JP3759765B2 - Liposome - Google Patents

Description

【００７６】
実施例２：イオジキサノールの封入
実施例１に記載の方法に従って、イオジキサノールを含有するリポソーム製剤を調製した。結果を表２に示す。
【００７７】
【表２】

実施例３．
実施例１で得られたリポソーム懸濁液を等張グルコースにより希釈して５０mg封入ヨウ素／mlの濃度とした。希釈の後、懸濁液は乳白色に変わった。この乳懸液の顕微鏡分析の際にリポソームの凝集体は観察されなかった。乳濁液を１年間貯蔵した後、封入されたイオジキサノールの量及びサイズ分布への影響は観察されなかった。
【００７８】
実施例４．
実施例３の乳濁液をラットに静脈内注射した。封入されたイオジキサノールの大部分が、注射後に肝臓に分布することを確認したが、脾臓にも高濃度のイオジキサノール濃度が検出された。注射後、組織に分布したイオジキサノールは時間と共に減少し、最終的にはこれら２つの器官中にイオジキサノールは見られなくなった。他のすべての器官のイオジキサノールのレベルは血中イオジキサノールのレベルと平行して低下した。注射されたイオジキサノールの大部分は尿中に回収された。
【００７９】
実施例５．
実施例３の乳濁液を多数の肝癌転移を有するラットに静脈内注射した。封入ヨウ素５０mg及び１００mg／kgの投与量において、それぞれ４２及び６２HUCT値のＸ線減衰が肝臓の正常領域で観察されたが、腫瘍転移部での減衰への影響は最小であった。肉視的観察により、検出された腫瘍が直径５mm未満であることが示された。
【００８０】
実施例６．
実施例３の乳濁液を、封入ヨウ素２００〜７５００mg／kg体重の投与量においてマウスの群に静脈内注射した。体重増加及び死亡を１４日間にわたり追跡した。分析の結果により、すべての動物は、リポソーム封入イオジキサノール３ｇ／kgの注射に対して生存したことが示された。これらの動物は、注射されなかった動物に比べて、観察期間にわたり体重増加の鈍化を示さなかった。リポソーム封入イオジキサノール４ｇ及び５ｇ／kgの投与量において、体重増加のわずかな投与量依存的減少が存在し、そして死が観察された。ＬＤ₅₀はリポソーム封入イオジキサノール５ｇ／kgと推定された。
【００８１】
実施例７．
ラットでの封入ヨウ素１００〜１０００mg／kgの投与量での実施例１の乳濁液の１回の又は反復（３注射／週、３週間）静脈内注射の後、種々の組織結合酵素の血中レベルを測定し、そしてすべての主要器官の組織を種々の時点で分析した。血清酵素について、食塩水を注射した対照動物と比べてわずかな効果のみが観察された。肝臓及び脾臓中の貪食細胞の投与量依存的な空胞変性の増加とは別に、リポソームによる組織学的変化は検出されなかった。貪食細胞の空胞変性の程度は、肝臓及び脾臓に分布するイオジキサノールの減少と平行して低下した。
【００８２】
実施例８．
次の成分：
イオジキサノール（全量） ４００mg／mL
封入されたヨウ素 ８０mg／mL
ソルビトール ２０mg／mL
トロメタモール（ＴＲＩＳ） ０．０９７mg／mL
ＥＤＴＡＮａ₂ Ｃａ ０．１mg／mL
水素添加ホスファチジルコリン ５１．２mg／mL
水素添加ホスファチジルセリン ５．１mg／mL
注射用水（添加して右の量にする） １mL（約０．９mL）
を含んで成る診断組成物。
【００８３】
方法
リン脂質をクロロホルム／メタノール／水（容量比（４：１：０．０２５）に溶解し、そして溶剤を蒸発させた（ロータリーエバポレーション）。イオジキサノール及びソルビトールの等張液を作り、そして６０〜７０℃に加熱し、そしてその後の工程の間この温度を維持した。リン脂質混合物を撹拌しながら加え、そしてリポソームを形成した。リポソームのサイズを調整するため、この調製物をホモジナイズした（Ｒｏｔｏｒ／Ｓｔａｔｏｒホモジナイザー）。次に、リポソームを、直列に配置された７枚のポリカーボネートフィルター（孔直径１μｍ）を通して押出した。
生成物を、イオジキサノール及びソルビトールの等張溶液で希釈し、そしてトロメタモール及びＥＤＴＡを添加した。生成物を濾過してガラスバイアルに入れ、そして１２１℃にて１５分間オートクレーブ滅菌した。
【００８４】
実施例９．イオトロラン含有リポソーム
イオジキサノールの代りにイオトロラン（ｉｏｔｒｏｌａｎ）を用い、実施例８に記載したのと同様にしてリポソームを調製した。
実施例１０．水素添加ホスファチジルグリセロールによるイオジキサノール含有リポソーム
水素添加ホスファチジルセリンの代りに水素添加ホスファチジルグリセロールを使用し、実施例８に記載したのと同様にしてリポソームを調製した。
【００８５】
実施例１１．ＭＲＩ造影剤（ガドリニウム）
水素添加ホスファチジルコリン及び水素添加ホスファチジルセリンの脂質フィルムを、実施例１に記載したようにして調製する。１０mlのＧｄ ＨＰＤＯ３Ａの０．５Ｍ溶液を６５℃に加熱する。この混合物を６５℃で加熱しながらミキサーにより１５分間攪拌する。この混合物を、孔サイズ１．０μｍのポリカーボネート膜フィルターを通して加圧下に濾過して多重層リポソーム（ＭＬＶ）を調製する。ＭＬＶをガラスバイアルに入れ、そして１２１℃にて２０分間オートクレーブ滅菌する。ＭＬＶはＧｄ ＨＰＤＯ３Ａを含有している。
【００８６】
実施例１２．ＭＲＩ造影剤（マンガン）
Ｇｄ ＨＰＤＯ３Ａの代わりに３，６−ビス〔Ｎ−（２，３−ジヒドロキシプロピル）−Ｎ−メチルカルバモイルメチル〕−３，６−ジアザスベリン酸のマンガンキレートの溶液（０．０７Ｍ）（ＷＯ ９３／２１９６０（Ｎｙｃｏｍｅｄｌｍａｇｉｎｇ ＡＳ）に従って調製）を用いて実施例１１に従ってリポソームを調製する。ＭＬＶはマンガンキレートを含有する。
【００８７】
実施例１３．ＭＲＩ造影剤（ジィスプロシウム）
Ｇｄ ＨＰＤＯ３Ａの代わりにＤｙ ＤＴＰＡ−ＢＭＡの溶液（０．５Ｍ）を用いて実施例１１に従ってリポソームを調製する。ＭＬＶはＤｙ ＤＴＰＡ−ＢＭＡを含有する。
【００８８】
実施例１４．ＭＲＩ造影剤（酸化鉄）
Ｇｄ ＨＰＤＯ３Ａの代わりに磁性酸化鉄の懸濁液（１０mM、粒子サイズ１０〜３０nm）を用いて実施例１１に従ってリポソームを調製する。ＭＬＶは磁性酸化鉄を含有する。
【００８９】
実施例１５．リポソーム性Ｇｄ ＨＰＤＯ３Ａ（ＨＥＰＣ−ＨＥＰＳ）
卵黄由来の水素添加ホスファチジルコリン（ＨＥＰＣ）６４０mgと実施例１に記載したようにしてＨＥＰＣから合成した水素添加ホスファチジルセリン（ＨＥＰＳ）６４mgとの前調製混合物を、２５０mM Ｇｄ ＨＰＤＯ３Ａ（ガドテリドール）を含有するグルコースの５％水溶液（等張溶液）１０mlを収容したバイアルに加えた。この混合物を６５℃にて３０分間撹拌し、そしてさらに１時間、この温度に保持した。このリポソーム溶液を５回凍結−融解処理し、そして６５℃にて２枚重ねの４００nmポリカーボネートフィルターを通して５回押出し、標記生成物を得た。
【００９０】
実施例１６．リポソーム性Ｇｄ ＨＰＤ３０Ａ（ＤＰＰＣ／ＤＰＰＧ）
ジパルミトイルホスファチジルコリン（ＤＰＰＣ）６４０mgと実施例１に記載したようにして調製したジパルミトイルホスファチジルグリセロール（ＤＰＰＧ）との脂質ブレンドを、２５０mM Ｇｄ ＨＰＤＯ３Ａを含有するグルコースの５％水溶液（等張液）１０mlを収容したバイアルに加えた。この混合物を５０℃にて３０分間撹拌し、そしてさらに１時間この温度で保持した。リポソーム溶液を５回凍結−融解処理し、そして５０℃にて２枚重ねの４００nmポリカーボネートフィルターを通して５回押出した。
【００９１】
実施例１７．リポソーム性Ｇｄ ＤＴＰＡ−ＢＭＡ
水溶液がＧｄ ＨＰ−ＤＯ３Ａの代りに２５０mM Ｇｄ ＤＴＰＡ−ＢＭＡ（ガドジアミド）を含有した他は、実施例１５と同じ方法を用いた。
実施例１８．リポソーム性Ｄｙ ＴＴＨＡ
溶液がＧｄ ＨＰ−ＤＯ３Ａの代りに２５０mM Ｄｙ ＴＴＨＡ（トリエチレンテトラミン六酢酸のジスプロシウム錯体）を含有した他は、実施例１５と同じ方法を用いた。
【００９２】
実施例１９．超音波造影剤
Ｇｄ ＨＰＤＯ３Ａの代わりに食塩水を用いて実施例８に従ってリポソームを調製する。混合物を窒素により１時間加圧する（７０psi)。過剰の窒素を除去し、そして生ずる混合物を、１．０μｍの孔サイズを有するポリカーボネート膜フィルターを通して濾過する。得られるＭＬＶは窒素ガスを含有する。
実施例２０．
実施例８の分散液をラットに注射（ｉ．ｖ）したところ、このラットは７５mgＩ（封入されたもの）／kgの４７ΔＨＵ及び１００mgＩ（封入されたもの）／kgの７０ΔＨＵにおいて、肝臓でＸ線減衰を与えた。
実施例２１．
実施例８の分散液を雄性及び雌性のラットに注射した。ラットにおける静脈投与のおよそのＬＤ５０は２０００mgＩ（封入されたもの）／kg体重であった。[0001]
[Industrial application fields]
The present invention relates to a diagnostic composition for injection comprising a liposome encapsulating one or more contrast agents.
[0002]
