Ｐｈａｒｍａｃｙ
，１５，１９７（１９８９）］でも製造可能である。The above manufacturing method is a manufacturing method using an ultrasonic treatment method [Huang, C.; :Biochemistry,
8,344 (1969)], but is not limited to this.
Freeze-thaw method [Pick, U. :Arch. Bioche
m. Biophys. , 212, 186 (1981)]
, surfactant removal method [Brunner, J.; et a
l:Biochem. Biophys Acta, 45
5, 322 (1976)], reversed-phase evaporation method [Szok
a, F. et al: Proc. Natl. Acad
．． Sci. USA, 75, 4194 (1978)], a method using an emulsifier [Talsma, H. et al. :
Drug Dev. andInd. Pharmacy
, 15, 197 (1989)].
【００１９】また、本脂肪小体にはｐＨ勾配を付与して
もよい。好ましくは脂肪小体外液のｐＨを２〜６、脂肪
小体内封溶液のｐＨを７〜１１、特にｐＨ９〜１０に調
整されたものがよい。更に好ましくは、脂肪小体内封溶
液のｐＨを弱酸と強塩基の組み合わせにてｐＨを７〜１
１に調整されたものがよい。脂肪小体外液は、各種公知
の緩衝剤、例えばクエン酸緩衝液、酢酸緩衝液、乳酸緩
衝液などによりｐＨ２〜６に調整する。特に好ましい外
液としてはクエン酸緩衝液が挙げられ、好ましいｐＨは
２〜６、更に好ましくはｐＨ３〜５である。緩衝液濃度
は、通常５〜５００ｍＭ、好ましくは２０〜３００ｍＭ
であり、浸透圧を調節するために庶糖等を加えてもよい
。[0019] Furthermore, a pH gradient may be applied to the present fat bodies. Preferably, the pH of the liquid outside the fat body is adjusted to 2 to 6, and the pH of the solution inside the fat body is adjusted to 7 to 11, particularly preferably 9 to 10. More preferably, the pH of the solution encapsulated in fat bodies is adjusted to 7 to 1 using a combination of a weak acid and a strong base.
The one adjusted to 1 is better. The extra-fat body fluid is adjusted to pH 2 to 6 using various known buffers, such as citrate buffer, acetate buffer, and lactic acid buffer. A particularly preferred external solution is a citric acid buffer, and its pH is preferably 2 to 6, more preferably 3 to 5. Buffer concentration is usually 5-500mM, preferably 20-300mM
Sucrose or the like may be added to adjust the osmotic pressure.
【００２０】脂肪小体内封溶液は、好適には弱酸と強塩
基にてｐＨ７〜１１に調整する。弱酸と強塩基の組み合
わせとしては、炭酸と炭酸ナトリウム、リン酸とリン酸
ナトリウム、ホウ酸とホウ酸ナトリウム、ピロリン酸と
ピロリン酸ナトリウムなどが挙げられる。特に好ましく
は炭酸と炭酸ナトリウムにて、ｐＨ９〜１０に調整され
たものが例示される。緩衝液濃度は、通常１００〜１０
００ｍＭ、好ましくは５００〜６００ｍＭである。[0020] The lipid body encapsulation solution is preferably adjusted to pH 7 to 11 using a weak acid and a strong base. Combinations of weak acids and strong bases include carbonic acid and sodium carbonate, phosphoric acid and sodium phosphate, boric acid and sodium borate, and pyrophosphoric acid and sodium pyrophosphate. Particularly preferred are those whose pH is adjusted to 9 to 10 using carbonic acid and sodium carbonate. Buffer concentration is usually 100-10
00mM, preferably 500-600mM.
【００２１】また、本発明製剤はアルブミン、デキスト
ラン、ビニル重合体、非イオン性界面活性剤、ゼラチン
、ヒドロキシエチル澱粉から選ばれた高分子物質を安定
化剤として配合してもよい。当該高分子物質安定化剤は
、ＰＧ類と共に脂肪小体内の空隙に取り込まれていても
よいし、またＰＧ類を取り込んだ脂肪小体製剤に配合（
即ち、脂肪小体外に配合）してもよい。もちろん脂肪小
体の内外ともに配合してもよいことはいうまでもない。
当該安定化剤の添加量は脂質１重量部に対して０．５〜
１０重量部、好ましくは１〜５重量部である。The preparation of the present invention may also contain a polymeric substance selected from albumin, dextran, vinyl polymer, nonionic surfactant, gelatin, and hydroxyethyl starch as a stabilizer. The polymer substance stabilizer may be incorporated into the voids within the fat body together with PGs, or may be incorporated into a fat body preparation incorporating PGs (
That is, it may be blended outside the fat body). Of course, it goes without saying that it may be incorporated both inside and outside the fat body. The amount of the stabilizer added is 0.5 to 1 part by weight of lipid.
