MXPA99003867A - Improvements in or relating to diagnostic/therapeutic agents - Google Patents

Abstract

Targetable diagnostic and/or therapeutically active agents, e.g. ultrasound contrast agents, comprising a suspension in an aqueous carrier liquid of a reporter comprising gas-containing or gas-generating material, said agent being capable of forming at least two types of binding pairs with a target.

Description

IMPROVEMENTS RELATED TO DIAGNOSTIC / THERAPEUTIC AGENTS DESCRIPTION OF THE INVENTION This invention relates to diagnostic and / or therapeutically active agents, more particularly to diagnostic and / or therapeutically active agents that incorporate portions that have affinity to sites and / or structures within the body so that imaging can be improved diagnostic and / or therapy of particular positions within the body. Of particular interest are diagnostic agents for use in ultrasound imaging, which will be referred to below as directed ultrasound contrast agents. It is well known that ultrasonic imaging comprises a potentially valuable diagnostic tool, for example in studies of the vascular system, particularly in cardiography and tissue microvasculature. Various contrast agents have been proposed to improve the acoustic images obtained in this manner, including suspensions of solid particles, emulsified liquid droplets, gas bubbles and encapsulated gases or liquids. It is generally accepted that low density contrast agents which are easily compressible are particularly efficient in terms of the acoustic backscattering they generate, and therefore considerable interest has been shown in the preparation of systems containing gas and that generate gas. Gas-containing contrast media are also known to be effective in magnetic resonance imaging (MR) imaging, for example, as susceptibility contrast agents which will act to reduce the intensity of the MR signal. Oxygen-containing contrast media also represent potentially useful magnetic MR contrast agents. Furthermore, in the field of X-ray imaging it has been observed that gases such as carbon dioxide can be used as negative oral contrast agents or intravascular contrast agents. The use of radioactive gases, for example radioactive isotopes of inert gases such as xenon, have also been proposed in scintigraphy, for example for blood accumulation imaging. The directed ultrasound contrast agents can be considered to comprise: (i) an indicator portion capable of interacting with the ultrasound irradiation to generate a detectable signal; (ii) one or more vectors that have affinity for particular target sites and / or structures within the body, for example, by specific cells or areas of pathology; and (iii) one or more linkers that connect to the indicator and the vectors, in case these are not directly linked. The molecules and / or the structure to which the contrast agent is intended to bind will then be referred to as the target or target. In order to obtain the specific image formation of a selected region / structure in the body, the objective must be present and available in this region / structure. Ideally, it will be expressed only in the region of interest, but usually it will also be present in other positions in the body, which generates possible background problems. The target may be a defined molecular species (ie, a target molecule) or an unknown molecule or a more complex structure (ie, an objective structure) which is present in the area from which an image is to be formed , and is capable of binding specifically or selectively to the given vector molecule. The vector is attached to the indicator portion in order to link these portions to the region / structure from which an image is to be formed. The vector can be specifically bound to a chosen target, or can only be selectively bound, also having affinity for a limited number of other molecules / structures, again creating possible background problems.

There is a body limited in the prior art in relation to directed ultrasound contrast agents. A) Yes, for example, US-A-5531980 is directed to systems in which the indicator comprises an aqueous suspension of air or gas microbubbles stabilized by one or more surfactants or film-forming surfactants present at least partially in lamellar or laminar form, the surfactants are linked to one or more vectors comprising "bioactive species designed for specific targeting purposes". It is established that the bubbles are not encapsulated directly by the surfactant material but rather it is incorporated in the liquid-filled liposomes which stabilizes the microbubbles. It will be appreciated that the lamellar or laminar surfactant material such as phospholipids present in such liposomes will inevitably present in the form of one or more lipid bilayers with the "tail to tail" lipophilic tails and the hydrophilic heads both inside and outside "see, for example, Schneider, M. in "Liposomes as drug carriers: 10 years of research" in Drug targeting, Nyon, Switzerland, 3 -5 October 1984, Buri, P. and Gumma, A. (Ed), Elsevier, Amsterdam 1984) EP-A-0727225 discloses directed ultrasound contrast agents in which the indicator comprises a chemical substance having a sufficient vapor pressure so that a portion thereof is gas at the body temperature of the subject. Chemistry is associated with a surfactant or an albumin carrier which includes a ligand of cell adhesion molecule based on protein, peptide or carbohydrate as a vector. s of contrast correspond to the phase shift colloid systems described in document O-A-9416739; and now it is recognized that the administration of such phase-shifting colloids can lead to the generation of microbubbles which grow uncontrollably, possibly to the extent that they cause potentially harmful emboli from, for example, the vasculature of the myocardium and brain (see, for example, Schwarz, Advances in Echo-Contrast [1994 (3)], pp. 48-49). WO-A-9320802 proposes that an enhancement of the tissue-specific ultrasonic image can be obtained using acoustically-reflective oligolamellar liposomes conjugated to tissue-specific ligands such as antibodies, peptides, lectins, etc. Liposomes are deliberately chosen as gas-free and thus do not have the advantageous echogenic properties of gas-based ultrasound contrast agents. Additional references to this technology, for example, in the targeting of fibrin, thrombi and atherosclerotic areas are found in publications by Alkanonyuksel, H. et al. in J. Pharm. Sci. (1996) 85 (5), 486-490; J ". Am. Coil, Cardiol. (1996) 27 (2) Suppl A, 298A; and Circulation, 68 Sci. Sessions, Anaheim 13-16 November 1995. There are also numerous publications that relate contrasts for ultrasound which refer to the possible use of monoclonal antibodies as vectors without providing significant practical details and / or materials that include indicators which they can be captured by the reticuloendothelial system and thus limit the imaging enhancement of organs such as the liver - see, for example, WO-A-9300933, WO-A-9401140, WO-A-9408627, WO -A-9428874, US-A-5088499, US-A-5348016 and US-A-5469854. In general, these directed contrast agents of the prior art are intended to improve contrast at specific sites in the body, for example tumor cells, by utilizing a vector to bind strongly to a target in order to obtain concentration in the target cells. In contrast to this principle of using a vector to bind with high affinity to a target, the present invention is based in part on the finding that diagnostic and / or therapeutically active agents with more favorable properties can be obtained by the use of multiple classes of target vector interactions (eg involving agents associated with a plurality of different vectors and / or with one or more vectors having affinity for different targets in the same or different types of cells). Thus, the binding of diagnostic and / or therapeutic agents that contain gas or that generate gas, for example, can be obtained from multiple pairs of binding between a vector with specificity for more than one receptor or between more than one vector with affinity for one or more types of objective, either low or high affinities. Such multiple junctions of the agent conjugated to vector to one or more molecules / target structures can result in advantageous targeting properties, for example, by improving the specificity of the target and / or by distinguishing interactions in a desired target area compared to the interactions of background with lower levels of molecules / structures similar to the target expressed elsewhere in the body. The use of a vector that binds with high affinity to a target is well known. However, the present invention is based on the finding that the desired binding of gas-containing and gas-generating diagnostic and / or therapeutic agents can be obtained by forming multiple binding pairs with low affinity between a type of vector and a type of objective, or by forming multiple binding pairs between one or more types of vectors and one or more types of objectives, with affinities either low or high. Therefore, the multiple binding of the agent to vector to one or more molecules / target structures can be advantageous targeting properties, for example, to improve the specificity of the target and / or to distinguish interactions in the desired target area from the interactions of background with lower concentrations of molecules / structures similar to the target expressed elsewhere in the body. Therefore, according to one aspect of the present invention, there is provided a diagnostic and / or therapeutically active agent which can be targeted, for example, a contrast agent for ultrasound, comprising a suspension in a carrier liquid. aqueous, for example, an injectable carrier liquid of an indicator comprising a gas-containing or gas-generating material, characterized in that the agent is capable of forming at least two types of binding pairs, ie, it is conjugated to minus two vectors or a vector capable of binding to at least two binding sites. An advantageous embodiment of the invention is based on the additional finding that adhesion limited to targets is a highly useful property for diagnostic and / or therapeutically active agents, property which can be obtained using vectors that are provided with temporary retention instead of a fixed adhesion to an objective. Therefore, such agents, instead of being fixedly retained at specific sites, for example can effectively show a delayed flow form along the vascular endothelium by virtue of their transient interactions with endothelial cells. Therefore, such agents can become concentrated in the walls of blood vessels, in the case of ultrasound contrast agents and provide improved echogenicity thereof in relation to the volume of the bloodstream, which lack anatomical characteristics. Therefore, they can allow an improved formation of the capillary system image, including the microvasculature and thus can. facilitate the distinction between normal and inadequately irrigated tissue, for example in the heart, and may also be useful for visualizing structures such as Kupffer cells, thrombi and atherosclerotic lesions or for visualizing areas of neovascularized and inflamed tissue. The present invention is suitable for imaging changes that occur in normal blood vessels which are located in areas of tissue necrosis. It will be appreciated that the binding affinities depend on numerous interactions as well as their strength. The density of the vector molecules on the surface of the indicator units can therefore be selected so that the degree of interactions between particular agents and objectives is appropriately adjusted. The term "multiple specificity" is also used to describe an injectable carrier liquid, or a gas containing or gas generating material consisting of one or more vectors with a specificity for one or more cell surface receptors while at the same time comprising a second element with specificity for a substrate or binding to the receptor system to which it induces a therapeutic response. Therefore, multiple specific imaging agents comprising a vector which is targeted, such as the antibodies against fibrin described by Lanza et al., Are included within the scope of the present invention. (Circulation, (1996) 94 (12), pp 3334), the peptides that bind to the atherosclerotic annexin V plaque such as YRALVDTLK, or any other known vector that is associated with fibrin clots, in combination with a drug or enzyme with fibrinolytic activity such as streptokinase, plasminogen activator (tPA), urokinase (uPA) or prourokinase (scuPA) which results in a localized therapeutic antithrombotic effect. This invention also extends to include vectors with increased specificity for tumor cells in combination with drug vectors or molecules that function as chemotherapeutic agents capable of inhibiting tumor growth. It is well known that many, if not all, target molecules are not expressed exclusively as target sites; a common situation is that such molecules are overexpressed by target cells or in an objective structure but that they are also expressed at lower levels elsewhere in the body. The use of indicators that have a multiplicity of vectors with relatively low affinity for the objective can be advantageous in this situation, since the indicator tends to concentrate in regions of high density by the objective, which allows a multiple union (and therefore both strong) to the indicator (for example, a gas-containing agent incorporates the folic acid and glutathione vectors for multiple specific binding to folic acid receptors and glutathione-S-transferase receptors respectively which are overexpressed as tumor cells). On the other hand, areas of low target density will not provide sufficient interaction with such low affinity vectors to bind the target. In such embodiments of the invention, low affinity vectors can be considered as having an association constant Ka for interaction with a target molecule or structure of less than 108 M "1, eg, less than 107 M" 1, preferably less than 106 M "1. A further embodiment of this invention is therefore based on the finding that the desired binding of gas-containing and gas generating diagnostic and / or therapeutic agents can be obtained by forming binding pairs With low affinity between more than one type of vector and one or more target types, therefore, multiple vectors can be used to increase specificity, so that the indicator will only bind to target cells or structures that express a combination particular of target molecules It may also be useful to select a plurality of vectors which bind to different parts, eg, epitopes or antigenic determinants, of an objective structure in order to provide an increased joining force. This can be particularly advantageous when the density of the objective is low. Advantageously, products comprising two or more vectors with different specificities can be used, that is, which bind to different target molecules on different cells, as "general purpose" agents for detection of a range of diseases, for example different forms of Cancer. Thus, for example, the use of such agents may allow the detection of metastases, which are often heterogeneous with respect to the expression of the target molecules (ie, antigens). Within the context of the present invention, the indicator unit will usually remain attached to the vectors. In another type of targeting procedure, sometimes referred to as pre-targeting, the vector (often a monoclonal antibody) is administered alone; Subsequently, the indicator is administered coupled to a portion which is capable of specifically binding to the vector molecule (when the vector is an antibody, the indicator can be coupled to an immunoglobulin-binding molecule, such as protein A or a antibody against immunoglobulin). An advantage of this protocol is that it can allow time for elimination of vector molecules which do not bind to their targets, which substantially reduces the background problems that are related to the presence of an excess of indicator conjugate. -vector. Within the context of the present invention, predirection can be designed with a specific vector, followed by indicator units that can be coupled to another vector and a portion which binds to the first vector. Within the context of the present invention, in some cases and in particular for the determination of blood perfusion rates in defined areas, for example in myocardium, it is of interest to measure the rate at which the ultrasound contrast agents attached to the target are displaced or released from the target. This can be obtained in a controlled manner by subsequent administration of a vector or other agent capable of displacing or releasing the contrast agent from the target. Useful vectors according to the invention include ligands for cell adhesion proteins, as well as cell adhesion proteins themselves when they have corresponding ligands on surfaces of endothelial cells. Examples of cell adhesion proteins include integrins, most of which bind to the amino acid sequence Arg-Gly-Asp (RGD). If desired, the vector can be targeted to specific cell adhesion proteins that are expressed primarily on activated endothelial cells such as those found or that are close to sites of inflammation or other pathological responses. Other vectors which can be used include proteins and peptides that bind to cell surface proteoglycans, which are protein complexes and sulfated polysaccharides found in most cells, including endothelial cells. Such proteoglycans contribute to the negative surface charge of all nucleated cells of vertebrate animals; this charge can also be exploited according to the invention by using positively charged vectors, for example, comprising cationic lipids which will interact electrostatically with the endothelial surface. A further aspect of the present invention is, for example, when a vector or vectors bind to the indicator or are non-covalently included in the indicator in a manner in which the vector or vectors are not readily exposed to the targets or receptors. . Therefore, increased tissue specificity can be obtained by applying an additional process to expose the vectors, for example, the agent is exposed after administration to external ultrasound to change the diffusing capacity of the portions containing the vectors. The indicator may be in any convenient form, for example it may be any suitable formulation of ultrasound contrast agent that contains gas or generates gas. Representative examples of such formulations include gas microbubbles stabilized (eg, at least partially encapsulated) by a surface membrane resistant to coalescence (eg gelatin, for example as described in WO-A-8002365), a filmogenic protein (for example an albumin such as human serum albumin, for example, as described in US-A-4718433, US-A-4774958, US-A-4844882, EP-A-0359246, WO-A- 9112823, WO-A-9205806, WO-A-9217213, WO-A-9406477 or WO-A-9501187), polymeric material (for example, a synthetic biodegradable polymer as described in EP-A-0398935, a elastic interfacial synthetic polymer membrane as described in EP-A-0458745, a microparticulate biodegradable polyaldehyde as described in EP-A-0441468, a microparticulate N-dicarboxylic acid derivative of a polycyclic polyamino acid-imide as describes in EP-A-0458079, or a biodegradable polymer as described in WO-A-9317718 or WO-A-9607434), a wall-forming, non-polymeric and non-polymerizable material (for example, as described in US Pat. WO-A-9521631), or a surfactant or surfactant (e.g., a polyoxyethylene-polyoxypropylene block copolymer surfactant such as Pluronic, a polymeric surfactant such as described in WO-A-9506518, or a film-forming surfactant such as a phospholipid, for example, as described in WO-A-9211873, WO-A-9217212, WO-A-9222247, WO-A-9428780 or WO-A-9503835). Other useful formulations of gas-containing contrast agents include solid systems containing gas, for example microparticles (especially microparticle aggregates) having a gas contained therein or otherwise associated with them (eg, being absorbed onto the gas). the surface thereof and / or contained within voids, cavities or pores therein, for example, as described in EP-A-0122624, EP-A-0123235, EP-A-0365467, WO-A -9221382, WO-A-9300930, WO-A-9313802, WO-A-9313808 or WO-A-9313809). It will be appreciated that the echogenicity of such microparticulate contrast agents can be derived directly from the contained / associated gas and / or gas (e.g. microbubbles) released from the solid material (e.g., upon dissolution of the microparticulate structure).