[Prior art]
Contrast agents are used in various fields of imaging to enhance image contrast, the most important of which are X-ray imaging, magnetic resonance imaging (MRI), ultrasound imaging and It is a nuclear medicine image (Nuclear Medicine Imaging) diagnosis.
[0003]
In X-ray diagnostic imaging including applications such as computed tomography (CT) and digital subtraction angiography (DSA), contrast is based on the difference in electron density. Currently used X-ray contrast agents are generally based on heavy elements, such as barium salts such as barium sulfate that can be used to enhance gastrointestinal visualization, gastrointestinal visualization and non- An iodine-based contrast agent that can be used for oral administration can be mentioned.
[0004]
Iodine X-ray contrast agents most commonly contain a phenyl iodide group and are typically carboxyl, carbamoyl, N-alkylcarbamoyl, N-hydroxyalkylcarbamoyl, acylamino, N-position at the 3- and / or 5-positions. -Having at least one 2,4,6-triiodophenyl group having a group selected from the group consisting of alkylacylamino and acylaminomethyl. This type of ionic X-ray contrast agent includes metrizoic acid, diatriazoic acid, iotalamic acid, oxaglic acid and salts of these acids.
[0005]
Nonionic iodinated contrast agents that are generally substantially less toxic than ionic contrast agents due to low osmotic pressure and consequently less hemodynamic effects include iohexol, iopentol, iopamidol, iodixanol, iopromide, iotrolan, and metrisamide Is mentioned. For example, so-called dimers such as iodixanol and iotrolane are included therein, and they can be formulated to be isotonic with blood at concentrations exceeding 300 mg I / ml by their isotonicity.
[0006]
Recently, parenteral X-ray contrast agents based on heavy metal clusters / chelates have attracted attention. See, for example, WO-A-9114460 and WO-A-9217215.
From a hydrophilic point of view, all of the aforementioned X-ray contrast agents have approximately the same extracellular biodistribution and thus exhibit similar clinical indications and are excreted in the kidney. Accordingly, attempts have been made to find an organ-specific contrast agent. For example, an iodinated phenyl group was bound to a polymer support such as starch in the hope of improving the blood half-life of iodine-based contrast agents.
[0007]
Possible liver contrast agents based on in vivo degradable particles have been proposed (see for example WO-A-8900988 and WO-A-9007491), ionic or non-ionic X-ray contrast agents Have been proposed (see below). Such formulations have not yet been marketed due to stability and toxicity issues, and have not yet reached the late clinical development stage, so the need for more stable, non-toxic and organ-specific X-ray contrast agents is still met. It has not been.
[0008]
The main contrast parameter of MRI that can be manipulated by contrast agents is the spin-lattice relaxation time (T₁ ) And spin-spin relaxation time (T₂ ). For example, paramagnetic chelates based on manganese (2+), gadolinium (3+) and iron (3+) have spin-lattice relaxation times (T₁ ), Thereby increasing the signal intensity. MRI contrast agents based on magnetic / superparamagnetic particles are spin-spin relaxation times (T₂ ) And causes a decrease in signal intensity.
[0009]
Large doses of dysprosium-based paramagnetic chelates and paramagnetic compounds will also reduce MR signal intensity. For a review of MRI contrast agents, some of which are in development or commercially available, see, for example, D.D.Stark and W.G.Bradley: Magnetic Resonance Imaging, Mosby 1992, Chapter 14.
[0010]
Hydrophilic chelates such as GdDTPA, GdDOTA, GdHPDO3A and GdDTPA-BMA are distributed extracellularly and excreted in the kidney. Such compounds are useful, for example, for visualizing lesions of the central nervous system. Other examples of organ- or tissue-specific drugs include MnDPDP, GdBOPA, GdEOB-DTPA, paramagnetic porphyrins, polymer compounds, granules and liposomes.