The amount is 10 parts by weight, preferably 1 to 5 parts by weight.
【００２２】また、糖類の添加は、浸透圧の調整および
、後述する凍結乾燥製剤を調製する際に有利である。
かかる糖類としては、単糖類（グルコース、マンノース
、ガラクトース、果糖等）、二糖類（ショ糖、麦芽糖、
乳糖等）、糖アルコール（マンニット、ソルビット、キ
シリット等）が好適なものとして例示されるが、これら
に限定されるものではない。その添加量は脂質１重量部
に対して０．５〜１０重量部、好ましくは１〜５重量部
である。[0022] Addition of saccharides is also advantageous in adjusting the osmotic pressure and in preparing the freeze-dried preparation described below. Such sugars include monosaccharides (glucose, mannose, galactose, fructose, etc.), disaccharides (sucrose, maltose,
Preferred examples include, but are not limited to, sugar alcohols (mannitol, sorbitol, xylitol, etc.). The amount added is 0.5 to 10 parts by weight, preferably 1 to 5 parts by weight, per 1 part by weight of lipid.
【００２３】上記製造方法により直径が約０．０２〜１
μの範囲のＰＧ類含有脂肪小体を含む粒子を提供でき、
ＰＧ類はほぼ完全に脂肪小体に取り込まれるため遊離物
の除去操作も不要である。このものは次いで、ペレット
状、懸濁状製剤として調製する。製剤化は医薬品の製法
において広く公知の方法に準ずる。かくして得られたＰ
Ｇ類含有脂肪小体組成物は、ＰＧ類の脂肪小体からの遊
離を優位に抑制し、しかもその粒子径は均一であり、極
めて微少であることから医療用製剤として好ましい。[0023] By the above manufacturing method, the diameter is about 0.02 to 1.
Particles containing PG-containing fat bodies in the μ range can be provided,
Since PGs are almost completely incorporated into fat bodies, there is no need to remove free substances. This is then prepared as a pellet or suspension formulation. Formulation follows a widely known method for manufacturing pharmaceuticals. Thus obtained P
The G group-containing fat body composition is preferable as a medical preparation because it significantly suppresses the release of PGs from the fat body, and its particle size is uniform and extremely small.
【００２４】また本脂肪小体は、液状製剤を凍結させた
後、減圧下で乾燥させ、凍結乾燥製剤としても提供され
るが、その調製法は公知の方法に従えばよい。本発明の
脂肪小体は、一般的に経口薬又は注射薬として使用され
、その投与量は成人の場合、ＰＧ類として１〜５０μｇ
、０．０２〜１ｎｇ／１ｋｇ／１回の量において投与さ
れる。凍結乾燥製剤は、その使用時に生理的に許容され
る水性溶媒によって溶解又は希釈して用いられるのが一
般的であるが、製剤上の工夫によって錠剤化、カプセル
化、腸溶性カプセル化してもよい。[0024] The present fat bodies can also be provided as a freeze-dried preparation by freezing the liquid preparation and then drying it under reduced pressure, which can be prepared by any known method. The fat bodies of the present invention are generally used as oral drugs or injection drugs, and the dosage for adults is 1 to 50 μg as PGs.
, in an amount of 0.02-1 ng/kg/dose. Freeze-dried preparations are generally dissolved or diluted with a physiologically acceptable aqueous solvent before use, but they may also be made into tablets, capsules, or enteric-coated capsules depending on the formulation. .
【００２５】[0025]
【実施例・実験例】以下の実施例および実験例は、特に
言及しない限り各々次の要領で行った。
１．脂肪小体の製造法（凍結乾燥融解法によるｐＨ勾配
を有する脂肪小体の製造法）：ジパルミトイルホスファ
チジルコリン（以下、ＤＰＰＣと略す：シグマ社製）７
０ｍｇとコレステロール（以下、ＣＨＯＬと略す：シグ
マ社製）３０ｍｇ（モル比５５：４５）を混合する。こ
の混合物をクロロホルム１ｍｌに溶解した後、２５ｍｌ
容の梨型フラスコに入れ、ロータリーエバポレータでク
ロロホルムを留去し、脂質薄膜を調製した。内壁に脂質
薄膜を形成したフラスコをデシケーターに入れ真空ポン
プで１時間吸引した。次に、フラスコに第１の緩衝液（
脂肪小体内封溶液と同組成）を１ｍｌ加え、ボルテクス
・ミキサーで攪拌して脂質懸濁液（ＭＬＶ）を調製した
。[Examples/Experimental Examples] The following Examples and Experimental Examples were carried out in the following manner unless otherwise specified. 1. Method for producing fat bodies (method for producing fat bodies having a pH gradient by freeze-drying and thawing method): Dipalmitoyl phosphatidylcholine (hereinafter abbreviated as DPPC: manufactured by Sigma) 7
0 mg and 30 mg of cholesterol (hereinafter abbreviated as CHOL; manufactured by Sigma) (molar ratio 55:45) are mixed. After dissolving this mixture in 1 ml of chloroform, 25 ml
The mixture was placed in a pear-shaped flask, and chloroform was distilled off using a rotary evaporator to prepare a lipid thin film. The flask with a thin lipid film formed on its inner wall was placed in a desiccator and suctioned for 1 hour using a vacuum pump. Next, add the first buffer solution (
A lipid suspension (MLV) was prepared by adding 1 ml of the same composition as the fat body encapsulation solution and stirring with a vortex mixer.