The descriptions of all the documents mentioned above in relation to formulations of gas-containing contrast agents are incorporated herein by reference. Gas microbubbles and other gas containing materials such as microparticles preferably have an initial average size that does not exceed 10 μm (for example 7 μm or less) in order to allow their free passage through the pulmonary system after administration -, for example, by intravenous injection. When using compositions containing phospholipids according to the invention, for example in the form of gas microbubbles stabilized with phospholipids, representative examples of useful phospholipids include lecithins (ie, phosphidylcholines), for example natural lecithins such as yolk lecithin. egg or lecithin from soybeans and synthetic or semi-synthetic lecithins such as dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or diastereoylphosphatidylcholine; phosphatidic acids; phosphate-di-ethanolamines; phosphatidylserines; phosphatidylglycerols; phosphatidylinositol; cardiolipins; sphingomyelins; fluorinated analogs of any of the foregoing; mixture of any of the above and mixtures with other lipids such as cholesterol. The use of phospholipids comprising predominantly (for example 75%) of molecules individually presenting a net total charge, for example a negative charge, for example as in the phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, phosphatidic acids and / or cardiolipins that occur naturally (eg soybean or egg yolk derivatives), semi-synthetic (eg partially or completely hydrogenated) and synthetic phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, phosphatidic acids and / or cardiolipins, can be particularly advantageous. Other exemplary lipids which can be used to prepare contrast agents containing gas include fatty acids, stearic acid, palmitic acid, 2-n-hexadecyl tartaric acid, oleic acid, or other acid-containing lipid structures. These lipid structures are considered particularly interesting when they are coupled by the formation of the amide bond to amino acids containing one or more amino groups. The amino acids modified with resulting lipids (for example dipalmitoilysin, diastereoyl-2,3-diaminopropionic acid) are considered useful precursors for the union of functionalized separating elements that have coupling sites for conjugation of one or more vector molecules. A further extension of this invention relates to the synthesis of lipopeptide structures comprising a lipid indicator attached to a linker portion (eg PEG, polyamino acid, alkyl halide, etc.), the linker is suitably functionalized for coupling to a more vector molecules. A particular preference is the inclusion of a positively charged linker element (for example two or more lysine residues) for anchoring the indicator element in the microbubble through the electrostatic interaction with the negatively charged membrane. Multiple specific targeting is attainable by mixing and "adding" phospholipid structures containing gas with one or more directed lipopeptide sequences. Multiple specificity can also be obtained by assembling more than one vector over a branched lysine core structure such as those described by Tam et. to the.

