![]() Improvements in or Relating to Contrast Agents
专利摘要:
Ultrasonic visualization of perfusion in a subject, in particular myocardium and other tissues, is carried out using a novel gas containing contrast agent formulation which promotes controllable and transient growth of the gas phase in vivo after administration, and thus can act as a deposited perfusion tracer. The formulation includes an injectable aqueous medium comprising a dispersion gas and an oil-in-water emulsion for injection comprising an oil-in-water emulsion containing a diffuseable component that can diffuse in vivo into the dispersion gas to promote its temporary growth. The materials present and the materials present on the surface of the dispersed oil phase have affinity for one another, for example as a result of having opposite charges. In cardiac perfusion imaging, the agent may be advantageously co-administered with vasodilators such as adenosine to increase the difference in return signal intensity of normal and impaired myocardial tissue, respectively. 公开号:KR20010042915A 申请号:KR1020007011727 申请日:1999-04-22 公开日:2001-05-25 发明作者:모르텐 에릭센;헬게 톨레스하우그;로알드 스쿠르트바이트;알란 쿠트베르트손;죠니 오스텐센;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 Related to It {Improvements in or Relating to Contrast Agents} The present invention relates to ultrasound imaging, more particularly to novel contrast agent formulations and their use in ultrasound imaging, such as visualization of tissue perfusion. Contrast agents, including dispersions of gas microbubbles, are well known to be efficient backscattering agents of ultrasound, particularly due to their compactness and low density. Such microbubble dispersions, if properly stabilized, can often advantageously enable low-dose, high-efficiency ultrasound visualization of, for example, pulsed and tissue microvascular systems. The use of ultrasonography to measure blood perfusion (i.e. blood flow per unit of tissue mass) can be used to detect tumor tissue, typically from other healthy vasculature, for example from healthy tissue, and for example to detect myocardial infarction. It is of great value in studying it. The problem with using existing ultrasound contrast agents in cardiac perfusion studies is that the information content of the obtained images is damaged by the attenuation caused by the contrast agent present in the ventricles of the heart. In the co-pending International Patent Publication No. 9817324, incorporated herein by reference, the ultrasound visualization of perfusion in human or animal subjects, particularly in the myocardium and other tissues, promotes controllable transient growth of the gas phase in vivo after administration. It has been disclosed that the formulation can be made and / or improved by a gas containing contrast agent formulation. Such contrast agent formulations are used to promote controllable and temporary stagnation of the gaseous phase, for example in the form of microbubbles, in the tissue microvascular system, thereby increasing the concentration of gas in such tissues and thus their echogenicity, for example proportional to blood retention. To increase. Such use of gas as a deposited perfusion tracer differs significantly from current proposals for intravenously administrable microbubble ultrasound contrast agents. Thus, microbubble growth generally needs to be avoided if microbubble growth is not regulated, which can lead to potentially dangerous tissue embolism. Thus, there may be a need to use compositions and gas mixtures selected to limit dosage and / or to prevent inward diffusion of blood gas into microbubbles to minimize bubble growth in vivo (eg, International Patent Publications). 9503835 and International Patent Publication No. 9516467). On the other hand, a composition comprising a gaseous phase dispersed according to International Patent Publication No. 9817324 may be dispersed through the inward diffusion of molecules of gas or vapor derived from the following material, hereinafter simply referred to as " diffusable component " Although co-administered with a composition comprising one or more substances capable of or generating in vivo gas or vapor pressure sufficient to promote controllable growth of the gas phase, the transport mechanisms other than diffusion are discussed in more detail below. It will be appreciated that it may be additionally or alternatively related to the work of the invention as such. Such simultaneous administration of a composition comprising a dispersed gas phase containing composition and a dispersible component with appropriate volatility has previously been proposed for the administration of only volatiles in the form of phase transfer colloids as described, for example, in WO 9416739. It can be contrasted with Thus, the contrast agent formulation of WO9817324 allows for the control of factors such as growth potential and / or rate of dispersed gas by selection of appropriate components of the co-administered composition, while Administration can lead to the development of microbubbles that are difficult to control and unevenly, or that at least a portion of the microbubbles may cause potentially dangerous embolism of the myocardium and vasculature and the brain, for example (Schwarz, Advances). in Echo-Contrast [1994 (3)], pp. 48-49). It has been found that the administration of phase transfer colloids alone cannot induce volatilization or constant in vivo volatilization of dispersed phases, resulting in gas or vapor microbubbles. Grayburn et al., J. Am. Coll. Cardiol. 26 (5) [1995], pp. 1340-1347, show that the preliminary activation of perfluoropentane emulsions is low enough to avoid hemodynamic side effects. It has been suggested that effective imaging doses may be necessary to achieve myocardial opacity in dogs. Activation techniques for such colloidal dispersions, including applying low specific gravity, are described in WO 9640282; Typically this involves partially filling the syringe with the emulsion and then forcibly recovering and releasing the plunger of the syringe to generate a temporary pressure change that causes the formation of gas microbubbles in the emulsion. This is inherently a slightly problematic technique that cannot provide a constant level of activation. Referring again to phase transfer colloids, U.S. Pat.No. 5536489 describes the use of emulsions of water-insoluble gas forming chemicals such as perfluoropentane as contrast agents for site specific imaging, where only those emulsions can be used in a particular body within the body to be imaged. It is mentioned that the application of ultrasonic energy to a location can generate a significant number of image enhancement gas microbubbles. However, our study found that emulsions of volatile compounds such as 2-methylbutane or perfluoropentane were sonicated at a sufficient energy level to provide a distinct contrast effect using a bicomponent contrast agent according to International Patent Publication No. 9817324. It has been found that it does not provide detectable echo improvement when in vitro or in vivo. The combination formulation of WO 9817324 is for simultaneous, separate or sequential use as a contrast agent in ultrasound imaging, i) an injectable aqueous composition with dispersed gas; And ii) a composition comprising a diffusible component that can diffuse in vivo with said dispersion gas to at least temporarily increase its size. The formulations can advantageously be used to visualize tissue perfusion in a subject, and increasing the size of the dispersing gas is useful for effecting gas enrichment or transient stagnation in the microvascular system of such tissues, thus increasing its reproducibility. Done. A particular advantage of the formulations is that the growth of the dispersing gas is appropriately in other forms, including by sonication, lower or higher frequency sonic energy than is typically used for medical ultrasonic imaging, shaking, vibration, electric fields or radiation. Can be induced or increased by particle bombardment by, for example, neutral particles, ions or atoms. This allows for particularly effective regulation of factors such as the onset and rate of growth of the dispersion gas, such that the growth is positioned on a particular side of the subject's body such that the gas is temporarily stagnated within the microvascular system of the target organ, for example within the myocardium. To make it possible. The present invention discloses in International Patent Publication No. 9817324 if two compositions are formulated such that the dispersing gas component and the diffusive component have affinity for each other as a result of, for example, attraction, electrostatic force or other physical or chemical (including biological) bonding. The efficacy of contrast agent formulations of the type described is based on the finding that it can be substantially enhanced. This is accomplished by formulating the dispersed gas component as a stabilized gas dispersion and formulating the diffusive component as a stabilized emulsion such that the material present on the surface of the dispersed gas has affinity for the material present on the surface of the dispersed diffusive component. Can be. Surface materials having affinity for one another can be materials such as, for example, surfactants that stabilize gas and diffusible component dispersions. In addition, surface materials having suitable mutual affinity may be mixed, chemically linked, or associated with the non-affinity stabilizing material in each dispersion. While not wishing to be based on theory, the affinity formed between the dispersing gas and the diffusive component increases the likelihood of interaction between them, for example, by 10-100 times or higher, so that the components lack such mutual affinity. It is believed that more disperse gas components are caused to grow for a given dose of two components in comparison. This is especially the case when ultrasound or similar activation is used to induce the growth of the dispersion gas. Here, it is believed that in situations where there is no significant affinity between the components, ultrasound can only induce a relatively low level of interaction with the dispersible and diffusive components of a substantial portion of the dispersed gas phase. However, the degree of interaction can be significantly increased by the use of gas and diffusive components having mutual affinity. Therefore, the contrast agent formulation according to the present invention can be used while providing an equivalent contrast effect at a significantly lower dosage than that proposed in WO9817324. This has a valuable meaning in terms of product stability, because it is any from its volatile components as described, for example, in J. Appl. Physiol. 40 (5) [1976], pp. 745-751. This is because the risk of embolism allows for the use of diffusive emulsions at negligible levels even after dilution with blood gases. Alternatively or in addition, the dosage of the gas dispersion can be reduced with possible advantages with regard to product stability and toxicity. Such dose reduction also allows for the initial disappearance of disperse gases from ventricular blood and thereby allows for more rapid visualization of stagnant gases in myocardial tissue, for example imaging available in applications such as ultrasound cardiac examination. You can also extend your time zone. It has also been found that the contrast agent formulations according to the present invention can easily facilitate effective imaging of tissues such as myocardium using conventional B-modal injection techniques. Thus, the ultrasound energy emitted by the scanner operating in the B-way is sufficient to induce the growth of the dispersed gas phase, which is then retained in the microvascular system and diagnostically useful for at least 5-10 minutes without ultrasound-induced deterioration. It can generate information. Such behavior is markedly different from that seen by existing gas containing contrast agents which are generally degraded relatively rapidly during sonication, thus requiring the use of more complex techniques, including discontinuous imaging, to achieve satisfactory visualization. According to one aspect of the invention i) a first composition which is an aqueous aqueous medium for injection comprising a dispersion gas and a substance for stabilizing the gas; And ii) an oil-in-water emulsion for injection comprising a dispersible component that can diffuse in vivo into the dispersion gas such that the oil phase at least temporarily increases its size, further comprising a substance for stabilizing said emulsion And a material present on the surface of the disperse gas phase and a material present on the surface of the disperse oil phase have affinity for each other, providing a complex preparation for simultaneous, separate or sequential use as a contrast agent in ultrasound imaging. do. The invention also i) injecting the first composition as defined above into the pulse system of the subject; ii) injecting said defined second composition into said subject before, during or after injecting said first composition; And iii) generating an ultrasound image of at least a portion of the object It provides a method of forming an improved image of a human or non-human animal subject comprising. The necessary affinity between the surface materials present in the first and second compositions, respectively, can be achieved by using them with opposite charges, for example, by interacting with each other and electrostatically bonding to each other. Thus, for example, as discussed in more detail below, one of the surface materials may be a cationic surfactant and the other may be an anionic surfactant. The charge difference between the surface materials can be achieved by incorporating the appropriate cationic and / or anionic additives as needed in the stabilizing material, for example the surfactant, present on the surface of either or both of the respective dispersed phases of the two compositions. It may be. Each surface material may also include stabilizers or additives containing special