Improvents in or Relating to Contrast Agents

专利摘要:
PURPOSE: Ultrasound imaging, more particularly to novel contrast agent preparations and their use in ultrasound imaging, for example in visualising tissue perfusion are provided. CONSTITUTION: A combined preparation for simultaneous, separate or sequential use as a contrast agent in ultrasound imaging, the preparation comprises: i) an injectable aqueous medium having gas dispersed therein; and ii) a composition comprising a diffusible component capable of diffusion in vivo into the dispersed gas so as at least transiently to increase the size thereof.
公开号:KR20000052652A
申请号:KR1019990703426
申请日:1997-10-21
公开日:2000-08-25
发明作者:죠니 오스텐센；모르텐 에릭센；지그문트 프리그스타트；폴 롱베트
申请人:조오지 디빈센조, 토브 아스 헬지, 에바 요한손；니코메드 이메이징 에이에스；
IPC主号:

专利说明:

Contrast Agents or Improvements {Improvents in or Relating to Contrast Agents}
Contrast agents that include microbubble dispersions of gases are particularly effective backscatterers of ultrasound because of their low density and easy compression of the microbubbles. Such microbubble dispersions can advantageously make ultrasound visualization of vascular and tissue microvascular systems very effective, often at low doses, as long as they are properly stabilized.
The use of ultrasonography to measure blood perfusion (eg, blood flow per unit of tissue mass) can be used, for example, to detect tumors, tumor tissues with blood vessel distributions that are typically different from healthy tissues, and for example myocardial infarction. It is worth researching the myocardium. A problem in the application of current ultrasound contrast agents to cardiac perfusion studies is that the information content of the obtained images degrades due to the attenuation caused by the contrast agent present in the heart ventricles.
The present invention is directed to the discovery that ultrasound visualization of perfusion in subjects, particularly myocardium and other tissues, can be achieved (achieved) or increased by gas-containing contrast agent formulations that promote controlled and transient growth of the gas phase in vivo after administration. It is based. Thus, such contrast agent formulations can be used to promote the controllable and temporary retention of gaseous phase, such as in microbubble form, in tissue microvascular systems, thereby increasing the concentration of gas in such tissues and thus increasing the responsiveness to plasma.
It will be appreciated that the use of such gas as a deposited perfusion tracker is significantly different from the current proposal for intravenous administrable microbubble ultrasound contrast agents. Thus, it is generally believed that if microbubble growth is not regulated, it may be necessary to inhibit it, since it can lead to potentially harmful tissue embolism. Thus, it may be necessary to use a gas mixture with a composition selected to inhibit (or inhibit) dosage and to minimize bubble growth in vivo by inhibiting the dispersion of blood gas into the interior of the microbubble (eg, See International Patent Publication Nos. 9503835 and 9516467).
In contrast, according to the present invention, a composition comprising a dispersed gas phase has a sufficient gas or vapor pressure to promote controlled growth of the dispersed gas phase in vivo through the internal diffusion of gas or vapor molecules derived therefrom, It will be appreciated that transport mechanisms other than diffusion may be additionally or alternatively included in the practice of the invention, as administered in combination with a composition comprising one or more substances capable of being generated, as described in more detail below.
Such simultaneous administration of a composition comprising a dispersed gas phase containing composition and a diffusion component with suitable volatility has been previously described for the sole administration of volatiles in the form of phase transfer colloids, as described in WO 9416739. This may be in contrast to the proposal. Thus, the contrast agent formulations of the present invention allow for the control of factors such as the growth rate and / or probability of dispersed gases by selecting suitable components of the co-administered composition, as described in detail below, Administration of a phase transfer colloid alone can result in the production of uncontrollable and unevenly growing microbubbles such that at least a portion of the microbubbles can result in myocardial vasculature and potentially dangerous embolism of the brain [eg, Schwarz Advances in Echo-Contrast (1994 (3)), Schwartz. 48-49].
In addition, administration of phase transfer colloids alone will produce gas or vapor microbubbles as they do not result in reproducible or consistent in vivo volatility of the dispersed phase. Grayburn et al. Am. Coll. Cardiol. 26 (5) [1995], pp. 1340-1347 argues that preliminary activation of perfluoropentane emulsion may be required at effective imaging doses low enough to prevent hemodynamic side effects in order to achieve myocardial clouding in dogs. Activation techniques for such colloidal dispersions, including the application of low pressure thereto, are described in WO 9640282, in particular, this is achieved by filling the syringe with an emulsion, then forcibly pulling it back and releasing the plunger of the syringe to release the emulsion. Creating a temporary pressure change that results in the formation of a gas microbubble within. This is inherently a harmful technique and may not provide a certain degree of activation.
In US Pat. No. 5536489, emulsions of water-insoluble gas-forming chemicals such as perfluoropentane can be used as contrast agents for position specific imaging, which emulsions can be applied to ultrasonic energy at specific locations in the body where imaging is required. It is described that it only produces a significant number of image increasing gas microbubbles. The inventors have found that emulsions of volatile compounds, such as 2-methylbutane or perfluoropentane, may be ex vivo or in vivo when sonicated at an energy level sufficient to provide a significant contrast effect using the bicomponent contrast agent according to the invention. It has been found that does not provide a detectable echo increase in.
The present invention relates to ultrasonic imaging, more particularly novel contrast agent formulations, and their use in ultrasonic imaging such as visualization of tissue perfusion.
According to one embodiment of the invention,
i) an injectable aqueous medium with gas dispersed therein; And
ii) a composition comprising a diffusion component that can diffuse into the dispersed gas in vivo and at least temporarily increase its size
Mixed formulations are provided for simultaneous, separate or continuous use as contrast agents in ultrasound imaging.
According to another embodiment of the invention,
i) injecting a physiologically acceptable aqueous medium in which gas is dispersed into the patient's vascular system;
ii) administering to the patient a composition comprising a diffusion component that can diffuse into the dispersed gas in vivo and at least temporarily increase its size prior to, during or after injection of the aqueous medium; And
iii) generating an ultrasound image in at least a portion of the patient
Provided is an improved image generation method in a human or non-human animal.
The method according to the invention can be advantageously used to visualize tissue perfusion in a patient, wherein an increase in the size of the dispersed gas is used to achieve an increase in gas or temporary retention in the microvascular system of such tissues to enhance its repercussions. Let's do it.
Any biocompatible gas may be present in the gas dispersion, as used herein, including any material (including mixtures) that is at least partially, for example substantially or completely gaseous (including steam). The term “gas” is formed at 37 ° C., the body temperature of normal humans. Thus, the gas may be, for example, air; nitrogen; Oxygen; carbon dioxide; Hydrogen; Helium, argon, xenon or krypton and silver inert gases; Sulfur fluorides such as sulfur hexafluoride, disulfer decafluoride or trifluoromethylsulfur pentafluoride; Selenium hexafluoride; Optionally halogenated silanes such as methylsilane or dimethylsilane; Alkanes such as methane, ethane, propane, butane or pentane, cycloalkanes such as cyclopropane, cyclobutane or cyclopentane, alkenes such as ethylene, propene, propadiene or butene, or alkynes such as acetylene or propene Low molecular weight hydrocarbons (eg, no more than 7 carbon atoms); Ethers such as dimethyl ether; Ketones; ester; Halogenated low molecular weight hydrocarbons (eg, up to 7 carbon atoms); Or mixtures of the foregoing. Preferably, at least some of the halogen elements in the halogenated gas are fluorine atoms; Thus the biocompatible halogenated hydrocarbon gases are bromochlorodifluoromethane, chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoro Ethane, chlorotrifluoroethylene, fluoroethylene, ethylfluoride, 1,1-difluoroethane and perfluorocarbons. Representative perfluorocarbons include perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane (eg optionally perfluoro in a mixture with other isomers such as perfluoro-iso-butane). Perfluoroalkanes such as rho-n-butane), perfluoropentane, perfluorohexane or perfluoroheptane; Perfluoropropene, perfluorobutene (eg perfluorobut-2-ene), perfluorobutadiene, perfluoropentene (eg perfluoropent-1-ene) or purple Perfluoroalkenes such as uro-4-methylpent-2-ene; Perfluoroalkynes such as perfluorobut-2-yne; And perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutane, perfluorotrimethylcyclobutane, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentane, perfluoro Perfluorocycloalkanes such as rocyclohexane, perfluoromethylcyclohexane or perfluorocycloheptane. Other halogenated gases include fluorinated (eg perfluorinated) ethers such as methyl chloride, fluorinated (eg perfluorinated) ketones such as perfluoroacetone and perfluorodiethyl ether. There is. Perfluorinated gases such as sulfur hexafluoride, and the use of perfluorocarbons such as perfluoropropane, perfluorobutane, perfluoropentane and perfluorohexane can be used for microbubbles containing such gases. This is particularly advantageous because it has been recognized as very stable in the bloodstream. It is also possible to use other gases with physicochemical properties that form highly stable microbubbles in the bloodstream.
The dispersed gas may be administered in any convenient form, for example using any suitable gas containing ultrasonic contrast agent formulation, such as a gas containing composition. Representative examples of such agents include aggregate resistant surface membranes (e.g., gelatin as described in WO8002365), membrane forming proteins (e.g., US Pat. No. 4,714,331, 4,477,58, 4,48,882). Albumin, such as human serum albumin, as described in European Patent Publication No. 0359246, International Patent Publication No. 9112823, No. 9205806, No. 9217213, No. 966477 or No. 950187), Polymeric materials (eg synthetic biodegradable polymers as described in EP 0398935, elastic interfacial synthetic polymer membranes as described in EP 0458745, particulates as described in EP 0441468). Biodegradable polyaldehydes, particulate N-dicarboxylic acid induction of polyamino acid-polycyclic imides as described in European Patent Publication No. 0458079 , Or biodegradable polymers as described in WO9317718 or WO9607434), nonpolymeric and nonpolymerizable wall forming materials (e.g. WO9521631), or surfactants (e.g., For example, polyoxyethylene-polyoxypropylene block copolymer surfactants such as Pluronic, a polymer surfactant described in WO9506518, or WO 9211873, WO 9217212, WO Microbubbles of gas stabilized (eg, at least partially encapsulated) by membrane forming surfactants, such as phospholipids, as described in headings 9222247, 9428780, 9503780, or 9729783. It includes.
Other useful gas-containing contrast agent formulations include, for example, European Patent Publication Nos. 022624, 023235, 00365467, International Patent No. 9,229,930, Particulates (especially aggregates of particulates) adsorbed on their surface and / or contained in spaces, cavities or voids, as described in headings 9313802, 9313808 or 9313809; There is the same gas-containing solid system. The echo of such particulate contrast agents can be derived directly from the contained (or bound) gas and / or from the gas (eg microbubbles) liberated (eg upon collapse of the particulate structure).
All disclosures of the foregoing documents relating to gas containing contrast agent formulations are incorporated herein by reference.
Other gas-containing materials, such as gas microbubbles and particulates, may be administered by, for example, intravenous injection, and then do not exceed 10 μm (eg, 7 μm or less) to allow them to pass freely through the lung system. It is desirable to have an initial average size. However, larger microbubbles may be produced when they contain a relatively hemolytic gas or a mixture of air, oxygen, nitrogen or carbon dioxide with one or more other diffuse gases such as one or more substantially insoluble non-diffusion gases such as perfluorocarbons. Can be used. Diffusion of the soluble and diffusible gas contents after administration to the outside is determined by the residual amount of insoluble and non-diffusing gases, and the resulting microbubbles can be selected to pass through the lung capillaries of the lung system to a size that can be selected. Will shrink rapidly.
Since the dispersed gas administered according to the invention causes growth in vivo by interaction with the diffusion component, the minimum size of the microbubbles, solid mixed gases, etc., when administered, typically exhibits significant interaction with the ultrasound. May be substantially smaller than the size deemed necessary to provide (typically about 1-5 μm at commonly used imaging frequencies); Thus, a portion of the dispersed gas may have a small size, for example 1 nm or less. Thus, the present invention may allow the use of gas containing compositions that have not been proposed to be used as ultrasonic contrast agents until now because of the small size of the dispersed gas portion.
When phospholipid-containing compositions are used according to the invention, for example in the form of phospholipid-stabilizing gas microbubbles, representative examples of useful phospholipids are natural lecithin, semisynthetic (eg, partially or Fully hydrogenated) lecithin and lecithin such as dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or synthetic lecithin such as distearoylphosphatidylcholine (ie, phosphatidylcholine); Phosphatidic acid; Phosphatidylethanolamine; Phosphatidylserine; Phosphatidylglycerol; Phosphatidylinositol; Cardiolipin; Sphingomyelin; Any of the above fluorinated homologs; Mixtures of any of the foregoing and with other fats such as cholesterol. Naturally produced (eg soy or egg yolk extract), semisynthetic (eg partially or fully hydrogenated) and synthetic phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid and / or cardiolipin (eg Particularly advantageous would be the use of phospholipids (e.g., at least 75%), which include significantly higher molecules (e.g., at least 75%) individually, such as those described in WO 9729783). Can be.
Representative examples of gas-containing particulate materials that may be useful in accordance with the present invention include carbohydrates (eg, hexoses such as glucose, fructose or galactose; disaccharides such as sucrose, lactose or maltose; arabinose, xyl Pentoses, such as los or ribose; α-, β-, and γ-cyclodextrins; starch, hydroxyethyl starch, amylose, amylopectin, glycogen, inulin, pullulan, dextran, carboxymethyl dextran, dextran phosphate Polysaccharides such as ketodextran, aminoethyldextran, alginate, chitin, chitosan, hyaluronic acid or heparin; and sugar alcohols including alditol such as mannitol or sorbitol), inorganic salts (e.g. sodium chloride) Organic salts (e.g., sodium citrate, sodium acetate or sodium tartrate), x-ray contrast agents (e.g., typically Crude, diatrizoic acid, iotalamic acid, iosaglylic acid, iohexel, iopentol, iomidol, iodicsanol, iopromide, metrizamide, iodipamide, meglumine iodipamide, meglumine N at carboxyl, carbamoyl, N-alkylcarbamoyl, N-hydroxyalkylcarbamoyl, acylamino, 3- and / or 5-positions in acetzoate and meglumine ditrizoate Commercially available carboxylic acid and nonionic amide contrast agents containing one or more 2,4,6-triiodophenyl groups with substituents such as -alkylacylamino or acylaminomethyl, and polypeptides and proteins (eg, gelatin Or albumin, such as human serum albumin).
Other gas-containing materials that may be useful in accordance with the present invention include gas-stabilized materials that are stabilized by metals (as described, for example, in US Pat. Nos. 36,744,61 and 35,088), gases stabilized by synthetic polymers. Containing Material [See, for example, US Pat. No. 3,751,494 or Farandand's Power Technology 22 (1979), pp. 11-16], a commercially available microsphere of the type Expancell (registered trademark) such as Expancel 551 DE [eg, Eur. Plast. News 9 (5) (1982), p. 39, Nonwovens Industry (1981), p. 21 and Mat. Plast. Elast. 10 (1980), p. 468], commercially available microspheres of the type Ropaque (see, eg, J. coatings Technol. 55 (707) (1983), p. 79], micro- and nano-sized gas-containing structures such as zeolites, and nano-sized open pore-containing chemical structures such as inorganic or organic aerogels, fullerenes, clathrates or nanotubes [eg, GE Gadd). By Science 277 (5328) (1997), pp. 933-936], and natural surfactant stabilized microbubble dispersions (for example, by d'Arrigo "Stable Gas-in-Liquid Emulsions, Studies in physical and theoretical chemistry" 40 As described in Elsevier, Amsterdam (1986).
A wide variety of diffusing components can be used in accordance with the present invention, including gases / vapors, volatile liquids, volatile solids and precursors capable of generating gas upon administration, which are intended to allow internal diffusion of gas or vapor molecules into dispersed gases. It must have or be able to generate sufficient gas or vapor pressure in vivo to facilitate it. It should be understood that a mixture of two or more diffusion components may be used in accordance with the present invention as needed, and the term "diffusion component" as used herein is to be understood to include such mixtures. Similarly, references to administration of diffusion components are intended to include administration of two or more such components, either as a mixture or as multiple dosages.
The composition comprising the diffusion component may take a suitable form and may be administered by any suitable method, and the route of administration depends in part on the area of the patient to be investigated. Thus, if it is desired to facilitate the temporary retention of gases, for example in the tissues of the gastrointestinal lining, oral administration of suitable compositions comprising diffusion components may be particularly desirable. In a representative embodiment of this use, the gas dispersion can be injected intravenously at a dosage similar to that used in echocardiograms, and the diffusion component is an orally administrable emulsion, for example, as described in further detail below. It can be formulated as an oil-in-water perfluorocarbon emulsion, and is used at a dosage of, for example, 0.2 to 1.0 μl perfluorocarbon / kg. After administration and dispensing of the two compositions, the growth of gaseous dispersion in capillary plasma of the gastric wall or barrier can enhance contour contrast from these regions. It should be understood that an inverse combination of orally administrable gas dispersions and intravenous injectable diffusion components may be useful for providing contour contrast from the lining or mucosa of the gastrointestinal system.
For example, such orally administrable gas dispersions or diffusion component compositions may be incorporated to mix with the composition or to introduce chemical groups or substances that adhere to components such as surfactants or other stabilizing moieties to promote adhesion to the walls of the gastrointestinal tract. When used, this may be desirable as it may stimulate growth of the dispersed gas phase by enhancing contact with the diffusion component. Examples of such adhesion promoters and materials have been previously described in connection with gastrointestinal X-ray contrast agents, acrylic esters as described in WO9722365, iodophenol sulfonates as described in US Pat. , And iodide phenyl esters as described in US Pat. No. 52,60049.
Inhalation of a suitably volatile diffusion component can be used to promote the growth of the administered gas dispersion immediately after passing through the lung capillaries, so that the gas remains temporarily in the capillaries of the myocardium. In such embodiments, the growth of dispersed gas may be further increased by excessively raising the lung pressure of the diffusion component, for example to below 0.5 bar, by using a respirator or exhaling the patient for resistance.