(Proc. Nati, Acad. Sci. USA, 1989, 86, 9084) or by incorporating multiple vectors in a linear sequence. Multiple specificity can also be obtained by using lipopeptides or phospholipids comprising combinational libraries synthesized by chemical synthesis as described by Lowe (Combinatorial Chemistry, Chemical Society Reviews, 1995, 309-317). Also within the scope of this invention are functionalized microbubbles that have one or more reactive groups for non-specific reaction with receptor molecules located on the cell surface. For example, microbubbles comprising a diol can bind to cell surface receptors via disulfide exchange reactions. The reversible nature of this covalent bond means that the flow of bubbles can be controlled by altering the redox environment. Similarly, "activated" microbubbles of membranes comprising active esters such as N-hydroxysuccinimide esters can be used to modify the amino groups that are found in a multiplicity of cell surface molecules. Representative examples of microparticulate gas-containing materials which may be useful according to the invention include carbohydrates (e.g. exoses such as glucose, fructose or galactose); disaccharides such as sucrose, lactose or maltose; pentoses such as arabinose, xylose or ribose; I heard-, ß- and y-cyclodextrins; polysaccharides such as starch, hydroxyethylstarch, amylose, amylopectin, glycogen, inulin, pullulan, dextran, carboxymethyldextran, dextran phosphate, ketodextran, aminoethyldextran, alginates, chitin, chitosan, hyaluronic acid or heparin; and sugar alcohols including alditols such as mannitol or sorbitol), inorganic salts (for example sodium chloride), organic salts (for example sodium citrate, sodium acetate or sodium tartrate), contrast agents for X-rays (for example any of the commercially available carboxylic acids and non-ionic amide contrast agents containing at least one group 2, 4 , 6-triiodophenyl having substituents such as carboxyl, carbamoyl, N-alkylcarbamoyl, N-hydroxyalkylcarbamoyl, acylamino, N-alkylacylamino or acylaminomethyl in the 3- and / or 5- positions, as in metrizoic acid, diatrizoic acid, yotalámico acid, yoxagolic acid, yohexol, yopentol, yopamidol, iodixanol, yopromide, metrizamide, iodipamide, meglumine iodipamide, meglumine acetrizoate and meglumine diatrizoate), and polypeptides and proteins (e.g. gelatin-or albumin such as human serum albumin). Any biocompatible gas may be present in the indicator or in the contrast agents according to the invention, as used herein, the term "gas" includes any substance (including mixtures) substantially or completely in gaseous form (including vapor) ) at the normal human body temperature of 37 ° C. Therefore, the gas can comprise air; nitrogen; oxygen; carbon dioxide; hydrogen; an inert gas such as helium, argon, xenon, or krypton; a sulfur fluoride such as sulfur hexafluoride, thiazulf decafluoride or trifluoromethylsulfur pentafluoride; Selenium hexafluoride; an optionally halogenated silane such as methylsilane or dimethylsilane, a low molecular weight hydrocarbon (for example containing up to 7 carbon atoms), for example an alkane such as methane, ethane, a propane, a butane or a pentane, a cycloalkane such as cyclopropane, cyclobutane or cyclopentane, an alkene such as ethylene, propene, -propadiene or a butene, or an alkylene such as acetylene or propyne; an ether such as dimethyl ether; a ketone; an ester; a halogenated hydrocarbon of low molecular weight (for example containing up to 7 carbon atoms); or a mixture of any of the foregoing. Advantageously, at least part of the halogen atoms in the halogenated gases are fluorine atoms, therefore the biocompatible halogenated hydrocarbon gases can be selected, for example, from bromochlorodifluoromethane, c 1 or od if 1 or more, c 1 or odi f luo r ome t an, bromotrifluoromethane, chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene, ethyl fluoride, 1,1-difluoroethane and perfluorocarbons, for example perfluoroalkanes such as perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes (e.g. perfluoro-n-butane, optionally in admixture with other isomers such as perfluoro-iso-butane), perfluoropentanes, perfluorohexanes and perfluoroheptans perfluoroalkenes such as perfluoropropene, perfluorobutenes (for example perfluorobut-2-ene) and perfluorobutadiene perfluoroalkynes such as perfluorobut-2- Non-perfluorocycloalkanes such as perfluorocyclobutane perf luoromethylcyclobutane, perfluorodimethylcyclobutanes perfluorotrimethyl-cyclobutanes, perfluorocyclopentane perfluoromethylcyclopentane, perfluorodimethylcyclopentanes perfluorocyclohexane, perfluoromethylcyclohexane and perfluorocycloheptane. Other halogenated gases include methyl chloride, fluorinated ketones (for example perfluorinated) such as perfluoroacetone and fluorinated ethers (for example perfluorinated) such as perfluorodiethylether. The use of perfluorinated gases, for example sulfur hexafluoride or perfluorocarbons such as perfluoropropane, perfluorobutanes and perfluoropentanes, can be particularly advantageous in view of the high stability recognized in the bloodstream of microbubbles containing such gases. The indicator can be manufactured by any convenient process, for example, when preparing formulations that contain gas or that generate gas. Representative examples include the preparation of a suspension of gas microbubbles by contacting a surfactant with a gas and mixing them in the presence of an aqueous carrier, as described in WO 9115244; or by atomizing a solution or dispersion of a wall-forming material in the presence of a gas in order to obtain hollow microcapsules, as described in EP 512693A1; the preparation of solid microspheres by a double emulsion process, as described in US 5648095; or a process for forming hollow microcapsules by spray drying as described in EP 681843A2; or by preparing gas-filled liposomes by stirring an aqueous solution comprising a lipid i in the presence of a gas as described in the document US 5469854. A process suitable for binding the desired vector to the indicator comprises a modification of the surface of the preformed indicator with a suitable linker using reactive groups on the surface of both the indicator and the vector. It may be particularly advantageous to mix the indicator material with the substance containing the vector at any stage of the process. Such a process will result in the incorporation or union of the vector to the indicator. An optional process step can remove the excess vector not bound to the indicator by washing the particles containing gas followed by separation, for example, by flotation. A preferred aspect is the use of lipopeptide structures incorporating functional groups such as thiol, maleimide, biotin, etc., which can be premixed if desired with other indicator molecules prior to the formation of the gas containing agents. The binding of the vector molecules can be carried out using linker reagents included below. The binding of an indicator unit to the desired vectors can be carried out by covalent or non-covalent means, which usually involve interaction with one or more functional groups located on the indicator and / or the vectors. Examples of chemically reactive functional groups which can be used for this purpose include amino, hydroxyl, sulfhydryl, carboxyl and carbonyl groups, as well as carbohydrate groups, vicinal diols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl, imidazolyl and groups phenolic The covalent coupling of the indicator and the vectors can therefore be carried out using linkers containing reactive portions capable of reaction with such functional groups. Examples of reactive portions capable of reaction with sulfhydryl groups include α-haloacetyl compounds of the type X-CH 2 CO- (where X = Br, Cl or I), which show particular reactivity by sulfhydryl groups but which may also be used for modify the imidazolyl, thioether, phenol and amino groups as described by Gurd, FRN in Methods Enzymol. (1967) 11, 532. The N-maleimide derivatives are also considered selective towards sulfhydryl groups, but may additionally be useful in the coupling of amino groups under certain conditions. The N-maleimides can be incorporated into linker systems for indicator-vector conjugation as described by Kitagawa, T. et al. in Chem. Pharm. Bull. (1981) 29, 1130 or are used as polymer crosslinkers for bubble stabilization as described by Kovacic, P. et al. in J. "Am. Chem. Soc. (1959) 81, 1887. Reagents such as 2-iminothiolane, for example, as described by Traut, R. et al., in Biochemistry (1973) 12, -3266, which introduces a thiol group through the conversion of an amino group, it can be considered as sulfhydryl reactants if the binding is produced through the formation of disulphide bridges.Therefore, reagents which introduce reactive disulfide bonds within either the indicator or the vectors may be useful, since the binding can be carried out around by disulfide exchange between the vector and the indicator, examples of such reagents include Ellman's reagent (DTNB), 4,4'-dithiodipyridine , methyl-3-nitro-2-pyridyl disulfide and methyl-2-pyridyl disulfide (described by Kimura, T. et al in Analyt. Biochem. (1982) 122, 271). Examples of reactive portions capable of reaction with amino groups include alkylating agents and acylating agents. representative include: i) a-haloacetyl compounds, which show specificity towards amino groups in the absence of reactive thiol groups and are of the type X-CH2CO- (where X = Cl, Br or I), for example as described by Wong , YH.H. in Biochemistry (1979) 24, 5337; ii) N-maleimide derivatives, which can react with amino groups either through a Michael-type reaction or through acylation by addition to the carbonyl group of the ring as described by Smyth, D.G. et al. in J. Am. Chem. Soc. (1960) 82, 4600 and Biochem. J. (1964) 91, 589; iii) aryl halides such as reactive nitrohaloaromatic compounds; iv) Aquil halides as described by McKenzie, J.A. et al. in J. Protein Chem. (1988) 7, 581; v) aldehydes and ketones capable of forming Schiff base with amino groups, the adducts formed are usually stabilized by reduction to provide a stable amine; vi) epoxide derivatives such as epichlorohydrin and bisoxiranes, which can react with amino, sulfhydryl or phenolic hydroxyl groups; vii) chloro-containing derivatives of s-triazines, which are highly reactive towards nucleophiles such as amino, sulfhydryl