groups, molecules, ligands or vectors that can interact via chemical bond interactions such as covalent bonds, hydrogen bonds or ionic bonds. Thus, surface materials are each, but not limited to, for example, antigens and antibodies or fragments thereof, lectin and carbohydrate containing groups, avidin / streptavidin and biotin or biotinyl groups, drugs and receptors, propagators and receptors, hormones and Receptors, peptides or proteins and complementary peptides or proteins, enzymes or inactive enzymes and substrate analogs or inhibitors, nucleic acid (DNA or RNA) sequences and complementary nucleic acid sequences, or chelators and ligands. In general, any biocompatible gas may be present in the gas dispersion used as the first composition in accordance with the present invention, and the term “gas” as used herein refers to any gas or vapor form at normal human body temperature of 37 ° C. At least partially, for example substantially or completely, of the substance (including the mixture). Thus, representative gases include air; nitrogen; Oxygen; carbon dioxide; Hydrogen; Inert gases such as helium, argon, xenon or krypton; Sulfur fluorides such as sulfur hexafluoride, decafluoride disulfide or pentafluorinated trifluoromethylsulfur; Selenium hexafluoride; Optionally halogenated silanes such as methylsilane or dimethylsilane; Low molecular weight hydrocarbons (e.g. containing up to 7 carbon atoms), for example, alkanes such as methane, ethane, propane, butane or pentane, cycloalkanes such as cyclopropane, cyclobutane or cyclopentane, ethylene, propene Alkenes such as propadiene or butene, or alkynes such as acetylene or propene; Ethers such as dimethyl ether; Ketones; ester; Halogenated low molecular weight hydrocarbons (eg containing up to 7 carbon atoms); Or mixtures of any of the above components. Advantageously, at least some of the halogen atoms in the halogenated gas are fluorine atoms, so that the biocompatible halogenated hydrocarbon gas is for example bromochlorodifluoromethane, chlorodifluoromethane, dichlorodifluoromethane, bromo Trifluoromethane, chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene, ethylfluoride, 1,1-difluoroethane and perfluorocarbon Can be selected from. Representative perfluorocarbons include perfluoroalkanes such as perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane (eg perfluoro-n-butane, optionally perfluoro Mixtures with other isomers such as rho-iso-butane), perfluoropentane, perfluorohexane or perfluoroheptane; Perfluoroalkenes such as perfluoropropane, perfluorobutene (eg perfluorobut-2-ene), perfluorobutadiene, perfluoropentene (eg perfluoropentene -1-ene) or perfluoro-4-methylpent-2-ene; Perfluoroalkynes such as perfluorobut-2-yne; And perfluorocycloalkanes such as perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutane, perfluorotrimethylcyclobutane, perfluorocyclopentane, perfluoromethylcyclopentane, Perfluorodimethylcyclopentane, perfluorocyclohexane, perfluoromethylcyclohexane, or perfluorocycloheptane. Other halogenated gases include methyl chloride, fluorinated (eg perfluorinated) ketones such as perfluoroacetone and fluorinated (eg perfluorinated) ethers such as perfluorodiethyl Ethers. The use of perfluorinated gases, for example sulfur hexafluoride and perfluorocarbons such as perfluoropropane, perfluorobutane, perfluoropentane and perfluorohexane, has led to the flow of microbubbles containing such gases It is particularly advantageous in terms of recognized high stability in the interior. Other gases with physicochemical properties that allow them to form highly stable microbubbles in the bloodstream can likewise be useful. The gas may be present in the first composition in the form of microbubbles, for example at least partially encapsulated or stabilized by a gas stabilizing material. This stabilizing material is for example a cohesive resistant surface membrane (e.g. gelatin as described in WO8002365), a filmogen protein (e.g. albumin, e.g. U.S. Pat. 4774958, U.S. Patent 4444882, European Patent Publication 0359246, International Patent Publication 9112823, International Patent Publication 9305806, International Patent Publication 9217213, International Patent Publication 9406477 or International Patent Publication 9501187 ), Polymeric materials (eg, synthetic biodegradable polymers as described in EP 0398935, elastic interfacial synthetic polymer membranes as described in EP 0458745, as described in EP 0441468). Fine particulate biodegradable polyaldehydes, particulate N-dicarboxy of polyamino acid-polycyclic imides as described in European Patent Publication No. 0458079 Acid derivatives, or biodegradable polymers as described in WO9317718 or WO9607434), nonpolymerizable and nonpolymerizable wall forming materials (such as described in WO9521631) or interfaces Active agents (eg, polyoxyethylene-polyoxypropylene block copolymer surfactants such as Pluronic, polymeric surfactants as described in WO9506518, or film forming surfactants, eg For example, phospholipids as described in International Patent Publication No. 9211873, International Patent Publication No. 9217212, International Patent Publication No. 9222247, International Patent Publication No. 9428780, International Patent Publication No. 9503835, or International Patent Publication No. 9729783). It may include. The first composition can also be contained in, or associated with, a gas-containing solid-state, for example, European Patent Publication No. 0122624, European Patent Publication No. 0123235, European Patent Publication No. 0365467, International Patent Absorbed on the surface of voids, cavities or pores therein, as described in Publication 921382, International Patent Publication 9300930, International Patent Publication 9313802, International Patent Publication 9313808 or International Patent Publication 9313809. And / or particulates (especially aggregates of particulates) having a gas contained therein. The echogenicity of such particulate contrast agents can be derived directly from the contained / associated gas and / or from the gas (eg microbubbles) liberated from the solid material (eg, upon dissolution of the particulate structure). It will be understood. The description of all the above documents relating to gas containing formulations is incorporated herein by reference. Gas microbubbles and other gas-containing materials, such as particulates, do not exceed 10 micrometers (eg, 7 micrometers or less) to allow free passage through the pulmonary artery following administration by, for example, intravenous injection. It is desirable to have an initial average size. However, for example, they contain larger mixtures of one or more relatively blood soluble or diffusible gases such as air, oxygen, nitrogen or carbon dioxide and one or more substantially insoluble and non-diffusing gases such as perfluorocarbons. Microbubbles may be used. Outward diffusion of soluble / diffusing gas content after administration will be determined by the amount of insoluble / non-diffusing gas present in such microbubbles and is selected to allow passage of the resulting microbubbles through the pulmonary capillaries of the pulmonary artery system. It will make it as easy to shrink as possible. Since the dispersing gas administered according to the present invention will grow in vivo through interaction with the diffusive component, the minimum size of the microbubbles, solids associated gases, etc. administered will provide a significant interaction with the ultrasound. It may generally be substantially smaller than the required size (typically about 1-5 μm at commonly used imaging frequencies), and therefore, the dispersion gas component may have a size as small as, for example, 1 nm or less. Thus, the present invention may enable the use of gas containing compositions that have not been proposed so far for use as ultrasonic contrast agents, for example, because of the small size of the dispersion gas component. When the phospholipid containing first composition is used according to the invention, for example in the form of phospholipid stabilized gas microbubbles, representative examples of useful phospholipids are lecithin (ie phosphatidylcholine), for example neutral lecithin, for example egg yolk Lecithin or soy lecithin, semisynthetic (eg partially or fully hydrogenated) lecithin and synthetic lecithins such as dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine; Phosphatidic acid; Phosphatidylethanolamine; Phosphatidylserine; Phosphatidylglycerol; Phosphatidylinositol; Cardiolipin; Spinogmyelin; Any of the above fluorinated analogs; Mixtures of any of the foregoing and with other lipids such as cholesterol. For example, as described in WO 9729783, for example, natural (eg, derived from soy or egg yolk), semisynthetic (eg, partially or fully hydrogenated) and synthetic phosphatidylserine, phosphatidyl The use of phospholipids comprising predominantly (eg, at least 75%) of molecules with individually net total charges, eg negative charges, such as glycerol, phosphatidylinositol, phosphatidic acid and / or cardiolipin, in particular It is advantageous. Representative examples of gas-containing particulate materials that may be useful in the first composition according to the invention include carbohydrates (eg hexoses such as glucose, fructose or galactose; disaccharides such as sucrose, lactose or horses). Tosses; pentoses such as arabinose, xylose or ribose; α-, β- and γ-cyclodextrins; polysaccharides such as starch, hydroxyethyl starch, amylose, amylopectin, glycogen, inulin, pullulan, Dextran, carboxymethyl dextran, dextran phosphate, ketodextran, aminoethyldextran, alginate, chitin, chitosan, hyaluronic acid or heparin; sugar alcohols including alditol such as mannitol or sorbitol), inorganic Salts (eg sodium chloride), organic salts (eg sodium citrate, sodium acetate or sodium tartrate), X-ray contrast agents (eg, Trizoic acid, ditrizoic acid, iotalamic acid, iosaglinic acid, iohexel, iopentol, iopamidol, iodixanol, iopromide, methazamide, iodipamide, meglumine iodipamide, meth Carboxyl, carbamoyl, N-alkylcarbamoyl, N-hydroxyalkylcarbamoyl, acyl at 3- and / or 5-positions, such as glutamine acetrizoate and meglumine ditrizoate Any commercially available carboxylic acid and nonionic amide contrast agent which typically contains one or more 2,4,6-triiodophenyl groups with substituents such as amino, N-alkylacylamino or acylaminomethyl), and polypeptides And proteins (eg, albumin such as gelatin or human serum albumin). Other gas-containing materials that may be useful in the first composition in accordance with the present invention include metal-stabilized gas-containing materials (eg, as described in US Pat. No. 36,744,61 or US Pat. No. 3,3528809), synthetic polymers. Gas-containing materials stabilized by (see, eg, US Pat. No. 3,751,494 or Farnand in Powder Technology 22 [1979], pp. 11-16), Expancel® type Commercially available microspheres, such as Xpancel 551 DE (see, 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 Ropaque® type (see, eg, J. Coatings Technol. 55 (707) [1983], p. 79), micro- and nano-sized gas-containing structures such as zeolites, inorganic or organic aerogels, containing nano-sized open pores Chemical structures such as fullerences, cladrates or nanotubes (for example described in GE Gadd in Science 277 (5328) [1997], pp. 933-936) and natural surfactant stabilized microparticles Bubble dispersions (e.g., described in d'Arrigo in "Stable Gas-in-Liquid Emulsions, Studies in physical and theoretical chemistry" 40-Elsevier, Amsterdam [1986]). The disperse oil phase in the second composition of the formulation according to the invention may comprise any suitable diffusive component which is at least partially insoluble in water and incompatible with water. The diffusive component in such emulsions is advantageously liquid at processing and storage temperatures, which may be as low as, for example, -10 ° C if the aqueous phase contains an appropriate antifreeze material, but at body temperature is gas or exhibits substantial vapor pressure. Suitable compounds can be selected from, for example, various emulsifiable low boiling liquids provided in International Publication No. 9416379, incorporated herein by reference. Specific examples of emulsifiable diffusible 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; Aliphatic hydrocarbons which may be saturated or unsaturated, straight or branched, for example n-butane, n-pentane, 2-methylpropane, 2-methylbutane, 2,2-dimethylpropane, 2,2-dimethylbutane, 2 , 3-dimethylbutane, 1-butene, 2-butene, 2-methylpropene, 1,2-butadiene, 1,3-butadiene, 2-methyl-1-butene, 2-methyl-2-butene, isoprene, 1-pentene, 1,3-pentadiene, 1,4-pentadiene, butenin, 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 being fluorine atoms, for example 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 difluoromethylether, 1-chloro-2,2,2-trifluoroethyl difluoromethyl ether, partially fluorinated alkanes (e.g., Penafluoropropane such as 1H, 1H, 3H-pentafluoropropane, nonafluorobutane such as hexafluorobutane, 2H-nonafluoro-t-butane, and decafluoro such as 2H, 3H-decafluoropentane Lopentane, and tridecafluorohexanes such as 1H-tridecafluorohexane, partially fluorinated alkenes (eg, 1H, 1H, 2H-heptafluoropent-1-ene; Lofenten and nonafluorohexene such as 1H, 1H, 2H-nonafluorohex-1-ene), fluorinated ethers (e.g., 1,1,2,2-tetrafluoroethyl methyl ether, 2, 2,3,3,3-pentafluoropropyl methyl ether, 1,1,2,3,3,3-hexafluoropropyl methyl ether or 2,2,3,3,3-pentafluoropropyl difluoro Romethyl ether), and more preferably, such as perfluorocarbons. Examples of perfluorocarbons include perfluoroalkanes such as perfluorobutane, perfluoropentane, perfluorohexane (eg perfluoro-2-methylpentane), perfluoroheptane, purple Fluorooctane, perfluorononane and perfluorodecane; Perfluorocycloalkanes such as perfluorocyclobutane, perfluorodimethylcyclobutane, perfluorocyclopentane and perfluoromethylcyclopentane; Perfluoroalkenes such as perfluorobutene (eg perfluorobut-2-ene or perfluorobuta-1,3-diene), perfluoropentene (eg perfluoro Pent-1-ene) and perfluorohexene (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. If necessary, the dispersible component may be formulated as part of a patented pharmaceutical emulsion, such as Intralipid®. In a further embodiment of the invention, the oil phase can be a mixture of two fluids, the first being for example a perfluorocarbon as discussed above, for example perfluorodimethylcyclobutane, the other being slightly more Volatile lipophilic "filling" materials with high water solubility, such as halogenated inhalation anesthetics or hydrocarbons. The purpose of the "filling" material is to nonspecifically increase the microbubble size. Following the onset of growth of the dispersed gas phase, the microbubbles will readily shrink after their initial growth as a result of the loss of the "filling" material by outward diffusion. Currently, residual microbubbles containing only a lower water solubility of the first volatile compound and blood gas will have a reduced size that can be controlled by appropriate selection of the initial ratio of the two volatile fluids in the diffusing component emulsion. Representative mixing ratios for the two fluids may be a 1: 9 perfluorocarbon: "fill" material. The emulsion stabilizer material may typically comprise one or more surfactants. It will be appreciated that the nature of such surfactants can significantly affect factors such as the growth rate of the dispersed gas phase. In general, a wide range of surfactants may be useful, for example selected from various surfactants described in European Patent Publication No. 0727225, which is incorporated herein by reference. Representative examples of useful surfactants include fatty acids (eg, straight chain saturated or unsaturated fatty acids containing 10 to 20 carbon atoms) and carbohydrates and triglyceride esters thereof, phospholipids (eg lecithin), fluorine-containing phospholipids, proteins ( For example, albumin such as human serum albumin), polyethylene glycol, block copolymer surfactants (e.g. polyoxyethylene-polyoxypropylene block copolymers such as Pluronics, elongated polymers, e.g. Acyloxyacyl polyethylene glycols, for example polyethylene glycol methyl ether 16-hexadecanoyloxy-hexadecanoate having a molecular weight of 2300, 5000 or 10000, fluorine-containing surfactants (eg, International features sold as Zonyl and Fluorad, or incorporated herein by reference Publication No. 9639197 as hereinbefore described to a call) and for example one or more fourth ammonium group, and one or more fat-based, for example, long chain (e. G., 10-30 C) a cationic surfactant comprising an alkyl or alkanoyl I can lift it. The use of cationic materials as surfactants or other stabilizers or as additives to stabilizers in the surface materials present in the diffusive component emulsions according to the invention is an anionic surface material in terms of the electrostatic interactions involved between the two surface materials. For example, negatively charged phospholipids such as natural (eg derived from soy or egg yolk), semisynthetic (eg partially or fully hydrogenated) or synthetic phospholipids such as phosphatidylserine, phosphatidylglycerol It may be particularly advantageous to use with gas dispersions containing phosphatidylinositol, phosphatidic acid and cardiolipin. Generally, at least some hydrophobic and / or substantially water-insoluble compounds having a wide range of cationic materials, for example basic nitrogen atoms, for example primary amines, secondary amines, tertiary amines and alkaloids, for example Pyrrolidine, piperidine, imidazole, pyridine, quinoline and alkyl- and aryl-guanidinium compounds can be used. Examples of representative cationic materials include lipophilic quaternary ammonium or pyridinium salts, such as dididodecyldimethylammonium bromide, cetyltrimethyl-ammonium chloride, cetylpyridinium chloride, cetyltrimethylammonium bromide, quaternium-26, Oleyltrimethylammonium chloride, cetylethyldimethyl-ammonium bromide, rapylium chloride, Halimide®, cetalconium chloride, 1,2-distearoyl-3-trimethyl-ammoniumpropane, betaine Cetyl ester or DC-cholesterol; Lipophilic secondary or tertiary amines such as diethylstearylamine, methylstearylamine, dimethylspingosin, esters of fatty alcohols with dimethylglycine, esters of fatty acids with dimethylethanolamine, fatty alcohols with sarcosine Esters or esters of fatty alcohols with N (2)-or N (6) -dimethyllysine; Amides of fatty acids with substituted di- or tri-amines, such as N-stearoyl-N'-dimethylaminopropylamine; Primary amines such as stearylamine or dodecylamine; Esters of fatty alcohols with amino acids, alanine, lysine, serine or threonine, for example alanine cetyl ester or lysine cetyl ester; Amides of fatty acids with di- or tri-amines, such as monostearoyldiaminopropane or monostearoyl-putrescine; Or positively charged phospholipids such as dialkyl-sn-glyceroethylphosphatidylcholine or phosphatidic acid, such as dipalmitoylphosphatidic acid or distearoylphosphatidic acid and amino alcohols such as lysine hydroxy Esters with ethylamide, hydroxylysine ethyl ester, 1,3-diamino-2-propanol or 2,4-diaminobenzyl alcohol. Positively charged atoms other than nitrogen, for example sulfur (for example as in sulfonium compounds), iodine (for example as in iodonium compounds), selenium or phosphorus (for example phosphoniums) Lipophilic cationic compounds, such as in compounds) and suitable positively charged metal complexes may be useful. Preferred cationic materials are endogenous compounds (e.g., spinosine, DL-dihydrospinosine, dimethylspinosine, phytospinosine or phycosin) or compounds that can be readily degraded into endogenous materials (e.g., Esters or amides of choline, ethanolamine, putrescine, lysine, arginine, glycine, sarcosine, dimethylglycine, carnitine, betaine or spermidine, such as cetyl betaine ester, or derivatives of common amino acids) do. The use of fluorine-containing cationic surfactants such as fluorinated positively charged phospholipids or fluorinated cationic surfactants sold under the trade name Zonyl may be advantageous. The second composition can be injected, for example intravenously, intramuscularly or subcutaneously, with the latter route being advantageous when one wishes to specifically limit the effect of the diffusive component to the particular target side of the subject. One example of a subcutaneous injectable composition includes nanoparticles as used in lymphatic contrast X-rays. The droplet size of the emulsion for intravenous injection should preferably be less than 10 μm, eg less than 7 μm and greater than 0.1 μm to facilitate unobstructed passage through the pulmonary artery. It may be advantageous to use first and second compositions comprising dispersed gas microbubbles and dispersed diffuser droplets, respectively, having substantially similar sizes, for example 1-7, for example 2-6 μm in diameter. . If desired, the dispersible component can be formulated as a microemulsion. Such systems are advantageous in terms of their thermodynamic stability and the fact that the diffusive components are uniformly distributed throughout the aqueous phase; Thus, the microemulsion may have the appearance of a solution but exhibit the properties of the emulsion with respect to the partial pressure of the dispersed phase. As noted above, the present invention makes it possible to use the dispersing component containing emulsions required so far at substantially low dosages. Phase transfer colloidal contrast agents, as described in WO 9416739, are typically administered in an amount corresponding to about 0.1 ml dispersed phase / kg body weight. International Publication No. 9817324 mentions that when the dispersible component is a perfluorocarbon formulated as an oil-in-water emulsion, it can typically be administered at a dose equivalent to 0.2-1.0 μl perfluorocarbon / kg body weight. have. However, the present invention provides a low dose of diffuse components, such as 1-100 nl diffuse components / weight, of at least 20 and possibly less than 200 times the images comparable to that observed in WO9817324. Kg, for example 20 nl diffusible component / kg body weight. It will be appreciated that although the volume is likely to increase by more than 100 times when the diffusing component of the emulsion is evaporated, the total dosage of the diffusing component at such dosages is generally insufficient to pose a risk of embolism. In addition, such dosages are below certain limits that gas bubbles can spontaneously generate in the low pressure vein compartments (eg, vena cava, right ventricle and pulmonary artery) of the blood circulation as a result of volatile diffuse components and blood gases that supersaturate the blood. There will be. In order to ensure maximum volatility of the diffusive component after administration and increase the growth of the dispersion gas, both of which are endothermic processes, the temperature of the first and / or second composition is manipulated prior to administration and / or the exothermic reactive component therein. It may be advantageous to include; It may be particularly advantageous to use such components which exothermicly react under the influence of ultrasonic radiation. The growth of the dispersed gas phase in vivo is effected, for example, by expansion of any encapsulation stabilizing material (if it has sufficient flexibility) and / or, for example, from the second composition to the growing gas-liquid interface. It may be accompanied by extraction of excess surfactant or other stabilizing material. However, stretching of the encapsulating material and / or its interaction with the ultrasound may substantially increase its porosity. Such breakdown of the encapsulating material has so far been found to lead to rapid loss of echogenicity through outward diffusion and dissolution of the resulting gas in many cases, while the inventors have found that the exposed gas when using the contrast agent formulation according to the invention It was found to exhibit substantially stability. Although not based on theoretical calculations, the present inventors have applied to a diffusive component that provides an internal pressure gradient to counteract the outward diffusion tendency of the microbubble gas, for example, in the form of free microbubbles. It is thought that, by the supersaturated environment generated by it, for example, it can be stabilized against collapse of micro bubbles. Since the exposed gas surface is substantially free of encapsulation material, the contrast agent formulation has specifically targeted acoustic wave properties, as evidenced by high backscattering and low energy absorption (e.g., indicated by high backscattering; damping ratio). To indicate; This effect of echo generation can continue for a significant period of time even during subsequent ultrasound irradiation. Therefore, the stabilizing effect of the co-administered diffusive components can be very advantageously used to increase both the duration and size of echogenicity of existing gas containing contrast agent formulations when these parameters may be insufficient when the contrast composition is administered alone. Can be. Thus, for example, the duration of the effect of an albumin based contrast agent is often severely limited by the breakdown of encapsulated albumin material as a result of changes in systolic pressure in the heart or venous system or as a result of ultrasound irradiation, but in accordance with the present invention It can be substantially improved by simultaneous administration. In an exemplary embodiment of the method of the invention, a composition comprising a dispersing gas component and a composition comprising an emulsifiable diffusive component, at least a portion of the dispersing gas passes through the lungs and is diffuseable after intravenous injection of both compositions. Rapid ingrowth after passage from the lungs through inward diffusion of the components is selected to be temporarily retained in the myocardium and thereby to enable ultrasound visualization of myocardial perfusion. When the concentration of volatile diffuse components in the blood stream is reduced, for example, the components may disappear from the blood, for example by removal through the lungs and divergence by the subject, by metabolism or by redistribution to other tissues. At that time, the diffusive component will typically diffuse out of the dispersion gas, which will shrink to its initial smaller size and ultimately flow freely once again in the bloodstream and is typically removed from it by the reticuloendothelial system. The substantially temporary pattern of echogenicity followed by the disappearance of the contrasting effect is markedly different from any reverberation characteristic exhibited by either of the two compositions when administered alone. It will be appreciated that the adjustment of the retention period of the dispersing gas may be made by appropriate adjustment of the dosage and / or dosage of the dispersing component and / or the nature and affinity of the formulation, in particular between the gas component and the diffusing component. Other capillary systems such as, but not limited to, kidneys, liver, spleen, thyroid, skeletal muscle, breast and prostate are similarly imaged. In general, the growth rate and / or range of the dispersion gas is characterized by the nature of the gas and gas stabilizing material and more particularly the emulsifiable diffusible component and the way in which it is formulated, for example the nature of the emulsion stabilizing material and the size of the emulsion droplets. Can be controlled by an appropriate choice. In the last situation, for a given amount of emulsified diffusive component, the reduction in droplet size can increase the delivery rate of the diffusive component compared to that from larger droplets, because of the higher surface area: volume ratio This is because more rapid release can occur from smaller droplets. Other parameters that allow for control include relative amounts, order of administration, time intervals between the two administrations and possible spatial