Intramuscular or subcutaneous injection of a suitably formulated diffusion component composition comprising, for example, a physiologically acceptable carrier liquid, can be advantageously used when it is desired to specifically limit the effect of the component to a particular target area of the patient. One example of a composition for subcutaneous injection is nanoparticles such as those used for lymphatic angiography. Subcutaneously injected diffusion components can be dissolved in the lymphatic system, which can result in the growth of intravenously injected gas dispersions to facilitate imaging of lymph nodes. Subcombinations of subcutaneously injected gas dispersions and intravenous injected diffusion components can similarly be used.
Intravenous injection of a suitably formulated diffusion component composition comprising a physiologically acceptable carrier liquid may be used to initiate gas growth in which the gas dispersion and components of the diffusion component composition are dispersed and It is selected to control variables such as growth rate and thus the tissue reverberation in the body part can be increased by the temporary retention of gas in the microvasculature, thus allowing considerable diversity in the practice of the present invention.
Suitable topical formulations may be applied to the skin to facilitate transdermal absorption of the diffusion components. Such administration can be used for imaging and / or treatment of skin, subcutaneous tissue, and adjacent areas and organs, for example for the purpose of peripheral circulation of body limbs such as legs.
Diffusion components for administration orally or by injection may include solutions or mixtures with water and / or one or more water-miscible and physiologically acceptable organic solvents such as ethanol, glycerol or polyethylene glycol; Dispersants in an aqueous medium, such as components of an oil phase or of an oil phase of an oil-in-water emulsion; Microemulsions, a system in which a substance is effectively dissolved within the hydrophobic interior of a surfactant micelle present in an aqueous medium; Or may be formulated as a mixture of particulates or nanoparticles adsorbed on the surface of the particulates or nanoparticles, and / or contained within a space, cavity or void of the particulates or nanoparticles, or dispersed in a suitable carrier liquid encapsulated in microcapsules.
If the diffusion component is administered as a solution, the in vivo partial pressure derived therefrom will depend on the concentration of the component in the bloodstream and the corresponding pressure of the pure component material according to Raoult's law in a system approaching the ideal . Thus, if the component has a low water solubility, it is preferred that it has a sufficient vapor pressure of at least 50 torr, preferably at least 100 torr, in pure form at normal body temperature. Examples of relatively water insoluble components having high vapor pressures are gases such as those exemplified above as possible microbubble gases.
Representative examples of better water soluble and water miscible diffusion components that can exhibit lower vapor pressures at body temperature include aliphatic ethers such as ethyl methyl ether or methyl propyl ether; Aliphatic esters such as methyl acetate, methyl formate or ethyl formate; Aliphatic ketones such as acetone; Aliphatic amides such as N, N-dimethylformamide or N, N-dimethylacetamide; And aliphatic nitriles such as acetonitrile.
However, it may be desirable to use a diffusion component formulated as an emulsion in a suitable aqueous medium (ie, as a stabilized suspension) that is substantially free from mixing with the water, in which the vapor pressure of the aqueous phase of the diffusion component is substantially This is because, even in very dilute emulsions, they are equal to the vapor pressure of the pure component material. In such embodiments, the diffusion component may be formulated as part of a proprietary registered pharmaceutical emulsion such as Intralipid (registered trademark, Pharmacia product).
The diffusion component in such emulsions is advantageously liquid at processing and storage temperatures, and may be as low as -10 ° C if the liquid phase contains a suitable underfloor material, while at body temperature it is gaseous or exhibits substantial vapor pressure. Suitable compounds can be selected from the various emulsifiable low boiling liquids described in International Patent Publication No. 9416379, supra, the disclosures of which are incorporated herein by reference. Specific examples of emulsifiable diffusion components include aliphatic ethers such as diethyl ether; Polycyclic oils or alcohols such as menthol, camphor or eucalyptol; Heterocyclic compounds such as furan or dioxane; n-butane, n-pentane, 2-methylpropane, 2-methylbutane, 2,2-dimethylpropane, 2,2-dimethylbutane, 2,3-dimethylbutane, 1-butene, 2-butene, 2-methyl Propene, 1,2-butadiene, 1,3-butadiene, 2-methyl-1-butene, 2-methyl-2-butene, isoprene, 1-pentene, 1,3-pentadiene, 1,4-pentadiene Aliphatic hydrocarbons, which may be saturated or unsaturated straight or branched chains such as butenine, 1-butyne, 2-butyne or 1,3-butadiin; Alicyclic hydrocarbons such as cyclobutane, cyclobutene, methylcyclopropane or cyclopentane; And halogenated low molecular weight hydrocarbons (eg, containing up to 7 carbon atoms). Representative halogenated hydrocarbons include dichloromethane, methyl bromide, 1,2-dichloroethylene, 1,1-dichloroethane, 1-bromoethylene, 1-chloroethylene, ethyl bromide, ethyl chloride, 1-chloropropene, 3- Chloropropene, 1-chloropropane, 2-chloropropane and t-butyl chloride. At least some of the halogen atoms are dichlorofluoromethane, trichlorofluoromethane, 1,2-dichloro-1,2-difluoroethane, 1,2-dichloro-1,1,2,2-tetrafluoroethane , 1,1,2-trichloro-1,2,2-trifluoroethane, 2-bromo-2-chloro-1,1,1-trifluoroethane, 2-chloro-1,1,2 -Trifluoroethyl difluoromethyl ether, 1-chloro-2,2,2-trifluoroethyl difluoromethyl ether, partially fluorinated alkanes (e.g., 1H, 1H, 3H-pentafluoro Pentafluoropropane, such as ropropane, hexafluorobutane, nonafluorobutane, such as 2H-nonafluoro-t-butane, and decafluoropentane, such as 2H, 3H-decafluoropentane), partially fluorine Oxidized alkenes (eg, heptafluoropentene such as 1H, 1H, 2H-heptafluoropent-1-ene, and nonafluorohexene such as 1H, 1H, 2H-nonafluorohex-1-ene) ), Fluorine Hydrocarbon ethers (eg 2,2,3,3,3-pentafluoropropyl methyl ether or 2,2,3,3,3-pentafluoropropyl difluoromethyl ether), and more preferably It is preferable that it is a fluorine atom in a perfluorocarbon. Examples of perfluorocarbons include perfluorobutane, perfluoropentane, perfluorohexane (eg, perfluoro-2-methylpentane), perfluoroheptane, perfluorooctane, perfluorononane And perfluoroalkanes such as perfluorodecane; Perfluorocycloalkanes such as perfluorocyclobutane, perfluorodimethyl-cyclobutane, perfluorocyclopentane and perfluoromethylcyclopentane; Perfluorobutene (for example perfluorobut-2-ene or perfluorobuta-1,3-diene), perfluoropentene (for example perfluoropent-1-ene) and purple Perfluoroalkenes such as luorohexene (eg, perfluoro-2-methylpent-2-ene or perfluoro-4-methylpent-2-ene); Perfluorocycloalkenes such as perfluorocyclopentene or perfluorocyclopentadiene; And perfluorinated alcohols such as perfluoro-t-butanol.
In addition, such emulsions may contain one or more surfactants to stabilize the dispersion, which may be the same or different from any surfactant (s) used to stabilize the gas dispersion. The nature of any of these surfactants can significantly affect factors such as the growth rate of the dispersed gas phase. In general, a wide range of surfactants selected from the various lists described in European Patent Publication No. 0743225, the disclosure of which is incorporated herein by reference, may be useful. Representative examples of useful surfactants include fatty acids (eg, straight-chain saturated or unsaturated fatty acids having 10 to 20 carbon atoms) and carbohydrates thereof, and triglyceride esters thereof, phospholipids (eg lecithin), fluorine-containing phospholipids, Protein (eg, albumin, such as human serum albumin), polyethylene glycol, and block copolymer surfactants (eg, polyoxyethylene-polyoxypropylene block copolymers such as pluronic, acyloxyacyl polyethylene glycol Such extended polymers, for example polyethylene glycol methyl ether 16-hexadecanoyloxy-hexadecanoate having a molecular weight of 2300, 5000 or 10000, and fluorine-containing surfactants [eg, zonyl ( Zonyl) and those sold under the trade names Fluorad, or described in International Patent Publication No. 9639197 (its The disclosure of which is incorporated herein by reference). Particularly useful surfactants include phosphatidylserine, phosphatidylglycerol, phosphatidyl, naturally occurring (eg, derived from soy or egg yolk), semisynthesized (eg, partially or fully hydrogenated) and synthesized Phospholipids include molecules with a total negative net charge, such as inositol, phosphatidic acid, and / or cardiolipin.
The droplet size of the diffusion component dispersed in the emulsion for intravenous injection should preferably be at most 10 μm, for example at most 7 μm and at least 0.1 μm so that passage through the lung system is unobstructed and easy.
In addition, as described above, the diffusion component that is not miscible with water may be formulated into a microemulsion. Such systems are advantageous due to their thermodynamic stability and the fact that the diffusion components are substantially uniformly dispersed throughout the aqueous phase; Thus, although the microemulsion has the appearance of a solution, it can exhibit the properties of the emulsion with respect to the partial pressure of the dispersed phase.
Gas precursors that can be used include any biocompatible component capable of generating gas in vivo, ie at body temperature and physiological pH. Representative examples include inorganic and organic carbonates and bicarbonates, and nitrogen generating materials such as pyrazoline, pyrazole, triazoline, diazoketone, diazonium salt, tetrazole and azide. In such a system, it should be understood that this is the finally generated gas which is the actual diffusion component.
After administration, in order to ensure maximum volatility of the diffusion component and increase the growth of the dispersed gas (both endothermic reaction), the temperature of the solution or suspension of the diffusion component and / or gas dispersion prior to administration is multiplexed (multiplexed) Or) incorporating an exothermic reactive component; The use of these components in an exothermic reaction under the influence of ultrasonic radiation can be particularly advantageous.
The growth of the dispersed gas phase in vivo can be achieved by the expansion of any encapsulating material (which has sufficient flexibility) up to the growing gas-liquid interface and / or by removing excess surfactant from the administered material. . However, stretching of the encapsulating material and / or the interaction of the material with ultrasound may substantially increase its porosity. This rupture of the encapsulating material has so far been found in many cases to result in rapid loss of reverberation through diffusion and dissolution of the exposed gas outwards, but we have found that when using the contrast agent formulation according to the invention, It was found that the gas was substantially stable. While not wishing to be bound by theoretical calculations, the inventors have found that the exposed gas in the form of free microbubbles can be stabilized against the collapse of microbubbles due to supersaturated conditions caused by diffusion components, It is believed to provide an inward pressure gradient that prevents the tendency to spread outward. Substantially in the absence of encapsulating material, the exposed gas surface causes the contrast agent to exhibit exceptionally desirable acoustic properties (eg, expressed in high backscatter: attenuation ratios), as evidenced by high backscattering and low energy absorption. Can do it; This echo effect can last for a considerable period of time, even while continuing the ultrasound irradiation.
Thus, the stabilizing effect of the diffusion components administered together may be used to significantly increase both the duration and the size of the echo of the gas-containing contrast agent formulation present if such variables may be insufficient when the contrast composition is administered alone. Can be. Thus, the duration of the albumin-based contrast agent effect is often severely limited by the aggregation of encapsulated albumin material as a result of changes in systolic pressure in the cardiac or vascular system, or as a result of ultrasound irradiation, but with a diffusion component in accordance with the invention May be substantially increased by administration together.
In an exemplary method embodiment of the present invention, a composition comprising a gas dispersion and a composition comprising a diffusion component suspension passes through the lungs after at least a portion of the dispersed gas passes through the internal diffusion of the diffusion components from the lungs. It is selected to enable ultrasound visualization of myocardial perfusion by performing rapid growth to temporarily remain in the myocardium. As the concentration of volatile diffuser components in the blood stream decreases, i.e., as the components are removed from the blood by removal through lungs and by redistribution of the patient to aerobic, metabolism or other tissues, the diffuser component is typically a dispersed gas. Will diffuse outward, thus shrinking to an earlier smaller size and ultimately flowing freely in the bloodstream and will typically be removed from it by the reticuloendothelial system. The type in which the contrast effect disappears after this substantial transient increase in echo is remarkably different from the echo characteristic exhibited by each of the two compositions when administered alone. Accordingly, it will be understood from these results that the residual duration of the dispersed gas can be achieved by appropriate adjustment of the dosage and / or formulation of the diffusion component.
Other capillary relationships such as but not limited to kidneys, liver, spleen, thyroid, skeletal muscle, breast, penis can be similarly imaged.
Factors such as the rate of growth and / or the extent of growth of the dispersed gas are generally determined by appropriate selection of the gas and any encapsulation stabilizing material, more specifically the nature of any surfactants used, and the components of the formulation into emulsions. The size of the dispersed phase droplets to be controlled by the proper choice of the nature of the diffusion component and the formulation manner; Thus, for a given amount of emulsified diffusion component, a decrease in droplet size may result in a faster rate of delivery of the diffusion component compared to the rate of movement from the larger droplet, since a larger surface area: volume ratio may result in faster release from smaller droplets. Can be increased. Other variables for control include the relative amounts to which the two compositions are administered (they are administered separately), the order of administration, the time interval between the two administrations, and the possible spatial isolation of the two administrations. In this regard, the inherent diffusivity of the diffusion components may allow for use in different ways in different parts of the body, for example for inhalation, intradermal, subcutaneous, intravenous, intramuscular or oral use, and dispersed gas It will be appreciated that possible dosage forms for the subject may be somewhat more limited.
Particularly important variables for the diffusion component are their solubility and diffusion (e.g., expressed in their diffusion constants) in water / blood, which is any rate of encapsulation of their transport rate and dispersed gas through the carrier liquid or blood. The permeability through the membrane will be measured. In addition, the gas generated by the diffusion component in vivo will affect its concentration as well as the rate of diffusion into the dispersed gas. Thus, according to Pick's law, together with the diffusion constant of the diffusible material in the surrounding liquid medium, the concentration gradient of the diffusion component with respect to the distance between each gas microbubble and the emulsion droplets is determined by a simple diffusion rate. Will be measured; The concentration gradient is measured by the solubility of the diffusion component in the surrounding medium and the distance between each gas microbubble and emulsion droplet.
The effective transport rate of the diffusing component is, if necessary, adjusted by the viscosity of the dispersed gaseous composition and / or the diffusing component composition, e.g., one or more such as, for example, X-ray contrast agents, polyethylene glycols, carbohydrates, proteins, polymers or alcohols. Biocompatibility viscosity enhancers can be mixed into the formulation to adjust. For example, it may be advantageous to inject the two compositions together as a relatively large volume of bolus (eg, having a volume of at least 20 ml for a 70 kg human subject), which would enter the right ventricle and lung capillaries of the heart. Because it will delay the complete mixing of the blood and the constituent until (the growth of the dispersed gas is initiated). The delay of dispersed gas growth can be maximized, for example, by using a carrier liquid that is not cooled and saturated with the gas and any other diffusion components described above.
As mentioned above, transport mechanisms other than diffusion will be included in the practice of the present invention. Thus, transport can occur via, for example, hydrodynamic flow in the surrounding liquid medium; This may be important in blood vessels and capillaries where high shear flow rates may occur. In addition, the transport of the diffusion component to the dispersed gas occurs, for example, as a result of the collision or near-collision process between the gas microbubbles and the emulsion droplets, resulting in adsorption and / or diffusion of the diffusion components on the microbubble surface. Results in the penetration of microbubbles, ie the formation of coalescence. In this case, the diffusion constant and the solubility of the diffusion component have a minimal effect on the transport rate, and the particle size of the diffusion component (e.g. droplet size when it is formulated into an emulsion) and the frequency of collisions between the microbubbles and the droplets are microscopic. It is a major factor controlling the rate and extent of bubble growth. Thus, for a given amount of emulsified diffusion component, a decrease in droplet size will increase the total number of droplets, thus reducing the average interparticle distance between gas microbubbles and emulsion droplets and thus impingement and / or coalescence By increasing the likelihood, the transport rate can be increased. It will be appreciated that the rate of transport that proceeds through the impingement process can be significantly increased by applying additional ultrasonic motion to the gas microbubbles and emulsion droplets of the diffusing component by applying ultrasonic energy. The motion of the collision process induced by the ultrasonic energy may be necessary for the initiation of coalescence of gas droplets and emulsion droplets in which a certain amount of energy collides with the movement for transport of the diffusion component in the carrier liquid and / or blood. May be different. Thus, it would be advantageous to select the size and mass of the emulsion droplets to produce sufficient impact force with the vibrating microbubbles to induce coalescence.
In addition, as described above, the permeability of any material encapsulating the dispersed gas phase is a variable that can affect the growth rate of the gas phase, and thus a diffusion component (e.g., a single layer) that readily permeates such encapsulation material. Or a polymer or surfactant membrane, such as one or more bilayers of membrane forming surfactants such as phospholipids). However, the inventors have found that substantially impermeable encapsulation materials may be used, which may be of lower or higher frequency (eg, in the range of 10 Hz to 1 Hz, preferably, than those typically used in medical ultrasound imaging). It is evident that continuous treatment of sonication, including sonication at 1 Hz to 10 MHz and mixed contrast agent formulations administered in accordance with the present invention, or simple or complex pulse patterns, will promote or increase the growth of dispersed gases. Because. This growth can be induced by ultrasonic irradiation used to conduct the study, or by preliminary local irradiation, which acts to effect the temporary retention of gases in the microvascular structure of a particular target organ. Alternatively, activation of dispersed gas growth can be induced by applying a sufficient amount of other forms of energy, such as shaking, vibration, electric fields, irradiation or particle impact, using neutral particles, ions or electrons.
Without wishing to be bound by theory, sonication may at least temporarily change the permeability of the encapsulating material, the diffusivity of the diffusion component in the surrounding liquid and / or the frequency of collisions between the emulsion droplets and the encapsulated microbubbles. This effect can be observed using extremely short ultrasonic pulses (eg, having a duration of about 0.3 ms in B-mode imaging, or about 2 ms in Doppler or second harmonic imaging). This suggests that ongoing ultrasonic irradiation continuously increases the parallel radiation of gas bubbles [Leighton, EG, "The Acoustic Bubble", Academic Press (1994), p. It is unlikely to be an example of rectified diffusion, and ultrasonic pulses can destroy the encapsulation membrane and increase the growth of the dispersed gas into the exposed gas phase through the internal diffusion of the diffusion component.