[0011]
Therefore, various paramagnetic metal ions and chelates are encapsulated in the liposome. For example, small unilamellar liposomes (SUV), large unilamellar liposomes (LUV) and multilamellar liposomes (MLV) with various lipid compositions, surface charges and sizes have been proposed as MRI contrast agents [eg, SESeltzer, Radiology171(1989) p. 19; S.E.Seltzer et al., Invest. Radiol.twenty three(1988) P.131; C. Tilcock et al., Radiology171(1989) p. 77; C. Tilcock et al., Biochim Biophys. Acta10222(1990) p.181; E.C.Unger et al., Invest. Radiol.twenty five(1990) p. 638; E.C. Unger et al., Invest.twenty three(1988) p.928; E.C.Unger et al., Radiology171(1989) p.81; E.C.Unger et al., Magn.Reson.Imaging7(1989) p.417 and J.Vion-Dury et al., J.Pharmacol.Exp.Ther.250(1989) p.1113]. However, despite a wealth of reports on liposomal MRI contrast agents, no such product is commercially available today and is not in late clinical development.
[0012]
Ultrasound image diagnosis is based on, for example, penetrating ultrasonic waves in a frequency range of 1 to 10 MHz into a human or animal subject through a transducer, and the ultrasonic waves interact with an interface of body tissue or body fluid. Yes. Ultrasound image contrast results from the differential reflection / absorption of sound waves at such interfaces. Results can be doubled using Doppler techniques including the use of Color Doppler to assess blood flow.
[0013]
It has long been understood that it would be advantageous to use contrast agents to amplify the differences in the acoustic properties of different tissues / fluids, and since indocyanine green was used in 1968 as the first ultrasound contrast agent Many other contrast-inducing agents have been tested. They include emulsions, solid granules, water-soluble compounds, free bubbles and various types of encapsulated gas-containing systems. It is generally accepted that low density contrast agents that are easily compressible in terms of the acoustic backscatter that the contrast agent produces are particularly effective. Thus, gas containing systems and gas generating systems tend to exhibit significantly greater efficacy than other types of contrast agents.
[0014]
Three ultrasound contrast agents are currently on the market or are in late clinical development. They include Echovist ™ based on gas-containing galactose microcrystals, Levovist ™ comprising gas-containing galactose microcrystals coated with fatty acids, and bubbles encapsulated by partially denatured human serum albumin Infoson ™. However, the clinical use of these agents is limited due to the short contrast half-life (ie relative loss of stability in vivo) and / or limited storage stability.
[0015]
Therefore, ultrasound contrast agents that combine excellent storage stability and in vivo stability (preferably at least several circulation passes in the case of cardiac and non-cardiac perfusion analysis), especially cardiac and non-cardiac perfusion studies There continues to be a need for ultrasound contrast agents for use.
[0016]
[Problems to be solved by the invention]
Despite the numerous publications that suggest the use of liposomal formulations and other techniques to increase the tissue or organ specificity of various types of contrast agents, this type of product that is either commercially available or in late clinical development There is currently no one. This is believed to be the result of problems related to low encapsulation rates, toxicity, poor storage stability and / or in vivo instability or short contrast half-life. Accordingly, there remains a need for contrast agents that solve these problems. In the field of ultrasound imaging, to contrast agents that exhibit sufficiently high in vivo stability for use in cardiac and non-cardiac perfusion, and formulations that are stable enough to allow efficient liver imaging. There are special requirements.
[0017]
In general, lipid soluble drugs are easily encapsulated in liposomes. On the other hand, water-soluble electrolytes among water-soluble drugs are due to the interaction between the charge of the drug and the charge of the charged lipid (JP-A-2-187370) or due to the pH gradient between the outside and inside of the liposome (WPI 88-272022 / 38). It can be encapsulated in the internal aqueous phase of the liposome.
[0018]
However, even if the above method is used, the amount of the encapsulated drug is not so large. In particular, when the drug is a water-soluble non-electrolyte substance, the above-mentioned means cannot be applied, and therefore it is not easy to efficiently encapsulate the water-soluble non-electrolyte in the liposome. As a means for efficiently encapsulating water-soluble non-electrolytes in the internal aqueous phase of liposomes, the reverse phase evaporation method (Proc. Natl. Acad. Sci. USA, 75 (9), 4194, 1978), ether An injection method (Id., 443, 629, 1975) was proposed.
[0019]
However, since such a method uses an ether having a low ignition point, it cannot be used for industrial production of a large amount of liposomes. Furthermore, the liposomes produced by these methods are mainly single membrane liposomes. Compared to multilamellar liposomes, unilamellar liposomes have the disadvantage of low stability in blood.
[0020]
International patent application W88 / 09165 describes an injectable liposome preparation comprising an X-ray contrast agent solution in liposomes and a buffered saline continuous phase in which the liposomes are suspended. Liposomes are formed in an aqueous medium containing an X-ray contrast agent, but for injection they must be isolated by centrifugation and resuspended in buffer. It would be very convenient if the liposome preparation could be autoclaved to prepare a sterile composition for injection. Preparing a formulation from the individual components of the mixture under aseptic conditions is expensive and unreliable. The liposome suspension can also be sterilized by filtration, but such a sterilization method reduces the particle size of the liposome, and as a result, the retention rate is also lowered.
[0021]
Furthermore, the autoclave treatment of the liposome suspension was not successful because it has a phase transition temperature (Tc) much lower than the autoclave temperature (121 ° C.). That is, the present inventors show that when a liposome preparation such as the liposome preparation of WO88 / 09165 having an X-ray contrast agent only inside the liposome is autoclaved, the X-ray contrast agent comes out of the liposome. I found it. We have also described the above by autoclaving the liposomes in the continuous phase such that the concentration of dissolved X-ray contrast agent outside the liposomes is substantially the same as that of the inner aqueous phase of the lipid layer. Found that the problem can be avoided. Such autoclave sterilization is usually performed in a sealed container that is maintained after cooling and storage until opened just prior to injection.
[0022]
The present invention also uses neutral lipids and charged lipids to form liposome membranes, and keeps the average particle size of the liposomes relatively small, for example, in the 50-300 nm size range, thereby increasing encapsulation efficiency, toxicity, Based on the knowledge that liposomal contrast agents can be improved for use in imaging in terms of manufacturing process and / or product lifetime. The liposomes carry a net negative charge or a net positive charge that provides electrostatic repulsion between the liposome membranes resulting in a high entrapment capacity of, for example, 5 ml / g or more.
[0023]
According to one aspect of the present invention, we provide a diagnostic composition for administration to a human or animal containing multilamellar liposomes and containing at least one contrast agent suspended in an aqueous medium. Wherein the composition may optionally co-exist with single membrane liposomes, the liposomes consisting of neutral phospholipids and charged phospholipids, the liposomes having an average particle size of 50-3000 nm, and Provided is a diagnostic composition characterized in that the concentration of contrast agent in the aqueous phase filling the interior of the liposome is the same as that in the aqueous medium in which the liposome is suspended.
[0024]
Accordingly, the composition of the present invention comprising liposomes filled with fluid is a common blood pool imaging requirement as a systemic contrast agent together with liposomes comprising a contrast agent that collects in the liver and spleen and aids in the imaging of these. An external continuous phase of a conventional contrast medium satisfying
The liposome suspension of the present invention has a high encapsulation capacity in the internal aqueous phase (eg, 5 ml / g or more, preferably 6 ml / g or more) and can therefore retain a large amount of water-soluble drug, And it is stable in blood and on storage and against autoclave sterilization.
[0025]
The composition of the present invention used for X-ray imaging diagnosis can incorporate, for example, any of the iodine-based X-ray contrast agents and heavy metal cluster / chelate X-ray contrast agents known in the art, for example, as described above.