separation of the two administrations when the two compositions are administered and they are administered separately. In this last aspect, it will be appreciated that the inherent diffusivity of the diffusive component allows it to be applied to other parts of the body by various routes, such as subcutaneously, intravenously or intramuscularly. Particularly important parameters relating to the diffusible component are to determine its rate of transport through the carrier liquid or blood, encapsulating its diffusivity (eg, indicated by its diffusion coefficient) and its solubility in water / blood, and the dispersion gas Its permeability through any membrane of stabilizing material. The pressure generated by the in vivo diffusing component will also affect its rate of diffusion into the dispersion gas, such as its concentration. Thus, according to the law of Pick's, the concentration gradient of the diffusive component, which is related to the distance between the individual gas microbubbles and the emulsion droplets, for example together with the diffusion coefficient of the diffusible material in the surrounding liquid medium, is determined by simple diffusion. The rate of delivery will be determined and the concentration gradient is determined by the solubility of the diffusive components in the medium of interest and the distance between the individual gas microbubbles and the emulsion droplets. Likewise, the water solubility of the diffusive component, vapor pressure and molecular size will prolong the life of the microbubbles by the influence of these parameters on the diffusion rate of the diffusive component. Thus, this allows for adjustment of the contrast period, which can optimally be 2 to 5 minutes. The effective rate of transport of the diffusive component can be adjusted, for example, by adjusting the viscosity of the dispersed gas phase composition and / or the diffusive component composition, for example at least one biocompatible viscosity increasing agent such as X-ray contrast agent, polyethylene It can be adjusted by including glycols, carbohydrates, proteins, polymers or alcohols in the formulation. For example, it may be advantageous to simultaneously inject the two compositions as a relatively high volume (eg, having a volume of at least 20 ml in the case of a 70 kg human subject), because this is due to the This is because it will delay the complete mixing of the components with the blood (and thus the onset of growth of the dispersing gas) until entering the right ventricle. The delay in growth of the dispersion gas can be maximized, for example, by using an unsaturated carrier liquid for the gas and any other diffusive component as so far defined as a result of cooling. As noted above, other transport mechanisms than diffusion can be included in the work of the present invention. Thus, for example, transport may occur via passive hydraulic flow in the surrounding liquid medium; This can be important in blood vessels and capillaries where high shear rate flow can occur. The transport of the diffusing component into the dispersion gas impinges the absorption of the diffusing component at the microbubble surface and / or the penetration of the diffusing component into the microbubbles, i.e. the collision between the gas microbubbles and the emulsion droplets, leading to a form of aggregation. Or as a result of nearby collision processes. In such cases, the diffusion coefficient and solubility of the diffusive component are key factors controlling the rate of delivery of the diffusive component, particle size (eg, droplet size when it is formulated as an emulsion) and rate and extent of microbubble growth. It has a minimal effect on the collision frequency between phosphorus microbubbles and droplets. Thus, for example, for certain amounts of emulsified diffusive components, a decrease in droplet size will lead to an increase in the total number of droplets, thus reducing the mean interparticle distance between gas microbubbles and emulsion droplets and causing collisions and / or ) Increase the rate of delivery by increasing the likelihood of aggregation. As noted above, the rate of delivery that proceeds through the collision process can be increased significantly if additional vibrational movement is imparted to the gas microbubbles and emulsion droplets of the diffusive component through the application of ultrasonic energy. The kinetics of the collision process induced by such ultrasonic energy may, for example, be due to the diffusion of the diffusive component in the carrier liquid and / or blood in that a particular energy level may be required to initiate the agglomeration of impingement gas microbubbles and emulsion droplets. It may be different from the dynamics of transport. Thus, it may be advantageous to select the size and mass of the emulsion droplets so as to generate a collision force with the vibrating microbubbles sufficient to induce agglomeration. In addition, the permeability of any stabilizing material that encapsulates the dispersed gas phase as described above is a parameter that can affect the growth rate of the gas phase, and it is any such encapsulating material (e.g., a polymer or surfactant membrane, e.g. For example, it may be desirable to select a diffusive component that readily permeates a single layer or one or more bilayers of membrane forming surfactants such as phospholipids. However, the inventors have discovered that substantially impermeable encapsulation materials may be used because it appears that ultrasonic- or other energy input-induced growth of the dispersion gas may occur despite the presence of such an impermeable material. While not wishing to be based on the theory, the ultrasonic waves can at least temporarily change the permeability of the encapsulating material, the diffusivity of the diffusing component in the surrounding liquid phase and / or the impingement force between the emulsion droplets and the encapsulating microbubbles. Since the effect can be observed using an extremely short ultrasonic pulse (e.g., about 0.3 ms in B-mode imaging or about 2 ms in Doppler or second harmonic imaging), it is possible It does not appear to be an example of rectified diffusion causing a constant increase in the equilibrium radius of (Leighton, EG-"The Acoustic Bubble", Academic Press [1994], p. 379), and ultrasonic pulses destroy the encapsulated membrane and thus expose the gas phase. It is possible to increase the growth of the dispersion gas through inward diffusion of the diffusive component into the furnace. If desired, the dispersing gas or diffusible component may comprise an azeotrope or may be selected such that the azeotropic mixture is formed in vivo when the dispersible component mixes with the dispersing gas. Such azeotrope formation can be achieved by the volatility of halogenated hydrocarbons such as fluorocarbons (including perfluorocarbons), for example liquids, such as fluorocarbons (including perfluorocarbons) that are liquid such that they can be administered in gaseous form at normal body temperatures of 37 ° C. under standard conditions. It can be used effectively to increase. This has the substantial advantage of effective echo life in vivo of contrast agents containing such azeotropic mixtures, because of the increased molecular weight of parameters such as water solubility, fat solubility, diffusivity and pressure resistance of compounds such as fluorocarbons. It is known to decrease accordingly. Contrast agents containing biocompatible azeotropic mixtures that are gaseous at 37 ° C. are described in International Publication No. 9847540, which is incorporated herein by reference. In general, the recognized natural resistance of azeotropic mixtures to the separation of constituents will increase the stability of contrast agent compounds containing the same ingredients during manufacture, storage and handling and also after administration. Azeotropic mixtures useful according to the invention can be selected, for example, by reference to azeotropic mixtures, by experimental studies and / or by theoretical predictions (see, for example, Tanaka in Fluid Phase Equilibria 24 (1985), pp. 187-203, by Kittel, C. and Kroemer, H. in Chapter 10 of Thermal Physics (WH Freeman & Co., New York, USA, 1980) or by Hemmer, PC in See Chapters 16-22 of Statistisk Mekanikk (Tapir, Trondheim, Norway, 1970)). An example of a literature of azeotropic mixtures that effectively reduces the boiling point of high molecular weight components below normal body temperature is 1,1,2-trichloro-1,2,2 described in US Pat. No. 40,550,49 having an azeotropic point of 24.9 ° C. 57:43 w / w mixture of -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 Patent 5599783, US Patent 55605647, US Patent 55605882, US Patent 55607616, US Patent 5607912, which are incorporated herein by reference. US Pat. No. 5,561,210, US Pat. No. 5,565,565 and US Pat. Simons et al. In J. Chem. Phys. 18 (3) (1950), pp. 335-346, describe the perfluoro-n-pentane (boiling point of 29 ° C.) and n-pentane (boiling point of 36 ° C.). It is reported that the mixture exhibits a clear deviation from Raoult's law and the effect is most pronounced for almost equimolar mixtures. In practice, it has been found that the boiling point of the azeotropic mixture is about 22 ° C. or less. Mixtures of perfluorocarbons and unsubstituted hydrocarbons generally exhibit useful azeotrope properties, and strong azeotrope effects have been observed for mixtures of such components with substantially similar boiling points. Examples of other perfluorocarbon: hydrocarbon azeotrope are mixtures of perfluoro-n-hexane (boiling point 59 ° C.) and n-pentane and azeotrope when the azeotrope has a boiling point between room temperature and 35 ° C. A mixture of perfluoro-4-methylpent-2-ene (boiling point 49 ° C.) and n-pentane when having a boiling point of ° C .; Other potentially useful azeotropic mixtures include mixtures of halotan and diethyl ether and two or more fluorinated gases, such as perfluoropropane and fluoroethane, perfluoropropane and 1,1,1-trifluoro Ethane or perfluoroethane and difluoromethane. Fluorinated gases, such as perfluoroethane, are known to be capable of forming azeotrope with carbon dioxide (see, for example, International Patent Publication No. 9502652). Thus, administration of such a gas containing contrast agent leads to in vivo formation of a three- or more component azeotrope with blood gases, such as carbon dioxide, thereby further increasing the stability of the dispersion gas. When two compositions of the complex contrast agent preparation according to the invention are administered simultaneously, they may be injected, for example, from a separate syringe via a suitable coupling means or may be premixed under controlled conditions, which preferably avoids premature dispersal gas. Can be. The composition for mixing prior to simultaneous administration may be advantageously stored in a suitable dual or multi-chamber device. Thus, for example, a dispersion gas-containing first composition or a dried precursor thereof (e.g., an amphiphilic material is predominantly (e.g., at least 75%, preferably substantially completely), individually For example, a composition comprising a lyophilized residue of a suspension of gas microbubbles in an amphiphilic material-containing aqueous medium, consisting essentially of a phospholipid comprising a molecule having a negative charge), a second composition comprising a diffusive component The syringe containing can be contained in a first chamber, such as a vial, which is hermetically sealed, and the syringe outlet is closed with a membrane or plug, for example, to avoid premature mixing. The operation of the syringe plunger breaks the membrane and causes the second composition to mix or mix with the first composition and to reconstruct its precursor; After any necessary or desired shaking and / or dilution, the mixture may be removed (eg, by syringe) and administered. Alternatively, the two compositions can be stored, for example, in a single sealed vial or syringe separated by a membrane or plug; Overpressure of gas or vapor may be applied to one or both compositions. For example, disruption of the membrane or plug by insertion of a hypodermic needle into a vial leads to mixing of the composition; This can be increased by manual shaking as needed, after which the mixture can be removed and administered. In another embodiment, for example, a vial containing a dried precursor for a first composition is equipped with a first syringe containing a redispersible fluid for the precursor and a second syringe containing a second composition, or membrane separation Equipped with a syringe containing a redispersible fluid for the latter in a vial containing the prepared second composition and a dried precursor for the first composition can likewise be used. In embodiments of the invention wherein two compositions are mixed before, during or after the preparation, the mixture will typically be stored at high pressure or reduced temperature such that the pressure of the diffusive component is insufficient to provide growth of the dispersion gas. Activation of the growth of the dispersion gas can be induced by the release of excess pressure or by heating to body temperature which will occur following administration of the mixture, or if necessary, by preheating the mixture immediately before administration. In embodiments of the invention in which the two compositions are administered separately, the timing between the two administrations can be used to affect the face of the body where the growth of the dispersed gas phase occurs primarily. Thus, for example, the second composition is first injected and the diffusive component is concentrated in the liver to improve imaging of the organ upon subsequent injection of the dispersed gas-containing first composition. If the stability of the gas dispersion is allowed, it is likewise first injected and concentrated in the liver and then the second composition containing the diffusive component is administered to increase its echogenicity. Representative