If desired, the dispersed gas or diffusion component may comprise an azeotropic mixture or may be selected such that the azeotropic mixture is formed in vivo when the diffusion component is mixed with the dispersed gas. The formation of such azeotropic mixtures is relatively high molecular weight of compounds such as halogenated hydrocarbons, such as fluorocarbons (including perfluorocarbons) which are liquid at normal temperature at 37 ° C. under standard conditions and can be administered in gaseous form at this temperature. It can be effectively used to increase volatility. It is known that variables such as water solubility, fat solubility, diffusivity, and pressure resistance of compounds such as fluorocarbons decrease with increasing molecular weight, which is why it is possible to determine the echogenic useful life of contrast agents containing such azeotropic mixtures in vivo. Substantially advantageous in view.
In general, the identified natural resistance of azeotropic mixtures to the separation of their constituents will enhance the stability of the contrasting agent components containing them during formulation, storage and processing and after administration.
The azeotropic mixtures useful according to the invention can be selected by experimental investigation and / or by theoretical prediction, with reference to the literature on azeotropic mixtures [see, for example, Fluid Phase Equilibria 24 (1985) by Tanaka. ), pp. Of Chapter 10 of Thermal Physics (WH Freeman & Co., New York, USA, 1980), or Hemer (PC) of 187-203, Kittel, C., and Kroemer, H. Chapters 16-22 of Statistisk Mekanikk (Tapir, Trondheim, Norway, 1970), the disclosure of which is incorporated herein by reference].
Examples of literature for azeotropic mixtures that effectively reduce the boiling point of higher molecular weight components below normal body temperature are described in US Pat. No. 40,55049 as 1,1,2-trichloro-1, which is described as an azeotropic mixture having a boiling point of 24.9 ° C. 57:43 weight / weight mixture of 2,2-trifluoromethane (boiling point 47.6 ° C.) and 1,2-difluoromethane (boiling point 29.6 ° C.). Other examples of halocarbon-containing azeotrope include European Patent Publication No. 0783017, US Pat. No. 5,559,383, 5,560,567, 5,560,582, 5,560,616, 5,560,812, 5,561,10,56,565. And 565621, the disclosures of which are incorporated herein by reference.
Simons et al., J. Chem. Phys. 18 (3) (1950), pp. 335-346 shows that mixtures of perfluoro-n-pentane (boiling point 29 ° C.) and n-pentane (boiling point 36 ° C.) show a significant deviation from Raoul's law; This effect is reported to be most pronounced in approximately equimolar mixtures. In fact, it has been found that the boiling point of the azeotropic mixture is about 22 ° C. or lower. Mixtures of perfluorocarbons and unsubstituted hydrocarbons can generally exhibit useful azeotropic mixture properties; Strong azeotropic mixture effects have been observed in mixtures of these components having substantially similar boiling points. Examples of other perfluorocarbon: hydrocarbon azeotrope mixtures include mixtures of perfluoro-n-hexane (boiling point 59 ° C.) and n-pentane (azeotropic mixture has a boiling point from room temperature to 35 ° C.), and perfluoro-4 A mixture of -methylpent-2-ene (boiling point 49 ° C.) and n-pentane (the azeotropic mixture has a boiling point of approximately 25 ° C.).
Other potentially useful azeotropic mixtures include mixtures of halotan and diethyl ether, and for example perfluoropropane and fluoroethane, perfluoropropane and 1,1,1-trifluoroethane, or perfluoro There is a mixture of two or more fluorinated gases such as ethane and difluoromethane.
It is known that fluorinated gases, such as perfluoroethane, can form an azeotrope with carbon dioxide (see International Publication No. 9502652). Thus, administration of a contrast agent containing such a gas can form a tertiary or higher azeotrope with a blood gas, such as carbon dioxide, in vivo to further enhance the stability of the dispersed gas.
When two components of the contrast agent formulations mixed according to the invention are administered simultaneously, they can be injected from separate syringes by suitable coupling means, or preferably premixed under controlled conditions to prevent premature microbubble growth. You can.
The composition for mixing prior to simultaneous administration may be advantageously stored in a suitable dual or multi-chamber device. Thus, it includes a liquefied portion of a gas dispersion composition or a precursor thereof dried (eg, a suspension of a gas microbubble in an amphoteric-containing aqueous medium, in particular the amphoteric material essentially consists essentially of the total net charge ( For example, consisting of a phospholipid comprising molecules each having a negative) (eg, at least 75%, preferably substantially 100%), in a first chamber such as a vial, wherein the diffusion component composition Sealingly connecting a syringe containing a; The syringe outlet may be sealed with a membrane or stopper to prevent premature mixing. The operation of the syringe plunger ruptures the membrane and mixes the diffusion component composition with the gas dispersion component or with its precursor and reconstitutes it; After any necessary or desired shaking and / or dilution, the mixture may be taken out and administered (by syringe).
Alternatively, the two compositions can be stored in a single sealed vial or syringe and separated by a membrane or stopper; Excessive pressure of gas or vapor may be applied to each or all of the compositions. Rupture of the membrane or plug by inserting a hypodermic needle into a vial leads to mixing of the composition; This may be facilitated by shaking by hand, if necessary, and then the mixture may be taken out and administered. A vial containing a dried precursor for the gas dispersion composition was installed with a first syringe containing a redispersible fluid for the precursor and a second syringe containing a diffusion component composition and separated into a membrane to diffuse component composition and gas dispersion. Other embodiments may similarly be used to install a syringe containing a redispersible fluid for the dried precursor in a vial containing the dried precursor for the composition.
In embodiments in which the gas dispersion composition and the diffusion component composition are mixed prior to administration, the mixture will be stored at the elevated temperature or low temperature during the preparation step or subsequently afterwards, typically where the pressure of the diffusion component is insufficient for growth of the dispersed gas. Activation of dispersed gas growth can be induced by simply mitigating excessive pressure, by heating up to body temperature after the mixture has been administered, or by preheating the mixture just prior to administration, if desired.
In embodiments of the invention where the gas dispersion composition and the diffusion component composition are administered separately, the time between the two administrations can be adjusted to affect the body region where the growth of the dispersed gas phase occurs markedly. Thus, by first injecting the diffusion component and concentrating the liver, imaging of the organ can be improved upon subsequent injection of the gas dispersion. If the stability of the gas dispersion is tolerated, it may be first administered to concentrate in the liver and then administered to the diffusion component to increase its reverberation.
Imaging modalities that can be used in accordance with the present invention include B-mode imaging (e.g., from the fundamental frequency of emitted ultrasonic pulses, from their half-harmonics or larger harmonics, or the sum of the emitted pulses and the frequencies derived from these harmonics). Or using amplitudes that vary over time of a signal envelope generated from a difference, preferably an image generated from a fundamental frequency or its second harmonic), color Doppler imaging, Doppler amplitude imaging, and such There are two-dimensional and three-dimensional imaging techniques, such as a combination of color Doppler imaging and Doppler amplitude imaging techniques and the other modalities described above. For a given dose of gas dispersion and diffusion component composition, the use of color Doppler imaging ultrasound to induce the growth of dispersed gas results in a stronger contrast effect during subsequent B-mode imaging as a result of using high intensity ultrasound. It turned out to provide. In order to reduce motor effects, successive burns of tissues such as the heart or kidneys can be collected with the aid of suitable synchronizing techniques (eg, patient respiratory movement or ECG methods). In addition, the contrast agent can be detected by measuring the change in resonance frequency or frequency absorption accompanied by the growth of the dispersed gas.
It will be appreciated that the dispersed gas content of the mixed contrast agent formulation according to the present invention tends to remain temporarily in the tissue at a concentration proportional to the local tissue perfusion rate. Thus, when using ultrasound imaging modalities, such as conventional imaging or harmonic B-mode imaging, in which the indication is derived directly from the return signal intensity, images of such tissue can be interpreted as perfusion diagrams where the displayed signal intensity is a function of local perfusion. . This is in contrast to images obtained using free-flowing contrast agents, where the local concentration of the contrast agent and the corresponding return signal intensity depend on the actual blood content rather than the perfusion rate of the local tissue.
In ventricular studies, if perfusion is derived from return signal intensity in accordance with this embodiment of the present invention, the patient may be increased in order to increase the identification and difference of burn intensity between any myocardial region with normally perfused myocardium and stenosis artery. It may be advantageous to apply physical or drug stress to the patient. As can be seen from radionuclide cardiac imaging, this stress induces vasodilation and an increase in blood flow in healthy myocardial tissue, whereas blood flow in non-perfused tissues with stenotic arteries is limited in capacity for arterial vasodilation. It is substantially unchanged since it is already consumed by the inherent self-regulation to increase blood flow.
The application of stress such as physical exercise or pharmacological stress by administration of adrenergic agonists can lead to discomfort such as chest pain in a group of patients suffering from heart disease, and thus adenosine, dipyridamole, nitroglycerin, isole Bide mononitrate, prazosin, doxazosin, dihydralazine, hydralazine, sodium nitroprusside, pentoxifylline, amelodipine, felodipine, isradipine, nifedipine, nimodipine, verapamil, diltiazem And vasodilating drugs selected from nitric oxide are preferred to increase perfusion of healthy tissues. In the case of adenosine, this increases coronary blood flow more than fourfold in healthy myocardial tissue, increasing the absorption and transient retention of the contrast agent according to the present invention, thus significantly increasing the difference in return signal strength between normal and overfused myocardial tissue. You can. Since essentially natural capture processes are involved, the residual of the contrast agent according to the invention is very effective; This can be contrasted with the absorption of radionuclide tracers such as thallium 201 and technetium cestamivi, which is limited by the short contact time between the tracer and the tissue, and thus during the total plasma distribution to the tracer to ensure an optimal effect. It may be necessary to maintain vasodilation (for example, 4 to 6 minutes in thallium synthogram imaging). In contrast, the contrast agents of the present invention do not suffer from such diffusion or transport limitations, and also because their residual in myocardial tissue can be quickly terminated by interruption of growth-induced ultrasound irradiation, cardiac perfusion according to this embodiment of the present invention. Vasodilation time required to achieve imaging can be very short, up to 1 minute. This will reduce any inconvenient period that can be caused to the patient by the administration of vasodilator drugs.
Adenosine is a particularly useful vasodilation drug in that the required vasodilation requires a short duration, and it is not only an endogenous substance but also has a very short duration of action for only 2 seconds, as evidenced by plasma half-life. Thus, vasodilation will be the strongest in the heart because drugs tend to reach distant tissue below the pharmacologically active concentration. Because of this short half-life, repeated injections or infusions of adenosine may be essential during cardiac imaging according to this embodiment of the invention; For example, it will be appreciated that initial administration of 150 μg / kg of adenosine may be performed substantially concurrently with administration of the contrast agent composition, followed by a slow injection of additional 150 μg / kg of adenosine over 20 seconds after 10 seconds. .
Contrast agent preparations according to the invention may be advantageous for use as delivery agents for bioactive moieties, such as therapeutic drugs (ie, agents which have a beneficial effect on certain diseases of living or non-human animals), in particular at the target site. have. Thus, the therapeutic compound, which may be present in the dispersed gas, may be connected to a portion of the encapsulation wall or matrix via covalent or ionic bonds, and to the spacer arm as needed, or may be physically mixed into such encapsulation material or matrix material. And; This final choice is particularly applicable to the field in which the therapeutic compound and the encapsulating or matrix material have similar properties or solubility.
The adjustable growth properties of the dispersed gas can be used to result in its temporary retention in the microvascular structure of the desired target area; Particularly advantageous is the use of ultrasonic irradiation to induce growth and thereby the residual of gas and mixed therapeutic compounds in the target structure. In addition, local injection of the gas dispersion composition, or more preferably the diffusion component composition as described above, can be used to concentrate the growth of the dispersed gas in the target area.
Therapeutic compounds that can be coupled to site-specific vectors that are affinity for a particular cell, structure, or pathological site, as needed, may include expansion or cleavage of encapsulated or matrix materials caused by the growth of dispersed gases, Release as a result of dissolution of the encapsulating or matrix material, or degradation of the microbubbles or particulates (e.g., induced by reversal or sonication of the concentration gradient of the diffusion component in the target region). When the therapeutic agent is chemically bound to the encapsulation wall or matrix, it may be advantageous for any spacer arm to which the bond is or is linked to contain one or more labile groups that cleave to release the drug. Representative examples of groups that can be cleaved include amides, imides, imines, esters, anhydrides, acetals, carbamates, carbonates, carbonate esters and hydrolysis and / or in vivo as a result of enzymatic action. There are disulfide groups that can be biodegradable.
Representative and non-limiting examples of drugs useful according to this embodiment of the present invention include vincristine, vinblastine, vindesine, busulfan, chlorambucil, spiroplatin, cisplatin, carboplatin, methotrexate, adriamycin , Mitomycin, bleomycin, cytosine arabinoside, arabinosyl adenine, mercaptopurine, mitotan, procarbazine, dactinomycin (antitinomycin D), daunorubicin, doxorubicin hydrochloride, taxol, plicamycin, Aminoglutetimide, esturamustine, flutamide, leuprolide, megestrol acetate, tamoxifen, testosterone, trirostane, amsacrine (m-AMSA), asparaginase (L-asparaginase) Anti-neoplastic agents such as etoposide, interferon a-2a and 2b, blood products such as hematoporphyrin or derivatives thereof; Biological response modifiers such as muramyl peptides; Antifungal agents such as ketoconazole, nystatin, griseofulvin, flu cytosine, myconazole or amphotericin B; Growth hormone, melanocyte stimulating hormone, estradiol, beclomethasone dipropionate, betamethasone, cortisone acetate, texamethason, flunisolide, hydrocortisone, methylprednisolone, paramethasone acetate, prednisolone, prednison, triamcinolone or flude Hormones or hormonal homologues such as locodison acetate; Vitamins such as cyanocobalamin or retinoids; Enzymes such as alkaline phosphatase or manganese superoxide dismutase; Anti-allergic agents such as amelexanox; Anticoagulants such as warfarin, fenprocomon or heparin; Antithrombotic agents; Circulating drugs such as propranolol; Metabolic synergists such as glutathione; antituberculous agents such as p-aminosalicylic acid, isoniazid, capreomycin sulfate, cyclohexine, etabutol, ethionamide, pyrazineamide, rifampin or streptomycin sulfate; Antibacterial agents such as acyclovir, amantadine, azidomidine, ribavirin or vidarabine; Vasodilators such as diltiazem, nifedipine, verapamil, erythritol tetranitrate, isosorbide dinitrate, nitroglycerin or pentaerythritol tetranitrate; Dapson, chloramphenicol, neomycin, sefachlor, cephadroxyl, separexin, cepradine, erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin, cyclacillin Antibiotics such as picloxacillin, hetacillin, methicillin, naphcillin, penicillin or tetracillin; Anti-inflammatory agents, such as diflunisal, ibuprofen, indomethacin, meclefenamate, mefenamic acid, naproxen, phenylbutazone, pyroxicam, tolmetin, aspirin or salicylate; Protoplasts such as chloroquine, metronidazole, quinine or meglumine antimonate; Antirheumatic agents such as penicillamine; Anesthetics such as peregreic; Opiates, such as codeine, morphine or opium; Ventricular glycosides such as deslanees, digitoxins, dioxins, digitalins or digitalis; Neuromuscular blocking antibodies such as atraccurium mesylate, galamine triethiodide, hexafluorenium bromide, metocurin iodide, pancuronium bromide, succinylcholine chloride, tubokurarin chloride or bekuronium bromide ; Amobarbital, Amobarbital Sodium, Aprobabital, Butabarbital Sodium, Chloral Hydrate, Etchlorbinol, Etynamate, Flurazzepam Hydrochloride, Glutetimide, Metotrimethrazine Hydrochloride, Methipril Sedatives such as ron, midazolam hydrochloride, paraaldehyde, pentobarbital, secobarbital sodium, debutal, temazepam or triazolam; Local anesthetics such as bupivacaine, chloroprocaine, ethidocaine, lidocaine, mepivacaine, procaine or tetracaine; General anesthetics and pharmaceutically acceptable salts such as dropperidol, etomidate, fentanyl citrate with dropperidol, ketamine hydrochloride, metohexal sodium or thiopental (e.g. acids such as hydrochloride or bromate) Addition salts or basic salts such as sodium, calcium or magnesium salts) or derivatives thereof (for example acetate); And radiochemicals, including beta-emitters. Antithrombotic agents such as vitamin K antagonists, agents with heparin type activity such as heparin and antithrombin III, dalteparin and enoxaparin; Platelet aggregation inhibitors such as ticlopidine, aspirin, dipyridamole, iloprost and absximab; And platelet enzymes such as streptokinase and plasminogen activator are of particular interest. Examples of other therapeutic agents include genetic materials such as hexane, RNA, and DNA of natural or synthetic organs, including recombinant RNA and DNA. DNA encoding a particular protein can be used to treat many different types of disease. For example, tumor deprivation factor or interleukin-2 can be provided for the treatment of advanced tumors; Thymidine kinase can be provided to treat uterine cancer or brain tumors; Interleukin-2 can be provided to treat neuroblastoma, malignant melanoma or kidney cancer; Interleukin-4 may be provided to treat tumors.
The contrast agent formulations according to the invention can be used as vehicles for imaging modalities other than ultrasound, for example X-rays, light imaging, magnetic resonance and, particularly preferably, contrast enhancing portions for scintogram imaging imaging agents. Growth control in the dispersed gas phase can be used to irradiate target organs or tissues with ultrasound to induce desired growth control and temporary retention of reagents such that these agents are placed in the intended area of the patient's body, which is a suitable ratio. It can be imaged using an ultrasound imaging modality.
In addition, the contrast agent formulations according to the invention can be used as vehicles for therapeutically active substances that do not necessarily need to be removed from the formulations in order to exhibit their therapeutic effect. Such formulations may contain radioactive atoms or ions, such as beta-emitters, which exhibit local radiation release effects and then exhibit gas phase growth dispersed at the target site and transient retention of reagents. It is to be understood that such agents are designed so that subsequent contraction of the dispersed gas and stoppage of the dispersed gas do not occur until the required therapeutic radiation dose is administered.