The iodine-based X-ray contrast agent in the composition of the present invention preferably contains 3 or more iodine atoms per molecule, and usually contains one or more iodinated phenyl groups. Nonionic contrast agents, particularly so-called nonionic dimers that are highly hydrophilic and produce low osmotic pressure even at high concentrations, such as iodixanol and iotrolane, are preferred. The concentration of the contrast agent in the composition can vary widely and will be influenced by factors such as the nature of the contrast agent, the intended route of administration of the final composition and clinical indicators.
[0026]
Typical concentrations will be in the range of, for example, 10-300 mg of encapsulated iodine (preferably 60-180 mg, more preferably 70-110 mg) per ml of composition. The concentration of total iodine per ml of composition is preferably 40 to 450 mg, more preferably 160 to 320 mg. The compositions of the invention are usually administered intravenously, and their dosage can vary widely as well, eg, depending on clinical indicators, but in general, typical doses for vascular or liver imaging are, for example, It can be in the range of 5-300 mg iodine per kg body weight.
[0027]
The heavy metal cluster / chelate in the liposomal formulation of the present invention preferably comprises at least two metal atoms having an atomic number greater than that of iodine. Representative contrast agents of this type include, for example, tungsten clusters / chelates described in WO-A-9114460 and WO-A-9217215. Nonionic clusters / chelates would be preferred.
[0028]
The composition of the present invention for MR imaging can contain, for example, any of the paramagnetic agents known in the art, such as those described above. Such paramagnetic contrast agents can be encapsulated in, for example, vesicles or covalently bound, or can be incorporated non-covalently into lipid membranes.
[0029]
Paramagnetic contrast agents can be, for example, physiologically acceptable paramagnetic metal salts or chelates, or can contain free radicals, preferably free radicals of the nitroxide type. Manganese (2+) salts are preferred when the paramagnetic agent is a free metal ion. The chelates are preferably based on manganese (2+), gadolinium (3+), dysprosium (3+) or iron (3+), and chelates as described in the literature (see for example WO-A-9110645) Agents such as NTA, EDTA, HEDTA, DTPA, DTPA-BMA, BOPTA, TTHA, NOTA, DOTA, DO3A, HP-DO3A, EOB-DTPA, TETA, HAM, DPDP, porphyrins and their derivatives can be included. .
[0030]
The concentration of such paramagnetic agent in the composition can be, for example, in the range of 1 mM to 0.5M. The MR imaging composition of the present invention is usually administered intravenously, and a typical dose would be, for example, 0.02 mmol of encapsulated metal chelate per kg body weight. As above, concentrations and doses will be influenced by factors such as the nature of the paramagnetic agent, the intended route of administration and clinical indicators.
[0031]
Liposomal formulations for MR imaging can also include superparamagnetic or ferromagnetic agents, such as those known in the art, eg, by encapsulation. Preferred agents of this type include magnetite, γ-Fe₂ O_Three , Mixed ferrites, and other iron-containing compounds with magnetism, including organic ferromagnetic materials. The encapsulated agent can be free or coated with, for example, dextran, fatty acids or other biologically harmless compounds known to stabilize magnetic materials. If the product is for parenteral use, the coating film must be degraded in vivo. The agent is conveniently in the form of an aqueous suspension of particles having a particle size in the range of 4 nm to 1 μm, preferably 4 to 40 nm.
[0032]
The concentration of such a diagnostic agent can be, for example, in the range of 0.01 mM to 0.1 M, and the dose will be affected by the nature of the drug, its biodistribution and clinical indicators. Typical doses for vascular imaging (including perfusion) or liver imaging can be, for example, in the range of 0.1-100 μmol iron encapsulated per kg body weight when administered intravenously.
Liposome compositions for ultrasound imaging can be used in any type of ultrasound technology, including Doppler technology. Such compositions preferably comprise liposomes in which a physiologically acceptable gas is encapsulated or liposomes in which a gas precursor is encapsulated or covalently bound.
[0033]
In general, any biocompatible gas can be present in the ultrasonic composition according to the present invention, for example air, nitrogen, oxygen, hydrogen, nitrous oxide, carbon dioxide, or more preferably water insoluble gas. For example, helium, argon, sulfur hexafluoride, and optionally low molecular weight hydrocarbons that may be fluorinated, such as perfluoroalkanes (C₂ ~ C₇ ), Acetylene or carbon tetrafluoride or mixtures thereof can be present. As used herein, the term “gas” includes any substance that is in gaseous form at a normal body temperature of 37 ° C., and thus a liquid boiling at low temperatures, such as diethyl ether or certain perfluoroalkanes. Include.
[0034]
Gas precursors include aminomalonates; carbonates and bicarbonates such as lithium carbonate, lithium bicarbonate, sodium carbonate, sodium bicarbonate, ammonium carbonate, calcium carbonate and magnesium bicarbonate; physiologically acceptable diazonium Compound; CO-O-CR¹ R² -O-CO-OR^Three Carbonate esters containing groups of the type; and β-keto acids.
[0035]
They can react in various ways to produce gas-containing liposomes. For example, carbonates and bicarbonates can generate carbon dioxide in vivo with an acidic pH value that is common in the body after administration; diazonium compounds can generate nitrogen, for example, when irradiated with UV light; Carbonate esters interact with nonspecific esterases in vivo resulting in the generation of carbon dioxide; β-keto acids will spontaneously decarboxylate.
[0036]
The ultrasound composition according to the present invention can be administered, for example, orally or parenterally, but may be advantageous for specific applications in direct administration into body cavities such as the fallopian tube. In general, however, intravascular administration by intravenous injection will probably be most commonly used to enhance vascular imaging, including heart and extracardiac perfusion. Because of its stability, most of the compositions so administered will eventually undergo uptake by the reticuloendothelial cell line, primarily into the liver, and provide good liver ultrasound contrast enhancement.
[0037]
Ease of compressibility is a desirable property of low density ultrasound contrast agents. Since the liposomes in the compositions of the present invention comprise an essentially non-solid matrix, they will exhibit a considerable degree of flexibility. This will thus increase compressibility and thereby increase the echogenicity of the gas filled ultrasound composition of the present invention.
[0038]
One essential component for membrane formation of the liposomes of the invention is a neutral phospholipid comprising a substantially saturated fatty acid residue. The number of carbon atoms in the fatty acid residue is usually at least 14, preferably at least 15, more preferably at least 16. When the number of carbon atoms in the fatty acid residue is less than 14, the ability of the liposome to accommodate the internal aqueous phase is low, and the stability of the liposome in blood after administration is also low. On the other hand, usually, when the number of carbon atoms in the fatty acid residue is 28 or more, biocompatibility is lowered, and a very high temperature is required during the production of liposomes.
[0039]
The term “substantially saturated” means that the fatty acid residues in the neutral phospholipids used are completely saturated (no CC double bonds are present) or have a degree of unsaturation. Means very low. For neutral phospholipids consisting of substantially saturated fatty acid residues, the degree of unsaturation indicated by the iodine value is 20 or less, and preferably the iodine value is 10 or less. If the degree of unsaturation is too high, the retention will be low and the liposomes will be easily oxidized and difficult to heat sterilize.
[0040]
Neutral phospholipids used in the present invention are, for example, neutral glycerophospholipids, such as partially or fully hydrogenated natural, such as soybean or egg yolk, or synthetic phosphatidylcholines, especially semi-synthetic or synthetic dipalmitoyl Preferred examples include phosphatidylcholine (DPPC) or distearoyl phosphatidylcholine (DSPC).