ultrasonic imaging techniques that may be useful in accordance with the present invention include: fundamental wave B-system imaging; Harmonic B-system imaging, including, for example, reception of low and second or high frequencies; Tissue Doppler imaging, optionally including selective reception of fundamental, harmonic or low frequency echo frequencies; Color Doppler imaging, optionally including selective reception of fundamental, harmonic or low frequency echo frequencies; Power Doppler imaging, optionally including selective reception of fundamental, harmonic or low-frequency echo frequencies; Power or color Doppler imaging using loss of correlation or apparent Doppler shift caused by changes in acoustic properties of contrast medium microbubbles, such as may be caused by spontaneous or ultrasonically induced destruction, fracture, growth, or aggregation; Pulse inverted imaging, optionally including selective reception of fundamental, harmonic or low harmonic echo frequencies and including more than two pulses emitted in each direction; Pulse inversion imaging using loss of correlation caused by changes in acoustic properties of contrast medium microbubbles, such as may be caused by spontaneous or ultrasonically induced disruption, fracture, growth, or aggregation; Pulse preliminary imaging described in the 1997 IEEE Ultrasonics Symposium, pp. 1567-1570; And ultrasonic imaging techniques based on a comparison of the echoes obtained with different emission output amplitudes or waveforms in order to detect nonlinear effects caused by the presence of gas microbubbles. For certain doses of gas dispersions and diffusive component compositions, the use of color Doppler imaging ultrasound to induce the growth of dispersed gas is probably a result of the use of higher intensity ultrasound, resulting in a stronger contrast effect during subsequent B-mode imaging. It was found to provide. In order to reduce the migration effect, sequential images of tissues such as the heart or kidneys can be collected with a suitable synchronization technique (eg gate controlled for the subject's ECG or respiratory movement). Measurement of the resonance frequency or frequency absorption change accompanying the growth of the dispersion gas may also be made generally to detect the contrast agent. It will be appreciated that the dispersion gas content of the composite contrast agent formulation according to the present invention will be likely to be temporarily stagnant in tissue at a concentration proportional to the local tissue perfusion rate. Thus, when the display uses an ultrasonic imaging modality, such as conventional or harmonic B-way imaging, in which the display is derived directly from the feedback signal intensity, the image of such tissue can be considered as a perfusion map in which the displayed signal intensity is a function of local perfusion. . This contrasts with images obtained with 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 cardiac studies, where perfusion maps are derived from return signal intensity in accordance with this embodiment of the invention, the characteristics indicative of the difference between the normally perfused myocardium and any myocardial region supplied by the stenosis artery and thus the image intensity It may be advantageous to subject the patient to physical or pharmacological stress to reinforce the difference. As is known from radionuclide cardiac imaging, such stress induces vasodilation and increased blood flow in healthy myocardial tissue, while blood flow in downstream perfusion tissue supplied by the stenosis arteries is intended to increase blood flow with limited aortic vasodilation capacity. Since it is already consumed by intrinsic self-regulation, it does not change substantially. Stressing physically or pharmacologically by administration of adrenergic agonists can potentially lead to discomfort, such as chest pain, in a group of patients suffering from heart disease, and therefore, by administration of vasodilators, It is desirable to increase perfusion. Representative vasodilators useful in accordance with the present invention include endogenous / metabolic vasodilators such as lactic acid, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, oxides of nitrate and hypercarbonate, hypoxia / hypoxia reduction or hyperemia Agents that cause; Phosphodiesterase inhibitors such as dipyridamole and sildenafil; Sympathetic activity inhibitors such as clonidine and methyldopa; Smooth muscle relaxants such as papaverine, hydralazine, dihydralazine and nitroprusside; Beta receptor agonists such as dopamine, dobutamine, arbutamine, albuterol, salmeterol and isoproterenol; Alpha receptor antagonists such as doxazosin, tetrazosin and prazosin; Organic nitrates such as glyceryl trinitrate, isosorbide dinitrate and isosorbide mononitrate; Angiotensin converting enzyme (ACE) inhibitors, benazepril, meptopril, enalapril, posinopril, ricinopril, quinapril and ramipril; Angiotensin II antagonists (or AT1 receptor antagonists) such as valsartan, losartan and candesartan; Calcium channel blockers, such as amlodipine, nicardipine, nimodipine, felodipine, isradipine, diltiazem, verapamil and nifedipine; Prostaglandins such as alprostadyl; And endothelial dependent vasodilators. The use of adenosine is particularly preferred because it is an endogenous substance and has a rapid but short-term vasodilating effect. This latter property is confirmed by the fact that it has a blood retention half-life of only a few seconds, thereby minimizing discomfort for the patient that can be exerted during vasodilation. The vasodilatory effect induced by adenosine will be the strongest in the heart because the drug will reach tissue farther than the pharmacologically active concentration, and therefore it is the best vasodilator in the field of heart rate recording use of the method of the present invention. In addition to arterial stenosis, other tissue / perfusion abnormalities that affect local vascular regulation can be detected in accordance with the present invention by inducing angiogenesis. Thus, for example, blood vessels with malignant tumor lesions may be poorly differentiated and thus exhibit an impaired response to vasoconstrictor compared to normal tissue; A deficiency of similar vasoconstrictive reactions can occur in tissues with severe inflammation. Observation of the response to vasoconstriction stimulation in terms of changes in signal intensity during the imaging procedure can provide useful diagnostic information. Representative examples of vasoconstrictors that may be useful in such embodiments include isoprenulin, epinephrine, norepinephrine, dopamine, metaramimin, prenalterol, ergotamine, dihydroergotamine, methylsergid and nitric acid production inhibitors, For example, analogs of L-arginine; Such drugs can be administered, for example, locally or systemically. For any purpose, it may be advantageous to administer two or more vasoactive substances together or in sequence. When two vasoactive substances are added, both may be vasodilators, both vasodilators, or one vasodilator and the other may be vasoconstrictor. When two vasodilators or two vasoconstrictors are used, local differences in signal intensity can be determined during a single experiment because they must differ in at least one property, such as tissue specificity or mechanism of action. When administered separately, vasoconstrictors may be administered first followed by vasodilators, or in reverse order. Administration of adenosine increases the coronary blood flow in healthy myocardial tissue by a four-fold excess, greatly increasing the absorption and transient congestion of the contrast agent according to the present invention, thus greatly reducing the difference in return signal strength between normal and perfused myocardial tissue. Increase. Since inherently a physical capture process is involved, the identity of the contrast agent according to the invention is very efficient, which is limited by the short contact time between the tracer and the tissue, and radionuclide tracers such as thallium 201 and technetium cestamibi It may be necessary to maintain vasodilation for the entire period of blood retention distribution (eg, 4-6 minutes for thallium scintillation) for the tracer to ensure optimal effects. The contrast agent of the present invention, on the other hand, is not subject to such diffusion or transport limitations, and because its identity in myocardial tissue may also be terminated quickly, for example by stopping growth-generating ultrasound irradiation, according to embodiments of the present invention, cardiac perfusion Vasodilation time required for imaging can be very short, for example, less than 1 minute. This will shorten any inconvenience periods that may be caused by the patient by the administration of vasodilators. Because of the short half-life of adenosine described above, repeated injections or infusions thereof may be necessary during cardiac imaging in accordance with embodiments of the present invention, for example initial administration of adenosine at 150 μg / kg is substantially concurrent with administration of the contrast agent composition. And then, after 10 seconds, additional adenosine of 150 μg / kg may be injected slowly, for example for a period of 20 seconds. Adenosine may be injected at a constant rate during the time interval between injection and precipitation of the contrast agent in the myocardium. Contrast agent formulations according to the invention can be advantageously used as delivery agents for biofunctional components such as therapeutic agents (e.g., agents which have a beneficial effect on certain diseases in living humans or non-human animals), particularly at the target site. have. Thus, for example, the therapeutic compound is in the first gas (eg, via covalent or ionic bonds, if necessary via a spacer arm) or connected to a portion of the stabilizing material or physically mixed with such stabilizing material. For example, in a dispersed gas; The latter choice is particularly available when the therapeutic compound and stabilizing material have similar polarity or solubility. The adjustable growth properties of the dispersion gas can be used to temporarily hold it in the microvascular system of the target area; The use of ultrasound irradiation to induce the growth and hence congestion of gases and related therapeutic compounds in the target construct is particularly advantageous. For example, topical injection of the gas-containing first composition, or more preferably the diffusive component-containing second composition, which has been described so far, may also be used to concentrate the growth of the dispersion gas within the target plane. If desired, therapeutic compounds that can be linked to site specific vectors having affinity for particular cells, structures or pathological sites are, for example, growth of dispersion gases, solubilization of stabilizing substances or gas-microbubbles or gas-containing particulates. May be released as a result of stretching or cleavage of the gas stabilizing material caused by disintegration of (e.g., induced by ultrasound or by inversion of the concentration gradient of the diffusive component in the target plane). When the therapeutic agent is chemically bound to a gas stabilizing material, it may be advantageous for the bond or any spacer arm associated therewith to contain one or more labile groups that degrade and release the agent. Representative degradable groups include, for example, disulfide groups, amides, imides, imines, esters, anhydrides, acetals, carbamates, carbonates and carbonates that can be biodegraded in vivo as a result of hydrolysis and / or enzymatic action. Carbonate esters. Representative and non-limiting examples of drugs useful according to embodiments of the present invention include anti-neoplastic agents such as 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, Estramustine, Plutamide, Leproprolide, Megestrol Acetate, Tamoxifen, Testosterone, Trilostane, Amsacrine (m-AMSA), Asparaginase (L- Asparaginase), etoposide, interferon a-2a and 2b, blood products such as hematoporphyrin or derivatives thereof; Biological response modifiers such as muramylpeptide; Antifungal agents such as ketoconazole, nystatin, griseofulvin, flucitocin, myconazole or amphotericin B; Hormones or hormonal analogues such as growth hormone, black blood stimulating hormone, estradiol, beclomethasone dipropionate, betamethasone, cortisone acetate, dexamethasone, flunisolidide, hydrocortisone, methylprednisolone, paramethasone acetate, prednisolone Prednisone, triamcinolone or fludrocortisone acetate; Vitamins such as cyanocobalamine or retinoids; Enzymes such as alkaline phosphatase or manganese peroxide dismutase; Anti-allergic agents such as amelexanox; Anticoagulants, such as warfarin, phenprocumon or heparin; Antithrombotic agents; Blood circulation agents such as propranool; Metabolic agents such as glutathione; Therapeutic agents for tuberculosis such as p-aminosalicylic acid, isoniazid, capreomycin sulfate, cycloseccin, ethambutol, ethionamide, pyrazinamide, rifampin or streptomycin sulfate; Antiviral agents such as acyclovir, amantadine, azidomidine, ribavirin or vidarabine; Vasodilators such as diltiazem, nifedipine, verapamil, erythritol tetranitrate, isosorbide dinitrate, nitroglycerin or pentaerythritol tetranitrate; Antibiotics such as dapsone, chloramphenicol, neomycin, cefachlor, cephadroxyl, ceparexin, ceparadine, erythromycin, clindamycin, linoomycin, amoxicillin, ampicillin, baccampillin, carbenicillin, diclox Saline, cyclacillin, picloxacillin, hetacillin, methicillin, naphcillin, penicillin or tetracillin; Anti-inflammatory agents, such as diflunisal, ibuprofen, indomethacin, meclefenamate, mefenamic acid, naproxen, phenylbutazone, pyricampam, tolmetin, aspirin or salicylate; Antiprotozoal agents such as chloroquinine, metronidazole, quinine or meglumine antimonate; Antirheumatic drugs such as penicillamine; Anesthetics such as paregory; Opiates such as codeine, morphine or opium; Cardiac glycosides