The contrast agent formulations according to the invention may also exhibit their own therapeutic properties. Thus, the dispersed gas can be concentrated in the capillary causing the tumor and act as a cytotoxic agent by blocking such capillary. Thus, localized embolism can be obtained by applying ultrasonic energy locally; This may be important by itself or in combination with other therapeutic means. In addition, the concentration of gas dispersed in the capillary can increase the absorption of ultrasonic energy in the overheat treatment; It can be used to treat liver tumors. Irradiation with ultrasound rays concentrated at a relatively high energy (eg 5 W) of 1.5 MHz may be suitable for this application.
It will be appreciated that the present invention extends to formulations comprising an aqueous medium having a gas dispersed therein, and a composition comprising a diffusion component as a general composition of matter, and can be extended to use for non-imaging purposes.
The following non-limiting examples serve to illustrate the invention.
Example 1-Formulation
a) perfluorobutane gas dispersion
Hydrogenated phosphatidylserine (100 mg) in 2% solution of propylene glycol in purified water (20 mL) was heated to 80 ° C. for 5 minutes and the resulting dispersion was cooled to room temperature overnight. Transfer 1 ml portions to 2 ml vials, flush each volume of top space with perfluorobutane gas, and use the vial for 45 seconds using an Espe CapMix® mixer for dental materials. Shaking to give a milky white microbubble dispersion with a volume average diameter of 5.0 μm when measured using a Coulter counter [all Coulter counter measurements have instruments having a 50 μm aperture and a measuring range of 1 to 30 μm. Using at room temperature; Isoton II was used as electrolyte.
b) dispersion of lyophilised perfluorobutane gas dispersion
Samples of the milky dispersion from Example 1 (a) were washed three times by centrifugation, and after removing the lower layer, an equal volume of 10% sucrose solution was added. The resulting dispersion was lyophilised, redispersed in purified water and obtained a milky microbubble dispersion with a volume average diameter of 3.5 μm as measured using a Coulter counter.
c) 2-methylbutane emulsion
Hydrogenated phosphatidylserine (100 mg) in purified water (20 mL) was heated to 80 ° C. for 5 minutes and the resulting dispersion was cooled to 0 ° C. overnight. 1 ml of the dispersion was transferred to a 2 ml vial, to which 200 μl of 2-methylbutane (boiling point 28 ° C.) was added. Subsequently, the vial was shaken for 45 seconds using Capmix to obtain an emulsion of the diffusion component, which was stored at 0 ° C. when not in use. The volume average diameter of the emulsion droplets was 1.9 μm as measured using a Coulter counter.
d) perfluoropentane emulsion
The method of Example 1 (c) was repeated except that 2-methylbutane was replaced with perfluoropentane (boiling point 29 ° C.). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
e) 2-chloro-1,1,2-trifluoroethyl difluoromethyl ether emulsion
The method of Example 1 (c) was repeated except that 2-methylbutane was replaced with 2-chloro-1,1,2-trifluoroethyl difluoromethyl ether (boiling point 55-57 ° C.). . The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
f) 2-bromo-2-chloro-1,1,1-trifluoroethane emulsion
The method of Example 1 (c) was repeated except that 2-methylbutane was replaced with 2-bromo-2-chloro-1,1,1-trifluoroethane (boiling point 49 ° C.). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
g) 1-chloro-2,2,2-trifluoroethyl difluoromethyl ether emulsion
The method of Example 1 (c) was repeated except that 2-methylbutane was replaced with 1-chloro-2,2,2-trifluoroethyl difluoromethyl ether (boiling point 49 ° C.). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
h) dispersion of gas containing polymer / human serum albumin particles
Gas-containing particles coated with human serum albumin of polymers prepared from ethylidene bis (16-hydroxyhexadecanoate) and adipoyl chloride, prepared according to example 3 (a) of WO9607434 Mg) was triturated in plaster and shaken for 24 hours on a laboratory shaker and dispersed in 0.9% aqueous sodium chloride (10 mL).
i) dispersion of gas-containing polymer / gelatin particles
Gelatin-coated gas-containing particles (100 mg) of polymers prepared from ethylidene bis (16-hydroxyhexadecanoate) and adipoyl chloride, prepared according to example 3 (e) of WO 9607434. Was ground in a plaster, shaken for 24 hours on a laboratory shaker and dispersed in 0.9% aqueous sodium chloride (10 mL).
j) 2-methylbutane emulsion
The method of Example 1 (c) was repeated except that the emulsion was diluted 10-fold before use and stored in an ice bath when not in use.
k) perfluoropentane emulsion
The procedure of Example 1 (d) was repeated except that the emulsion was diluted 10-fold before use and stored in an ice bath when not in use.
l) perfluoropentane emulsion
Hydrogenated phosphatidylserine (100 mg) in purified water (20 mL) was heated to 80 ° C. for 5 minutes and the resulting dispersion was cooled to 0 ° C. overnight. 1 ml of the dispersion was transferred to a 2 ml vial, to which 100 μl of perfluoro-n-pentane (boiling point 29 ° C.) was added. Subsequently, the vial was shaken for 75 seconds using Capmix to obtain an emulsion of the diffusion component, which was stored at 0 ° C. when not in use. The volume average diameter of the emulsion droplets was 2.9 μm as measured using a Coulter counter.
m) perfluorobutane emulsion
The method of Example 1 (l) was repeated except that perfluoropentane was replaced with perfluorobutane (boiling point-2 ° C.). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
n) perfluoropentane emulsion prepared by sonication
Hydrogenated phosphatidylserine (500 mg) in purified water (100 mL) was heated to 80 ° C. for 5 minutes and the resulting dispersion was cooled to room temperature overnight. 10 ml of the dispersion was transferred to a 30 ml vial, to which perfluoropentane (1 ml) was added. The resulting mixture was sonicated for 2 minutes to obtain a dispersion of diffusion components with droplets having an average diameter of less than 1 μm.
o) perfluoropentane emulsions
The method of Example 1 (l) was repeated except that the volume of the used perfluoropentane was reduced to 60 μl. The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
p) perfluoropentane emulsion
The method of Example 1 (l) was repeated except that the volume of the used perfluoropentane was reduced to 20 μl. The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
q) perfluoropentane: perfluoro-4-methylpent-2-ene (1: 1) emulsion
Example 1 (except that perfluoropentane was replaced with a mixture of 50 μl of perfluoropentane (boiling point 29 ° C.) and 50 μl of perfluoro-4-methylpent-2-ene (boiling point 49 ° C.) The method of l) was repeated. The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used. The volume average diameter of the emulsion droplets was 2.8 μm as measured using a Coulter counter.
r) perfluoropentane: 1H, 1H, 2H-heptafluoropent-1-ene (1: 1) emulsion
Except for replacing perfluoropentane with a mixture of 50 μl perfluoropentane (boiling point 29 ° C.) and 50 μl of 1H, 1H, 2H-heptafluoropent-1-ene (boiling point 30 to 31 ° C.), The method of Example 1 (l) was repeated. The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
s) distearoylphosphatidylcholine: perfluoropentane emulsion stabilized by distearoylphosphatidylcholine (1: 1)
The method of Example 1 (l) was repeated except that hydrogenated phosphatidylserine was replaced with a mixture of distearoylphosphatidylcholine (50 mg) and distearoylphosphatidylserine, sodium salt (50 mg). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
t) distearoylphosphatidylcholine: perfluoropentane emulsion stabilized by distearoylphosphatidylcholine (3: 1)
The method of Example 1 (l) was repeated except that hydrogenated phosphatidylserine was replaced with a mixture of distearoylphosphatidylcholine (75 mg) and distearoylphosphatidylserine, sodium salt (25 mg). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
u) distearoylphosphatidylcholine: perfluoropentane emulsion stabilized by distearoylphosphatidylglycerol (3: 1)
The method of Example 1 (l) was repeated except that hydrogenated phosphatidylserine was replaced with a mixture of distearoylphosphatidylcholine (75 mg) and distearoylphosphatidylglycerol, sodium salt (25 mg). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
v) hydrogenated phosphatidylcholine: perfluoropentane emulsion stabilized by hydrogenated phosphatidylserine (11: 1)
The method of Example 1 (l) was repeated except that hydrogenated phosphatidylserine was replaced with 100 mg of a mixture of hydrogenated phosphatidylcholine and hydrogenated phosphatidylserine (11: 1). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
w) distearoylphosphatidylcholine: perfluoro-4-methylpent-2-ene emulsion stabilized by distearoylphosphatidylcholine (3: 1)
Hydrogenated phosphatidylserine was replaced with a mixture of distearoylphosphatidylcholine (75 mg) and distearoylphosphatidylserine, sodium salt (25 mg), and perfluoropentane was replaced with perfluoro-4-methylpent-2- The method of Example 1 (l) was repeated except that it was replaced with yen. The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
x) distearoylphosphatidylcholine: perfluoropentane stabilized with distearoylphosphatidylserine (3: 1): perfluoro-4-methylpent-2-ene (1: 1) emulsion
Example 1 (w), except that perfluoro-4-methylpent-2-ene was replaced with a mixture of 50 μl perfluoropentane and 50 μl perfluoro-4-methylpent-2-ene The method was repeated. The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
y) distearoylphosphatidylcholine: perfluoropentane stabilized with distearoylphosphatidylglycerol (3: 1): perfluoro-4-methylpent-2-ene (1: 1) emulsion
The method of Example 1 (x) was repeated except that distearoylphosphatidylserine sodium salt was replaced with distearoylphosphatidylglycerol sodium salt. The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
z) perfluorodecalin: perfluorobutane emulsion
Hydrogenated phosphatidylserine (100 mg) in aqueous glycerol (5.11%) / propylene glycol (1.5%) (20 mL) was heated to 80 ° C. for 5 minutes and the resulting dispersion was cooled to 0 ° C. overnight. 1 ml of the dispersion was transferred to a 2 ml vial, to which 100 μl of perfluorodecalin (boiling point 141 to 143 ° C.) was added and saturated with perfluorobutane (boiling point −2 ° C.). Subsequently, the vial was shaken for 60 seconds using Capmix to obtain an emulsion of the diffusion component which was stored at 0 ° C. when not in use.
aa) perfluorodecalin: perfluoropropane emulsion
The method of Example 1 (z) was repeated except that perfluorodecalin saturated with perfluorobutane was replaced with perfluorodecalin saturated with perfluoropropane (boiling point -39 ° C). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
ab) perfluorodecalin: sulfur hexafluoride emulsion
The method of Example 1 (z) was repeated except that perfluorodecalin saturated with perfluorobutane was replaced with perfluorodecalin saturated with sulfur hexafluoride (boiling point -64 ° C.). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
ac) pentafluoropentane emulsions stabilized with Fluorad FC-170C
1 mL of a dispersion of fluoride FC-170C (200 mg) in purified water (20 mL) was transferred to a 2 mL vial, to which 100 μl perfluoro-n-pentane was added. Subsequently, the vial was shaken for 75 seconds using Capmix to obtain an emulsion of the diffusion component, which was stored at 0 ° C. when not in use.
ad) Pluronic F68: perfluoropentane emulsion stabilized with fluoride FC-170C
100 μl of 10% Pluronic F68 solution was added to 200 μl of 1% fluoride FC170C and 700 μl of purified water. The resulting mixture was transferred to a 2 ml vial and 100 μl of perfluoro-n-pentane was added thereto. Subsequently, the vial was shaken for 75 seconds using Capmix to obtain an emulsion of the diffusion component, which was stored at 0 ° C. when not in use. Subsequently, the sample of the emulsion was transferred to a twisted plastic vial (2.8 mL) and sonicated in a water bath for 2 minutes (pulse sonication: 1 per second). The volume average diameter of the sonicated emulsion droplets was 0.99 μm as measured by Coulter counter.
ae) Pluronic F68: perfluoropentane emulsion stabilized with fluoride FC-170C and prepared by homogenization
1 ml of 10% Pluronic F68 solution was added to 2 ml of 1% fluoride FC170C and 7 ml of purified water, followed by 1 ml of perfluoro-n-pentane to the resulting mixture. The dispersion thus obtained was then homogenized by stirrer / fixed homogenizer at 23000 rpm for 2 minutes. The resulting emulsion was transferred to a twisted plastic vial (10 ml) and then sonicated in a water bath for 2 minutes (pulse sonication: 1 per second).
af) perfluoropentane emulsion
Hydrogenated phosphatidylserine (250 mg) in purified water (100 mL) was heated to 80 ° C. for 5 minutes and the resulting dispersion was cooled to 0 ° C. overnight. 1 ml of the dispersion was transferred to a 2 ml vial, to which 100 [mu] l of perfluoropentane was added. The vial was shaken for 75 seconds using Capmix to obtain an emulsion of the diffusion component which was stored at 0 ° C. when not in use.
ag) dispersion of lyophilized perfluorobutane gas dispersion
Samples of the milky dispersion from Example 1 (a) were washed three times by centrifugation, and after cooling water was removed, the same volume of 10% sucrose solution was added. The resulting dispersion was lyophilised and then redispersed in purified water to give a milky microbubble dispersion having a volume average diameter of 2.6 μm when measured using a Coulter counter.
ah) perfluoropropane gas dispersion
The method of Example 1 (a) was repeated except that the perfluorobutane gas was replaced with a perfluoropropane gas. The resulting milky microbubble dispersion had a volume average diameter of 2.6 μm as measured using a Coulter counter.
ai) perfluoropentane emulsion
Hydrogenated phosphatidylserine (100 mg) in purified water (100 mL) was heated to 80 ° C. for 5 minutes and the resulting dispersion was cooled to 0 ° C. overnight. 1 ml of the dispersion was transferred to a 2 ml vial, to which 100 [mu] l of perfluoropentane was added. The vial was shaken for 75 seconds using Capmix to obtain an emulsion of the diffusion component which was stored at 0 ° C. when not in use.
aj) Brij 58: perfluoropentane emulsion stabilized with fluoride FC-170C and prepared by shaking
Breeze 58 (400 mg) was added to a solution of 0.1% fluoride FC-170C (10 mL) and stirred at room temperature for 1 hour. 1 ml of the resulting solution was transferred to a 2 ml vial, to which perfluoropentane (100 μl) was added. Subsequently, the vial was shaken for 75 seconds using Capmix to obtain an emulsion of the diffusion component, which was stored at 0 ° C. when not in use.
ak) Breeze 58: perfluoropentane emulsion stabilized with fluoride FC-170C and prepared by sonication
Breeze 58 (400 mg) was added to a solution of 0.1% fluoride FC-170C (10 mL) and stirred at room temperature for 1 hour. Perfluoropentane (1 mL) was then added, and the resulting mixture was sonicated for 2 minutes to give an emulsion of small droplets of diffusion component. The emulsion was stored at 0 ° C. when not used.
al) perfluoro-4-methylpent-2-ene emulsion
The method of Example 1 (l) was repeated except that perfluoropentane was replaced with perfluoro-4-methylpent-2-ene (boiling point 49 ° C.). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
am) 1H, 1H, 2H-heptafluoropent-1-ene emulsion
The method of Example 1 (l) was repeated except that perfluoropentane was replaced with 1H, 1H, 2H-heptafluoropent-1-ene (boiling point 30-31 ° C.). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
an) perfluorocyclopentene emulsion
The method of Example 1 (l) was repeated except that perfluoropentane was replaced with perfluorocyclopentene (boiling point 27 ° C.). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
ao) perfluorodimethylcyclobutane emulsion
The method of Example 1 (l) was repeated except that perfluoropentane was replaced with perfluorodimethylcyclobutane (mixture of 1,2- and 1,3-isomers, boiling point 45 ° C.). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
ap) perfluorohexane: emulsion of n-pentane azeotrope
Perfluoro-n-hexane (boiling point 59 ° C.) 4.71 g (0.014 mol) of [Fluorochem Ltd.] and n-pentane (boiling point 36 ° C.) [Fluka AG] 0.89 g ( 0.012 mole) was mixed in a vial to obtain an azeotropic mixture which appeared to readily boil at 35 ° C. In another vial, hydrogenated phosphatidylserine (100 mg) in purified water (20 mL) was heated to 80 ° C. for 5 minutes and the resulting dispersion was cooled to room temperature. 1 ml of the phospholipid dispersion was transferred to a 2 ml vial, to which 100 [mu] l of the azeotropic mixture was added. The vial was then shaken for 45 seconds using Capmix to obtain an emulsion of the diffusion component and stored at room temperature when not in use.
aq) perfluorodimethylcyclobutane emulsion
Example 1 (except that perfluoropentane was replaced with perfluorodimethylcyclobutane (1,1-isomers greater than 97%, 1,2- and 1,3-isomers balanced) The method of l) was repeated. The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
ar) perfluorohexane emulsion
The method of Example 1 (l) was repeated except that perfluoropentane was replaced with perfluorohexane (boiling point 57 ° C.). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
as) perfluorodimethylcyclobutane emulsion stabilized with fluorinated surfactant
Hydrogenated phosphatidylserine was perfluorinated distearoylphosphatidylcholine (5 mg / ml) or a mixture of perfluorinated distearoylphosphatidylcholine and hydrogenated phosphatidylserine (3: 1, total lipid concentration 5 mg / ml) The method of Example 1 (aq) was repeated except that it was replaced with. The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
at) 2,2,3,3,3-pentafluoropropyl methyl ether emulsion
The method of Example 1 (l) was repeated except that perfluoropentane was replaced with 2,2,3,3,3-pentafluoropropyl methyl ether (boiling point 46 ° C.). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
au) 2H, 3H-decafluoropentane emulsion
The method of Example 1 (l) was repeated except that perfluoropentane was replaced with 2H, 3H-decafluoropentane (boiling point 54 ° C.). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
av) perfluorodimethylcyclobutane emulsion stabilized with lysophosphatidylcholine
The method of Example 1 (aq) was repeated except that hydrogenated phosphatidylserine was replaced with lysophosphatidylcholine. The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
aw) hydrogenated phosphatidylserine: perfluorodimethylcyclobutane emulsion stabilized with lysophosphatidylcholine (1: 1)
The method of Example 1 (aq) was repeated except that hydrogenated phosphatidylserine was replaced with a mixture of hydrogenated phosphatidylserine and lysophosphatidylcholine (1: 1). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
ax) Perfluorodimethylcyclobutane emulsion stabilized with polyethylene glycol 10,000-based surfactants
Example 1, except that the hydrogenated phosphatidylserine dispersion was replaced with a solution of α- (16-hexadecanoyloxy-hexadecanoyl) -ω-methoxypolyethylene glycol 10,000 (10 mg / ml) in water. The method of (aq) was repeated. The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
ay) perfluorodimethylcyclobutane emulsion stabilized with polyethylene glycol 10,000-based surfactants
Example 1, except that the hydrogenated phosphatidylserine dispersion was replaced with a solution of α- (16-hexadecanoyloxy-hexadecanoyl) -ω-methoxypolyethylene glycol 10,000 (20 mg / ml) in water. The method of (aq) was repeated. The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
az) Microbubbles filled with perfluorobutane encapsulated by phosphatidylserine and RGDC-Mal-polyethylene glycol 2000-distearoylphosphatidylethanolamine
To a mixture of phosphatidylserine (4.5 mg) and Mal-polyethylene glycol 2000-distearoylphosphatidylethanolamine (0.5 mg) in a vial was added a solution of 1.4% propylene glycol / 2.4% glycerol in water (1 mL). The dispersion was heated to 80 ° C. for 5 minutes, cooled to room temperature and flushed with perfluorobutane gas. The vial was shaken for 45 seconds using Capmix and then placed on a roller table. After centrifugation, the cooling water was exchanged with the RGDC solution in sodium phosphate buffer at pH 7.5 and the vial was left on the roller table for several hours.
ba) Microbubbles filled with perfluorobutane encapsulated by phosphatidylserine and dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000
To a vial containing phosphatidylserine and dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 (ratio 10: 1), a 2% propylene glycol solution in water was added to obtain a lipid concentration of 5 mg / ml. The dispersion was heated to 80 ° C. for 5 minutes, then cooled to room temperature and the top space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds using Capmix and then placed on a roller table. Washed by centrifugation, the cooling water was removed and the same volume of water containing 10% sucrose was added. The resulting dispersion was lyophilised and then redispersed with water to give a milky white microbubble dispersion.