【００２６】次に、ドライアイス上で凍結後、３７℃温
浴中で融解するという操作を５回繰り返し、脂肪小体（
ＬＵＶ）を調製した。[0026] Next, the procedure of freezing on dry ice and thawing in a 37°C hot bath was repeated five times to obtain fat bodies (
LUV) was prepared.
【００２７】次に、０．２μｍのＮｕｃｌｅｐｏｒｅフ
ィルター（ＣｏｓｔａｒＣｏｒｐｏｒａｔｉｏｎ）を用
いて１０回濾過を行い、粒子径を均一にした。次に前記
調製に用いたのと同じ緩衝液３ｍｌに脂肪小体１００μ
ｌを加えたサンプルに、第２の緩衝液〔脂肪小体外液と
同組成、８００ｍＭ蔗糖含有２０ｍＭクエン酸（ｐＨ３
．０）を使用〕を５ｍｌ加え、超遠心（５００００ｒＰ
ｍ、３０分間）を２回行い、膜透過ｐＨ勾配を有した脂
肪小体を調製した。次に、風乾したＰＧ類に上記脂肪小
体を加え６０℃、１０分間インキュベーションした。
以上の工程によりＰＧ類含有脂肪小体を調製した。なお
、ＭＬＶは多重ラメラ小胞を、ＬＵＶは粒子径の大きな
単層ラメラ小胞を示す。Next, filtration was performed 10 times using a 0.2 μm Nuclepore filter (Costar Corporation) to make the particle size uniform. Next, 100 μl of fat bodies were added to 3 ml of the same buffer solution used in the above preparation.
A second buffer [same composition as the fat body extracellular fluid, 20mM citric acid containing 800mM sucrose (pH 3)]
．． 0)] and ultracentrifuged (50,000 rP).
m, 30 minutes) twice to prepare fat bodies with a transmembrane pH gradient. Next, the above fat bodies were added to the air-dried PGs and incubated at 60°C for 10 minutes. A PG-containing fat body was prepared by the above steps. Note that MLV indicates a multilamellar vesicle, and LUV indicates a unilamellar vesicle with a large particle size.
【００２８】２．ＰＧ類の封入効率の測定：トリチウム
標識ＰＧ類を利用して、限外濾過法にて測定した。即ち
、限外濾過膜（Ａｍｉｃｏｎ製ＡＰ−１）上に捕捉され
たＰＧ類を脂肪小体に取り込まれたＰＧ類とし、限外濾
過膜を通過したＰＧ類をフリーのＰＧ類とした。そして
、全体のＰＧ類に対する捕捉されたＰＧ類の割合を以て
ＰＧ類の封入効率とした。即ち、ＰＧ類の封入効率は、
図１に示した如き手法を経て、限外濾過膜の放射活性お
よび濾液の放射活性を各々測定し、下式によって求めた
ものである。（限外濾過膜の放射活性）÷（限外濾過膜
の放射活性＋濾液の放射活性）×１００（放射活性の測
定）サンプリングした液１００μｌに液体シンチレータ
ー１０ｍｌを加え、良く攪拌した後、放射活性を測定し
た。2. Measurement of encapsulation efficiency of PGs: Measured by ultrafiltration using tritium-labeled PGs. That is, PGs captured on the ultrafiltration membrane (AP-1 manufactured by Amicon) were considered to be PGs incorporated into fat bodies, and PGs that had passed through the ultrafiltration membrane were considered to be free PGs. Then, the ratio of captured PGs to the total PGs was defined as the encapsulation efficiency of PGs. That is, the encapsulation efficiency of PGs is
The radioactivity of the ultrafiltration membrane and the radioactivity of the filtrate were each measured using the method shown in FIG. 1, and calculated using the following formula. (Radioactivity of ultrafiltration membrane) ÷ (Radioactivity of ultrafiltration membrane + Radioactivity of filtrate) x 100 (Measurement of radioactivity) Add 10ml of liquid scintillator to 100μl of the sampled liquid, stir well, and then add radioactivity. was measured.