such as deslaneeside, digitoxin, digoxin, digitalin or digitalis; Neuromuscular blockers such as atraccurium mesylate, galamine triethided, hexafluorenium bromide, metokurin iodide, pancuronium bromide, succinylcholine chloride, tubokurarin chloride or bekuronium bromide ; Sedatives such as amobarbital, amobarbital sodium, aprobarbital, butabarbital sodium, chloral hydrate, ethchlorbinol, ethinamate, flulazepam hydrochloride, glutetidemide, methotrimreprazine Hydrochloride, metyprilon, midazolam hydrochloride, paraldehyde, pentobarbital, secobarbital sodium, debutal, temazepam or triazolam; Local anesthetics such as bupivacaine, chloroprocaine, ethidocaine, lidocaine, mepivacaine, procaine or tetracaine; General anesthetics, for example, dropperidol, etomidate, fentanyl citrate with dropperidol, ketamine hydrochloride, methohexyl sodium or thiopental and pharmaceutically acceptable salts thereof (eg, acid additions) Salts such as hydrochloride or hydrobromide or base salts such as sodium, calcium or magnesium salts) or derivatives (eg acetate); And radiochemicals including, for example, β-ray emitters. Of particular importance are antithrombotic agents, for example agents having heparin and heparin like activity, such as antithrombin III, dalteparin and enoxaparin; Platelet aggregation inhibitors such as ticlopidine, aspirin, dipyridamole, iloprost and absikmab; And thrombolytic enzymes such as streptokinase and plasminogen activators. Other examples of therapeutic agents include genetic materials such as natural or synthetic nucleic acids, RNA and DNA, including recombinant RNA and DNA. DNA encoding a particular protein can be used to treat many different types of diseases. For example, tumor necrosis factor or interleukin-2 can be provided to treat advanced cancer; Thymidine kinase can be provided to treat ovarian cancer or brain tumors; Interleukin-2 can be provided to treat neuroblastoma, malignant melanoma or kidney cancer; Interleukin-4 can be provided to treat cancer. Contrast agent preparations according to the invention can be used as vehicles for contrast enhancement components for imaging modalities other than ultrasound, such as, for example, X-rays, photoimaging, magnetic resonance and more preferably scintillation imaging agents. Controlled growth of the dispersed gas phase can be used to place such agents within the subject's body within the subject's body using ultrasound irradiation of the target organ or tissue to induce the desired controlled growth and transient retention of the agent, followed by appropriate It may be imaged using a non-ultrasound imaging method. The contrast agent formulations according to the invention can also be used as vehicles for therapeutically active substances which do not necessarily require release from the formulations in order to exert their therapeutic effect. Such formulations may include radioactive atoms or ions, such as, for example, β-ray emitters that exhibit local radiation emission effects following growth and transient stagnation of the dispersed gas phase of the formulation at the target site. It will be appreciated that such formulations should preferably be designed such that continued contraction and stagnation of the dispersing gas is not stopped until the desired therapeutic radiation dose is administered. The contrast agent formulations according to the invention may also exhibit therapeutic properties by themselves. Thus, for example, the formulation can be used therapeutically by intravenous injection of a high dose of the formulation followed by exposure of the tumor-causing artery to topical ultrasound irradiation. Next, the growing gas phase can block blood circulation to the tumor. Thus, by applying topical ultrasound energy it may be possible to enable controlled and also localized embolism, which may be important as is or with other therapeutic means. The concentration of disperse gas in capillaries may also increase the absorption of ultrasound energy in the treatment of hyperthermia; It can be used, for example, in the treatment of liver cancer. Other tissues that can be treated in this way include breast, thyroid and prostate. For example, irradiation with a relatively high energy (eg 5W) focused ultrasound beam at 1.5 Hz may be suitable for such applications, for example. The following non-limiting examples are intended to illustrate the invention. <Production example 1> Perfluorobutane Gas Dispersion with Negatively Charged Surface Material Hydrogenated phosphatidylserine (5 mg / ml in 1% w / w propylene glycol solution in purified water) and perfluorobutane gas were immediately homogenized at 7800 rpm and about 40 ° C. to form a creamy white microbubble dispersion. The dispersion was fractionated to substantially remove small microbubbles (<2 mu m), and aqueous sucrose was added to adjust the volume of the dispersion to a predetermined microbubble concentration to obtain a sucrose concentration of 92 mg / ml. A portion of 2 ml of the formed dispersion was filled into a 10 ml flat bottom vial specially designed for freeze drying and the contents were freeze dried to give a white porous cake. The lyophilization chamber was filled with perfluorobutane and the vial was sealed. Prior to use, water was added to the vial and shaken by hand slowly for several seconds to obtain a perfluorobutane microbubble dispersion. The concentration of microbubbles in the dispersion was 1.1% v / v and the median microbubbles size was 2.7 μm. <Production example 2> Perfluorobutane Gas Dispersion with Positively Charged Surface Material 2 ml of a 1 ml dispersion of 1,2-distearoyl-3-trimethylammoniumpropane (1 mg / ml) and distearoylphosphatidylcholine (4 mg / ml) in a 2% w / v propylene glycol solution in purified water Placed in a vial. The space portion was flushed with perfluorobutane gas and the vial was shaken for 45 seconds using an Espe CapMix® mixer for dental materials. The milky white microbubble dispersion formed was washed three times by centrifugation, the bottom portion was removed, and then a volume of purified water was added. The concentration of microbubbles in the formed dispersion was 4.9% v / v and the median microbubbles size was 3.2 μm. <Production example 3> Perfluorobutane Gas Dispersion with Biotinylated Surface Material Distearoylphosphatidylserine (4.5 mg) and biotin-dipalmitoylphosphatidylethanolamine (0.5 mg) were weighed into clear 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 space portion was flushed with perfluorobutane gas. The vial was sealed and shaken for 45 seconds using an ESP Capmix® mixer and then placed on a roller table for 16 hours. The microbubble dispersion formed was washed extensively with deionized water. <Production example 4> Perfluorodimethylcyclobutane emulsion with positively charged surface material 1 ml of a dispersion of didodecyldimethylammonium bromide (5 mg / ml in purified water) was placed in a 2 ml vial to which 100 μl of perfluorodimethylcyclobutane (boiling point 45 ° C.) was added. The vial was sealed and shaken for 75 seconds using ESP Capmix® to obtain an emulsion of the diffusive component, which was stored at 0 ° C when not in use. The emulsion was washed three times by centrifugation, the bottom portion was removed and then the same volume of purified water was added. The concentration of droplets in the emulsion was 6.2% v / v and the median droplet size was 2.3 μm. <Production example 5> Perfluorohexane emulsion with positively charged surface material 100 μl of perfluorohexane (boiling point 57 ° C.) of 1 ml of a dispersion of 1,2-distearoyl-3-trimethylammoniumpropane (1 mg / ml) and distearoylphosphatidylcholine (4 mg / ml) in purified water. Was placed in a 2 ml vial added. The vial was sealed and shaken for 75 seconds using an ESP Capmix® mixer to obtain an emulsion of the diffusing component, which was stored at 0 ° C. when not in use. The emulsion was washed three times by centrifugation, the bottom portion was removed and then the same volume of purified water was added. The concentration of droplets in the emulsion was 2.9% v / v and the median droplet size was 2.9 μm. <Production example 6> Perfluorodimethylcyclobutane emulsion with negatively charged surface material 1 ml of a dispersion of hydrogenated phosphatidylserine (5 mg / ml in purified water) was placed in a 2 ml vial to which 100 μl of perfluorodimethylcyclobutane (boiling point 45 ° C.) was added. The vial was sealed and shaken for 75 seconds using an ESP Capmix® mixer to obtain an emulsion of the diffusing component, which was stored at 0 ° C. when not in use. The emulsion was washed three times by centrifugation, the bottom portion was removed and then the same volume of purified water was added. The concentration of droplets in the emulsion was 6.9% v / v and the median droplet size was 2.7 μm. <Production example 7> Perfluorodimethylcyclobutane emulsion with avidinylated surface material Distearoylphosphatidylserine (4.5 mg) and biotin-dipalmitoylphosphatidylethanolamine (0.5 mg) were weighed into clear vials and 1.0 ml of a solution of 2% propylene glycol was added. The mixture was then cooled to room temperature after heating to 80 ° C. 100 [mu] l of perfluorodimethylcyclobutane was added, the vial was sealed and shaken for 75 seconds using an ESP Capmix® mixer to obtain an emulsion of the diffusive component. Diluted samples of tanning (100 μl tanning in 1 mL water) were incubated with excess avidin and placed on a roller table. The diluted emulsion was extensively washed with water and concentrated by centrifugation. <Production example 8> Perfluorodimethylcyclobutane emulsion with positively charged surface material 1,2-Distearoyl-3-trimethylammoniumpropane (73 mg) and distearoylphosphatidylcholine (641 mg) were placed in a 250 mL round bottom flask and chloroform (100 mL) was added. The flask was heated with hot tap water until a clear solution was obtained, and then the flask was placed on a rotavapor and evaporated at 350 mbar using a water bath temperature of 45 ° C. to remove chloroform. To remove traces of residual solvent, the samples were exposed overnight at about 20 mbar vacuum. MilliQ water (143 mL) was then added and the flask was placed on a rotavapor again and spun at full speed while immersed in an 80 ° C. water bath. After about 25 minutes, the samples were transferred to appropriate vials and placed in an overnight refrigerator to cool. A portion of the sample was transferred to a 2 ml chromatography vial and 100 μl of perfluorodimethylcyclobutane (boiling point 45 ° C.) was added to each vial. The vial was shaken for 75 seconds on ESP Capmix® and the sample was immediately ice cooled. The contents of the vial were collected in larger vials and the emulsion was characterized for size distribution and total particle volume concentration using a Coulter counter. The median droplet size was 2.67 μm, confirming that the emulsion is suitable for injection. Particle volume concentration measurements were used to adjust the concentration to about 1% v / v dispersed phase using MilliQ water. Emulsions were stored in the refrigerator until use. <Production example 9> Perfluoromethylcyclopentane emulsion with positively charged surface material The procedure of Preparation Example 8 was repeated except that perfluoromethylcyclopentane (boiling point 48 ° C.) was used instead of perfluorodimethylcyclobutane. Coulter counter analysis indicates that the median droplet size of the emulsion is 2.63 μm. <Production example 10> Perfluoro-2-methylpentane emulsion with positively charged surface material The procedure of Preparation 8 was repeated except that perfluoro-2-methylpentane (boiling point 50-57 ° C.) was used instead of perfluorodimethylcyclobutane. Coulter counter analysis indicates that the median droplet size of the emulsion is 2.72 μm. <Production example 11> Perfluorohexane emulsion with positively charged surface material The procedure of Preparation 8 was repeated except that perfluorohexane (boiling point 58-60 ° C.) was used instead of perfluorodimethylcyclobutane. Coulter counter analysis indicates that the median droplet size of the emulsion is 2.54 μm. <Production example 12> Positively charged lipopeptides: synthesis of palmitoyl-Lys (palmitoyl) -Lys-Lys-Ahx-Lys-Arg-Lys-Arg-Lys-Arg-NH 2 , where Ahx = aminohexanoic acid Lipoproteins were synthesized on a 0.25 mmol scale on an ABI 433A automated peptide synthesizer starting with Rink amide resin using a 1 mmol amino acid cartridge. All amino acids and palmitic acid were previously activated using O-benzotriazol-1-yl-N, N, N'-N'-tetramethyluronium hexafluorophosphate (HBTU) before coupling. Trifluoroacetic acid (TFA) containing 5% phenol, 5% triisopropylsilane and 5% water was simultaneously removed from the resin for 2 hours to obtain 150 mg of crude product. A 30 mg aliquot of the crude was subjected to preparative HPLC (Vydac 218TP1022 column) for 40 minutes using a gradient of 70-100% B (A = 0.1% TFA / water and B = acetonitrile) at a flow rate of 9 ml / min. Purified. After freeze drying, 19 mg of pure material was obtained (analytical HPLC: gradient 70-100% B, where B = acetonitrile and A = 0.01% TFA / water; column-Vydac 218TP54: detection-UV 214 nm; product retention Time = 11 min) Additional product characterization was performed using MALDI mass spectrometry: M + H theory 1845, found 1850. <Production example 13> Positively charged lipopeptides: synthesis of palmitoyl-Dpr (palmitoyl) -Arg-Arg-Lys-NH 2 , where Dpr = diaminopropionic acid Fat proteins were synthesized on a 0.25 mmol scale on an ABI 433A automated peptide synthesizer starting with a link amide resin using a 1 mmol amino acid cartridge. All amino acids and palmitic acid were previously activated using HBTU before coupling. 