and hydroxyl groups; viii) s-triazine-based aziridines detailed above, for example as described by Ross, W.C.J. in Adv. Cancer Res. (1954) 2, 1, which react with nucleophiles such as amino groups by ring opening; ix) Escharic acid diethyl esters as described by Tietze, L.F. in Chem. Ber. (1991) 124, 1215; and x) a-ha or alkyl ethers which are more reactive alkylating agents than the normal alkyl halides due to the activation caused by the ether oxygen atom, for example, as described by Benneche, T. et al. in Eur. J. Med. Chem. (1993) 28, 463. Representative acyl-reactive acylating examples include: i) isocyanates and isothiocyanates, particularly aromatic derivatives, which form stable urea and thiourea derivatives respectively, and have been used for protein cross-linking as described by Schick, AF et al. in J. Biol. Chem. (1961) 236, 2477; ii) sulfonyl chlorides, which have been described by Herzig, D.J. et al. in Biopolymers (1964) 2, 349 and which may be useful for the introduction of a fluorescent indicator group in the linker; iii) acid halides; iv) active esters such as nitrophenyl esters or N-hydroxysuccinimidyl esters; v) acid anhydrides such as symmetrical mixed N-carboxyanhydrides; vi) other reagents useful for the formation of the amide bond such as described in Bodansky, M. et al. in 'Principies of Peptide Synthesis' (1984) Springer-Verlag; vii) acylazides, for example, wherein the azide group is generated from a preformed hydrazide derivative using sodium nitrite, for example, as described by Wetz, K. et al. in Anal. Biochem. (1974) 58, 347; viii) azalactones bound to polymers such as bisacrylamide, for example as described by Rasmussen, J.K. in. Reactive Polymers (1991) 16, 199; and ix) imidoesters, which form stable amidines upon reaction with amino groups, for example, as described by Hunter, M.J. and Ludwig, M.L. in J ". Am. Chem. Soc. (1962) 84, 3491. Carbonyl groups such as aldehyde functions can be reacted with weak protein bases at such a pH that the functions of the nucleophilic protein side chain are protonated Weak bases include 1,2-aminothiols such as those found in the N-terminal cysteine residues, which selectively form stable 5-membered thiazolidine rings with aldehyde groups, for example as described by Ratner, S. et al. J. Am. Chem. Soc. (1937) 59, 200. Other weak bases such as phenylhydrazones can be used, for example as described by Heitzman, H. et al., In Proc. Nati. Acad. Sci. USA (1974 71, 3537. Aldehydes and ketones can also be reacted with amines to form Schiff bases which can advantageously be stabilized through reductive amination.Alkoxylamino portions react easily with ketones and aldehydes to produce stable alkoxamines. s, for example, as described by Webb, R. et al. in Bioconjugate Chem. (1990) 1, 96. Examples of reactive portions capable of reaction with carboxyl groups include diazo compounds such as diazoacetate esters and diazoacetamides which react with high specificity to generate ester groups, for example, as described by Herriot RM in Adv. Protein Chem. (1947) 3, 169. Carboxylic acid modifying reagents such as carbodiimides, which react through formation of O-acylurea followed by amide bond formation can also be used in a useful manner; the linkage can be facilitated through an amine or can result in direct coupling of vector-receptor. Useful water soluble carbodiimides include l-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide (CMC) and l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), for example as described by Zot , HG and Puett, D. in J. Biol. Chem. (1989) 264, 15552. Other useful carboxylic acid modifying agents include isoxazolium derivatives such as reagent K from Woodwards; chloroformates such as p-nitrophenyl chloroformate; carbonyldiimidazoles such as 1,1'-carbonyldiimidazole and N-carbalkoxydihydroquinolines such as N- (ethoxycarbonyl) -2-ethoxy-1,2-dihydroquinoline. Other potentially useful reactive moieties include vicinal diones such as p-phenylenediglyoxal, which can be used to react with guanidinyl gro for example as described by Wagner et al. in Nucleic acid Res. (1978) 5, 4065; and diazonium salts, which can undergo electrophilic substitution reactions, for example as described by Ishizaka, K and Ishizaka T. in J. Immunol. (1960) 85, 163. Bis-diazonium compounds are easily prepared by Treatment of aryldiamines with sodium nitrite in acid solutions It will be appreciated that the functional groin the indicator and / or vector can be converted, if desired, to other functional groprior to the reaction, for example to confer additional reactivity or selectivity Examples of methods useful for this purpose include the conversion of amides to carboxylic acids using reagents such as dicarboxylic anhydrides; conversion of amines to diols using reagents such as N-actylhomocysteine thiolactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane or succinyl derivatives. idyl containing thiol The conversion of thiols to carboxylic acids using reagents such as a-haloac stages, the conversion of thiols to amines using reagents such as ethylene imine or 2-bromoethylamine; the conversion of carboxylic acids to amines using reagents such as carbodiimides followed by diamines; and the conversion of alcohols to thiols using reagents such as tosyl chloride followed by transesterification with thiol acetate hydrolysis to thiol with sodium acetate. Vector-receptor coupling can also be carried out using enzymes as zero-length cross-linking agents; thus, for example, transglutaminase, peroxidase and xanthine oxidase have been used to produce cross-linked products. Inverse proteolysis can also be used for crosslinking by amide bond formation. Non-covalent vector-receptor coupling, for example, can be carried out by electrostatic charge interactions, for example, between an indicator with a polylysinyl functionality and a vector with polyglutamyl functionality, through chelation in the form of metal complexes stable or through high affinity binding interaction such as avidin-biotin binding. Polylysine, coated non-covalently to a negatively charged membrane surface can also increase the non-specific affinity of a microbubble for a cell by charge interactions. Alternatively, the vectors can be coupled to a protein or known peptide sequence which binds to phospholipids. In many cases, a single phospholipid molecule can be attached to a protein such as a translocase, while other proteins can be attached to surfaces consisting mainly of phospholipid head groand thus can be used to bind vectors to microspheres of phospholipids; an example of such a protein is β2-glycoprotein I (Chonn, A., Semple, S.C. and Cullis, P.R., Journal of Biological Chemistry (1995) 270, 25845-25849). Proteins that bind to phosphatidylserine have been described, for example by Igarashi, K. et al. in Journal of Biological Chemistry 270 (49), 29075-29078. Annexins are a class of proteins that bind to phospholipids, many of which bind particularly avidly to phosphatidylserine (reviewed in Raynal, P. and HB Pollard.) Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins. "Biochim Biophys, Acta 1197: 63-93) A conjugate of a vector can also be used with such a protein that binds to phosphatidylserine to bind the vector to encapsulated phosphatidylserine microbubbles. the amino acid sequence of a binding protein is known, the portion that binds to phospholipids can be synthesized or isolated and can be used for conjugation with a vector, and in this way the biological activity which can be located in another is avoided It is also possible to obtain molecules that bind specifically to the surface (or in the "membrane") of the microspheres by direct analysis of the molecular theses looking for molecules that bind to microspheres. For example, phage libraries displaying small peptides can be used for such selection. The selection can be made by simply mixing the microspheres and the phage display library and eluting the phages that bind to the floating microspheres. If desired, the selection can be made under "physiological conditions" (for example in blood) to eliminate peptides which cross-react with blood components. An advantage of this type of process is that only the binding molecules which do not destabilize the microspheres are those that are selected, since only bound molecules that are in contact with intact floating microspheres will ascend to the top. It is also possible to introduce a certain kind of "tension" during the selection procedure (eg pressure) to ensure that the destabilizing binding portions are not selected. In addition, selection can be made under cutting conditions, for example, by first allowing the phage to react with the microspheres and then allowing the microspheres to pass through a surface coated with phage antibodies, under flowing conditions. In this way, it is possible to select binders which can withstand cutting conditions that occur in vivo. The binding portions identified in this way can be coupled (chemically via peptide synthesis, or at the DNA level using recombinant vectors) to a vector molecule, which is a general tool for attaching any molecule or vector to the microspheres. A vector which comprises or is coupled to a lipopeptide peptide or linker which contains an element capable of mediating the membrane insertion, may also be useful. An example is described by Leenhouts, J. M. et al. in Febs Letters (1995) 370 (3), 189-192. Non-bioactive molecules consisting of known groups of anchor / signal membrane insertion can also be used as vectors for certain applications, an example being the hydrophobic segment Hl of the Na subunit, K-ATPase, described by Xie, Y and Morimoto, T. in J. Biol. Chem. (1995) 270 (20), 11985-11991. The anchoring group can also be fatty acids or cholesterol. The coupling can also be carried out using avidin or streptavidin, which has four sites of high binding affinity for biotin. Therefore, avidin can be used to conjugate the vector to the indicator if both the vector and the indicator are biotinylated. The examples are described by Bayer, E.A. and Wilchek, M. in Methods Biochem. Anal (1980) 26, 1. This method also allows for extending to include indicator-to-indicator binding, a process which can encourage the association of bubbles and consequently a potentially increased echogenicity. Non-covalent coupling can also utilize the bifunctional nature of bispecific immunoglobulins.