【００２９】３．リン脂質（ＰＣ）の定量：リン脂質測
定用試薬（酵素剤）（デンカ生研社製）を用いて測定し
た。3. Quantification of phospholipid (PC): Measured using a phospholipid measurement reagent (enzyme agent) (manufactured by Denka Seiken Co., Ltd.).
【００３０】実施例１・実験例１
封入効率の検討：ＰＧ類として、ＰＧＥ１を使用し、前
記脂肪小体の製造法に準じて製造した脂肪小体であって
、内封溶液５００ｍＭ炭酸（ｐＨ１０．０）、外液８０
０ｍＭ庶糖含有２０ｍＭクエン酸（ｐＨ３．０）とし、
脂肪小体へのＰＧＥ１の封入効率を調べた。その結果は
図２に示した通りである。なお、ＰＧＥ１５μｇに対し
、脂肪小体をＰＣ量として０．０７５ｍｇ使用し、２５
℃でインキュベーションした。また、ＥＰＣは卵黄ホス
ファチジルコリンのことであり、ＥＰＣ／ＣＨＯＬリポ
ソームはＤＰＰＣ／ＣＨＯＬリポソームと同様の方法に
て調製した。Example 1/Experimental Example 1 Examination of encapsulation efficiency: PGE1 was used as the PG, and fat bodies were produced according to the method for producing fat bodies described above. .0), external fluid 80
20mM citric acid (pH 3.0) containing 0mM sucrose,
The encapsulation efficiency of PGE1 into fat bodies was investigated. The results are shown in FIG. In addition, for 15 μg of PGE, 0.075 mg of adipose body was used as the PC amount, and 25
Incubated at ℃. Further, EPC refers to egg yolk phosphatidylcholine, and EPC/CHOL liposomes were prepared in the same manner as DPPC/CHOL liposomes.
【００３１】図２の結果から明らかなように、ＥＰＣ／
ＣＨＯＬの組成の脂肪小体は２０分間インキュベーショ
ンするとＰＧＥ１の封入効率は６０％に達し平均値であ
る７０％にほぼ等しくなった。これに対し、ＤＰＰＣ／
ＣＨＯＬの組成の脂肪小体は６０分間インキュベーショ
ンしてもＰＧＥ１の封入効率は４０％であり、７０％の
平均値になるには４時間を要した。これは膜の流動性が
ＰＧＥ１の脂肪小体への取り込み速度に多大な影響を与
えていることを意味する。As is clear from the results in FIG.
When adipose bodies having the composition of CHOL were incubated for 20 minutes, the encapsulation efficiency of PGE1 reached 60%, which was almost equal to the average value of 70%. On the other hand, DPPC/
Even after incubation for 60 minutes in adipose bodies having the composition of CHOL, the encapsulation efficiency of PGE1 was 40%, and it took 4 hours to reach an average value of 70%. This means that membrane fluidity greatly influences the rate of PGE1 uptake into fat bodies.
【００３２】実施例２・実験例２
脂肪小体へのＰＧＥ１の保持能に及ぼす希釈の影響：Ｐ
Ｇ類として、ＰＧＥ１を使用して前記脂肪小体の製造法
に準じてｐＨ勾配法にて、ＰＧＥ１５μｇに対し脂肪小
体のＰＣ量として７．５ｍｇを用いて脂肪小体を製造し
た。なお、ＥＰＣ／ＣＨＯＬは２５℃、１時間、ＤＰＰ
Ｃ／ＣＨＯＬは６０℃、１０分間のインギュベーション
で脂肪小体へＰＧＥ１を封入した。次に、この脂肪小体
を８００ｍＭ庶糖含有２０ｍＭクエン酸で１０倍および
１００倍希釈し、脂肪小体へのＰＧＥ１保持率を求めた
。かくして、脂肪小体へのＰＧＥ１の保持能に及ぼす希
釈の影響を調べ、その結果を図３に示した。なお、本測
定においては希釈後、直ちに限外濾過（２０℃）に付し
た。図３の結果から明らかなように、１００倍希釈する
と、ＥＰＣ／ＣＨＯＬに比べＤＰＰＣ／ＣＨＯＬの方が
ＰＧＥ１保持率は高かったExample 2/Experimental Example 2 Effect of dilution on retention ability of PGE1 in fat bodies: P
As Class G, PGE1 was used to produce fat bodies using a pH gradient method according to the above method for producing fat bodies, using 15 μg of PGE and 7.5 mg of PC in the fat bodies. In addition, EPC/CHOL is 25℃, 1 hour, DPP
C/CHOL encapsulated PGE1 into fat bodies by incubation at 60°C for 10 minutes. Next, this fat body was diluted 10 times and 100 times with 20 mM citric acid containing 800 mM sucrose, and the retention rate of PGE1 in the fat body was determined. Thus, the effect of dilution on the ability of PGE1 to be retained in fat bodies was investigated, and the results are shown in FIG. In this measurement, the sample was immediately subjected to ultrafiltration (20° C.) after dilution. As is clear from the results in Figure 3, when diluted 100 times, the PGE1 retention rate was higher in DPPC/CHOL than in EPC/CHOL.