50 mg of crude product was obtained by simultaneously removing peptide and side chain protecting groups from the resin for 2 hours in TFA containing 5% phenol, 5% triisopropylsilane and 5% water. The crude was subjected to preparative HPLC (Vydac 218 TP1022 column) for 40 minutes using a gradient of 90-100% B (A = 0.1% TFA / water and B = 0.1% TFA / acetonitrile) at a flow rate of 9 ml / min. Purification by After lyophilization, 5 mg of pure material was obtained (analytical HPLC: gradient 80-100% B, where A = 0.1% TFA / water and B = 0.1% TFA / acetonitrile; column-Vydac 218TP54: detection-UV 214 Nm; product retention time = 15 min.) Additional product characterization was performed using MALDI mass spectrometry, M + H theory 1021, found 1022. <Production example 14> a) hexadecanoic acid 2-tert-butoxycarbonylaminoethyl ester N-Boc-ethanolamine (1.6 g, 10 mmol) and palmitoyl chloride (3.28 g, 12 mmol) were dissolved in dichloromethane (25 mL) and triethylamine (1.68 mL, 12 mmol) was added with stirring. The reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with 100 mL of dichloromethane and transferred to the extraction vessel, washed with 1 x 10 mL 1M sodium hydrogen carbonate and 2 x 25 mL water, dried and the solvent was then removed in vacuo. The crude product was purified by column chromatography on silica. Identity: TLC (one spot) and MALDI (M + 1). b) hexadecanoic acid 2-aminoethyl ester hydrochloride Hexadecanoic acid 2-tert-butoxycarbonylaminoethyl ester (1.1 g, 2.7 mmol) obtained from a) was dissolved in 4M hydrogen chloride / dioxane (10 mL) with stirring. After a few minutes a white precipitate began to form. After 30 minutes TLC showed the starting material was fully converted. The white precipitate was collected by filtration, washed with dioxane on the filter and dried in vacuo. Identity: TLC (one spot) and MALDI (M + 1). <Production example 15> a) 4-hexadecanoylaminobutylcarbamic acid tert-butyl ester Boc-1,4-diaminobutane (1 g, 5.3 mmol) and palmitoyl chloride (1.64 g, 6 mmol) were dissolved in dichloromethane (25 mL). Triethylamine (0.64 mL, 6 mmol) was added and the reaction mixture was stirred overnight, then diluted with dichloromethane to 150 mL, transferred to an extraction vessel and transferred to 1 × 10 mL 1M sodium hydrogen carbonate and 2 × 25 mL water. Wash, dry and then remove the solvent in vacuo. The crude product was dissolved in chloroform (25 mL) and placed in an overnight refrigerator. Pure product was isolated as sticky crystals. Identity: TLC (one spot) and MALDI (M + 1). b) hexadecanoic acid 4-aminobutyl amide hydrochloride 4-hexadecanoylaminobutylcarbamic acid tert-butyl ester (1 g, 2.3 mmol) obtained from above a) was dissolved in 4M hydrogen chloride / dioxane (10 mL) with stirring. After a few minutes white crystals began to precipitate. The reaction mixture was diluted with dioxane (10 mL) and stirring continued for 4 h, at which time TLC showed complete conversion of starting material. The white precipitate was collected by filtration, washed with dioxane on the filter and dried in vacuo. Identity: TLC (one spot) and MALDI (M + 1). <Production example 16> a) tert-butoxycarbonylaminoacetic acid hexadecyl ester Boc-Gly-OH (1.74 g, 10 mmol) and 1-hexadecanol (2.5 g, 10 mmol) were dissolved in dichloromethane (30 mL) and dimethylaminopyridine (30 mg, catalytic amount) was added. Dicyclohexylcarbodiimide (2.1 g, 10 mmol) dissolved in dichloromethane (10 mL) was added dropwise with stirring for 10 minutes and the reaction mixture was stirred overnight at room temperature. Precipitated dicyclohexylurea was removed by filtration and the organic phase was diluted to 150 ml with dichloromethane. The organic phase was extracted with 1 x 5 ml 1 M sodium hydrogen carbonate and 2 x 10 ml water, dried and the solvent was then removed in vacuo. The crude product was used for the next step without further purification. Identity: TLC (one spot) and MALDI (M + 1). b) aminoacetic acid hexadecyl ester hydrochloride Tert-butoxycarbonylaminoacetic acid hexadecyl ester (2 g, 5 mmol) obtained from above a) was dissolved in dioxane (20 mL). 4M hydrogen chloride / dioxane (10 mL) was added and the reaction mixture was stirred at room temperature. After 30 minutes a white precipitate began to form. Diethyl ether (50 mL) was added and the reaction mixture was stirred overnight at room temperature, after which the precipitate was collected by filtration and washed with diethyl ether. TLC showed that the starting material was completely converted, but the product was contaminated with a small amount of 1-hexadecanol. Pure product was obtained by column chromatography on silica. Identity: TLC (one spot) and MALDI (M + 1). <Production example 17> Methylaminoacetic acid hexadecyl ester hydrochloride 4M hydrogen chloride / dioxane (10 mL) was added to the reaction vessel containing N-methylglycine (100 mg, 1.1 mmol) and 1-hexadecanol (1 g, 4.1 mmol). The slurry was stirred for several days at room temperature. After 4 days, the reaction mixture was homogeneous and TLC showed that the amino acid was fully converted. The solvent was removed in vacuo and the crude product was purified by column chromatography on silica. Identity: TLC (one spot) and MALDI (M + 1). <Production example 18> Dimethylaminoacetic acid hexadecyl ester hydrochloride 4M hydrogen chloride / dioxane (10 mL) was added to the reaction vessel containing N, N-dimethylglycine hydrochloride (150 mg, 1.1 mmol) and 1-hexadecanol (1.33 g, 5.5 mmol). The slurry was stirred at room temperature. After 3 weeks, the reaction mixture was homogeneous and TLC showed that the amino acid was fully converted. The solvent was removed in vacuo and the crude product was purified by column chromatography on silica. Identity: TLC (one spot) and MALDI (M + 1). <Production Example 19-36> Emulsions with positively charged surface material Distearoylphosphatidylcholine (90 mg) and the cationic additive (10 mg) in Table 1 below were placed in a 50 ml round bottom flask and chloroform (10 ml) was added (in Preparation Example 31, methanol ( 1 ml) was added to chloroform). The flask was heated with hot tap water until a clear solution was obtained, and then the flask was placed on a rotavapor and evaporated at 350 mbar using a water bath temperature of 45 ° C. to remove chloroform. To remove traces of residual solvent, the samples were exposed overnight at about 20 mbar vacuum. MilliQ water (20 mL) was then added and the flask was placed on a rotavapor again and spun at full speed while immersed in an 80 ° C. water bath. After about 10 minutes, the samples were transferred to appropriate vials and placed in an overnight refrigerator to cool. Some 1 ml of each sample was transferred to a 2 ml chromatography vial and perfluorodimethylcyclobutane (100 μl) was added to each vial. The vial was shaken for 75 seconds on ESP Capmix® and the sample was immediately ice cooled. The emulsion is collected in larger vials and the emulsion is characterized for size distribution and total particle volume concentration using a Coulter counter; The median droplet size is shown in Table 1 below. Particle volume concentration measurements were used to adjust the concentration of each emulsion to about 1% v / v dispersed phase using MilliQ water. Emulsions were stored in the refrigerator until use. Manufacturing example numberCationic additivesMedium droplet size (μm) 19DC-cholesterol3 201,2-distearoyl ethylphosphocholine2.4 21Benzylcetyldimethylammonium chloride2.4 22Cetyltrimethylammonium bromide2.6 23Cetylpyridinium Chloride2.5 24Palmitoyl-Dpr (palmitoyl) -Arg-Arg-Lys-NH 2 (Manufacturing Example 13)3.6 25Myristoyl Choline Chloride2.9 26Hexadecanoic acid 2-aminoethyl ester (Preparation 14)2.5 27Hexadecanoic acid 4-aminobutyl amide (Preparation 15)2.3 28Amino Acetic Acid Hexadecyl Ester (Preparation 16)2.4 29Cetyl carnitine ester2.4 30Psychosin2.5 31D-spinosine sulfate2.7 32Phytospingosin2.4 33DL-dihydrospinosine2.9 34Brominated Didodecyldimethylammonium2.4 35Methylaminoacetic acid hexadecyl ester (Preparation 17)3.1 36Dimethylaminoacetic acid hexadecyl ester (Preparation 18)3.5 <Production example 37> Positively charged lipopeptides: perfluorodimethyl containing palmitoyl-Lys (palmitoyl) -Lys-Lys-Ahx-Lys-Arg-Lys-Arg-Lys-Arg-NH 2 , where Ahx = aminohexanoic acid Cyclobutane emulsion Positively charged lipopeptide palmitoyl-Lys (palmitoyl) -Lys-Lys-Ahx-Lys-Arg-Lys-Arg-Lys-Arg-NH 2 (from distearoylphosphatidylcholine (90 mg) and Preparation Example 12 10 mg) was placed in a 50 mL round bottom flask and chloroform (10 mL) was added. The flask was heated with hot tap water until a clear solution was obtained, after which the flask was placed on a rotavapor and evaporated to remove chloroform. To remove traces of residual solvent, the samples were exposed to vacuum overnight. MilliQ water (20 mL) was then added and the flask was placed on a rotavapor again and spun at full speed while immersed in an 80 ° C. water bath. After about 10 minutes, the samples were transferred to appropriate vials and placed in an overnight refrigerator to cool. A portion of the sample was transferred to a 2 ml chromatography vial and 100 [mu] l of perfluorodimethylcyclobutane was added to each vial. The vial was shaken for 75 seconds using ESP Capmix® and the sample was immediately ice cooled. The contents of each vial were collected in larger vials and the emulsion was characterized for size distribution and total particle volume concentration using a Coulter counter. Particle volume concentration measurements were used to adjust the concentration of each emulsion to about 1% v / v dispersed phase using MilliQ water. Emulsions were stored in the refrigerator until use. <Example 1> In vivo imaging of dog heart 20 kg Monel dogs were anesthetized, a median sternotomy was performed, and the preretina was removed. Midline shortening B-mode imaging of the heart was performed through a low-attenuation 30 mm silicone rubber spacer using an ATL HDI-3000 scanner equipped with a P3-2 transducer. The frame rate was 40 Hz and the mechanical index was 1.1. a) [comparative example] Imaging with negatively charged perfluorobutane gas dispersion and negatively charged perfluorodimethylcyclobutane emulsion Of the perfluorobutane gas dispersion obtained from Preparation Example 1 corresponding to 0.2 μl gas / kg of body weight and the perfluorodimethylcyclobutane emulsion from Preparation Example 6 corresponding to 0.4 μl perfluorodimethylcyclobutane / kg of body weight. An amount was injected intravenously into dogs. It can be seen that the substantial rise in echo intensity from the myocardium started 20 seconds after injection and lasted for 10 minutes. The disappearance of the contrast agent effect from blood retention occurred earlier than the disappearance of the myocardial contrast effect. b) Imaging with negatively charged perfluorobutane gas dispersion and positively charged perfluorodimethylcyclobutane emulsion (high dosage) Of the perfluorobutane gas dispersion obtained from Preparation Example 1 corresponding to 0.2 μl gas / kg of body weight and the perfluorodimethylcyclobutane emulsion from Preparation Example 4 corresponding to 0.1 μl perfluorodimethylcyclobutane / kg of body weight. An amount was injected intravenously into dogs. The resulting myocardial contrast effect lasted for 20 minutes much more strongly than that observed in (a) above. c) Imaging with negatively charged perfluorobutane gas dispersion and positively charged perfluorodimethylcyclobutane emulsion (low dose) The procedure described in Example 1 (b) was repeated except that the dose of perfluorodimethylcyclobutane emulsion was reduced to an amount equivalent to 0.02 μl perfluorodimethylcyclobutane / kg body weight. The myocardial contrast effect of the result was comparable to that observed in Example 1 (a). <Example 2> In Vivo Imaging of Dog Heart (Contrast Low Dose) a) Imaging with negatively charged perfluorobutane gas dispersion and positively charged perfluorohexane emulsion The procedure described in Example 1 (b) was repeated except that the perfluoromethylcyclobutane emulsion was replaced with a fixed amount of perfluoronucleic acid emulsion from Preparation Example 5 corresponding to 0.02 μl perfluorohexane / kg body weight. It was. The myocardial contrast effect of the result was comparable to that observed in Example 1 (a). b) [comparative example] Imaging with negatively charged perfluorobutane gas dispersion and negatively charged perfluorodimethylcyclobutane emulsion The procedure described in Example 1 (a) was repeated except that the dose of perfluorodimethylcyclobutane emulsion was reduced to an amount equivalent to 0.02 μl perfluorodimethylcyclobutane / kg body weight. Only a faint myocardial contrast effect could be observed. c) [comparative example] Imaging using a positively charged perfluorobutane gas dispersion and a positively charged perfluorohexane emulsion To the dog, a certain amount of perfluorobutane gas dispersion obtained from Preparation Example 2 corresponding to 0.2 μl gas / kg body weight and a perfluorohexane emulsion obtained from Preparation Example 5 corresponding to 0.02 μl perfluorohexane / kg body weight Simultaneous intravenous injection was imaged as in Example 1. As a result, only a faint myocardial effect could be observed. d) Imaging with a positively charged perfluorobutane gas dispersion and a negatively charged perfluorodimethylcyclobutane emulsion A certain amount of the perfluorobutane gas dispersion obtained from Preparation Example 2 corresponding to 0.2 µl gas / kg of body weight and a certain amount of the perfluorodimethylcyclobutane emulsion obtained from Preparation Example 6 corresponding to 0.02 µl perfluorohexane / kg of body weight Dogs were simultaneously intravenously injected and imaged as in Example 1. The myocardial contrast effect of the result was comparable to that observed in Example 1 (a). <Example 3 (Comparative Example)> Imaging of dog heart using only positively charged perfluorohexane emulsion A certain amount of perfluoronucleic acid emulsion from Preparation Example 5 corresponding to 0.02 μl perfluorohexane / kg body weight was intravenously injected into dogs and imaged as in Example 1. As a result, no blood retention or myocardial contrast effect was observed. <Example 4 (comparative example)> Imaging of Dog Heart Without Preliminary Ultrasound The procedure of Example 1 (c) was repeated except that the ultrasound scanner was switched off for the