These molecules can bind specifically to two antigens, and in this way bind them. For example, either biospecific IgG or b (ab) '2 biospecific fragments under chemical engineering can be used as binding agents. Heterobifunctional biospecific antibodies have also been reported for binding to two different antigens, for example, as described by Bode, C. et al. in J. Biol. Chem. (1989) 264, 944 and by Staerz, -U.D. et al. in Proc. Nati Acad. Sci. USA (1986) 83, 1453. Similarly, any indicator and / or vector containing two or more antigenic determinants (for example as described by Chen, Aa et al., In Am. ". Pathol. (1988) 130, 216) can cross-link antibody molecules and can lead to the formation of cross-linked assemblies of multiple bubbles of potentially increased echogenicity.The so-called zero-length bonding agents, which induce direct covalent attachment of two reactive chemical groups without introducing additional binder material ( example as in the formation of an amide bond induced using carbodiimides or enzymatically), it can be used, if desired, to the extent that agents such as biotin / avidin systems are used which induce a non-covalent binding of indicator-vector and agents which include hydrophobic or electrostatic interactions.

However, most commonly, the binding agent will comprise two or more reactive portions, for example, as described above, connected by a separating element. The presence of such a separator allows the bifunctional linkers to react with specific functional groups within a molecule or between two different molecules, resulting in a binding between these two components and the introduction of an extrinsic linker material derived within the indicator-vector conjugate. . The reactive portions in a linker can be the same (homobifunctional agents) or different (heterobifunctional agents or, when several different reactive portions are present, heteromultifunctional agents), which provides a variety of potential reagents that can be covalently bound between any chemical species, either intramolecularly or intermolecularly. The nature of the intrinsic material introduced by the binding agent can be the critical presentation on the target capacity and the overall stability of the final product. Therefore it may be desirable to introduce labile bonds, for example, containing separating arms which are biodegradable or chemically sensitive or which incorporate enzymatic cleavage sites. Alternatively, the separator may include polymeric components, for example, to act as surfactants and improve the stability of the bubble. The separator may also contain reactive portions, for example, as described above to improve surface crosslinking, or it may contain a trace element such as a fluorescent probe, a spin marker or radioactive material. The spacer elements typically may consist of aliphatic chains which effectively separate the reactive portions of the linker by distances of between 5 and 30 A. They may also comprise macromolecular structures such as poly (ethylene glycols). Such polymeric structures, hereinafter referred to as PEG, are simple and neutral polyethers which have been given much attention in biotechnical and biomedical applications (see, for example, ilton Harris, J. (ed.) "Poly (ethylene glycol). chemistry biotechnical and biomedical applications "Plenum Press, New York, 1992). PEGs are soluble in most solvents, even in water, and are highly hydrated in aqueous environments, with two to three molecules of water attached to each segment of ethylene glycol; this has the effect of preventing the adsorption of either other polymers or proteins on the surfaces modified with PEG. It is known that PEG are not toxic and are not harmful to active proteins or cells, and at the same time it is known that the covalently linked PEGs are non-immunogenic and non-antigenic. In addition, PEGs can be easily modified and can bind to other molecules with only a small effect on their chemistry. Their solubility and advantageous biochemical properties are evident from the many possible uses of the PEGs and copolymers thereof, which include block copolymers such as PEG-polyurethanes and PEG-polypropylenes. Appropriate molecular weights for the PEG separators used according to the invention, for example, may be between 120 Daltons units and 20 kDaltons. The main mechanism for the uptake of particles by the cells of the reticular endothelial system (RES) is the opsonization by plasma proteins in blood; this marks the foreign particles which are then captured by the RES. The biological properties of the PEG spacer elements used according to the invention can serve to increase the circulation time of the contrast agent in a manner similar to that observed by the pegylated liposomes (see, for example, Klibanov, AL et al. FEBS Letters (1990) 268, 235-237 and Blume, G. and Cevc, G. in Biochim, Biophys, Acta (1990) 1029, 91-97). Other potentially useful protein modifications which can be made to the vectors include partial or complete deglycosylation by neuraminidase, endoglycosidases or periodate, since deglycosidation often results in a lower uptake by liver, spleen, macrophages, etc., whereas Protein neoglycosylation often results in increased uptake by the liver and macrophages; the preparation of forms truncated by proteolytic rupture leads to a reduced size and a shorter average duration in circulation; and cationization (cation formation) for example as described by Kumagi et al. in J. Biol. Chem. (1987) 262, 15214-15219; Triguero et al. in Proc. Nati Acad. Sci. USA (1989) 86, 4761-4765; Pardridge et al. in J. Pharmacol. Exp. Therap. (1989) 251, 821-826 and Pardridge and Boado, Febs Lett. (1991) 288, 30-32. Increased coupling efficiency can also be obtained for areas of interest using antibodies attached to the terminal part of the PEG separators (see, for example, Maruyama, K. et al., In Biochim, Bipohys, Acta (1995) 1234, 74-80. and Hansen, CB et al., in Biochim, Biophys, Acta (1995) 1239, 133-144). In some cases it is considered advantageous to include a PEG component as a stabilizer together with a vector or vectors or directly to the indicator in the same molecule where PEG does not serve as a spacer. Other representative spacer elements include structural type polysaccharides such as polygalacturonic acid, glycosaminoglycans, heparinoids, cellulose and marine polysaccharides such as alginates, chitosans and carrageenans; storage type * polysaccharides such as starch, glycogen, dextran and aminodextrans; polyamino acids and methyl and ethyl esters thereof, and in homo- and co-polymers of lysine, glutamic acid and aspartic acid; and polypeptides, oligonucleotides and oligosaccharides which may or may not contain cleavage sites by enzymes. In general, the spacer elements may contain separable groups such as vicinal glycol, - azo, sulfone, ester, thioester or disulfide groups. Separators containing biodegradable methylene diester or diamide groups of the formula - (Z) m.Y.X.C (R1R2) .X.Y. (Z) n- [where X, and Z are selected from -0-, -S-, and -NR- (where R is hydrogen or an organic group); each Y is a carbonyl, thiocarbonyl, sulfonyl, phosphoryl group or a similar acid-forming group: m and n are each zero or 1; and R1 and R2 are each hydrogen, an organic group or a group -X.Y. (Z) m-, or together form a divalent organic group] may also be used; as discussed in, for example, WO-A-9217436 such groups are easily biodegraded in the presence of esterases, for example in vivo, but are stable in the absence of such enzymes. Therefore, they can be advantageously linked to therapeutic agents to allow slow release thereof.

Poly [N- (2-hydroxyethyl) methacrylamides] are potentially useful separating materials by virtue of their low degree of interaction with cells and tissues (see for example Volfova, I., Rhohova, B .. and VR and Vetvicka, P. in J. Bioact, Comp.Polymers (1992), 7, 175-190). Working on a similar polymer consisting mainly of a closely related 2-hydroxypropyl derivative is shown to be subject to endocytosis by the mononuclear phagocyte system only to a low degree (see Goddard, P., Williamson, I., Bron, J., Hutchkinson, LE, Nicholls, J. and Petrak, K. in "Bioct., Compat. Polym. (1991) 6, 4-24.) Other potentially useful polymeric separator materials include: i) methyl methacrylate copolymers with methacrylic acid, these can be erodible (see Lee, PI in Pharm. Res. (1993) 10, 980) and carboxylate substituents that can cause a greater degree of expansion compared to neutral polymers; ii) block copolymers of polymethacrylates with biodegradable polyesters (see, for example, San Román, J. and Guillen-Garcia, P. in Biomaterials (1991) 12, 236-241); iii) cyanoacrylates, ie 2-cyanoacrylic acid ester polymers - these are biodegradable and have been used in the form of nanoparticles for selective drug delivery (see Forestier, F., Gerrier, P., Chaumard, C, S Quero , AM, Couvreur, P. and Labarre, C. in J. Antimicrob, Chemoter (1992) 30, 173-179); iv) polyvinyl alcohols which are soluble in water and are generally considered to be biocompatible (see for example Langer, R. in J. Control, Reléase (1991) 16, 53-60); v) vinylmethylether copolymers with maleic anhydride, which have been established to be bioerodible (see Finne, U., Hannus, M. and Urtti, A. in-Jpt. J. Pharm. (1992) 78. 237-241); vi) polyvinyl pyrrolidones, for example with molecular weight less than about 25,000, which are rapidly filtered by the kidneys (see Hespe, W., Meier, AM and Blankwater, YM in Arzeim. -Forsch. / Drug Res. (1977) 27 , 1158-1162); vii) polymers and copolymers of short chain aliphatic hydroxy acids such as glycolic, lactic, butyric, valeric and caproic acids (see for example Carli, Ft in Chim. Ind. (Milan) (1993) 75, 494-9), which includes copolymers which incorporate aromatic hydroxy acids in order to increase their rate of degradation (see Imasaki, K., Yoshida, M., Fukuzaki, H., Asano, M., Kumakura, M., Mashimo, T., Yamanaka, H and Nagai, T. in Int. J. Pharm. (1992) 81, 31-38); i viii) polyesters that consist of alternating units of ethylene glycol and terephthalic acid, for example DacronBR, which are not degradable but are highly biocompatible; ix) block copolymers comprising biodegradable segments of aliphatic hydroxy acid polymers (see, for example Younes, H., Nataf, PR, Cohn, D., Appelbaum, YJ, Pizov, G. and Uretzky, G. in Biomater. Cells Artif. Organs (1988) 16, 705-719), for example together with polyurethanes (see Kobayashi, H., Hyon, SH and Ikada, Y. in "Water-curable and biodegradable prepolymers" - ". Biomed. Res. (1991) 25, 1481-1494) x) polyurethanes which are known to be well tolerated in implants, and which can be combined with "soft", flexible segments, for example comprising poly (tetramethylene glycol) , poly (propylene glycol) or poly (ethylene glycol)) and aromatic "hard" segments, for example comprising 4,4'-methylenebis (phenylene isocyanate) (see for example Ratner, BD, Johnston, AB and Lenk, TJ in J). Biomed, Mater: Res: Applied Biomaterials (1987) 21, 59-90; Sa Da Costa, V. et al., In J. Coil, Interface Sci. (1981) 80, 445-452 and Affrossman, S. et al. in Clinical Materials (1991) 8, 25-31); xi) poly (1,4-dioxan-2-ones), which can be considered as biodegradable esters in view of their ester linkages (see for example Song, CX, * Cui, XM and Schindler, A. in Afed. Biol. Eng. Comput. (1993) 31, S147-150), and which may include glycolide or glycolide units to improve their absorption capacity (see Bezwada, RS, Shalaby, SW and Newman, HDJ in Agricul tural and synthetic polymers: Biodegradability and utilization (1990) (ed Glass, JE and Swift, G.), 167-174 - ACS symposium Series, # 433, Washington DC, USA - American Chemical Society); xii) polyanhydrides such as copolymers of sebasic acid (octanedioic acid) with bis (4-carboxy-phenoxy) propane, which has been shown in studies in rabbits (see Brem, H., Kader, A., Epstein, JI, Tamargo , RJ, Domb, A., Langer, R. and Leong, KW in Sel. Cancer Ther. (1989) 5, 55-65) and in studies in rats (see Tamargo, R., Epstein, JI, Reinhard, CS, Chasin, M. and Brem, H. in J. Biomed, Mater. Res. (1989) 23, 253-266) which are useful for controlled release of drugs in the brain without evident toxic effects; xiii) biodegradable polymers containing ortho ester groups, which have been used for controlled release in vivo (see Maa, Y.F. and Heller, J. in J. Control, Reléase (1990) 14, 21-28); and xiv) polyphosphazenes, which are inorganic polymers consisting of alternating atoms of phosphorus and nitrogen (see Crommen, J.H., Vandorpe, J. and Schacht, E.H. in J. Control, Reléase (1993) 24, 167-180). The following table includes binding agents and agents for protein modification which may be useful in the preparation of addressable contrast agents according to the invention.