【００３３】実施例３・実験例３
脂肪小体へのＰＧＥ１の保持能に及ぼす希釈の影響：Ｐ
Ｇ類として、ＰＧＥ１を使用し、前記脂肪小体の製造法
に準じてｐＨ勾配法にて構成脂質の異なる２種の脂肪小
体を製造し、これを８００ｍＭ庶糖含有２０ｍＭクエン
酸緩衝液にて２００倍希釈後、３７℃インキュベーショ
ンして脂肪小体のＰＧＥ１保持能を調べた。その結果は
図４に示した通りである。なお、ＰＧＥ１５μｇに対し
脂肪小体のＰＣ量を０．７５ｍｇとした。希釈前は両脂
肪小体のＰＧＥ１封入効率はＥＰＣ／ＣＨＯＬが９８．
９％、ＤＰＰＣ／ＣＨＯＬは９７．６％であった。この
ものを２００倍希釈するとＥＰＣ／ＣＨＯＬのＰＧＥ１
保持率は３２．８％となり、これに対しＤＰＰＣ／ＣＨ
ＯＬのＰＧＥ１保持率は５２．７％となり、３７℃で４
時間インキュベーションしても、この差は維持されてい
た。Example 3/Experimental Example 3 Effect of dilution on retention ability of PGE1 in fat bodies: P
As Class G, PGE1 was used to produce two types of fat bodies with different constituent lipids by the pH gradient method according to the above-mentioned method for producing fat bodies, and these were added to a 20mM citrate buffer containing 800mM sucrose. After 200-fold dilution, the mixture was incubated at 37°C and the ability of the fat bodies to retain PGE1 was examined. The results are shown in FIG. Note that the amount of PC in the fat body was 0.75 mg for 15 μg of PGE. Before dilution, the PGE1 encapsulation efficiency of both fat bodies was 98 for EPC/CHOL.
9%, and DPPC/CHOL was 97.6%. If you dilute this 200 times, EPC/CHOL PGE1
The retention rate was 32.8%, whereas DPPC/CH
The PGE1 retention rate of OL was 52.7%, which was 4 at 37℃.
This difference was maintained even after time incubation.
【００３４】実施例４・実験例４
脂肪小体からのＰＧＥ１の放出性：ＰＧ類として、ＰＧ
Ｅ１を使用し、前記脂肪小体の製造法に準じてｐＨ勾配
法にて脂肪小体を製造した。なお、ＰＧＥ１５μｇに対
して脂肪小体のＰＣ量は７．５ｍｇとした。かくして製
造された脂肪小体を、各緩衝液〔２０ｍＭクエン酸緩衝
液（ｐＨ４．５）、１００ｍＭへペス緩衝液（ｐＨ７．
４）、２０％ヒト血清アルブミン含有１００ｍＭヘペス
緩衝液（ｐＨ７．４）〕で１７倍希釈し、ヒト血清アル
ブミン（ＨＳＡ）存在下における、脂肪小体からのＰＧ
Ｅ１の放出性、即ち脂肪小体へのＰＧＥ１保持能を庶糖
密度勾配遠心法で調べた。Example 4/Experimental Example 4 Release of PGE1 from fat bodies: As PGs, PG
Using E1, fat bodies were produced by a pH gradient method according to the method for producing fat bodies described above. Note that the amount of PC in the fat body was 7.5 mg for 15 μg of PGE. The thus produced fat bodies were soaked in each buffer [20mM citrate buffer (pH 4.5), 100mM Hepes buffer (pH 7.
4) PG from fat bodies in the presence of human serum albumin (HSA) diluted 17 times with 100 mM Hepes buffer (pH 7.4) containing 20% human serum albumin.
The release property of E1, ie, the ability to retain PGE1 in fat bodies, was investigated by sucrose density gradient centrifugation.