bb) Microbubbles filled with perfluorobutane encapsulated by phosphatidylserine and distearoylphosphatidylethanolamine-polyethylene glycol 5000
To a vial containing phosphatidylserine and distearoylphosphatidylethanolamine-polyethylene glycol 5000 (ratio 10: 1), a 2% propylene glycol solution in water was added to obtain a lipid concentration of 5 mg / ml. The dispersion was heated to 80 ° C. for 5 minutes, then cooled to room temperature and the top space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds using Capmix and then placed on a roller table. Washed by centrifugation, the cooling water was removed and the same volume of water containing 10% polyethylene glycol was added. The resulting dispersion was lyophilised and then redispersed to give a milky white microbubble dispersion.
bc) Microbubbles filled with perfluorobutane encapsulated by phosphatidylserine and dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000
A solution of 2% propylene glycol in water was added to a vial containing phosphatidylserine and dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 (ratio 10: 1) to obtain a lipid concentration of 5 mg / ml. The dispersion was heated to 80 ° C. for 5 minutes, then cooled to room temperature and the top space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds using Capmix and then placed on a roller table. After washing by centrifugation, cooling water was removed, the same volume of water was added to give a milky white microbubble dispersion.
bd) Microbubbles filled with perfluorobutane encapsulated by phosphatidylserine and distearoylphosphatidylethanolamine-polyethylene glycol 5000
To a vial containing phosphatidylserine and distearoylphosphatidylethanolamine-polyethylene glycol 5000 (ratio 10: 1), a 2% propylene glycol solution in water was added to obtain a lipid concentration of 5 mg / ml. The dispersion was heated to 80 ° C. for 5 minutes, then cooled to room temperature and the top space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds using Capmix and then placed on a roller table. After washing by centrifugation, cooling water was removed, the same volume of water was added to give a milky white microbubble dispersion.
be) Perfluorodimethylcyclobutane emulsion stabilized by phosphatidylserine and dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000
The method of Example 1 (aq) was repeated except that hydrogenated phosphatidylserine was replaced with a mixture of hydrogenated phosphatidylserine and dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 (ratio 10: 1). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
bf) Perfluorodimethylcyclobutane emulsion stabilized by phosphatidylserine and distearoylphosphatidylethanolamine-polyethylene glycol 5000
The method of Example 1 (aq) was repeated except that hydrogenated phosphatidylserine was replaced with a mixture of hydrogenated phosphatidylserine and distearoylphosphatidylethanolamine-polyethylene glycol 5000 (ratio 10: 1). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
bg) microbubbles filled with lyophilised perfluorobutane redispersed in an emulsion
Samples of the milky dispersion prepared as described in Example 1 (bp) were washed three times by centrifugation, and after cooling water was removed, the same volume of 10% sucrose solution was added. The resulting dispersion was lyophilised and then redispersed in an emulsion prepared as described in Example 1 (aq) immediately before use.
bh) avidinylated perfluorodimethylcyclobutane emulsion droplets
Distearoylphosphatidylserine (4.5 mg) and biotin-dipalmitoylphosphatidylethanolamine (0.5 mg) were weighed into clean vials and 1.0 ml of a 2% propylene glycol solution was added. After heating to 80 ° C., the mixture was cooled to room temperature. 100 μl of perfluorodimethylcyclobutane was added and the vial was shaken for 75 seconds using Capmix to obtain an emulsion of the diffusion component. Diluted samples of the emulsion (100 μl of the emulsion in 1 mL of water) were incubated with excess avidin and placed on a roller table. The diluted emulsion was then washed with excess water and concentrated by centrifugation.
bi) 1H-tridecafluorohexane emulsion
The method of Example 1 (l) was repeated except that perfluoropentane was replaced with 1H-tridecafluorohexane (boiling point 71 ° C.). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
bj) perfluoropentane emulsion
The method of Example 1 (l) was repeated except that perfluoropentane was replaced with perfluoroheptane (boiling point 80-85 ° C.). The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
bk) Perfluorodimethylcyclobutane emulsions with phosphatidylserine and fluorescent streptavidin
Distearoylphosphatidylserine (4.5 mg) and biotin-dipalmitoylphosphatidylethanolamine (0.5 mg) were weighed into clean vials and 1.0 ml of a 2% propylene glycol solution was added. After heating to 80 ° C., the mixture was cooled to room temperature. 100 μl of perfluorodimethylcyclobutane was added and the vial was shaken for 75 seconds using a Capmix mixer to obtain an emulsion of the diffusion component. Diluted samples of the emulsion (100 μl of the emulsion in 1 mL of water) were incubated with excess fluorescent streptavidin in phosphate buffer and placed on a roller table. The diluted emulsion was then washed with excess water and concentrated by centrifugation.
bl) dispersion of lyophilised perfluorobutane gas dispersion
Samples of the milky dispersion prepared as described in Example 1 (a) were washed three times by centrifugation, and after cooling water was removed, the same volume of 10% sucrose solution was added. The resulting dispersion was lyophilised and redispersed in purified water immediately before use.
bm) perfluorodimethylcyclobutane emulsion stabilized by sterile phosphatidylserine
The method of Example 1 (aq) was repeated except that hydrogenated phosphatidylserine was replaced with hydrogenated phosphatidylserine sterilization solution. The emulsion of the diffusion components thus obtained was stored at 0 ° C. when not used.
bn) perfluoropropane gas dispersion
The method of Example 1 (a) was repeated except that the perfluorobutane gas was replaced with a perfluoropropane gas.
bo) Distributed Echovist
EcoBeast granules (Schering AG) (0.25 g) were added to an emulsion (1.15 mL) prepared as described in Example 1 (aq).
bp) perfluorobutane gas dispersion
Hydrogenated phosphatidylserine (500 mg) was added to a solution of 1.5% propylene glycol / 5.11% glycerol in water (100 mL) and heated to 80 ° C. for 5 minutes, after which the resulting dispersion was cooled to ambient temperature. Transfer to a 1 ml volume of 2 ml vial, flush each volume above the headspace with perfluorobutane gas, shake the vial for 45 seconds using Capmix and then place the vial on the roller table.
bq) Preparation of biotinylated perfluorobutane microbubbles
Distearoylphosphatidylserine (4.5 mg) and biotin-dipalmitoylphosphatidylethanolamine (0.5 mg) were weighed into clean vials and 1.0 ml of a solution of 1.4% propylene glycol / 2.4% glycerol was added. After heating to 78 ° C., the mixture was cooled to room temperature and the upper space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds using a Capmix mixer and then left on the roller table for 16 hours. The resulting microbubbles were washed thoroughly with deionized water.
br) aerogels
Pyrolyzed resorcinol-formaldehyde airgel particles (provided by Dr. Pekala, Lawrence Livermore National Laboratory) at 300 μl water, 1 drop of pH 9 buffer, and 1% Pluronic F68 5-10 drops were added. The airgel particles precipitated quickly but did not aggregate.
bs) small bubble
A rubber tube with an internal diameter of 8 mm and a length of approximately 20 cm was left to stand vertically, the bottom end was closed, and [Ystral (registered trademark) rotationally fixed homogenization manufactured according to Example 1 (a). To prepare a microbubble dispersion] was charged with a microbubble dispersion. Two hours later, a syringe connected to the cannula was inserted into a rubber tube near the bottom and a 1 ml fraction of the microbubble dispersion fractionated by size was collected. Coulter counter analysis showed that the average diameter of the microbubble dispersion thus obtained was 1.2 μm.
bt) 5% albumin: perfluorobutane gas dispersion stabilized by 5% dextrose (1: 3)
20% human serum was diluted with 5% purified water. 5 ml of diluted albumin sample was further diluted with 5% glucose (15 ml) and the resulting mixture was transferred to a vial. The top space was flushed with perfluorobutane gas and the vial was sonicated for 80 seconds to yield a milky microbubble dispersion.
bu) Dispersion of Buckminsterfullerene C ₆₀
Buchminsterfullerene C ₆₀ was added to 2.5% human serum albumin (1 mL) in 2 mL vials and shaken for 75 seconds using Capmix.
bv) sulfur hexafluoride gas dispersion
Distearoylphosphatidylcholine: dipalmitoylphosphatidylglycerol (10: 1) stabilized microbubbles were prepared as described in Example 5 of WO9409829. Thus, 50 mg of distearoylphosphatidylcholine, 5 mg of dipalmitoylphosphatidylglycerol and 2.2 g of polyethylene glycol were dissolved in 22 ml of t-butanol at 60 ° C., the solution was rapidly cooled to −77 ° C. and lyophilised overnight I was. 100 mg of the resulting powder was placed in a vial and the upper space was evacuated and filled with sulfur hexafluoride. 1 ml of purified water was added immediately before use to obtain a microbubble dispersion.
bw) 2-methylbutane emulsion
Hydrogenated phosphatidylserine (100 mg) in purified water (20 mL) was heated to 80 ° C. for 5 minutes and the resulting dispersion was cooled overnight in a refrigerator. 1 ml of the dispersion was transferred to a 2 ml vial, to which 100 μl of 2-methylbutane was added. The vial was shaken for 75 seconds using Capmix to obtain an emulsion of the diffusion component and then stored at 0 ° C. when not in use.
bx) liquefied perfluorobutane gas dispersion in aqueous sodium bicarbonate
Samples of the milky dispersion from Example 1 (a) were washed three times by centrifugation, and after cooling water was removed, the same volume of 10% sucrose solution was added. The resulting dispersion was lyophilised and then redispersed in 0.1 M sodium bicarbonate solution.
by) perfluorobutane gas dispersion
Perfluorobutane gas dispersion was prepared as in Example 1 (a). The dispersion was washed three times with purified water by centrifugation and the cooling water was removed to give a milky microbubble dispersion.
bz) perfluorobutane gas dispersion with iron oxide particles
To 1 mL of perfluorobutane gas dispersion prepared as described in Example 1 (by) was added 1 mL of purified water. The pH was raised to 11.2 with ammonium hydroxide and the dispersion was heated at 60 ° C. for 5 minutes. Uncoated iron oxide particles (0.3 mL, 4.8 mg / Fe mL) were added and the dispersion was left for 5 minutes. The pH was dropped to 5.9 with hydrochloric acid to give a brown dispersion, which after a while formed an upper layer with brown particles, a clear, uncolored underlayer, with no precipitation.
ca) perfluorobutane gas dispersion with iron oxide particles
0.3 ml of uncoated iron oxide particles (4.8 mg Fe / ml) at pH 7 were added to 1 ml of perfluorobutane gas dispersion prepared as in Example 1 (by) to give a brown precipitate which was left to brown. A microbubble, an upper layer with a clear lower layer solution was formed, with no precipitation.
cb) [Comparative Example]
0.3 ml of uncoated iron oxide particles (4.8 mg Fe / ml) was added to 1 ml of hydrogenated phosphatidylserine solution in purified water (5 mg / ml) to give a brown precipitate which was left to obtain a brown precipitate.
cc) perfluorobutane gas dispersion with iron oxide particles coated with oleic acid
1.3 mmol (0.259 g) of FeCl ₂ 4H ₂ O and 2.6 mmol (0.703 g) of FeCl ₃ 6H ₂ O were dissolved in 10 mL of purified water, and 1.5 mL of ammonium hydroxide was added thereto. The resulting iron oxide particles were washed five times with purified water (25 mL). Dilute ammonium hydroxide was added to the particles and the suspension was heated to 80 ° C. Oleic acid (0.15 g) was added and the dispersion was left at ambient temperature for 5 minutes. Purified water (10 mL) was added and the pH was dropped to 5.4 using hydrochloric acid. The dispersion was sonicated for 15 minutes, then the cooling water was removed and the particles suspended in 2-methylbutane (5 mL) to produce a fine black dispersion.
25 mg of distearoylphosphatidylcholine and 2.5 mg of dimyristoylphosphatidylglycerol were dissolved in 11 ml of t-butanol at 60 ° C., and 0.1 ml of iron oxide particles from above were added together with 1.1 g of polyethylene glycol 4000. The dispersion was heated at 60 ° C. for 10 minutes, cooled rapidly to −77 ° C. and lyophilised. 100 mg of lyophilisate was introduced into a 2 ml vial, then discharged and flushed twice with perfluorobutane gas. Subsequently, the lyophilisate was dispersed in 1 ml of purified water and washed twice with purified water by centrifugation while removing the cooling water and the precipitate. After standing, the resulting dispersion was light gray in color with a floating top layer.
Example 2-In Vitro Characterization of Microbubble Growth by Microscopy / Visual Observation
a)
Dilute one drop of perfluorobutane gas dispersion from Example 1 (a) at about 4 ° C. into one drop of purified water supersaturated at about 4 ° C. on a microscope objective cooled to about 4 ° C. The magnification was observed. It was observed that the size of the microbubbles varied from 2 to 5 μm. Subsequently, the temperature was raised to about 40 ° C., where a significant increase in microbubble size was observed, with larger microbubbles increasing in size to maximum. The number of microbubbles decreased significantly after about 5 minutes.
b) [control]
One drop of 2-methylbutane emulsion from Example 1 (c) cooled in an ice bath to about 0 ° C. was placed on a microscope objective lens cooled to about 0 ° C. and observed at a magnification of 400 times. It was observed that the size of the oily droplets of the emulsion varied from 2 to 6 μm. Then, temperature was raised to about 40 degreeC. No formation of microbubbles was observed.
c)
The perfluorobutane gas dispersion sample from Example 1 (a) (0.5 mL) was diluted with purified water (50 mL) and cooled to 0 ° C. A portion of this diluted dispersion (1 mL) was mixed with a portion (100 μl) of 2-methylbutane emulsion from Example 1 (c). The resulting mixture 1 drop was placed on a microscope objective maintained at 0 ° C. by heating and cooling steps and covered with a cover glass at 0 ° C. The temperature of the objective lens was gradually raised to 40 ° C. using the heating and cooling steps. Rapid and substantial growth of the microbubbles was observed by microscopy and confirmed by size and distribution measurements performed using the Malvern Mastersizer.
d) [comparative example]
The perfluorobutane gas dispersion sample from Example 1 (a) (0.5 mL) was diluted with purified water (50 mL) and cooled to 0 ° C. in an ice bath. A portion of the diluted dispersion (1 mL) was mixed with 100 μl of 5 mg / ml dispersion of hydrogenated phosphatidylserine in purified water at 0 ° C. The resulting mixture 1 drop was placed on a microscope objective lens cooled to 0 ° C. and observed at 400 × magnification. It was observed that the size of the microbubbles varied from 2 to 5 μm. Subsequently, the temperature was raised to about 40 ° C., although a significant increase in microbubble size was observed here, although the increase was not greater or faster than that observed in Example 2 (c).
e)
The perfluorobutane gas dispersion sample from Example 1 (a) was diluted (1: 1) with purified water and cooled to 0 ° C. 1 drop of 2-chloro-1,1,2-trifluoroethyl difluoromethyl ether emulsion from Example 1 (e) diluted on a microscope objective lens maintained at 0 ° C. by heating and cooling steps. It was added to the dispersion and covered with a cover glass at 0 ° C. The temperature of the objective lens was gradually raised to 40 ° C. using the heating and cooling steps. Rapid and substantial growth of the microbubbles was observed by microscopy.
f)
Samples of the perfluorobutane gas dispersion from Example 1 (a) were diluted with purified water (1: 1) and cooled to 0 ° C. 1 drop of 2-bromo-2-chloro-1,1,1-trifluoroethane emulsion from Example 1 (f) was microbubbles diluted on a microscope objective lens maintained at 0 ° C. by heating and cooling steps. It was added to the dispersion and covered with a cover glass at 0 ° C. The temperature of the objective lens was gradually raised to 40 ° C. using the heating and cooling steps.
Rapid and substantial growth of the microbubbles was observed by microscopy.
g)
Samples of the perfluorobutane gas dispersion from Example 1 (a) were diluted with purified water (1: 1) and cooled to 0 ° C. 1 drop of 1-chloro-2,2,2-trifluoroethyl difluoromethyl ether emulsion from Example 1 (g) was diluted on a microscope objective lens maintained at 0 ° C. by heating and cooling steps. It was added to the bubble dispersion and covered with a cover glass at 0 ° C. The temperature of the objective lens was gradually raised to 40 ° C. using the heating and cooling steps. Rapid and substantial growth of the microbubbles was observed by microscopy.
h)
1 drop of polymer / human serum albumin particulate dispersion from Example 1 (h) and 1 drop of perfluoropentane emulsion from Example 1 (k) were placed on a microscope objective lens warmed to 50 ° C. The magnification was observed. As mixed droplets, significant growth of microbubbles was observed.
i)
1 drop of polymer / human serum albumin particulate dispersion from Example 1 (h) and 1 drop of 2-methylbutane emulsion from Example 1 (j) were placed on a microscope objective lens warmed to 40 ° C. The magnification was observed. As mixed droplets, significant, rapid and massive growth of microbubbles was observed.
j)
1 drop of polymer / gelatinous particulate dispersion from Example 1 (i) and 1 drop of perfluoropentane emulsion from Example 1 (k) were placed on a microscope objective lens warmed to 50 ° C. at a magnification of 400 times Observed. As mixed droplets, significant growth of microbubbles was observed.
k)
1 drop of the polymer / gelatinous particulate dispersion from Example 1 (i) and 1 drop of 2-methylbutane emulsion from Example 1 (j) were placed on a microscope objective lens warmed to 40 ° C. at 400 × magnification. Observed. As mixed droplets, significant, rapid and massive growth of microbubbles was observed.
l) [Comparative Example]
One drop of perfluoropentane emulsion from Example 1 (k) was placed on a microscope objective lens warmed to 50 ° C. and observed at 400 times magnification. No microbubble formation was observed.
m) [comparative example]
One drop of 2-methylbutane emulsion from Example 1 (j) was placed on a microscope objective lens warmed to 40 ° C. and observed at 400 × magnification. No microbubble formation was observed.
n) [comparative example]
One drop of dispersion of polymer / human serum albumin microparticles from Example 1 (h) was placed on a microscope objective lens warmed to 40 ° C. and observed at 400 × magnification. No significant change was observed.
o) [comparative example]
One drop of dispersion of polymer / gelatin fine particles from Example 1 (i) was placed on a microscope objective lens warmed to 50 ° C. and observed at 400 times magnification. No significant change was observed.
p)
Human droplet albumin stabilized air microbubble dispersion 1 droplet prepared as described in US Pat. No. 4,714,33 and 2-methylbutane emulsion 1 droplet from Example 1 (j) at 20 ° C. on a microscope objective lens. Placed in and observed at a magnification of 400 times. Significant growth of microbubbles was observed as mixed droplets.
q)
Samples of the perfluorobutane gas dispersion from Example 1 (a) were diluted with purified water (1: 1) and cooled to 0 ° C. 1 drop of perfluorodecalin / perfluorobutane emulsion from Example 1 (z) was added to the microbubble dispersion diluted on a microscope objective lens maintained at 0 ° C. using a heating and cooling step and free at 0 ° C. Covered with a cover. The temperature of the objective lens was gradually increased to 40 ° C. using the heating and cooling steps. Rapid and substantial microbubble growth was observed by microscope.
r)
Samples of the perfluorobutane gas dispersion from Example 1 (a) were diluted with purified water (1: 1) and cooled to 0 ° C. 1 drop of perfluorodecalin / perfluoropropane emulsion from Example 1 (aa) was added to the microbubble dispersion diluted on a microscope objective lens maintained at 0 ° C. using a heating and cooling step and free at 0 ° C. Covered with a cover. The temperature of the objective lens was gradually increased to 40 ° C. using the heating and cooling steps. Rapid and substantial microbubble growth was observed by microscope.
s)
Samples of the perfluorobutane gas dispersion from Example 1 (a) were diluted with purified water (1: 1) and cooled to 0 ° C. 1 drop of perfluorodecalin / sulfur hexafluoride emulsion from Example 1 (ab) was added to the diluted microbubble dispersion on a microscope objective lens maintained at 0 ° C. using a heating and cooling step and free at 0 ° C. Covered with a cover. The temperature of the objective lens is gradually increased to 40 ° C. using the heating and cooling steps, and after 4 to 5 minutes, although the increase is less and less rapid than that observed in Examples 2 (q) and 2 (r) However, an increase in microbubble size was observed here.