first two minutes after injection. The contrast effect in the myocardium after switching the scanner back on was very short compared to what was observed with the same imaging method at the same time after injecting only the perfluorobutane gas dispersion. <Example 5> Imaging of dog heart with biotinylated perfluorobutane gas dispersion and avidinylated perfluorodimethylcyclobutane emulsion Of the perfluorobutane gas dispersion obtained from Preparation Example 3 corresponding to 0.02 μl gas / kg of body weight and the perfluorodimethylcyclobutane emulsion from Preparation Example 7 corresponding to 0.02 μl perfluorodimethylcyclobutane / kg of body weight. An amount was injected intravenously into dogs. Cardiac imaging was performed with a Vingmed CFM-750 scanner using midline axial projection. The scanner was adjusted to maximize ultrasound exposure to the imaged tissue area using a combination of continuous high frame rate imaging and peak power (7 on a scale of 0-7). After injection, early contrast enhancement was observed in both ventricles of the heart. Constant elevations in contrast improvement were observed in all regions of the myocardium, up to an increased intensity approaching the maximum white level on the screen. The contrast period of the tissue is about 30 minutes, while the contrast effect in blood retention decreases to near baseline within 5 minutes of injection, the image appears with little blood retention attenuation, and a complete and extremely sharp improvement in peripheral contrast of the myocardium. woke up. The contrast effect in the myocardium near the transducer did not seem to fade despite consecutive high-intensity ultrasound exposures. <Example 6-9> Imaging of dog heart with negatively charged perfluorobutane gas dispersion and positively charged emulsion from Preparation Example 8-11 19 kg Monel dogs were anesthetized, a median sternotomy was performed, and the preretina was removed. Midline shortening B-mode imaging of the heart was performed through a low-attenuation 30 mm silicone rubber spacer using an ATL HDI-3000 scanner equipped with a P3-2 transducer. The frame rate was 40 Hz and the mechanical index was 1.1. A constant amount of perfluorobutane gas dispersion obtained from Preparation Example 1 corresponding to 0.2 μl gas / kg body weight and an amount of one of the emulsions obtained from Preparation Example 8-11 corresponding to 0.02 μl volatile oil / kg body weight were intravenously administered to the dog. My injection It can be seen that the substantial rise in echo intensity from the myocardium started 20 seconds after injection in each case, and the peak intensity was more than that observed in Example 1 (a). Ultrasound intensity in the myocardium 90 seconds after injection was corrected for baseline and the results of myocardial contrast enhancement (MCEs) are shown in Table 2 below. Example numberDiffuse componentsBaseline calibrated MCE (dB) 6Perfluorodimethylcyclobutane7.9 7Perfluoromethylcyclopentane4.8 8Perfluoro-2-methylpentane6.6 9Perfluorohexane7.5 A substantial increase in myocardial opacity was observed when there was little contrast agent in the ventricles, indicating that the observed contrast enhancement was due to suppressed microbubbles in the myocardium. The duration of contrast effect varied from about 5 minutes to about 20 minutes, depending on factors such as water solubility and vapor pressure of the volatile oil. Table 3 below shows the MCE half time for each experiment. Example numberDiffuse componentsMCE Half Time (min) 6Perfluorodimethylcyclobutane2.9 *7Perfluoromethylcyclopentane1.9 8Perfluoro-2-methylpentane6.9 9Perfluorohexane7.4 *-Average of two measurements, 2.4 and 3.4 minutes respectively <Example 10-27> Imaging of dog heart with negatively charged perfluorobutane gas microbubbles and positively charged emulsion from Preparation Examples 19-36 24 kg Monel dogs were anesthetized, a median sternotomy was performed, and the preretina was removed. Midline shortening B-mode imaging of the heart was performed through a low-attenuation 30 mm silicone rubber spacer using an ATL HDI-3000 scanner equipped with a P3-2 transducer. The frame rate was 40 Hz and the mechanical index was 1.1. When testing the contrast agent containing the emulsion according to Preparation Examples 19-28, a certain amount of the perfluorobutane gas dispersion obtained from Preparation Example 1 corresponding to 0.2 µl gas / kg of body weight and 0.02 µl perfluorodimethylcyclobutane / kg of body weight A certain amount of perfluorodimethylcyclobutane emulsion corresponding to the dog was injected intravenously at the same time. For contrast agents comprising an emulsion obtained from Preparation Examples 29-36, the corresponding doses of gas and perfluorodimethylcyclobutane were 0.35 and 0.04 μl / ml, respectively. It can be seen that the substantial rise in echo intensity from the myocardium started 20 seconds after injection and lasted for 10 minutes, in which case the peak intensity was above that observed in Example 1 (a). About 2 minutes after injection, ultrasound opacity in the myocardium was corrected for baseline and the results of myocardial contrast enhancement (MCEs) are shown in Table 4 below. A substantial increase in myocardial opacity was observed when there was little contrast agent in the ventricles, indicating that the observed contrast enhancement was due to suppressed microbubbles in the myocardium. Example numberCationic additivesBaseline-Calibrated MCE (dB) 10DC-cholesterol11.78 111,2-distearoyl ethylphosphocholine17.03 12Benzylcetyldimethylammonium chloride10.43 13Cetyltrimethylammonium bromide11.46 14Cetylpyridinium Chloride11.16 15Palmitoyl-Dpr (Palmitoyl) -Arg-Arg-Lys-NH 2 10.64 16Myristoyl Choline Chloride10.29 17Hexadecanoic acid 2-aminoethyl ester14.41 18Hexadecanoic acid 4-aminobutyl amide12.74 19Aminoacetic acid hexadecyl ester16.14 20Cetyl carnitine ester12.11 21Psychosin13.56 22D-spinosine sulfate13.44 23Phytospingosin13.56 24DL-dihydrospinosine17.05 25Brominated Didodecyldimethylammonium9.54 26Methylaminoacetic acid hexadecyl ester15.50 27Dimethylaminoacetic acid hexadecyl ester15.33 <Example 28> Imaging of dog heart with negatively charged perfluorobutane gas dispersion and positively charged emulsion from Preparation Example 37 20 kg Monel dogs were anesthetized, a median sternotomy was performed, and the preretina was removed. Midline shortening B-mode imaging of the heart was performed through a low-attenuation 30 mm silicone rubber spacer using an ATL HDI-3000 scanner equipped with a P3-2 transducer. The frame rate was 40 Hz and the mechanical index was 1.1. Of the perfluorobutane gas dispersion obtained from Preparation Example 1 corresponding to 0.1 μl of gas / kg of body weight and the perfluorodimethylcyclobutane emulsion obtained from Preparation Example 37 corresponding to 0.04 μl of perfluorodimethylcyclobutane / kg of body weight. An amount was injected intravenously into dogs. Elevation and duration of echo intensity from the myocardium were measured.
权利要求:
Claims (27) [1" claim-type="Currently amended] i) a first composition which is an aqueous aqueous medium for injection comprising a dispersion gas and a substance for stabilizing the gas; And ii) an oil-in-water emulsion for injection comprising a dispersible component that can diffuse in vivo into the dispersion gas such that the oil phase at least temporarily increases its size, further comprising a substance for stabilizing said emulsion And a substance present on the surface of the dispersed gas phase and a substance present on the surface of the dispersed oil phase have affinity for each other. [2" claim-type="Currently amended] The process of claim 1 wherein the dispersion gas is air, nitrogen, oxygen, carbon dioxide, hydrogen, inert gas, sulfur fluoride, selenium hexafluoride, optionally halogenated silane, optionally halogenated low molecular weight hydrocarbon, ketone, ester or any of the above components A composite formulation comprising a mixture of. [3" claim-type="Currently amended] The combination formulation of claim 2, wherein the gas comprises sulfur hexafluoride or perfluorocarbons. [4" claim-type="Currently amended] 4. The combination formulation of claim 3, wherein the perfluorocarbon is perfluoropropane, perfluorobutane or perfluoropentane. [5" claim-type="Currently amended] 5. The composite formulation of claim 1, wherein the dispersion gas is stabilized by agglomerated resistant surface membranes, pilmogen proteins, polymeric materials, nonpolymerized and nonpolymerizable wall forming materials or surfactants. [6" claim-type="Currently amended] 6. The combination formulation of claim 5, wherein said surfactant comprises at least one phospholipid. [7" claim-type="Currently amended] 7. The combination formulation of claim 6, wherein at least 75% of the surfactants comprise phospholipid molecules with net total charge individually. [8" claim-type="Currently amended] 8. The combination formulation of claim 7, wherein at least 75% of the surfactant comprises at least one phospholipid selected from phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid and cardiolipin. [9" claim-type="Currently amended] The combination formulation of claim 8, wherein at least 80% of the phospholipids comprise phosphatidylserine. [10" claim-type="Currently amended] The method according to any one of claims 1 to 9, wherein the diffusive component is 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 or a mixture of any of the above components. It contains a composite formulation. [11" claim-type="Currently amended] The combination formulation of claim 10, wherein the diffusive component comprises at least one perfluorocarbon. [12" claim-type="Currently amended] The method of claim 11, wherein the perfluorocarbon (s) comprises at least one perfluoroalkane, perfluoroalkene, perfluorocycloalkane, perfluorocycloalkene and / or perfluorinated alcohol Combination formulation. [13" claim-type="Currently amended] 13. The combination formulation of claim 12, wherein the diffusive component comprises at least one perfluoropentane, perfluorohexane, perfluorodimethylcyclobutane and / or perfluoromethylcyclopentane. [14" claim-type="Currently amended] The co-formulation according to any one of claims 1 to 13, wherein the dispersible component emulsion is stabilized by phospholipids or lipopeptide surfactants. [15" claim-type="Currently amended] The combination formulation according to claim 1, wherein the first and second compositions each contain a surface material with opposite charge. [16" claim-type="Currently amended] The combination formulation of claim 15, wherein the first composition contains anionic surface material and the second composition contains cationic surface material. [17" claim-type="Currently amended] The method of claim 16 wherein the anionic material is a negatively charged phospholipid and the cationic material is a lipophilic quaternary ammonium salt, a lipophilic pyridinium salt, a lipophilic first, second or tertiary amine, optionally substituted di- Or fatty acid amide of tri-amine, fatty alcohol ester of amino acid or positively charged phospholipid or lipopeptide. [18" claim-type="Currently amended] 18. The combination formulation of claim 17, wherein the cationic material is present as an additive to the stabilizing material of the second composition. [19" claim-type="Currently amended] The combination formulation according to any one of claims 1 to 18, further comprising a vasodilator and / or vasoconstrictor. [20" claim-type="Currently amended] The combination preparation according to claim 19, wherein said vasodilator is adenosine. [21" claim-type="Currently amended] The combination preparation according to any one of claims 1 to 18, further comprising a therapeutic agent. [22" claim-type="Currently amended] 19. The combination formulation of any one of claims 1 to 18, further comprising a contrast enhancement component for imaging other than ultrasound. [23" claim-type="Currently amended] i) injecting the first composition as defined in claim 1 into the pulse system of a human or non-human animal subject; ii) injecting into said subject a second composition as defined in claim 1 before, during or after injecting said first composition; And iii) forming an ultrasound image of at least a portion of the object A method of forming an improved image of a human or non-human animal subject comprising a. [24" claim-type="Currently amended] The method of claim 23, wherein the microbubble growth from the contrast agent is activated in the subject by external activation. [25" claim-type="Currently amended] The method of claim 24, wherein the external activation comprises ultrasonic irradiation. [26" claim-type="Currently amended] 26. The method of any one of claims 23 to 25, wherein the vasodilator or vasoconstrictor is coadministered to the subject. [27" claim-type="Currently amended] The method of claim 26, wherein the vasodilator is adenosine.
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同族专利:
公开号 | 公开日 HU0102878A2|2002-03-28| NO20005250D0|2000-10-19| AU3617299A|1999-11-08| CA2329175A1|1999-10-28| AT375806T|2007-11-15| CN1306442A|2001-08-01| JP2002512206A|2002-04-23| IL139147D0|2001-11-25| BR9909822A|2000-12-19| US20040146462A1|2004-07-29| EP1073473B1|2007-10-17| NO20005250L|2000-12-18| WO1999053963A1|1999-10-28| PL343464A1|2001-08-13| GB9808599D0|1998-06-24| ZA200005789B|2001-07-30| DE69937341D1|2007-11-29| HU0102878A3|2002-12-28| EP1073473A1|2001-02-07|
引用文献:
公开号 | 申请日 | 公开日 | 申请人 | 专利标题
法律状态:
1998-04-22|Priority to GBGB9808599.6A 1998-04-22|Priority to GB9808599.6 1999-04-22|Application filed by 조오지 디빈센조, 토브 아스 헬지, 에바 요한손, 니코메드 이메이징 에이에스 1999-04-22|Priority to PCT/GB1999/001221 2001-05-25|Publication of KR20010042915A
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申请号 | 申请日 | 专利标题 GBGB9808599.6A|GB9808599D0|1998-04-22|1998-04-22|Improvements in or realting to contrast agents| GB9808599.6|1998-04-22| PCT/GB1999/001221|WO1999053963A1|1998-04-22|1999-04-22|Improvements in or relating to contrast agents| 相关专利
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