【００３５】実施例５・実験例５
ｐＨ勾配を持たないリポソームからのＰＧＥ１の放出性
ＰＧ類としてＰＧＥ１を用い、脂質として水素添加ホス
ファチジルコリン（以下Ｈ−ＥＰＣと略す）７００ｍｇ
を用い、前記脂肪小体（リポソーム）製造法に準じて製
造した脂肪小体であって、内封溶液、外液とも８００ｍ
Ｍ蔗糖を含有する２０ｍＭクエン酸（ｐＨ４．５）とし
、脂肪小体０．５ｍｌ中のＰＣ量７．５ｍｇに対してＰ
ＧＥ１５μｇを混合し、６０℃で１分間加熱した。この
リポソームをＨ−ＥＰＣリポソームとする。対照として
実施例２で調製したｐＨ勾配を持つＥＰＣ／ＣＨＯＬリ
ポソームを用いて、両者を８００ｍＭ蔗糖を含有する２
０ｍＭクエン酸（ｐＨ４．５）で２０００倍希釈し、３
７℃でインキュベーションして、経時的に脂肪小体中の
ＰＧＥ１保持率を調べた。その結果を図６に示した。
図６の結果から明らかなように、ｐＨ勾配を持たないＨ
−ＥＰＣリポソームの方がｐＨ勾配を持つＥＰＣ／ＣＨ
ＯＬリポソームよりＰＧＥ１の放出速度が遅かった。Example 5/Experimental Example 5 Release of PGE1 from liposomes without pH gradient PGE1 was used as the PG, and 700 mg of hydrogenated phosphatidylcholine (hereinafter abbreviated as H-EPC) was used as the lipid.
The fat corpuscles were produced according to the above-mentioned method for producing liposomes, and both the internal and external solutions were 800 m
20mM citric acid (pH 4.5) containing M sucrose, and P for 7.5mg of PC in 0.5ml of fat body.
15 μg of GE was mixed and heated at 60° C. for 1 minute. This liposome is referred to as H-EPC liposome. As a control, EPC/CHOL liposomes with a pH gradient prepared in Example 2 were used, and both were mixed with 2 containing 800 mM sucrose.
Diluted 2000 times with 0mM citric acid (pH 4.5),
After incubation at 7°C, the retention rate of PGE1 in fat bodies was examined over time. The results are shown in FIG. As is clear from the results in Figure 6, H
-EPC liposome has a pH gradient of EPC/CH
The release rate of PGE1 was slower than that of OL liposomes.
【００３６】〔蔗糖密度勾配遠心法〕蔗糖で密度勾配を
設けた中にリポソームを加え、リポソームに封入された
ＰＧ類とフリーのＰＧ類を分離するものである。本法を
適用することにより、限外濾過法では分析が不可能なリ
ポソームのＰＧＥ１保持能に及ぼす蛋白質（例えばヒト
血清アルブミン（ＨＳＡ））の影響も調べることができ
る。本方法は具体的に以下の通りである。超遠心用チュ
ーブ中で０．８Ｍ蔗糖を含むバッファー８００μｌに分
析すべきリポソームサンプル５０μｌを加える。ｐＨを
変えたり、ＨＳＡを加えたりした分析すべきバッファー
８００μｌにリポソームサンプル５０μｌを加え、最終
蔗糖濃度を８００ｍＭに調整する。この液を超遠心用チ
ューブに入れ、この上から５００ｍＭ蔗糖含有２０ｍＭ
クエン酸バッファーを４．１５ｍｌ、静かに重層する。
次に２３０，０００ｇで１６時間、４℃下で超遠心処理
する。超遠心終了後、底部から１ｍｌ／フラクションで
分取する。そして全体のＰＧ類に対する上層３ｍｌのＰ
Ｇ類の割合をもってＰＧ類の封入効率とした。その結果
から明らかなように、ｐＨ４．５のクエン酸緩衝液で希
釈した場合に較べ、ｐＨ７．４のへペス緩衝液で希釈す
ると２種の脂肪小体はともに多少ＰＧＥ１保持率が低下
した。２０％ＨＳＡを含有するへペス緩衝液で希釈する
と、ＥＰＣ／ＣＨＯＬ脂肪小体のＰＧＥ１保持率が３０
．２％であるのに対し、ＤＰＰＣ／ＣＨＯＬ脂肪小体の
ＰＧＥ１保持率は５２．６％と２０％以上高かった。[Sucrose density gradient centrifugation method] Liposomes are added to a density gradient made of sucrose, and PGs encapsulated in the liposomes and free PGs are separated. By applying this method, it is also possible to investigate the influence of proteins (for example, human serum albumin (HSA)) on the PGE1 retention ability of liposomes, which cannot be analyzed by ultrafiltration. This method is specifically as follows. Add 50 μl of the liposome sample to be analyzed to 800 μl of buffer containing 0.8 M sucrose in an ultracentrifuge tube. Add 50 μl of the liposome sample to 800 μl of the buffer to be analyzed with varying pH or adding HSA, and adjust the final sucrose concentration to 800 mM. Pour this solution into an ultracentrifuge tube, and add 20mM containing 500mM sucrose on top.
Gently overlay with 4.15 ml of citrate buffer. It is then ultracentrifuged at 230,000g for 16 hours at 4°C. After ultracentrifugation, collect 1 ml/fraction from the bottom. and P of the upper layer 3 ml for the total PG.