t)
Samples of the perfluorobutane gas dispersion from Example 1 (a) were diluted with purified water (1: 1) and cooled to 0 ° C. Pluronic F68-stabilized perfluoropentane emulsion 1 droplets from Example 1 (ad) were added to a microbubble dispersion diluted on a microscope objective lens maintained at 0 ° C. using a heating and cooling step, and 0 ° C. Covered with a glass cover. The temperature of the objective lens was gradually increased to 40 ° C. using the heating and cooling steps. Rapid and substantial microbubble growth was observed by microscope.
u)
1 drop of perfluorobutane gas dispersion from Example 1 (a) and 1 drop of Breeze 58: Fluorad FC-170C-stabilized perfluoropentane emulsion from Example 1 (aj) were warmed to 40 ° C. It was placed on a microscope objective lens and observed at a magnification of 400 times. After a while, slow microbubble growth was observed.
v)
1 drop of perfluorobutane gas dispersion from Example 1 (a) and 1 drop of Breeze 58: Fluorad FC-170C-stabilized perfluoropentane emulsion from Example 1 (ak) were warmed to 40 ° C. It was placed on a microscope objective lens and observed at a magnification of 400 times. After a while, microbubble growth was observed.
w)
1 drop of perfluorobutane gas dispersion from Example 1 (a) and 1 drop of perfluoro-4-methylpent-2-ene emulsion from Example 1 (al), warmed to 40 ° C. Leave on and observe at 400x magnification. After a while, slow microbubble growth was observed.
x)
1 drop of perfluorobutane gas dispersion from Example 1 (a) and 1 drop of 1H, 1H, 2H-heptafluoropent-1-ene emulsion from Example 1 (am) It was placed on the objective lens and observed at a magnification of 400 times. Significant and rapid microbubble growth was observed as mixed droplets.
y)
1 drop of perfluorobutane gas dispersion from Example 1 (a) and 1 drop of perfluorocyclopentene emulsion from Example 1 (an) were placed on a microscope objective lens warmed to 40 ° C. The magnification was observed. Significant, rapid and massive microbubble growth was observed as mixed droplets.
z)
400 μl of the perfluorobutane gas dispersion prepared as described in Example 1 (b) was transferred to a 2 mL vial at room temperature and 100 μl of the azeotropic mixture emulsion of Example 1 (ap) was added. One drop of the microbubble / emulsion mixture was placed on a microscope objective lens maintained at 20 ° C. using the heating and cooling steps. The temperature of the objective lens was rapidly increased to 37 ° C. using the heating and cooling steps. Substantial, spontaneous, rapid microbubble growth was observed.
aa)
Biotinylated microbubble 1 droplets prepared as described in Example 1 (bq) were added to emulsion 1 droplets prepared as described in Example 1 (bh) on a microscope objective lens warmed to 60 ° C. or lower, and 400 Observation was made at fold magnification. Significant growth of microbubbles and accumulation of microbubbles in aggregated emulsion droplets were observed.
ab)
Microbubbles prepared as described in Example 1 (bq) were prepared using the fluorescent streptavidin prepared as described in Example 1 (bk) to detect the adhesion of streptavidin to biotinylated microbubbles. Analysis can be performed by the same fluid cytometry as used.
ac)
1 droplet of EcoBeast Dispersion prepared as described in Example 1 (bo) was placed on the objective lens for microscopic observation and maintained at 37 ° C. using the heating and cooling steps. The sample was covered with a glass lid and placed under the microscope. Significant bubble growth was observed.
ad)
1 droplet of the airgel dispersion from Example 1 (br) was placed on the objective lens for microscopic observation and maintained at 37 ° C. using the heating and cooling steps. The sample was covered with a cover glass and placed under the microscope. One drop of 2-methylbutane emulsion (obtained from Example 1 (c) above, except 100 μl of 2-methylbutane was used instead of 200 μl) was added to the edge of the lid glass to allow the emulsion to permeate into the aerogel dispersion. . While increasing the temperature to approximately 60 ° C, microbubbles were produced from the airgel particles.
ae) [comparative example]
Airgel dispersion 1 droplets from Example 1 (br) were placed on the objective lens for microscopic observation and maintained at 20 ° C. using the heating and cooling steps. The sample was covered with a lid glass, placed under a microscope, and the temperature was raised to 60 ° C. No microbubble growth was observed.
af)
Microbubble 1 droplets from Example 1 (bs) were placed on the objective lens for microscopic observation. The sample was covered with a lid glass and placed under a microscope equipped with heating and cooling steps to maintain the sample temperature at 20 ° C. One drop of 2-methylbutane emulsion from Example 1 (c) was added to the edge of the lid glass to allow the emulsion to permeate the microbubble dispersion. No growth of microbubbles was observed during the mixing step. Subsequently, the temperature was increased to 40 ° C. at which time substantial microbubble growth was observed.
ag) [comparative example]
One drop of microbubble dispersion from Example 1 (bs) was placed on the objective lens for microscopic observation. The sample was covered with a lid glass and placed under a microscope equipped with heating and cooling steps to maintain the sample temperature at 20 ° C. When the temperature increased to 40 ° C., no growth of microbubbles was observed.
ah)
To the EcoBeast granules (Schering AG) on the microscope objective lens, 1 drop of solvent for the EcoBeast granules was added at ambient temperature. One drop of 2-methylbutane emulsion prepared as in Example 1 (bw) was added and irradiated at a magnification of 100 times. A significant increase in microbubbles was observed as mixed droplets.
ai)
Levovist® 1 drop prepared for injection and 2-methylbutane emulsion 1 drop prepared as in Example 1 (bw) were placed on a microscope objective lens at ambient temperature and magnified 400 times Was investigated. Significant, rapid and massive growth of microbubbles was observed as mixed droplets.
aj)
1 drop of perfluorobutane gas dispersion from Example 1 (br) and 1 drop of 2-methylbutane emulsion prepared as in Example 1 (bw) were placed on a microscope objective lens at ambient temperature, 400 times It was investigated at a magnification of. Significant, rapid and massive growth of microbubbles was observed as mixed droplets.
ak)
One drop of 2-methylbutane emulsion prepared as in Example 1 (by) was added at 40 ° C. to 1 drop of Buchminsterfullerene C ₆₀ dispersion from Example 1 (bu) on a microscope objective lens. Significant, rapid and massive growth of microbubbles was observed as mixed droplets.
al)
One drop of 2-methylbutane emulsion prepared as in Example 1 (bw) was added at 40 ° C. to one drop of Sulfur hexafluoride gas dispersion from Example 1 (bv) on a microscope objective lens. Significant, rapid and massive growth of microbubbles was observed as mixed droplets.
am)
One drop of 0.5 M hydrochloric acid was added to one drop of perfluorobutane gas dispersion in aqueous sodium bicarbonate from Example 1 (bx) on a microscope objective lens at ambient temperature. Significant, rapid and massive growth of microbubbles was observed as mixed droplets.
an)
A 2-methylbutane emulsion 1 drop prepared as in Example 1 (bw) was added to 1 drop of perfluorobutane gas dispersion with iron oxide particles from Example 1 (bz) on a microscope objective lens at ambient temperature. . Significant, rapid and massive growth of microbubbles was observed as mixed droplets.
ao)
A 2-methylbutane emulsion 1 drop prepared as in Example 1 (bw) was added to 1 drop of perfluorobutane gas dispersion with iron oxide particles from Example 1 (ca) on a microscope objective lens at ambient temperature. . Significant, rapid and massive growth of microbubbles was observed as mixed droplets.
ap) [comparative example]
One drop of 2-methylbutane emulsion prepared as in Example 1 (bw) was added to one drop of iron oxide particle dispersion from Example 1 (cb) on a microscope objective lens at ambient temperature. No formation of microbubbles was observed.
aq)
A 2-methylbutane emulsion 1 droplet prepared as in Example 1 (bw) was subjected to perfluorobutane gas dispersion 1 with iron oxide particles coated with oleic acid from Example 1 (cc) on a microscope objective lens at ambient temperature. Was added to the droplets. Significant, rapid and massive growth of microbubbles was observed as mixed droplets.
ar)
1 ml of the microbubble prepared as in Example 1 (bp) was diluted with 50 ml of water. 2 drops of diluted dispersion were added to 1 drop of soda water on the microscope objective lens at ambient temperature. Spontaneous microbubble growth was observed as mixed droplets.
as)
0.4 μl of the biotinylated microbubble dispersion prepared according to Example 1 (bq) and 0.02 mL of the perfluorodimethylcyclobutane emulsion prepared as described in Example 1 (bh) were continued at 37 ° C. It was continuously added to a beaker containing 200 ml of isotone with stirring. The mixture was incubated for 20 seconds. Ultrasonic beams pulsed at 2.5 MHz (10 Hz repetition frequency, 100 μJ in each pulse) were irradiated into the solution and observed under intense metering against a black background. Bright streaks of larger bubbles were immediately observed in the beam path.
at)
A microbubble dispersion 1 drop prepared as in Example 1 (bl) was placed on an objective lens for microscopy. The sample was covered with a lid glass, placed under a microscope equipped with heating and cooling steps, and the temperature was maintained at 20 ° C. One drop of perfluorodimethylcyclobutane emulsion prepared in Example 1 (as) was added to the edge of the lid glass to allow the emulsion to permeate the microbubble dispersion. When increasing the temperature to approximately 60 ° C., substantial microbubble growth was observed.
Example 3 In Vitro Microbubble Size and Distribution Properties
a) Measurement using a Marlbourne mastersizer
After mixing with the diffusion component, changes in growth and distribution size of the microbubbles were analyzed using a Marlburn mastersizer 1002 equipped with a 45 mm lens and measuring range from 0.1 to 80 μm. The sample cell contained isotone II (150 mL) and was connected to a thermostat bath operable over a temperature range of 9-37 ° C. A perfluorobutane gas dispersion sample from Example 1 (a) (110 μl) was added to the sample cell and equilibrated, followed by the addition of a portion of the 2-methylbutane emulsion from Example 1 (c) (500 μl). It was. The isotone II solution was pumped through the mastersizer and thermostat bath and passed through the measuring cell every 30 seconds. The measurement was repeated for 3 minutes every 30 seconds. The temperature of the isotone II solution was gradually increased and further measurements were performed. In addition, perfluorobutane gas dispersion and 2-methylbutane emulsion were analyzed separately using similar conditions. Analysis alone of the perfluorobutane gas dispersion showed that 82% of the microbubbles had a size of less than 9.9 μm at 9 ° C., and this ratio decreased to 31% when the temperature was increased to 37 ° C. This temperature change correspondingly increased the proportion of microbubbles with a size of 15 to 80 μm from 8% to 42%.
After mixing the perfluorobutane gas dispersion and the 2-methylbutane emulsion at 9 ° C., a slight increase in microbubble size was observed. Increasing the temperature to 25 ° C. resulted in significant growth of the microbubbles, with about 81% of the microbubbles having a size of 15 to 80 μm. In addition, the temperature was increased to grow microbubbles up to the size beyond the measuring range of the tool.
The microbubbles were rapidly grown by mixing the perfluorobutane gas dispersion and 2-methylbutane emulsion at 37 ° C., and after a measurement period of 1 minute 30 seconds, 97% of the microbubbles had a size of 15 to 80 μm.
b) Measurement with Coulter Multisizer
Changes in the growth and size distribution of the microbubbles after mixing with the diffusion components were analyzed using Coulter Multisizer II with a 50 μm aperture and a measurement range of 1 to 30 μm. Two components of each sample were added to each cell containing 200 ml of isotone II preheated to 37 ° C. and measurement was performed at this temperature. The size distribution of the mixture was measured immediately after introduction of the sample and after 1.5 minutes. Subsequently, the sample cell was exposed to ultrasound for 1 minute using a 2.25 MHz transducer connected to a pulse generator (the amount of energy was 100 μJ).
b) (i)
The perfluorobutane gas dispersion from Example 1 (ag) and the perfluoropentane emulsion from Example 1 (l) were mixed to form rapid and substantial growth of microbubbles after exposure to ultrasound. Total volume concentration increased from 3% to approximately 16%.
b) (ii)
The perfluorobutane gas dispersion from Example 1 (ag) and the perfluorobutane emulsion from Example 1 (m) were mixed to form rapid and substantial growth of microbubbles. Total volume concentration increased from approximately 1% to approximately 6%.
b) (iii)
The perfluorobutane gas dispersion from Example 1 (ag) and the perfluoropentane emulsion from Example 1 (p) were mixed to form rapid and substantial microbubble growth after exposure to ultrasound. Total volume concentration increased from 1% to approximately 4%.
b) (iv)
The perfluorobutane gas dispersion from Example 1 (ag) and the perfluoropentane emulsion from Example 1 (af) were mixed to form rapid and substantial growth of microbubbles after exposure to ultrasound. Total volume concentration increased from approximately 2% to approximately 8%.
b) (v)
Perfluorobutane gas dispersion from Example 1 (ag) and perfluoropentane / perfluoro-4-methylpent-2-ene emulsion from Example 1 (q) were mixed and exposed to ultrasound. It formed rapid and substantial growth of microbubbles. Total volume concentration increased from 2% to approximately 4%.
b) (vi)
Perfluorobutane gas dispersion from Example 1 (ag) and perfluoropentane / 1H, 1H, 2H-heptafluoropent-1-ene emulsion from Example 1 (r) were mixed to provide rapid and substantial The growth of microbubbles was formed. Total volume concentration increased from 2% to approximately 4.5%.
b) (vii)
The perfluorobutane gas dispersion from Example 1 (ag) and the perfluoropentane emulsion from Example 1 (s) were mixed to form rapid and substantial microbubble growth after exposure to ultrasound. Total volume concentration increased from 2% to approximately 13%.
b) (viii)
The perfluorobutane gas dispersion from Example 1 (ag) and the perfluoropentane emulsion from Example 1 (t) were mixed to form rapid and substantial microbubble growth after exposure to ultrasound. Total volume concentration increased from 2% to approximately 13%.
b) (ix)
The perfluorobutane gas dispersion from Example 1 (ag) and the perfluoropentane emulsion from Example 1 (u) were mixed to form rapid and substantial growth of microbubbles after exposure to ultrasound. Total volume concentration increased from 3% to approximately 15%.
b) (x)
The perfluorobutane gas dispersion from Example 1 (ag) and the perfluoropentane emulsion from Example 1 (v) were mixed to form rapid and substantial microbubble growth after exposure to ultrasound. Total volume concentration increased from 3% to approximately 22%.
b) (xi)
The perfluorobutane gas dispersion from Example 1 (ag) and the perfluoropentane emulsion from Example 1 (ai) were mixed to form rapid and substantial microbubble growth after exposure to ultrasound. Total volume concentration increased from approximately 3% to approximately 8%.
b) (xii)
Perfluorobutane gas dispersion from Example 1 (ag) and perfluoropentane: perfluoro-4-methylpent-2-ene emulsion from Example 1 (x) were mixed and exposed to ultrasound. It formed rapid and substantial growth of microbubbles. Total volume concentration increased from 2% to approximately 7.5%.
b) (xiii)
Perfluorobutane gas dispersion from Example 1 (ag) and perfluoropentane: perfluoro-4-methylpent-2-ene emulsion from Example 1 (y) were mixed and exposed to ultrasound It formed rapid and substantial growth of microbubbles. Total volume concentration increased from 2.5% to approximately 7%.
b) (xiv)
The perfluorobutane gas dispersion from Example 1 (ag) and the perfluoropentane emulsion from Example 1 (ac) were mixed to form rapid and substantial growth of microbubbles. The increase in microbubble size was larger and faster than that observed in Example 3 (b) (xv). Total volume concentration increased from 3.5% to approximately 53%.
b) (xv)
The perfluorobutane gas dispersion from Example 1 (ag) and the perfluoropentane emulsion from Example 1 (ae) were mixed to form rapid and substantial growth of microbubbles. Total volume concentration increased from 7% to approximately 19%. Microbubbles were further grown by exposure to ultrasound, which was shown by an increase in total volume concentration of approximately 54.5%.
b) (xvi)
Perfluoropropane gas dispersion from Example 1 (ah) and perfluoropentane emulsion from Example 1 (l) were mixed to produce a fast but not as large as observed in Example 3 (b) (i) The growth of bubbles formed. Total volume concentration increased from 3% to approximately 4.5%.
b) (xvii)
The perfluorobutane gas dispersion from Example 1 (ag) and the perfluoropentane emulsion from Example 1 (o) were mixed to form rapid and substantial growth of microbubbles after exposure to ultrasound. Total volume concentration increased from 1% to approximately 8%.
b) (xviii)
Samples of the perfluorohexane emulsion prepared as described in Example 1 (ar) had a total droplet concentration of 8.6% by volume and a droplet size of 2.6 μm.
b) (xix)
The 2,2,3,3,3-pentafluoropropyl methyl ether emulsion sample prepared as described in Example 1 (at) had a total droplet concentration of 4.3% by volume and a droplet size of 1.5 μm.
b) (xx)
The 2H, 3H-decafluoropentane emulsion samples prepared as described in Example 1 (au) had a total droplet concentration of 5.6% by volume and a droplet size of 1.9 μm.
b) (xxi)
The pentafluoroheptane emulsion samples prepared as described in Example 1 (bj) had a total droplet concentration of 8.5% by volume and a droplet size of 2.2 μm.
c) Measurement with Coulter multisizer (140 μm aperture)
Changes in growth and size distribution of the microbubbles after mixing with the diffusion components were analyzed using Coulter Multisizer II with 140 μm aperture. The measuring range was 10-80 micrometers. Bubble dispersions and emulsion droplets were added to each cell containing 200 ml of preheated Isotone II. The measurement was performed at 37 ° C. The size distribution of the mixture was measured immediately after mixing and after 3 minutes. The sample solution was then exposed to ultrasound for 1 minute using a 2.25 MHz converter connected to a pulse generator. The amount of energy was 100 μJ. The size distribution of the mixture was measured 1 and 3 minutes after exposure to ultrasound.
c) (i)
182 μl of heptafluoropent-1-ene emulsion prepared as described in Example 1 (am) was added to 400 μl of perfluorobutane gas dispersion prepared as described in Example 1 (bl), followed by micro The bubbles immediately increased in size and in the size range 10-80 μm the total volume concentration increased from low to about 60% by volume within 1 minute.
c) (ii)
After adding 70 μl of a perfluorodimethylcyclobutane emulsion prepared as described in Example 1 (av) to 330 μl of a perfluorobutane gas dispersion prepared as described in Example 1 (bl), the microbubbles were The size increased substantially after exposure to ultrasound. In the size range 10-80 μm, the total volume concentration increased from low concentrations to about 14 volume percent within 3 minutes.
c) (iii)
After adding 71 μl of the perfluorodimethylcyclobutane emulsion prepared as described in Example 1 (aw) to 330 μl of the perfluorobutane gas dispersion prepared as described in Example 1 (bl), the microbubbles were The size increased substantially after exposure to ultrasound. In the size range 10-80 μm, the total volume concentration increased from low concentrations to about 8.6 volume percent within 3 minutes.
c) (iv)
After adding 105 μl of a perfluorodimethylcyclobutane emulsion prepared as described in Example 1 (ax) to 300 μl of a perfluorobutane gas dispersion prepared as described in Example 1 (bl), the microbubbles were The size increased substantially after exposure to ultrasound. In the size range 10-80 μm, the total volume concentration increased from 3.2% by volume to about 4.8% by volume within 3 minutes.
c) (v)
After adding 105 μl of a perfluorodimethylcyclobutane emulsion prepared as described in Example 1 (ay) to 300 μl of a perfluorobutane gas dispersion prepared as described in Example 1 (bl), the microbubbles were The size increased substantially after exposure to ultrasound. In the size range 10-80 μm, the total volume concentration increased from 1.5% to about 2.2% by volume within 3 minutes.
c) (vi)
After resuspending lyophilic perfluorobutane microbubbles in perfluorodimethylcyclobutane prepared as described in Example 1 (bg), an immediate increase in microbubble size occurred. In the size range 10-80 μm, the total volume concentration increased from low concentrations to about 60% by volume within 1 minute.