[0041]
Another essential component for membrane formation of the liposomes of the present invention is a charged phospholipid comprising a substantially saturated fatty acid residue. The number of carbon atoms in the fatty acid residue is usually at least 14, preferably at least 15, more preferably at least 16. When the number of carbon atoms in the fatty acid residue is less than 14, the ability of the liposome to accommodate the internal aqueous phase is low, and the stability of the liposome in blood after administration is also low. On the other hand, usually, when the number of carbon atoms in the fatty acid residue is 28 or more, biocompatibility is lowered, and a very high temperature is required during the production of liposomes.
[0042]
The term “substantially saturated” means that the fatty acid residues of the charged phospholipid used are completely saturated (no CC double bonds are present) or have a very high degree of unsaturation. Means low. For charged phospholipids composed of fatty acid residues that are substantially saturated, the degree of unsaturation indicated by the iodine value is 20 or less, and preferably the iodine value is 10 or less. If the degree of unsaturation is too high, the retention will be low and the liposomes will be easily oxidized and difficult to heat sterilize.
[0043]
The charged phospholipid used in the present invention is, for example, a glycerophospholipid that is positively or negatively charged. Negatively charged phospholipids include, for example, phosphatidylserine, such as partially or fully hydrogenated natural, eg, soy or egg yolk, or semisynthetic phosphatidylserine, especially semisynthetic or synthetic dipalmitoyl phosphatidylserine. (DPPS) or distearoylphosphatidylserine (DSPS);
[0044]
Phosphatidylglycerols, such as partially or fully hydrogenated natural (eg from soy or egg yolk) or semisynthetic phosphatidylglycerols, especially semisynthetic or synthetic dipalmitoyl phosphatidylglycerol (DPPG) or distearoyl phosphatidylglycerol (DSPG) Phosphatidylinositol, eg partially or fully hydrogenated natural (eg derived from soy or egg yolk) or semi-synthetic phosphatidylinositol, in particular semi-synthetic or synthetic dipalmitoyl phosphatidylinositol (DPPI) or distearoyl phosphatidylinositol ( DSPI);
[0045]
Phosphatidic acid, such as partially or fully hydrogenated natural (eg from soy or egg yolk) or semi-synthetic phosphatidic acid, especially semi-synthetic or synthetic dipalmitoyl phosphatidic acid (DPPA) or distearoyl phosphatidic acid (DSPA) Is mentioned. Such charged phospholipids are usually used alone, but two or more kinds may be used. However, when two or more kinds are used, it is desirable from the viewpoint of preventing aggregation of liposomes to use between negatively charged phospholipids or between positively charged phospholipids.
[0046]
Preferred examples of the positively charged phospholipid include an ester of phosphatidic acid and amino alcohol, such as an ester of dipalmitoyl phosphatidic acid or distearoyl phosphatidic acid and hydroxyethylenediamine.
According to the present invention, the weight ratio of neutral phospholipid to charged phospholipid is usually 200: 1-3: 1, preferably 60: 1-4: 1, more preferably 40: 1-5: 1. For example, about 10: 1.
[0047]
The liposome of the present invention can contain various optional compounds in addition to the two essential components described above. For example, vitamin E (α-tocopherol) and / or vitamin E acetate as an antioxidant can be added in an amount of 0.01 to 2 mol%, preferably 0.1 to 1 mol% with respect to the total amount of lipid. .
In the diagnostic composition comprising the liposome of the present invention comprising such a lipid, the total lipid concentration is generally 20 mg / ml to 100 mg / ml, preferably 40 mg / ml to 90 mg / ml, more preferably 50 mg / ml to 80 mg. / Ml is preferable from the viewpoint of improving the retention rate of the contrast agent in the liposome.
[0048]
The aforementioned contrast agent is preferably encapsulated in the liposome in the form of an isotonic solution or suspension (in terms of physiological osmotic pressure in the body) so that the liposome is stably maintained in the body after administration. As the medium, water, a buffer solution such as Tris-HCl buffer solution, phosphate buffer solution, citrate buffer solution, or the like can be used.
[0049]
The preferred pH range at room temperature is 6.5 to 8.5, more preferably 6.8 to 7.8. When the X-ray contrast agent is a non-ionic contrast agent bearing multiple hydroxyl groups, such as iohexol, iodixanol or iopamidol, the buffer is preferably a negative temperature coefficient, as described in US Pat. No. 4,278,654. It is what has. The amine buffer has the required properties and is preferably TRIS. This type of buffer has a low pH at the autoclave temperature, which increases the stability of the X-ray contrast agent in the autoclave while returning to a physiologically more acceptable pH at room temperature.
[0050]
To obtain an isotonic solution or suspension, the contrast agent is dissolved or suspended in the medium at a concentration that provides the isotonic solution. For example, if the contrast agent alone cannot provide an isotonic solution due to the poor solubility of the contrast agent, other non-toxic water-soluble substances such as salts such as sodium chloride, mannitol, glucose, etc. are formed so that an isotonic solution is formed. Sugars such as sucrose and sorbitol can be added to the medium.
[0051]
As mentioned above, one advantage of the composition of the present invention is that it withstands autoclaving. They have good encapsulation capacity and encapsulation rate due to their lipid composition.
Moreover, as will be described later, the diagnostic composition comprising the liposome of the present invention has an excellent contrast effect and is also excellent in terms of side effects.
[0052]
Next, the manufacturing method of the liposome of this invention is demonstrated. The liposome of the present invention can be produced by a conventional method used for the formation of multilamellar liposomes. As those methods, the Bangham method (J. Mol. Dial.13, 238-252, 1965), polyhydric alcohol method (Japanese Patent Publication 4-36734), lipid dissolution method (Japanese Patent Publication 4-36735), and mechanochemical method (Japanese Patent Publication 4-28412).
[0053]
In general, the phospholipid as described above is dissolved in a volatile organic solvent such as chloroform, methanol, dichloromethane, ethanol, or a mixed solvent of the organic solvent and water, and then the solvent is removed. The target multilamellar liposome can be produced by mixing with an aqueous phase containing an agent and shaking or stirring. Regarding the solvent removal step in the production method, when evaporation is used as a removal means, the production method becomes a bangham method, and when spray drying is used, the production method becomes a spray drying method, and further freeze-drying. It is also possible to use.
[0054]
In the liposome production method as described above, the amount of the solvent used for the lipid is not particularly limited as long as it is an amount capable of sufficiently dissolving the lipid. In order to remove the solvent from the resulting mixture of lipid and solvent by evaporation, the solvent may be evaporated in a conventional manner, specifically under reduced pressure, and optionally in an inert gas atmosphere. Specific examples of the solvent used include volatile organic solvents as described above and mixed solvents of 10 parts by volume of the organic solvent and at most 1 part by volume of water.
[0055]
In order to remove the solvent by lyophilization, it can be removed by freezing at a temperature below the freezing point of the solvent using the freezing temperature, generally at -30 to -50 ° C, and then at a reduced pressure of about 0.005 to 0.1 Torr. A solvent may be selected and removed. Furthermore, in order to remove the solvent by spray drying, the air pressure is generally 1.0 kg / cm.² The air volume is 0.35cm^Three The inlet temperature at this time may be equal to or higher than the boiling point of the solvent used. For example, when chloroform is used as the solvent, the temperature may be 60 to 90 ° C., and the solvent may be removed in accordance with a conventional method.