The ratio of G was defined as the encapsulation efficiency of PG. As is clear from the results, the PGE1 retention rate of both types of fat bodies decreased somewhat when diluted with Hepes buffer of pH 7.4, compared to when diluted with citrate buffer of pH 4.5. When diluted with Hepes buffer containing 20% HSA, PGE1 retention in EPC/CHOL fat bodies was 30%.
．． 2%, whereas the PGE1 retention rate in DPPC/CHOL fat bodies was 52.6%, which was more than 20% higher.
【００３７】[0037]
【発明の効果】本発明の脂肪小体組成物は、脂肪小体か
らのＰＧ類の遊離が抑制され、持続性を有するという特
有の効果を有するものである。Effects of the Invention The fat body composition of the present invention has the unique effect of suppressing the release of PGs from fat bodies and having a sustained effect.
【図１】分析に用いた限外濾過の原理図である。FIG. 1 is a diagram of the principle of ultrafiltration used in analysis.
【図２】膜流動性のＰＧＥ１の保持能への影響を示すグ
ラフである。FIG. 2 is a graph showing the influence of membrane fluidity on PGE1 retention ability.
【図３】脂肪小体へのＰＧＥ１の保持能に及ぼす希釈の
影響を示すグラフである。FIG. 3 is a graph showing the effect of dilution on the ability of PGE1 to be retained in fat bodies.
【図４】脂肪小体へのＰＧＥ１の保持能に及ぼす希釈の
影響を示すグラフである。FIG. 4 is a graph showing the effect of dilution on the ability of PGE1 to be retained in fat bodies.
【図５】脂肪小体からのＰＧＥ１の放出性を示すグラフ
である。FIG. 5 is a graph showing the release of PGE1 from fat bodies.
【図６】脂肪小体からのＰＧＥ１の放出性を示すグラフ
である。FIG. 6 is a graph showing the release of PGE1 from fat bodies.
Claims (5)
、ジアシルグリセロリン脂質、ジアシルグリセロ糖脂質
、アルキルグリセロリン脂質、アルキルグリセロ糖脂質
、リゾリン脂質の群から選ばれた少なくとも一種の脂質
を含有し、該脂質がジアシルグリセロリン脂質又はジア
シルグリセロ糖脂質の際には、そのアシル基が飽和脂肪
酸由来であり、アルキルグリセロリン脂質又はアルキル
グリセロ糖脂質の際には、ジアルキル型の場合、その両
方のアルキル基が飽和炭化水素由来であり、アルキルア
シル型の場合、アルキル基が飽和炭化水素由来であり且
つアシル基が飽和脂肪酸由来であり、リゾリン脂質の際
にはアシル型の場合、そのアシル基は飽和脂肪酸由来で
あり、アルキル型の場合、そのアルキル型は飽和炭化水
素由来であることを特徴とするプロスタグランジン含有
脂肪小体組成物。1. Contains at least one type of lipid selected from the group of diacylglycerophospholipids, diacylglyceroglycolipids, alkylglycerophospholipids, alkylglyceroglycolipids, and lysophospholipids as a lipid for forming fat bodies, When the lipid is a diacylglycerophospholipid or a diacylglyceroglycolipid, its acyl group is derived from a saturated fatty acid, and when it is an alkylglycerophospholipid or an alkylglyceroglycolipid, when it is a dialkyl type, both alkyl groups are derived from a saturated fatty acid. It is derived from a saturated hydrocarbon, and in the case of an alkylacyl type, the alkyl group is derived from a saturated hydrocarbon and the acyl group is derived from a saturated fatty acid, and in the case of a lysophospholipid, in the case of an acyl type, the acyl group is derived from a saturated fatty acid. and in the case of an alkyl type, the alkyl type is derived from a saturated hydrocarbon.
アシルグリセロリン脂質である請求項１記載の組成物。2. The composition according to claim 1, wherein the lipid for forming fat bodies is a diacylglycerophospholipid.
請求項１又は請求項２記載の組成物。3. The composition according to claim 1 or 2, wherein the saturated fatty acid has 14 to 22 carbon atoms.
体内封溶液のｐＨを７〜１１に調整された請求項１〜３
のいずれかに記載の組成物。4. Claims 1 to 3, wherein the pH of the liquid outside the fat body is adjusted to 2 to 6, and the pH of the solution inside the fat body is adjusted to 7 to 11.
The composition according to any one of.