c) (vii)
76 μl of 1H-tridecafluorohexane emulsion prepared as described in Example 1 (bi) was added to 400 μl of perfluorobutane gas dispersion prepared as described in Example 1 (bl), followed by microbubbles. The size increased immediately and the total volume concentration in the size range 10-80 μm increased from low to about 20% by volume within 3 minutes.
c) (viii)
After adding 63 μl of the perfluorodimethylcyclobutane emulsion prepared as described in Example 1 (bm) to 741 μl of the perfluorobutane gas dispersion prepared as described in Example 1 (bl), the microbubbles were The size immediately increased, and in the size range 10-80 μm, the total volume concentration increased from low to about 2% by volume within 3 minutes.
c) (ix)
After adding 67 μl of a perfluorodimethylcyclobutane emulsion prepared as described in Example 1 (aq) to 56 μl of a perfluoropropane gas dispersion prepared as described in Example 1 (bn), the microbubbles were The size increased after exposure to ultrasound. In the size range 10-80 μm, the total volume concentration increased from low concentrations to about 2.7 volume percent within 1 minute.
Example 4 In Vitro Measurement of Auditory Attenuation
a)
A sample (1 μl) of the perfluorobutane gas dispersion from Example 1 (a) was suspended in isotone II (55 mL) at 37 ° C., and the acoustic attenuation was subjected to pulse-echo techniques at 3.5 and 5.0 MHz center frequencies. Measured as a function of time using two broadband converters. After 20 seconds, the diffusion component was added to the suspension and the measurement continued for an additional 120 seconds.
a) (i)
After adding 100 [mu] l of the 2-methylbutane emulsion from Example 1 (c), the attenuation index immediately increased by 4 or more, and accurate quantification was not possible because the attenuation exceeded the maximum value measurable by the system. The effect lasted for 50 seconds and was accompanied by a complete change in the form of attenuation spectra indicating a significant increase in microbubble size.
a) (ii)
Adding 20 μl of 2-methylbutane emulsion from Example 1 (c) resulted in a gradual increase in attenuation that rapidly dropped after reaching a maximum value of 3-4 times the initial value after 40 seconds. In addition, a complete change in the shape of the attenuation spectrum indicated a significant increase in the microbubble size.
a) (iii)
Adding 5 μl of 2-methylbutane emulsion from Example 1 (c) resulted in a gradual increase in attenuation that slowly reached the initial value after reaching a maximum of at least about 50% of the initial value after 30 seconds. The shift to lower resonance frequencies in the attenuation spectrum has been shown to increase the microbubble size normally.
a) (iv)
Add 500 μl of 2-chloro-1,1,2-trifluoroethyl difluoromethyl ether emulsion from Example 1 (e) slowly after reaching the maximum of at least about 50% of the initial value after 20 seconds. This resulted in a gradual increase in attenuation that dropped to the initial value. The shift to lower resonance frequencies in the attenuation spectrum has been shown to increase the microbubble size normally.
a) (v)
Adding 500 μl of perfluoropentane emulsion from Example 1 (d) resulted in a small increase in attenuation. The shift to lower resonance frequencies in the attenuation spectrum was shown to slightly increase the microbubble size.
For the control, adding 500 μl of water produced an indistinguishable change in attenuation.
b)
A 2-methylbutane emulsion sample (100 μl) from Example 1 (c) was added to Isotone II (55 mL) at 37 ° C. and auditory attenuation was measured as described in (a) above. After 20 seconds, a sample (1 μl) of perfluorobutane gas dispersion from Example 1 (a) was added to the suspension and suspension was continued for an additional 120 seconds. After addition of the gas dispersion, the attenuation rapidly increased, after 20 seconds the maximum measured concentration of the system was reached and started to decrease after 50 seconds. Attenuation spectra indicated the presence of large microbubbles.
For comparison, when 100 μl of water is used instead of 2-methylbutane emulsion, the attenuation increases rapidly after addition of the gas dispersion, and after 40 seconds, at a stable level of ¼ of that measured using 2-methylbutane emulsion. Reached. Attenuation was maintained at this level for the remainder of the 120 second measurement period. Attenuation spectra indicated the presence of less microbubbles.
Example 5 In Vivo Imaging of Canine Heart Using Perfluorobutane Gas Dispersion [Comparative Example]
A syringe containing an amount of perfluorobutane gas dispersion from Example 1 (b) corresponding to 2 μl of gas content was prepared and 20 kg of chest opened using a catheter with the contents inserted into the upper limb vein. Was injected into dogs. Imaging of the heart was performed using a Vingmed CFM-750 scanner using short axis imaging. One image was obtained at each terminal cardiac contraction by adjusting the scanner to reach the animal's ECG. A few seconds after injection, bright contrast was observed in the right ventricle and similar brightness contrast was seen in the left ventricle after 4-5 seconds, but substantial attenuation temporarily masked the back of the heart. Offline digital post-scattering intensity analysis was performed based on sinusoidal data recorded from the scanner. Simple and transient peaks of contrast increase in the left ventricle, starting at 3 seconds after the onset of the contrast and lasting for 10 seconds, were evident in a representative region before left ventricular myocardium.
Example 6 In Vivo Imaging of Dog Heart Using 2-Methylbutane Emulsion [Comparative Example]
A syringe containing 1.0 ml of 2-methylbutane emulsion from Example 1 (c) was prepared and the contents were injected into the animals described in Example 5. Imaging of the heart was performed as described in Example 5. No contrast effect was observed.
Example 7 In Vivo Imaging of Dog Heart Using Perfluorobutane Gas Dispersion and 2-Methylbutane Emulsion
Syringes were prepared as in Examples 5 and 6, and the contents of each syringe were simultaneously injected into dogs via a Y-piece connector and the catheter described in Example 5. Imaging of the heart was performed as described in Example 5. The echogenicity increase of the ventricles was similar to that observed in Example 5. In the left ventricular myocardium, echo intensity increased monotonously within 30 seconds after the arrival of contrast feedback into the coronary artery circulation. In the myocardium the contrast effect disappeared completely after 5 minutes.
Example 8 In Vivo Imaging of Dog Heart Using Perfluoropentane Emulsion
A syringe containing 0.5 ml of perfluoropentane emulsion from Example 1 (d) was prepared and the contents were injected into the animal as in Example 5. Imaging of the heart was performed as described in Example 5. No signal with increased echo was observed in any area of the image.
Example 9 In Vivo Low-Intensity Imaging of Dog Heart Using Perfluorobutane Gas Dispersion and Perfluoropentane Emulsion
Syringes were prepared as in Examples 5 and 8, and the contents of each syringe were simultaneously injected into a 20 kg mongrel with open chest via a catheter inserted into the Y-fragment connector and the upper limb vessels. Cardiac imaging was performed using a Bingmed CFM-750 scanner using short axis centerline imaging. The scanner was adjusted to minimize auditory output by lowering the ejection force to a value of 1 (on a scale ranging from 0 to 7) and allowing the animal to reach the ECG to obtain only one image at each terminal cardiac contraction. The observed contrast increase is as described in Example 5, but with a slight extension of time in the myocardium.
Example 10 In Vivo High-Intensity Imaging of Dog Heart Using Perfluorobutane Gas Dispersion and Perfluoropentane Emulsion
The experiment of Example 9 was repeated except that the scanner output was adjusted to minimize ultrasound exposure to the imaged tissue area. This was done using a mixture of continuous high frame imaging and best output (on a scale in the range 0-7). After injection, a strong and high contrast increase was observed in both ventricles of the heart. A steady rise in contrast increase was observed in all regions of the myocardium below the increasing intensity up to the maximum whiteness on the screen. While the duration of tissue contrast was approximately 30 minutes, the contrast effect in congestion was reduced to baseline within 5 minutes of injection, showing burns with little congestion attenuation, and a complete and extremely bright peripheral contrast increase of the myocardium. The contrast effect in the transducer-like myocardium did not appear to disappear despite successive high intensity ultrasound exposures.
Example 11 In Vivo High-Intensity Imaging of Canine Heart Using Perfluorobutane Gas Dispersion and Perfluoropentane Emulsion
The perfluoropentane emulsion used was prepared as in polyethylene glycol 10000 methyl ether 16-hexadecanoyloxyhexadecanoate (200 mg, Example 2 (k) of WO9607434 in purified water (20 mL). Solution), transfer 1 ml aliquots of this solution into 2 ml vials, add perfluoropentane (200 [mu] l) and shake the vial for 45 seconds using Capmix, 0 ° C. when not in use. The method of Example 10 was repeated except that the emulsion was prepared by storing the emulsion. The observed contrast increase in blood and myocardial tissue was as described in Example 5.
Example 12 In Vivo Imaging of Dog Kidneys
The same materials and injection methods as described in Example 9 were used. The left kidney of the dog was imaged through the intact abdominal wall using the same high power tool conditions as in Example 10. The central structure of the kidney containing the supply artery was included in the burn. Twenty seconds after injection, a steady increase in renal soft tissue contrast increase was observed, reaching a peak of extremely bright intensity after 1-2 minutes. Four minutes after injection, the transducer was moved to image the right kidney. First, the kidney had a normal, unexpanded appearance. However, upon application of this high intensity ultrasound, a slight increase in echo intensity was observed after several minutes, although less than the size observed in the left kidney.
Example 13 In Vivo Imaging of Dog Heart with Perfluorobutane Gas Dispersion and Reduced Amounts of Perfluoropentane Emulsion
The method of Example 10 was repeated except that the dose of perfluoropentane emulsion was reduced to ⅓. The peak intensity of myocardial contrast increase can be compared with that observed in Example 10, but the duration of tissue contrast was reduced from 30 minutes to less than 10 minutes.
Example 14 In Vivo Imaging of the Thoracic Hearts of Dog Hearts Using Perfluorobutane Gas Dispersion and Perfluoropentane Emulsions
The method of Example 10 was repeated with the chest closed. Myocardial contrast increase could be compared with that observed in Example 10.
Example 15 Color Doppler In Vivo Imaging of Dog Heart Using Perfluorobutane Gas Dispersion and Perfluoropentane Emulsion
The method of Example 10 was repeated except that a scanner (color Doppler mode) was used in the left ventricle for the first minute after injection to initiate microbubble growth. Myocardial contrast increase was stronger than that observed in Example 10.
Example 16 In Vivo Imaging of Dog Heart Using Perfluorobutane Gas Dispersion and Perfluoro-4-methylpent-2-ene Emulsion
0.5 ml of isotonic reconstituted perfluorobutane gas dispersion prepared according to Example 1 (ag) and 66 μl of perfluoro-4-methylpent-2-ene emulsion from Example 1 (al) were run. Injection was as described in Example 10. The resulting myocardial contrast increase can be compared in terms of strength as observed in Example 10, but with a duration of 6-8 minutes.
Example 17 In Vivo Imaging of Congestive Regions of the Dog Heart Using Perfluorobutane Gas Dispersion and Perfluoropentane Emulsion
A tributary of the dog's bent coronary artery was temporarily ligated for 2 minutes and then injected with a contrast agent as described in Example 10. The contrast increase of currently congested myocardium was substantially stronger than that of the surrounding normal tissue.
Example 18 In Vivo Imaging of Dog Heart with Perfluorobutane Gas Dispersion and Perfluorodimethylcyclobutane Emulsion
0.5 ml of isotonic reconstituted perfluorobutane gas dispersion prepared according to Example 1 (ag) and 66 μl of perfluorodimethylcyclobutane emulsion from Example 1 (ao) are described in Example 10. Injection was as follows. The resulting strong myocardial contrast increase could be compared with that observed in Example 16.
Example 19-In Vivo Imaging of Dog Heart with Perfluorobutane Gas Dispersion and Perfluorodimethylcyclobutane Emulsion
0.5 ml of isotonic reconstituted perfluorobutane gas dispersion prepared according to Example 1 (bl) and 66 μl of perfluorodimethylcyclobutane emulsion from Example 1 (aq) are described in Example 10. Injection was as follows. The resulting strong myocardial contrast increase could be compared with that observed in Example 16.
Example 20-"Particle to Particle" Targeting In Vivo
0.02 μl / kg of avidinylated perfluorobutane microbubbles prepared according to Example 1 (bq), and 0.02 μl / kg of perfluorodimethylcyclobutane emulsion prepared as described in Example 1 (bh). Kg anesthetized mongrel was injected intravenously simultaneously and the heart was imaged by ultrasound as described in Example 10. Myocardial echogenicity increase was similar to that observed in Example 10, except that the attenuation peak in the right ventricle blood was very less pronounced.
Example 21-In Vivo Targeting of the Rabbit Heart with Perfluorobutane Gas Dispersion and Perfluorodimethylcyclobutane Emulsion
Perfluorodimethyl from Example 1 (aq) and syringe containing perfluorobutane microbubble dispersion (volume average diameter 3.0 μm) corresponding to 1 μl of gas content prepared as in Example 1 (bl) A syringe containing 105 μl of cyclobutane emulsion was further prepared. The contents of both syringes were injected simultaneously into 5 kg rabbits using a catheter inserted into the ear vein. B-mode imaging of the heart was performed using an ATL HDI-3000 scanner with a P5-3 probe and short axis imaging of the centerline of both thorax openings. The results were comparable to those observed in Example 18.
Example 22-Ultrasonic Degradation Induced Drug Delivery
3 kg of anesthetized New Zealand black rabbit was prepared with 0.04 ml of perfluorodimethylcyclobutane emulsion prepared as described in Example 1 (aq) and perfluorobutane gas suspension prepared as described in Example 1 (bl) at the same time. Intravenous injection with 0.12 mL of water, left kidney was imaged using an ATL HDI-3000 scanner with P5-3 probe, and the scanner was adjusted for maximum output. Significant bubble growth and accumulation inside the renal soft tissue was observed. Subsequently, 160 mg of FITC-dextran (molecular weight 2,000,000) was dissolved in 5 ml of water, injected intravenously, continued with ultrasound imaging for an additional 5 minutes at the same site, and the scanner powered Doppler to maximize auditory output. Switched to mode. Subsequently, the animals were sacrificed, both kidneys removed and tested with ultraviolet light. An increase in fluorescence was observed at points of 50-100 μm in hepatocytes in the area of the left heart exposed for imaging ultrasound in the presence of microbubbles. In this regard, each of these points was nephron without intravascular fluorescence.
Example 23-Albunex (registered trademark) as a gas dispersion
0.3 ml / kg of Albunex and 1.5 μl / kg of perfluorodimethylcyclobutane emulsion prepared as described in Example 1 (aq) were injected intravenously into 20 kg of anesthetized male mongrel and Imaging by ultrasound as described. Myocardial increase was as described in Example 10.
Example 24 Microbubbles Targeted in Imaging of Rabbit Hearts
0.1 μl / kg of the microbubble prepared as described in Example 1 (az) was intravenously administered to the rabbit and the heart of the rabbit was imaged by ultrasound using an ATL HDI-3000 scanner with a P5-3 probe. A weak but persistent increase in myocardial echo was observed. After 3 minutes, 1.5 μl / kg of a perfluorodimethylcyclobutane emulsion prepared as described in Example 1 (aq) was injected. A slight increase in echo intensity from the myocardium from which high frequency sound waves were fired was observed.
Example 25-In Vivo Imaging of Rat Heart with Perfluorobutane Gas Dispersion and Perfluorodimethylcyclobutane Emulsion
The experiment described in Example 19 was performed in rats and comparable results were obtained.
Example 26-In Vivo Imaging of Dog Heart with Perfluorobutane Gas Dispersion and Perfluorohexane Emulsion
Example 10 was prepared from 0.1 μl / kg of perfluorohexane emulsion prepared as described in Example 1 (ar) and 0.2 μl / kg of perfluorobutane microbubble suspension prepared as described in Example 1 (bl). The dogs were injected at the same time as described. Myocardial contrast effect was comparable to that observed in Example 10.
Example 27-In Vivo Imaging of Dog Heart with Perfluorobutane Gas Dispersion and Heptafluoropent-1-ene Emulsion
0.3 μl / kg of perfluorobutane microbubble suspension prepared as described in Example 1 (bl) and 0.15 mL of heptafluoropent-1-ene emulsion prepared as described in Example 1 (am) were run. The dogs were injected at the same time as described in Example 10. A relatively weak myocardial contrast effect was observed, but stronger and longer lasting than that observed in Example 5.
Example 28 In Vivo Imaging of Dog Heart with Perfluorobutane Gas Dispersion and Sterile Phospholipid Perfluoromethylcyclobutane Emulsion
0.3 μl / kg of perfluorobutane microbubble suspension prepared as described in Example 1 (bl) and 0.3 μl / kg of perfluorodimethylcyclobutane emulsion prepared as described in Example 1 (bm). The dogs were injected at the same time as described in Example 19. Myocardial contrast effects comparable to those described in Example 19 were observed.
Example 29-In Vivo Imaging of Dog Heart with Perfluoropropane Gas Dispersion and Perfluoromethylcyclobutane Emulsion
Example 19 was prepared with 0.17 ml of perfluoropropane microbubble suspension prepared as described in Example 1 (bn) and 0.3 μl / kg of perfluorodimethylcyclobutane emulsion prepared as described in Example 1 (aq). The dogs were injected at the same time as described. Myocardial contrast effects comparable to those described in Example 19 were observed.
Example 30-In Vivo Imaging of Dog Stomach with Perfluorobutane Gas Dispersion and Perfluoromethylcyclobutane Emulsion
20 ml of a perfluorodimethylcyclobutane emulsion prepared as described in Example 1 (aq) was fed to the anesthetized dog through the gastrointestinal tract. Subsequently, a small amount of perfluorobutane microbubble dispersion (dosage range: 0.1-0.2 μl gas / kg) prepared as described in Example 1 (a) was injected intravenously. Ultrasonic imaging transducers are used on the abdominal wall, and the growth of localized microbubbles in gastric wall capillaries provides improved contrast and improves the contour of mucosal elevation.
Example 31-In Vivo Imaging of Dog Gastrointestinal Tract Using Perfluorobutane Gas Dispersion and Perfluoromethylcyclobutane Emulsion
The perfluorobutane dispersion prepared as described in Example 1 (a) was fed to the anesthetized dog through the gastrointestinal tract. The dispersion was evenly distributed into the upper ventricle and confirmed by ultrasound imaging. A small amount of perfluorodimethylcyclobutane emulsion (dosage range: 0.2-1 μl perfluorocarbon / kg) prepared as described in Example 1 (aq) was injected intravenously. Maintaining the ultrasonic transducer in the desired area; The growth of microbubbles in the upper fluidized bed adjacent to the mucosal surface provides improved contrast and improves the contour of mucosal elevation.
Example 32-In Vivo Imaging of Canine Heart with Perfluorobutane Gas Dispersion and Perfluoromethylcyclobutane Emulsion and Coadministered Adenosine
An obstructive snare was placed around the main tributary of the 22 kg left anterior downward coronary artery with the chest open, and the ultrasound transit time flowmeter immediately placed downstream of the obturator and then adjusted to stabilize about 14 to 10 ml / min. A 25% fluid reduction was formed. Subsequently, each of (i) the amount of perfluorobutane microbubble dispersion prepared as in Example 1 (bl), corresponding to 4.4 μl of gas content, (ii) 33 on dispersed perfluorodimethylcyclobutane The amount of perfluorodimethylcyclobutane emulsion from Example 1 (aq), corresponding to [mu] l, and (iii) the contents of three syringes each containing 3.0 mg of adenosine dissolved in 0.9% saline as simultaneous ringback. Intravenous injection was initiated 10 seconds later and slowly injected over 20 seconds with 3.0 mg of adenosine dissolved in 0.9% saline. The left ventricle of the heart was imaged using an ATL HDI-3000 scanner with a P5-3 probe, followed by continuous ultrasound for 1 minute at maximum power to induce microbubble growth, and then using B-mode imaging. Myocardium was tested. A very noticeable difference in the degree of gray grading was observed between the stenosis area (brighter than baseline recording) and the normal area (very brighter than baseline recording).