Heterobifunctional binding agents Notes: (l) = yodable; (2) = fluorescent Bonding agents homobifunctional í Biotination agents Notes: DPPE = dipalmitoylphosphatidylethanolamine; LC = long chain Agents for protein modification Binding agents used in accordance with the invention will generally produce binding of a vector to an indicator or of an indicator to another indicator with some degree of specificity, and may also be used to bind one or more therapeutically active agents. Ultrasound imaging modalities which can be used in accordance with the invention include two-dimensional and three-dimensional imaging techniques such as B-mode imaging (e.g. using amplitude of time variation of the signal envelope). generated from the fundamental frequency of the emitted ultrasound pulse, from subharmonics or from higher harmonics thereof or from the sum or difference of frequencies derived from the emitted pulse and such harmonics, images generated from the fundamental frequency or the second harmonic thereof being preferred), color Doppler image formation and Doppler amplitude image formation, and combinations of these latter two with any of the previous (technical) modalities. Surprisingly, signals of the second harmonic from the targeted monolayer microspheres are found to be excellent when used in accordance with the present invention. To reduce the effects of movement, successive images of tissues such as the heart or kidney can be collected with the aid of suitable synchronization techniques (for example synchronization with the ECG or the subject's respiratory movement). The measurement of changes in the resonance frequency or in the frequency absorption which accompanies the suppressed or delayed microbubbles can also be usefully performed to detect the contrast agent. The present invention provides a tool for the therapeutic delivery of drugs in combination with vector-mediated direction of the product to the desired site. By "therapeutic" and "medicament" is meant an agent that has a beneficial effect on a specific disease in a living human or non-human animal. Although combinations of drugs and contrast agents by ultrasound have been proposed, for example in the documents, WO-A-9428873 and WO-A-9507072, these products lack vectors that have affinity for particular sites and thus show a comparatively poor specific retention at desired sites before or during drug release. The therapeutic compounds used in accordance with the present invention can be encapsulated within the microbubbles or can be attached to or incorporated into the encapsulating walls. Therefore, the therapeutic compound can be bound to a part of the wall, for example, through covalent or ionic bonds, or it can be physically mixed in the encapsulating material, particularly if the drug has a polarity or solubility similar to that of the drug. of the membrane material, so as to prevent it from leaking out of the product before it is intended to act on the body. The release of the medicament can only be initiated by wet contact with the blood after administration or as a consequence of any other internal or external influence, for example dissolution processes catalyzed by enzymes or the use of ultrasound. The destruction of gas-containing microparticles using external ultrasound is a well-known phenomenon with respect to ultrasound contrast agents, for example, as described in WO-A-9325241; the rate of release may vary based on the type of therapeutic application, using a specific amount of ultrasound energy from the transducer. The therapeutic agent can be covalently bound to the encapsulating membrane surface using a suitable binding agent, for example, as described herein. Thus, for example, initially a phospholipid or lipopeptide derivative to which the drug binds via a selectively separable or biodegradable binder can be prepared followed by incorporation of the material into the microbubble. Alternatively, lipidated drug molecules which do not require processing to release an active medicament are incorporated directly into the membrane. The active lipidated drug can be released by increasing the resistance of the ultrasound beam. Exemplary drug delivery systems suitable for use in the present invention include any known therapeutic drug or active analogs thereof containing diol groups which are coupled to microbubbles containing diol under oxidative conditions which provides disulfide bonds. In combination with a vector or vectors, microbubbles modified with drug / vector are allowed to accumulate in the target tissue. The administration of an agent releases the drug molecule the directed microbubble in the vicinity of the target cell which increases the local concentration of the drug and improves the therapeutic effect. The product can also be prepared without the therapeutic substance, if desired. The medicine can then be attached or coated on the microbubbles before use. Thus, for example, the therapeutic substance can be added to a suspension of microbubbles in an aqueous medium and can be agitated in order to bind or adhere the therapeutic substance to the microbubbles. Other drug delivery systems include vector-modified phospholipid membranes, added with lipopeptide-like structures comprising a poly-L-lysine or poly-D-lysine chain in combination with a targeting vector. When applied to gene therapy / antisense technologies with particular emphasis on the delivery of the drug mediated by the receptor, the microbubble carrier is condensed with DNA or RNA via electrostatic interaction with the polycation. This method has the advantage that the vector or vectors used for directed delivery does not bind directly to the polylysine carrier portion. The polylysine chain is also more firmly anchored in the microbubble membrane due to the presence of lipid chains. The use of ultrasound to increase the effectiveness of supply has also been considered useful. Alternatively, the free polylysine chains are first modified with drug or vector molecules and then condense on the negative surface of directed microbubbles. Representative and non-limiting examples of medicaments useful in accordance with the invention include antineoplastic agents such as vinicristin, vinblastine, vindesine, busulfan, chlorambucil, espiroplatin, cisplatin, carbopla ina, methotrexate, adriamycin, mitomycin, bleomycin, cytosine arabinoside, arabinosil adenine mercaptopurine, mitotane, procarbazine, dactinomycin (antinomycin D), daunorubicin, doxorubicin hydrochloride, taxol, plicamycin, aminoglutethimide, estramustine, flutamide, leuprolide, megestrol acetate, tamoxifen, testolactone, trilostane, amsacrine (m-AMSA), asparaginase ( L-asparaginase), etoposide, interferon a-2a and 2b, blood products such as hematoporphyrins or derivatives of the above, -modifiers of the biological response such as muramyl peptides; antifungal agents such as ketoconazole, nystatin, griseofulvin, flucytosine, miconazole or amphotericin B; hormones or hormone analogues such as growth hormones, melanocyte stimulating hormone, is radiol, beclomethasone dipropionate, betamethasone, cortisone acetate, dexamethasone, flunisolide, hydrocortisone, methylprednisolone, parametasone acetate, prednisolone, prednisone, triamcinolone or acetate of fludrocortisone; vitamins such as cyanocobalamin or retinoids; enzymes such as alkaline phosphatase or manganese superoxide bismutase; antiallergic agents such as amelexanox; tissue factor inhibitors such as monoclonal antibodies and Fab fragments thereof, synthetic peptides, non-compound peptides that down-regulate the expression of tissue factor; platelet inhibitors such as GPIa, GPIb and GPIIb-IIIa, ADP receptors, thrombin receptors, von Willebrand factor, prostaglandins, aspirin, ticlopidine, clopigogrel and reopro; inhibitors of the coagulation target proteins such as: Fila, FVa, FVIIa, FVIIIA, FlXa, tissue factor, hepatin, hirudin, hirulog, argatroban, DEGR-rFVIIa and annexin V; fibrin formation inhibitors and fibrionolysis promoters such as t-PA, urokinase, plamin, streptokinase, rt plasminogen activator and rStafilokinase; antiangiogenic factors such as medroxyprogesterone, pentosan polysulfate, suramin, taxol, thalidomide, angiostatin, interferon alpha, metalloproteinase inhibitors, platelet factor 4, somtostatin, thrombospondin; circulatory drugs such as propranolol; metabolic enhancers such as glutathione; antituberculous agents such as p-aminosalicylic acid, isoniazid, capreomycin sulfate, cycloosexin, ethanbutol, ethionamide, pyrazinamide, rifampin or streptomycin sulfate; antivirals such as acyclovir, amantadine, azidothymidine, ribavirin or vidarabine; blood vessel dilator agents such as diltiazem, nifedipine, verapamil, erythritol tetranitrate, isosorbide dinitrate, nitroglycerin or pentaerythritol tetranitrate; antibiotics such as dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil, cephalexin, cephradine, erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin, cyclacillin, picloxacillin, hetacycline, methicillin, nafcillin, penicillin, polymyxin or tetracycline; anti-inflammatories such as diflunisal, ibuprofen, indomethacin, meclefanamate, mefenamic acid, naproxen, phenylbutazone, piroxicam, tolmetin, aspirin or salicylates; antiprotozoals such as chloroquine, metronidazole, quinine or meglumine antimonate; antirheumatics such as penicillamine; narcotics such as paregoric, opiates such as codeine, morphine or pyo; cardiac glycosides such as deslaneside, digitoxin, digoxin, digitalin or digitalis; neuromuscular blockers such as atracurium mesylate, galamina triethyodide, hexafluorenium bromide, methocurinium iodide, pancoronium bromide, succinylcholine chloride / tubocurarine chloride or vecuronium bromide; sedatives such as amobarbital, amobarbital sodium, apropbarbital, sodium butabarbital, doral hydrate, etclorovinol, ethinamate, flurazepam hydrochloride, glutethimide, methotrimeprazine hydrochloride, metiprilon, midazolam hydrochloride, paraldehyde, pentobarbital, secobarbital sodium, talbutal, temazepam or triazolam; local anesthetics such as bupivacaine, -chloroprocaine, etidocaine, lidocaine, mepivacaine, procaine or tetracaine; general anesthetics such as droperidol, etomidate, fentanyl citrate with droperidol, ketamine hydrochloride, sodium methohexital or thiopental and pharmaceutically acceptable salts (for example acid addition salts such as hydrochloride or hydrobromide salts or base salts such as sodium salts) , calcium or magnesium) or derivatives (eg acetates) thereof. Other examples of therapeutic substances include genetic material such as nucleic acids, RNA and DNA of natural or synthetic origin, including RNA and recombinant DNA. The DNA that codes for certain proteins can be used in the treatment of many different types of diseases. For example, tumor necrosis factor or genes for interleukin-2 can be provided to treat advanced cancers; genes for thymidine kinase can be provided to treat ovarian cancer or brain tumors; genes for interleukin-2 can be provided to treat neuroblastoma, malignant melanoma or kidney cancer; and genes for interleukin-4 can be provided to treat cancer. The lipophilic derivatives of drugs attached to the wall of the microbubbles through hydrophobic interactions can show therapeutic effects as part of the microbubble or after the release of the microbubble, for example by the use of ultrasound. If the drug does not possess the desired physical properties, a lipophilic group can be introduced to anchor the drug to the membrane. Preferably a lipophilic group can be introduced so as not to influence the in vivo potency of the molecule, or the lipophilic group can be separated by releasing the active medicament. Lipophilic groups can be introduced by various chemical means that depend on the functional groups available in the drug molecule. The covalent coupling can be carried out using functional groups in the drug molecule capable of reaction with appropriately functionalized lipophilic compounds. Examples of lipophilic portions include branched and unbranched alkyl chains, cyclic compounds, aromatic residues and aromatic and non-aromatic cyclic fused systems. In some cases, the lipophilic portion will consist of a properly functionalized steroid, such as cholesterol and related compounds. Examples of functional groups particularly suitable for derivatization (derivatization) include nucleophilic groups such as amino, hydroxy and sulfhydryl groups. Suitable processes for the lipophilic derivatization (formation of derivatives) of any drug containing a sulfhydryl group such as captopril may include direct alkylation, for example reaction by an alkyl halide under basic conditions and formation of thiol ester by reaction with a carboxylic acid activated. Representative examples of derivatization of any drug having carboxylic functions, such as atenolol and chlorambucil, include amide and ester formation by coupling amines and alcohols respectively, which possess necessary physical properties. A preferred aspect is the binding of cholesterol to a therapeutic compound by forming a degradable ester linkage. A preferred application of the present invention relates to angiogenesis, which is the formation of new blood vessels by branching existing vessels. The primary stimuli for this process may be an inadequate supply of nutrients and oxygen (hypoxia) to cells in a tissue. Cells can respond to secrete angiogenic factors, of which there are many; an example is the vascular endothelial growth factor. These factors initiate the secretion of proteolytic enzymes which decompose the basement membrane proteins, as well as inhibitors which limit the action of these potentially harmful enzymes. The combined effect of binding loss and receptor signals for angiogenic factors is to cause the endothelial cells to move, multiply and rearrange themselves, and eventually synthesize a basement membrane around the new vessels. Tumors can initiate angiogenesis when they reach the size of millimeters in order to maintain their high growth rate. Since angiogenesis is accompanied by characteristic changes in endothelial cells and in their environment, this process is a promising target for therapeutic intervention. The transformations that accompany angiogenesis are also very promising for diagnosis, malignant diseases are a preferred example, but the concept also shows great promise in inflammation and a variety of diseases related to inflammation. These factors are also involved in the revascularization of infarcted parts of the myocardium, which occurs if the stenosis is released within a short period. Numerous known receptors / targets associated with angiogenesis are provided in the subsequent tables. The use of the targeting principles described in the present description allows angiogenesis to be detected by most of the imaging modalities in use in medicine. Improved contrast-enhanced ultrasound may have additional advantages, the contrast medium being microspheres which are restricted to the interior of blood vessels. Even if the target antigens are found in many types of cells, the microspheres will bind exclusively to endothelial cells. The so-called promedications can also be used in agents according to the invention. Therefore, drugs can be derivatized to alter their physicochemical properties and to adapt them for inclusion in the indicator; such derivatized drugs can be considered as promedications and are usually inactive until the separation of the derivatizing group which regenerates the active form of the drug. By directing a microbubble filled with gas containing an activating enzyme of promedicamento to areas of pathology, images directed towards the enzymes can be formed, making it possible to visualize the moment in which the microbubbles are properly attached to the pathology area and at the same time when They have disappeared from areas that are not objective. In this way, the optimal time for injection of a single prodrug can be determined. Another alternative is to incorporate the promedicamento, the activating enzyme of promedicamento and the vector in the same microbubble in a system where the promedicamento will only be activated after a certain external stimulus. Such stimuli may be, for example, a tumor-specific protease as described above, or the discharge of the bubbles by external ultrasound after the desired addressing has been obtained. The therapeutic substances can be easily administered according to the invention to diseased or necrotic areas, which include the heart and vasculature in general, and to the liver, spleen and kidneys and other regions such as the lymphatic system, the body cavities or the gastrointestinal system. . The products according to the present invention can be used for therapeutic delivery directed either in vivo or in vitro. In the latter context, the products can be useful in in vi tro systems such as equipment for diagnosing different diseases or characterization of different components in blood or tissue samples. Techniques similar to those used to attach certain blood components or cells to polymer particles (eg, monodisperse magnetic particles) in vi tro to separate them from a sample can be used in the present invention, using the low density of the indicator units in agents of the present invention for carrying out the separation of the gas-containing material by flotation and repeated washing.