[0056]
The lipid residue thus obtained is mixed with a contrast medium-containing aqueous solution at a temperature equal to or higher than the phase transition temperature (Tc) of the used lipid, and then vigorously or gently shaken or stirred at a temperature equal to or higher than the Tc. And a preparation thereof.
The electrolyte ion concentration of the aqueous solution to be used is desirably as small as possible from the standpoint of retention, and specifically, the total ion concentration including cations and anions other than drugs is about 40 mM or less, more preferably about 20 mM. The following is desirable.
In general, the weight average particle diameter and the number average particle diameter are used to display the particle diameter of the liposome. From the viewpoint of the retention capacity of the liposome, it is desirable to represent the weight average particle diameter.
[0057]
The weight average particle diameter of the liposome of the present invention is generally 50 nm to 3,000 nm, preferably 150 nm to 1000 nm, more preferably 200 nm to 500 nm. In order to obtain the above desired particle size, an extrusion filtration method (Biochim. Biophys. Acta 557, 9 (1979)) in which large-sized liposomes are filtered through a filter having a predetermined pore size is used. Use it.
[0058]
The liposome of the present invention contains liposomes having multiple membranes at a certain ratio. In general, when initially formed by the above method, the liposome is multilamellar, but according to the preferred technique described above, the liposome suspension is, for example, one or more microporous filters having a pore size of about 1 micron. And the outer lipid layer is removed leaving a mixture of unilamellar liposomes and multilamellar liposomes. In general, the ratio of multilamellar liposomes is 30% by weight or more, preferably 40% by weight or more. Multilamellar liposomes have several advantages in terms of stability, but relatively large proportions of unilamellar liposomes are acceptable. This is because unilamellar liposomes provide a larger encapsulation capacity than multilamellar liposomes.
[0059]
The liposome thus prepared exists as a suspension in an aqueous medium (external solution) and is used as a diagnostic composition. The solution of the contrast agent that was not encapsulated in the liposome during the formation of the liposome is present in the external solution. Alternatively, the solution present outside the liposome can be replaced with another external solution, but the concentration of contrast agent in the internal and external solutions should be the same. In any case, the external liquid (dispersion medium) is preferably isotonic with respect to the internal aqueous phase of the liposome.
The electrolyte ion concentration in the liposome suspension thus prepared is preferably as small as possible from the viewpoint of storage stability and stability during heat sterilization. Generally, cations and anions other than drugs are preferably contained. The total ion concentration is preferably about 40 mM or less, more preferably about 20 mM or less.
[0060]
The encapsulation capacity of the liposomes of the present invention is at least 5 ml / g lipid, preferably at least 6 ml / g.
When encapsulating iodine-based X-ray contrast agents by the method of the present invention, the initial solution in the aqueous solution is reduced to reduce cost and potential toxicity problems by providing a product having a relatively high iodine / lipid ratio. A higher contrast agent concentration is desirable. However, according to the present invention, the preferred range of iodine to lipid weight ratio is 1.3 to 1.45. This is lower than some prior art ratios. In the case of WO88 / 09165, the lower limit is 1.5.
[0061]
The most preferred X-ray contrast composition of the present invention comprises hydrogenated phosphatidylcholine as neutral phospholipid and hydrogenated phosphatidylserine as charged lipid, preferably in a ratio of 10: 1. Such a composition can have an encapsulation capacity of greater than 7 ml / g. The most preferred X-ray contrast agent for use in such compositions is iodixanol. The aqueous medium inside and outside the liposome is preferably about 400 mg / ml iodixanol and an osmotic pressure regulator such as sorbitol, EDTANa₂Contains a stabilizer such as Ca and a TRIS buffer (pH about 7.4).
[0062]
A liposome composition for MR imaging can be obtained by this method using an aqueous solution of a paramagnetic MR contrast agent such as GdHPD03A or Fe having a particle size of about 10 nm, for example._Three O_Four Can be prepared by encapsulating an aqueous suspension of a superparamagnetic MRI contrast agent such as Such suspensions can be prepared, for example, by controlled precipitation of magnetite from a mixture of iron (2+) and iron (3+) salts.
A liposome composition in which a contrast agent is covalently bonded to a liposome membrane can be produced, for example, using a method similar to that described in US-A-5135737.
[0063]
Gas-containing compositions for ultrasound imaging can be prepared in various ways, for example using techniques similar to those described for the preparation of gas-containing unilamellar liposomes and multilamellar liposomes. Such techniques are described, for example, in US-A-4544545, US-A-4900540, WO-A-9109629 and WO-A-9115244. For example, a solution of a pH sensitive gas precursor is encapsulated and then the pH of the system is changed to facilitate the production of gas inside the liposome.
[0064]
In such cases, it may be advantageous to incorporate one or more ionophores in the membrane of the vesicle to help transport hydrogen or hydroxide ions through the membrane and thereby promote pH changes. May be. See for example WO-A-9109629 mentioned above.
Alternatively, the carbonate solution may be encapsulated with non-specific human esterase and the resulting liposomes may be incubated at 37 ° C. for 5 days, for example. Thereafter, the floating carbon dioxide-containing vesicles are separated and appropriately formulated.
[0065]
Another method for producing gas-containing vesicles involves applying an external pressure of gas to a preformed liposome suspension enclosing an aqueous medium. In general, the gas will be applied at a very high pressure, for example at least about 5 atmospheres.
[0066]
Another method is to disperse the selected phospholipids in an aqueous medium or a mixture of water-soluble bioreceptive organic solvents and water known to stabilize phospholipids such as glycerol and polyethylene glycol. And stirring the solution vigorously in the presence of a selected gas or gas mixture. Alternatively, liposomes can be formed by sonicating the phospholipid solution in the presence of a selected gas or gas mixture.
Any gas, such as air, nitrogen, oxygen, hydrogen, nitrous oxide, carbon dioxide, or more preferably a water-insoluble gas such as helium, xenon, argon, hexafluoride to produce the ultrasound contrast agent of the present invention. Sulfur and optionally fluorinated hydrocarbons such as methane, acetylene or carbon tetrafluoride, or mixtures thereof can be used.
In general, the compositions of the present invention used in any diagnostic imaging system can be modified with substances such as polyethylene glycol to increase the circulation half-life of the liposomes if desired. This may be particularly advantageous in applications involving cardiovascular imaging.
The following non-limiting examples are intended to illustrate the present invention.
[0067]
【Example】
Experiment
(1) Measurement of particle size
The weight average particle diameter of the obtained liposome was measured by a quasi-elastic light scattering method using a dynamic light scattering measuring device DLS-700 (Otsuka Electronics Co., Ltd.).
[0068]
(2) Measurement of enclosed capacity
The volume of the internal aqueous phase of the liposome was calculated by the ratio of iodixanol, which is a water-soluble non-electrolyte contained in the liposome preparation. That is, when the volume of the internal aqueous phase in a 1 ml liposome preparation is 0.4 ml, the encapsulation rate of iodixanol is 40%. Encapsulation volume is defined as the volume of the internal aqueous phase per gram of lipid. For example, when the lipid concentration of the liposome preparation is 0.056 g / ml and the encapsulation rate of iodixanol in the liposome is 40%, 0.056 g of lipid contains 0.4 ml of the internal aqueous phase in 1 ml of liposome. Therefore, the encapsulation capacity is 71 ml / g lipid.