权利要求:
Claims (29)
[1" claim-type="Currently amended] i) an injectable aqueous medium with gas dispersed therein; And
ii) a composition comprising a diffusion component that can diffuse into the dispersed gas in vivo and at least temporarily increase its size
Mixed formulations for simultaneous, separate or continuous use as contrast medium in ultrasound imaging.
[2" claim-type="Currently amended] The method of claim 1 wherein the dispersed gas is air, nitrogen, oxygen, carbon dioxide, hydrogen, inert gas, sulfur fluoride, selenium hexafluoride, optionally halogenated silanes, low molecular weight hydrocarbons, ketones, esters, halogenated low molecular weight hydrocarbons or these Mixed formulations comprising a mixture of.
[3" claim-type="Currently amended] 3. The mixed formulation of claim 2, wherein said gas comprises perfluorinated ketone, perfluorinated ether or perfluorocarbon.
[4" claim-type="Currently amended] 4. The mixed formulation of claim 3, wherein said perfluorocarbon comprises perfluoroalkane, perfluoroalkene or perfluorocycloalkane.
[5" claim-type="Currently amended] 3. The mixed formulation of claim 2, wherein said gas comprises sulfur hexafluoride or perfluoropropane, perfluorobutane or perfluoropentane.
[6" claim-type="Currently amended] The mixed formulation of claim 1, wherein the dispersed gas is stabilized by agglomerated resistant surface membranes, membrane forming proteins, polymeric materials, nonpolymeric and nonpolymerizable wall forming materials, or surfactants. .
[7" claim-type="Currently amended] The mixed formulation of claim 6, wherein said surfactant comprises at least one phospholipid.
[8" claim-type="Currently amended] 8. The blended formulation of claim 7, wherein at least 75% of the surfactant materials each comprise a phospholipid molecule having a total net charge.
[9" claim-type="Currently amended] The mixed formulation of claim 8, wherein at least 75% of the film forming surfactant material comprises at least one phospholipid selected from phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid and cardiolipin.
[10" claim-type="Currently amended] The mixed formulation of claim 9, wherein at least 80% of the phospholipids are phosphatidylserine.
[11" claim-type="Currently amended] The blended formulation of claim 1, wherein the composition comprising the diffusion component is formulated for skin, subcutaneous, intramuscular, intravenous administration or for inhalation.
[12" claim-type="Currently amended] The blended formulation of claim 1, wherein the composition comprising the diffusion component further comprises a carrier liquid.
[13" claim-type="Currently amended] 13. The mixed formulation of claim 12, wherein the diffusion component is dispersed in the aqueous carrier liquid in the form of an oil-in-water emulsion or a microemulsion.
[14" claim-type="Currently amended] The mixed formulation of claim 13, wherein the diffusion component comprises an aliphatic ether, a polycyclic oil, a polycyclic alcohol, a heterocyclic compound, an aliphatic hydrocarbon, an alicyclic hydrocarbon, or a halogenated low molecular weight hydrocarbon.
[15" claim-type="Currently amended] 15. The mixed formulation of claim 14, wherein the diffusion component comprises perfluorocarbons.
[16" claim-type="Currently amended] 16. The mixed formulation of claim 15, wherein the perfluorocarbon comprises perfluoroalkane, perfluoroalkene, perfluorocycloalkane, perfluorocycloalkene or perfluorinated alcohol.
[17" claim-type="Currently amended] The mixed formulation of claim 16, wherein the diffusion component comprises perfluoropentane, perfluorohexane, or perfluorodimethylcyclobutane.
[18" claim-type="Currently amended] 18. The mixed formulation of any one of claims 13-17, wherein the emulsion is stabilized by phospholipid surfactants.
[19" claim-type="Currently amended] 19. The mixed formulation of claim 18, wherein at least 75% of said phospholipid surfactants comprise molecules individually having a total net charge.
[20" claim-type="Currently amended] The mixed formulation of claim 19, wherein at least 75% of the phospholipid surfactants are selected from phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid and cardiolipin.
[21" claim-type="Currently amended] The mixed formulation of claim 20, wherein at least 80% of the phospholipid surfactants comprise phosphatidylserine.
[22" claim-type="Currently amended] 22. The mixed formulation of any one of claims 1 to 21, further comprising a vasodilating drug.
[23" claim-type="Currently amended] 23. The mixed formulation of claim 22, wherein said vasodilator drug is adenosine.
[24" claim-type="Currently amended] 22. The mixed formulation of any one of claims 1 to 21, further comprising a therapeutic agent.
[25" claim-type="Currently amended] 22. The mixed formulation of any one of claims 1 to 21, further comprising contrast enhancing residues for imaging methods other than ultrasound.
[26" claim-type="Currently amended] i) injecting a physiologically acceptable aqueous medium in which gas is dispersed into the patient's vascular system;
ii) administering to said patient a composition comprising a diffusion component that can diffuse into said dispersed gas in vivo and at least temporarily increase its size prior to, during or after injection of said aqueous medium; And
iii) generating an ultrasound image in at least a portion of the patient
Including, human or non-human animal enhanced image generation method.
[27" claim-type="Currently amended] The method of claim 26, wherein the composition comprising the diffusion component is administered by skin, subcutaneous, intramuscular, intravenous or by inhalation.
[28" claim-type="Currently amended] The method of claim 26 or 27, wherein the vasodilating drug is concurrently administered to the patient.
[29" claim-type="Currently amended] The method of claim 28, wherein said vasodilator drug is adenosine.

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1996-10-21|Priority to GBGB9621884.7A

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1996-10-21|Priority to GB9621884.7

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1997-04-23|Priority to GB9708239.0

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1997-04-23|Priority to GBGB9708239.0A

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1997-10-21|Application filed by 조오지 디빈센조, 토브 아스 헬지, 에바 요한손, 니코메드 이메이징 에이에스

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2000-08-25|Publication of KR20000052652A

优先权:

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申请号 | 申请日 | 专利标题

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GBGB9621884.7A|GB9621884D0|1996-10-21|1996-10-21|Improvements in or relating to contrast agents|

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GB9621884.7|1996-10-21|

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GB9708239.0|1997-04-23|

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GBGB9708239.0A|GB9708239D0|1997-04-23|1997-04-23|Improvements in or relating to contrast agents|