The vectors which can be used profitably in the generation of multiple specific bliming contrast agents according to the invention include the following: i) Antibodies, which can be targeted as vectors for a wide range of purposes, and which they may have advantageous properties such as very high specificity, high affinity (if desired), the possibility of modifying the affinity according to the need, etc. Whether the antibodies are bioactive or not depends on the specific combination of vector / target. Both conventional and genetically engineered antibodies can be used, the latter allowing the engineering treatment of antibodies to particular needs, for example, when considering affinity and specificity. The use of human antibodies may be preferred to avoid possible immune reactions against the vector molecule. An additional useful class of antibodies comprises the so-called biospecific antibodies, ie, antibodies having specificity for two different target molecules in an antibody molecule. Such antibodies may be useful, for example, in promoting the formation of bubble groups and may also be used for various therapeutic purposes, for example, to transport toxic portions towards the target. Various aspects of the biospecific antibodies are described by McGuinness, B. T. et al. in Nat. Biotechnol (1996) 14, 1149-1154; by George, A.J. et al. in J ". Immunol. (1994) 152, 1802-1811, by Bonardi et al., in Cancer Res. (1993) 53, 3015-3021, and by French, RR et al., in Cancer Res. (1991) 51, 2353-2361 ii) Cell adhesion molecules, their receptors, cytokines, growth factors, peptide hormones, and pieces thereof.These vectors are based on normal biological protein-protein interactions with receptors of the target molecule, and thus in many cases they will generate a biological response by binding with the targets and therefore may be bioactive, this may be a concern that is not important with vectors which are targeted to proteoglycans iii) Agonists / antagonists that are not peptides or binders not bioactive receptors for cell adhesion molecules, cytokines, growth factors and peptide hormones This category may include non-bioactive vectors which are not agonists or antagonists but which nevertheless can for a useful addressing capability. iv) Oligonucleotides and modified oligonucleotides which bind DNA or RNA through the Watson-Crick base pair or other types of base pairing. DNA is usually only present in the extracellular space as a consequence of cellular damage, so that such oligonucleotides, which are usually not bioactive, can be useful, for example, by targeting necrotic regions, which are associated with many pathological conditions different Oligonucleotides can also be designed to bind to proteins that specifically bind to DNA or RNA, for example, transcription factors which are often highly overexpressed or activated in tumor cells or in immune or endothelial activated cells. Combination libraries can be used to select oligonucleotides which bind specifically to possible target molecules (from proteins to caffeine) and which can therefore be used as vectors for targeting. v) Drugs that bind to DNA which can behave similarly to oligonucleotides but which can show activity and / or biological toxic effects if they are captured by the cells. vi) Various small molecules, which include bioactive compounds that are known to bind to biological receptors of various kinds. Such vectors or their targets can be used to generate non-bioactive compounds that join the same objectives. vii) Vector molecules of combinational libraries can be selected without necessarily knowing the exact molecular objective, by functionally selecting (in vitro, ex vivo or in vivo) binding molecules to a region / structure from which an image is to be obtained. viii) Several small molecules, including bioactive compounds that are known to bind to biological receptors of various kinds. Such vectors or their targets can be used to generate non-bioactive compounds that join the same objectives. ix) Proteins or peptides which bind to glycosaminoglycan side chains, aparano sulfate, which include glucosoaminoglycan-binding portions of larger molecules, since binding to such glycosaminoglycan side chains does not result in a biological response. Proteoglycans are not found in red blood cells, and therefore undesirable adsorption to these cells is eliminated. Other peptide and lipopeptide vectors thereof of particular interest for directed ultrasound imaging are included below: peptides that bind to atherosclerotic plaque such as YRALVDTLK, YA FRETLEDTRDRMY and RALVDTEFKVKQEAGAK; peptides that bind to thrombi such as NDGDFEEIPEEYLQ and GPRG; peptides that bind to platelets such as PLYKKIIKKLLES; and cholecystokinin, a-melanocyte-stimulating hormone, enterotoxin-1, thermostable, vasoactive intestinal peptide, synthetic alpha-M2 peptide for the third determinant region of heavy chain complementarity and analogs thereof for targeting tumors. The following tables identify the various receptors which can be directed by particular types of vectors and consequent areas of use for steerable ultrasound contrast agents, according to the invention, which contain such vectors.

Protein and peptide vectors - antibodies a.) Heider, K. H., M. Sproll, S. Susani, E. Patzelt, P.

Beaumier, E. Ostermann, H. Ahorn, and G. R. Adolf. 1996. "Characterization of a high-affinity monoclonal antibody specific for CD44v6 as a candidate for the disease of squamous cell carcinomas." Cancer Immunology I munotherapy 43: 245-253. b) I. Roitt, J. Brostoff, and D. Male. 1985 Immunology, London: Goer Medical Publishing, p. 4.7 c). Stromblad, S., and D. A. Cheresh. 1996. "Integrins, angiogenesis and vascular cell survival". Chemistry & Biology 3: 881-885.

Protein vectors and peptide cell adhesion molecules, etc.