シル基が不飽和脂肪酸由来の脂質化合物、アルキル基が
不飽和炭化水素由来である脂質化合物を併用してなる請
求項１〜４のいずれかに記載の組成物。5. As the lipid, a lipid compound in which the acyl group in the lipid compound is derived from an unsaturated fatty acid, and a lipid compound in which the alkyl group in the lipid compound is derived from an unsaturated hydrocarbon are used in combination. Composition of.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3250166A JPH04356421A (en) | 1991-03-06 | 1991-06-26 | Fat spherule composition containing prostaglandins |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6807991 | 1991-03-06 | ||
| JP3-68079 | 1991-03-06 | ||
| JP3250166A JPH04356421A (en) | 1991-03-06 | 1991-06-26 | Fat spherule composition containing prostaglandins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04356421A true JPH04356421A (en) | 1992-12-10 |
Family
ID=26409318
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3250166A Pending JPH04356421A (en) | 1991-03-06 | 1991-06-26 | Fat spherule composition containing prostaglandins |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04356421A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995012388A1 (en) * | 1993-11-04 | 1995-05-11 | The Liposome Company, Inc. | Methods of treatment using unilamellar liposomal arachidonic acid metabolite formulations |
| US5811118A (en) * | 1987-05-22 | 1998-09-22 | The Liposome Company, Inc. | Methods of treatment using unilamellar liposomal arachidonic acid metabolite formulations |
| WO1999016426A3 (en) * | 1997-10-01 | 1999-06-17 | Biomira Usa Inc | Multilamellar coalescence vesicles (mlcv) containing biologically active compounds |
| US5925375A (en) * | 1987-05-22 | 1999-07-20 | The Liposome Company, Inc. | Therapeutic use of multilamellar liposomal prostaglandin formulations |
| EP1523977A1 (en) * | 1997-10-01 | 2005-04-20 | Biomira Usa Inc. | Multilamellar coalescence vesicles (MLCV) containing biologically active compounds |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59222410A (en) * | 1983-06-01 | 1984-12-14 | Terumo Corp | Pharmaceutical preparation of liposome for keeping drug |
| JPS61100518A (en) * | 1984-10-22 | 1986-05-19 | Ono Pharmaceut Co Ltd | Lyophilized ribosome pharmaceutical preparation comprising dimyristoylphosphatidylcholine as main component |
| JPH02196713A (en) * | 1988-09-28 | 1990-08-03 | Yissum Res Dev Co Of Hebrew Univ Of Jerusalem | Liposome filling amphipathic particle effectively and discharging controllably |
| JPH02504027A (en) * | 1987-05-22 | 1990-11-22 | ザ リポソーム カンパニー，インコーポレイテッド | Prostaglandin-lipid preparations |
| JPH04503953A (en) * | 1988-12-29 | 1992-07-16 | ベンソン，ブラッドリー ジェイ． | Pulmonary administration of pharmaceutically active substances |
-
1991
- 1991-06-26 JP JP3250166A patent/JPH04356421A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59222410A (en) * | 1983-06-01 | 1984-12-14 | Terumo Corp | Pharmaceutical preparation of liposome for keeping drug |
| JPS61100518A (en) * | 1984-10-22 | 1986-05-19 | Ono Pharmaceut Co Ltd | Lyophilized ribosome pharmaceutical preparation comprising dimyristoylphosphatidylcholine as main component |
| JPH02504027A (en) * | 1987-05-22 | 1990-11-22 | ザ リポソーム カンパニー，インコーポレイテッド | Prostaglandin-lipid preparations |
| JPH02196713A (en) * | 1988-09-28 | 1990-08-03 | Yissum Res Dev Co Of Hebrew Univ Of Jerusalem | Liposome filling amphipathic particle effectively and discharging controllably |
| JPH04503953A (en) * | 1988-12-29 | 1992-07-16 | ベンソン，ブラッドリー ジェイ． | Pulmonary administration of pharmaceutically active substances |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5811118A (en) * | 1987-05-22 | 1998-09-22 | The Liposome Company, Inc. | Methods of treatment using unilamellar liposomal arachidonic acid metabolite formulations |
| US5925375A (en) * | 1987-05-22 | 1999-07-20 | The Liposome Company, Inc. | Therapeutic use of multilamellar liposomal prostaglandin formulations |
| WO1995012388A1 (en) * | 1993-11-04 | 1995-05-11 | The Liposome Company, Inc. | Methods of treatment using unilamellar liposomal arachidonic acid metabolite formulations |
| WO1995012389A1 (en) * | 1993-11-04 | 1995-05-11 | The Liposome Company, Inc. | Multilamellar liposomal arachidonic acid metabolite formulations |
| WO1999016426A3 (en) * | 1997-10-01 | 1999-06-17 | Biomira Usa Inc | Multilamellar coalescence vesicles (mlcv) containing biologically active compounds |
| US6544549B1 (en) | 1997-10-01 | 2003-04-08 | Biomira Usa Inc. | Multilamellar coalescence vesicles (MLCV) containing biologically active compounds |
| EP1523977A1 (en) * | 1997-10-01 | 2005-04-20 | Biomira Usa Inc. | Multilamellar coalescence vesicles (MLCV) containing biologically active compounds |
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