[0069]
The encapsulation rate of iodixanol in the liposome preparation was measured by gel filtration. That is, the liposome preparation was subjected to gel filtration through a gel filtration column (carrier: Pharmacia, Sephadex G50; column diameter 16 mm; column height 300 mm) using physiological saline as a mobile phase, and the eluate was fractionated (2.5 ml). / Fraction). 0.8 ml was collected from each fraction, 2 ml of methanol was added and stirred, then 1 ml of chloroform was added and stirred, and the whole was once dissolved.
[0070]
Chloroform (3500 rpm, 10 minutes, room temperature) was added to the mixture by stirring with 1 ml of chloroform, 1.2 ml of distilled water and further stirring, and then cooling with a refrigerated centrifuge (KUBOTA Corporation KR-702 type). The iodixanol concentration recovered in the upper layer (water and methanol phase) of the separated two layers was measured by absorbance at 246 nm. When the iodixanol concentration in the eluate was high, 0.8 ml was collected after appropriately diluted with distilled water and extracted with methanol, chloroform and distilled water.
[0071]
The amount of iodixanol in the liposome fraction eluted in the void volume was divided by the total amount of iodixanol collected in the eluate (the amount of iodixanol collected up to 25 fractions) to obtain the retention rate of the main drug. The end point of liposome fractionation was the fraction at which the iodixanol concentration was minimized.
[0072]
Example 1: Encapsulation of X-ray contrast agent iodixanol
A flask containing 0.640 g of hydrogenated phosphatidylcholine (HEPC) derived from egg yolk, 0.064 g of hydrogenated phosphatidylserine (HEPS) synthesized from HEPC, and 60 ml of a mixture of chloroform, methanol and water (weight ratio 100: 20: 0.1) Mixed in. The mixture was heated on a hot water bath (65 ° C.) to dissolve the phospholipids and the resulting solution was heated at 60 ° C. in a rotary evaporator to evaporate the solvent.
[0073]
The residue was further dried under vacuum for 2 hours to form a lipid film. Iodixanol (1,3-bis (acetylamino) -N, N'-bis [3,5-bis (2,3-dihydroxypropylaminocarbonyl) -2,4,6-triiodophenyl] -2-hydroxypropane 0.4 g / ml) and an aqueous solution containing sucrose (0.05 g / ml) are heated to 65 ° C., 10 ml of the heated solution is mixed with a lipid film, and the mixture is heated with a mixer while heating to 65 ° C. Stir for minutes. This mixture was filtered once under pressure through a polycarbonate membrane filter having a pore size of 1.0 μm to prepare multilamellar liposomes (MLV).
[0074]
The MLV thus prepared was placed in a glass vial and autoclaved at 121 ° C. for 20 minutes. The results are shown in Table 1.
The storage capacity was measured in the same manner as in the experimental example (3). The weight average particle size was measured in the same manner as in the experimental example (1).
[0075]
[Table 1]

[0076]
Example 2: Encapsulation of iodixanol
A liposome formulation containing iodixanol was prepared according to the method described in Example 1. The results are shown in Table 2.
[0077]
[Table 2]

Example 3
The liposome suspension obtained in Example 1 was diluted with isotonic glucose to a concentration of 50 mg encapsulated iodine / ml. After dilution, the suspension turned milky white. Liposome aggregates were not observed during microscopic analysis of the milk suspension. After storing the emulsion for 1 year, no effect on the amount and size distribution of encapsulated iodixanol was observed.
[0078]
Example 4
Rats were intravenously injected with the emulsion of Example 3. Although most of the encapsulated iodixanol was confirmed to be distributed in the liver after injection, high concentrations of iodixanol were also detected in the spleen. After injection, iodixanol distributed in the tissue decreased with time and eventually no iodixanol was found in these two organs. The level of iodixanol in all other organs decreased in parallel with the level of blood iodixanol. Most of the iodixanol injected was recovered in the urine.
[0079]
Example 5 FIG.
The emulsion of Example 3 was injected intravenously into rats with multiple liver cancer metastases. At doses of 50 mg and 100 mg / kg of encapsulated iodine, X-ray attenuation of 42 and 62 HUCT values was observed in the normal region of the liver, but the effect on attenuation at the tumor metastasis was minimal. Macroscopic observation showed that the detected tumor was less than 5 mm in diameter.
[0080]
Example 6
The emulsion of Example 3 was injected intravenously into groups of mice at doses of encapsulated iodine of 200-7500 mg / kg body weight. Weight gain and death were followed over 14 days. The results of the analysis showed that all animals survived the 3 g / kg injection of liposome-encapsulated iodixanol. These animals showed no slowing of weight gain over the observation period compared to animals that were not injected. At doses of 4 g and 5 g / kg of liposome-encapsulated iodixanol, there was a slight dose-dependent decrease in body weight gain and death was observed. LD₅₀Was estimated to be 5 g / kg of liposome-encapsulated iodixanol.
[0081]
Example 7
After single or repeated (3 injections / week, 3 weeks) intravenous injection of the emulsion of Example 1 at a dose of 100-1000 mg / kg of encapsulated iodine in rats, blood of various tissue-bound enzymes Medium levels were measured and tissues of all major organs were analyzed at various time points. Only a slight effect on serum enzymes was observed compared to control animals injected with saline. Apart from a dose-dependent increase in vacuolar degeneration of phagocytes in the liver and spleen, no histological changes due to liposomes were detected. The degree of vacuolation of phagocytic cells decreased in parallel with the reduction of iodixanol distributed in the liver and spleen.
[0082]
Example 8 FIG.
Next ingredient:
Iodixanol (total amount) 400mg / mL
Encapsulated iodine 80mg / mL
Sorbitol 20mg / mL
Trometamol (TRIS) 0.097mg / mL
EDTANa₂Ca 0.1 mg / mL
Hydrogenated phosphatidylcholine 51.2mg / mL
Hydrogenated phosphatidylserine 5.1 mg / mL
Water for injection (added to the right amount) 1mL (approx. 0.9mL)
A diagnostic composition comprising:
[0083]
Method
Phospholipids were dissolved in chloroform / methanol / water (volume ratio (4: 1: 0.025) and the solvent was evaporated (rotary evaporation). An isotonic solution of iodixanol and sorbitol was made and 60-70 C. and maintained at this temperature during subsequent steps The phospholipid mixture was added with stirring and liposomes were formed.This preparation was homogenized (Rotor / Stator) to adjust the size of the liposomes. Homogenizer) The liposomes were then extruded through 7 polycarbonate filters (pore diameter 1 μm) arranged in series.
The product was diluted with an isotonic solution of iodixanol and sorbitol and trometamol and EDTA were added. The product was filtered into glass vials and autoclaved at 121 ° C. for 15 minutes.
[0084]
Example 9 Liposomes containing iotrolane
Liposomes were prepared as described in Example 8 using iotrolan instead of iodixanol.
Example 10 Iodixanol-containing liposomes with hydrogenated phosphatidylglycerol
Liposomes were prepared as described in Example 8 using hydrogenated phosphatidylglycerol instead of hydrogenated phosphatidylserine.
[0085]
Example 11 MRI contrast agent (gadolinium)
A lipid film of hydrogenated phosphatidylcholine and hydrogenated phosphatidylserine is prepared as described in Example 1. 10 ml of a 0.5 M solution of Gd HPDO3A is heated to 65 ° C. The mixture is stirred with a mixer for 15 minutes while being heated at 65 ° C. This mixture is filtered under pressure through a polycarbonate membrane filter with a pore size of 1.0 μm to prepare multilamellar liposomes (MLV). Place MLV in glass vial and autoclave at 121 ° C. for 20 minutes. MLV contains Gd HPDO3A.
[0086]
Example 12 MRI contrast agent (manganese)
Solution of manganese chelate (0.07M) of 3,6-bis [N- (2,3-dihydroxypropyl) -N-methylcarbamoylmethyl] -3,6-diazasuberic acid instead of Gd HPDO3A (WO 93/21960) Liposomes are prepared according to Example 11 using (prepared according to Nycomedelmaging AS). MLV contains a manganese chelate.
[0087]
Example 13 MRI contrast agent (Dysprosium)
Liposomes are prepared according to Example 11 using a solution of Dy DTPA-BMA (0.5 M) instead of Gd HPDO3A. MLV contains Dy DTPA-BMA.
[0088]
Example 14 MRI contrast agent (iron oxide)
Liposomes are prepared according to Example 11 using a suspension of magnetic iron oxide (10 mM, particle size 10-30 nm) instead of Gd HPDO3A. MLV contains magnetic iron oxide.
[0089]
Example 15. Liposomal Gd HPDO3A (HEPC-HEPS)
A pre-prepared mixture of 640 mg of hydrogenated phosphatidylcholine (HEPC) derived from egg yolk and 64 mg of hydrogenated phosphatidylserine (HEPS) synthesized from HEPC as described in Example 1 was added to glucose containing 250 mM Gd HPDO3A (gadoteridol). A vial containing 10 ml of a 5% aqueous solution (isotonic solution) was added. The mixture was stirred at 65 ° C. for 30 minutes and held at this temperature for an additional hour. The liposome solution was freeze-thawed 5 times and extruded 5 times through a double 400 nm polycarbonate filter at 65 ° C. to give the title product.
[0090]
Example 16 Liposomal Gd HPD30A (DPPC / DPPG)
A lipid blend of 640 mg of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) prepared as described in Example 1 is added to 10 ml of a 5% aqueous solution of glucose (isotonic solution) containing 250 mM Gd HPDO3A. Added to the containing vial. The mixture was stirred at 50 ° C. for 30 minutes and held at this temperature for an additional hour. The liposome solution was freeze-thawed 5 times and extruded 5 times through two 400 nm polycarbonate filters at 50 ° C.
[0091]
Example 17. Liposomal Gd DTPA-BMA
The same method as in Example 15 was used, except that the aqueous solution contained 250 mM Gd DTPA-BMA (Gadodiamide) instead of Gd HP-DO3A.
Example 18 Liposomal Dy TTHA
The same method was used as in Example 15 except that the solution contained 250 mM Dy TTHA (dysprosium complex of triethylenetetramine hexaacetic acid) instead of Gd HP-DO3A.
[0092]
Example 19. Ultrasound contrast agent
Liposomes are prepared according to Example 8 using saline instead of Gd HPDO3A. The mixture is pressurized with nitrogen for 1 hour (70 psi). Excess nitrogen is removed and the resulting mixture is filtered through a polycarbonate membrane filter having a pore size of 1.0 μm. The resulting MLV contains nitrogen gas.
Example 20.
When rats were injected (iv) with the dispersion of Example 8, they were X-rayed in the liver at 75 mg I (encapsulated) / kg 47ΔHU and 100 mg I (encapsulated) 70 kg HU. Attenuated.
Example 21.
The dispersion of Example 8 was injected into male and female rats. The approximate LD50 for intravenous administration in rats was 2000 mg I (encapsulated) / kg body weight.

Claims (21)

A diagnostic composition containing multilamellar liposomes for administration to a human or an animal, wherein the composition may coexist with a monolayer liposome, and the liposome contains at least one contrast agent. And is suspended in an aqueous medium containing the contrast agent, wherein the contrast agent is selected from X-ray contrast agents and ultrasound contrast agents containing one or more phenyl iodide groups or heavy metal clusters or chelates is, the ultrasound contrast agent is a compound which is liquid, solid or semi-solid which generates a gas in vivo, the liposomes comprise neutral phospholipid and a charged phospholipid, the average particle diameter of the liposome is 50 a to 3000 nm, and the concentration of the contrast agent in the aqueous phase to meet the interior of the liposomes are substantially the same as that of the aqueous medium liposomes are suspended therein, Diagnostic composition characterized and. The diagnostic composition according to claim 1, wherein the aqueous medium in which the liposomes are suspended is isotonic . The neutral phospholipid and / or charged phospholipid comprises substantially at least one fatty acid residue is胞和containing at least 14 carbon atoms, diagnosis according to claim 1 or 2 Composition. 4. The neutral phospholipid and / or the charged phospholipid comprises at least one substantially hydrated fatty acid residue containing no more than 28 carbon atoms . 5. a diagnostic composition according to claim.   The diagnostic composition according to any one of claims 1 to 4, wherein the neutral phospholipid is phosphatidylcholine.   The diagnostic composition according to any one of claims 1 to 5, wherein the charged phospholipid is phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, or an ester of phosphatidic acid and amino alcohol.   The diagnostic composition according to claim 5 or 6, wherein the phosphatidyl group is a synthetic or semi-synthetic dipalmitoyl phosphatidyl or distearoyl phosphatidyl group.   The diagnostic composition according to any one of claims 1 to 7, wherein the liposome has an average particle diameter of 150 nm to 1000 nm.   The diagnostic composition according to any one of claims 1 to 8, wherein the encapsulation capacity of the liposome is at least 5 ml / g.   The diagnostic composition according to claim 9, wherein the encapsulation capacity of the liposome is at least 6 ml / g.   The diagnostic composition according to any one of claims 1 to 10, wherein a weight ratio of the neutral phospholipid to the charged phospholipid is 60: 1 to 4: 1.   The diagnostic composition according to any one of claims 1 to 11, wherein the total lipid concentration is 20 mg / ml to 100 mg / ml.   The diagnostic composition according to any one of claims 1 to 12, wherein the concentration of total lipid is 50 mg / ml to 80 mg / ml. The X-ray contrast agent, one or more, including iodide phenyl or heavy metal clusters or chelates is X- line contrast agent, the diagnostic composition according to claim 13. 15. The diagnostic composition according to claim 14 , wherein the contrast agent is iodixanol. The diagnostic composition according to claim 1 , wherein the contrast agent is an ultrasonic contrast agent containing a liquid, solid, or semi-solid compound that forms a gas in a living body. 17. The diagnostic composition according to claim 16 , wherein the total lipid concentration is at least 20 mg / ml. Claims 1 to 1 for use in diagnostic imaging 17 The diagnostic composition according to any one of the above. The method for producing a diagnostic composition according to claim 1, wherein neutral phospholipids and charged phospholipids are dissolved in a solvent, the solvent is removed to obtain a residue, and the residue is mixed with an aqueous solution containing a contrast agent. And forming a liposome encapsulating a contrast agent.   20. The method according to claim 19, wherein the liposome formed as described above is extruded through a membrane filter to reduce the particle size of the liposome. A method for producing a sterile diagnostic composition by autoclaving the diagnostic composition according to any one of claims 1 to 17 .