Integrins receptors, by cells in the integrin immune system, for example VLA-1, vessel wall example VLA-2, VLA-3, etc. laminin, VLA-4, VLA-5, collagen, VLA-6, ßra? , ß ^ a, fibronectin, ß1av, LFA-1, VCAM-l, Mac-1, CD41a, thrombospondin, etc. vitronectin, etc. Proteoglycan Molecule Adhesion of N-CAM Nerve (Homophilic) Cells (N-CAM) RGD- Integogen Peptides Angiogenesis Ve ctors that comprise growth factors / peptide hormones, and fragments thereof Various proteins and peptide vectors Vectors comprising agonists / antagonists that are not cytokine peptides / growth factors / peptide hormones / cell adhesion molecules Vectors comprising anti-angiogenic factors that comprise anaerogenic factors Vector molecules different from known anqioenetics factors with known affinity for receptors associated with angiogenesis endoglin tumors, inflammation endosialin tumors, inflammation endostatin tumors, M [fragments of inflammation collagen] antigen tumors, inflammation-related factor VII fibrinopeptide tumors, ZC inflammation basic factor of tumors, growth of inflammation fibroblasts tumor factor, growth of inflammation hepatocyte factor tumors, growth inflammation insulin-like interleukins tumors, for example: IL-8 inflammation inhibitory factor tumors, from leukemia inflammation tumor inhibitors, for example, batimastat metalloproteinase inflammation Antibodies tumors, for example: to angiogenetic inflammation monoclonal factors or their receptors, or component of the phyllolithic system Receptors / objectives associated with angiogenesis integrins: Tumors of D, P ß, and ßs. inflammation integrin vß3, integrin a6ßl t receptor of laminin integrins 6, integrins β, integrin a2ßl f integrin av / 33, integrin a5 subunit of integrin receptor avß5, fibronectin fibrin receptors Molecule-1 and -2 tumors, adhesion intercellular inflammation product of the gene tumors, Jaggred inflammation Ly-6 tumors, activation protein of N inflammation lymphocyte metalloproteinases tumors, matrix inflammation MHC class II tumors, inflammation product of the gene tumors, Notch inflammation Osteopontin tumors PECAM tumors, alias CD31 inflammation tumor receptor, ZC Plasminogen inflammation activator Oligonucleotides n Modified oligonucleotide vectors Nucleoside v nucleotide vectors Receptors comprising DNA binding drugs Receptors comprising protease substrates Receptors comprising protease inhibitors Combination library vectors Carbohydrate vectors lipidiums small molecule vectors References to the preceding tables A. Auerbach, W., and R. Auerbach. 1994"Angiogenesis inhibition: a review". Pharmac. Ther. 63: 265-311. B. Barinaga, M. 1997. "Designing Therapies That Target Tumor Blood Vessels. "Science 275 (Jan. 24): 482-484 C. Folkman, J., P. B. eisz, M.. Joullié,.

Li, and. R. Ewing. 1989. "Control of Angiogenesis With Synthetic Heparin Substitutes. "Science 243: 1490-1493, D. Fox, S. B., and A. L. Harris, 1997." arkers of tumor angiogenesis: Clinical applications in prognosis and anti-angiogenic therapy. "Investigational New Drugs 15 (1): -28.

E. Gasti, G., T. Hermann, 'M. Steurer, J. Zmija, E. Gunsilius, C. Unger, and A. Kraft. May 1997. "Angiogenesis as a target for tumor treatment". Oncology 54 (3): 177-184. F. Griffioen, A. W., M. J. H. Coenen, C. A. Damen, S. M. M. Hellwig, D. H. J. Vanweering, W. Vooys, G. H. Blij am, and G. Groenewegen. 1 August 1997. "CD44 is involved in tumor angiogenesis, an activation antigen on human endothelial cells". Blood 90 (3): 1150-1159. G. Hlatky, L., P. Hahnfeldt, and C. N. Coleman. 1996. "Vacular endothelial growth factor: environmental controls and effects in angiogenesis". Brit. J. Cancer 74 (Suppl XXVII): S151-S156. H. aragoudakis, M. E., E. Pipili-Synethos, E. Sakkoula, D. Panagiotopoulos, N. Craniti, and J. M. Atsoukas. 1996. "Inhibition of TRAP-induced angiogenesis by the tripeptide Phe-Pro-Arg, a thrombin-receptor-derived peptide analogue". Letters in Peptide Science 3: 227-232. I. Nguyen, M. 1997. "Angiogenic factors as tumor markers". Investigational New Drugs 15 (1): 29-37. J. Ono, M., H. Izumi, S. Yoshida, D. Gtot, S. Jimi, N. Kawahara, T. Shono, S. Ushiro, M. Ryuto, K. Kohno, Y. Sato, and M. Kuwano. 1996. "Angiogenesis as a new target for cancer treatment". Cancer Chemoter. Pharmacol 38 (Suppl.): S78-S82.

K. Passe, T. J., D. A *. Bluemke, and S. S. Siegelman. June 1997. "Tumor angiogenesis: Tutorial on implications for imaging". Radiology 203 (3): 593-600. L. Saclarides, T. J. February 1997. "Angiogenesis in colorectal cancer". Surgical Clinics of North America 77 (1): 253. M. Sage, E. H. May 1997. "Pieces of eight: Bioactive fragments of extracellular proteins as regulators of angiogenesis". Trends in Cell Biology 7 (5): 182-186. N. Sagi-Assif, 0., A. Traister, B. Z. Katz, R.

Anavi, M. Eskenazy, and I. P. Witz. 1996. "TNF and anti-Fas antibodies regullate Ly-6E.l expression by tumor cells: A possible link between angiogenesis and Ly-6E.l". Immunology Letters 54: 207-213. O. Strawn, L. M., G. Mc Ahon, H. App, R. Schreck, WR Kuchler, MP Longhi, TH Hui, C. Tang, A. Levitzki, A. Gazit, I. Chen, G. Keri, L. Orfi, W. Risau, I. Flame, A. Ullirch, KP Hirth, and LK Shawyer. 1996. "Flk-1 as a Target for Tumor Growth Inhibition". Cancer Res. 56: 3340-3545. P. Stromblad, S., and D. A. Cheresh. December 1996.

"Cell adhesion and angiogenesis". Trends in Cell Biology 6 (12): 462-468. Q. Stromblad, S., andD. A. Cheresh November 1996. "Integrins, angiogenesis and vascular cell survival". Chemistry & Biology 3 (11): 881-885.

R. Volpert, O., D. Jackso ?, N. Bouck, and D. I. H.

Linzer. September 1996. "The insulin-like growth factor The mannose 6-phosphate receptor is required for proliferin-induced angiogenesis. "Endocrinology 137 (9): 3871-3876, S. Yoshida, 0. M., T. Shono, H. Izumi, T-Ishibashi, H. Suzuki , Kuwano, 1997. "Involvement of Interleukin-8, Vascular Endothelial Growth Factor, and Basic Fibroblast Growth Factor in Tumor Necrosis Factor Alpha-Dependent Angiogenesis." Mol Cell. Biol. 17: 4015-4023. AB, MS Pepper, GA McMahon, F.

Nguyen, R. ontesano, and T. aciag. 1996. "An Antisense Oligonucleotide to the Notch Ligand Jagged Enhances Fibroblast Growth Factor-induced Angiogenesis < in vitro > J. Biol. Chem. 271 (Dec. 20): 32499-3502. U. Albini, A., R. Soldi, D. Giunciuglio, E. Giraudo, R. Benelli, R. Primo, D. Noonan, M. Salió, G. Camussi, W. Rockl, and F. Bussolino. 1996. "The angiogenesis induced by HIV-1 Tat protein is mediated by the Flk-1 / KDR receptor on vacular endothelial cells". Nature Medicine 2 (12 (Dec.)): 1371-1374. V. Ferrara,? 1996. "The biology of vascular endothelial growth factor". in Molecular, Cellular and Clinical Aspects of Angiogenesis, ed. M. E. Maragoudakis. New York: Plenum Press. X. Jackson, R.L., S.J. busch, and A.J. Cardin. 1991. "Glycosaminoglycans: Molecular Properties, Protein Interactions, and Role in Physiological Processes". Physiological Reviews 71 (2): 481-435. Y. Kinsella, M. G., C. K. Tsoi, H. T. Jarvelainen, and T. N. Wight. 1997. "Selective expression and processing of biglycan during migration of bovine aortic endothelial cells - The role of endogenous basic fibroblast growth factor".

Journal of Biological Chemistry 272: 318-325. Z. Folkman, J. 1996. Tumor angiogenesis and tissue factor. Nature Medicine 2, 167-8 ZA. Relf, M., S. LeJeune, P.A. Scott, S. Fox, K. Smith, R. Leek, A. Moghaddam, R. Whitehouse, R. Bicknell and A: L. Harris. 1997. Expression of the angiogenic factors vascular endothelial cell growth factor, acidic and basic fibroblast growth factor, tumor growth factor beta-1, platelet-derived endothelial cell growth factor, placenta growth factor and pleiotrophin in human primary breast cancer and its relation to angiogenesis§. Cancer Res. 57, 963-9. ZB Carmeliet, P., L. Moons, M. Dewerchin, N. Mackman, T. Luther, G. Breier, V. Ploplis, M. Muller, A. Nagy, E. Plow, R. Gerard, T. Edgington, W Risau, D. Collen. 1997. Ann, N. Y. Acad. Sci. 811, 191-206.

ZC Van Hinsbergh, P. Koolwijk, R. Haanemaijer. 1997. "Role of fibrin and plasminogen activators in repair-associated angiogenesis: in vitro studies with human endothelial cells" EXS 79, 391-411. Passe, T.J., D.A. Bluemke and S.S. Siegelman 1997.

Radiology 203: 593-600. The following non-limiting examples serve to illustrate the concept of multiple receptor specificity. Other combinations of vectors, separators and indicators and conjugation technologies that lead to multiple vector incorporation are also considered relevant or relevant to this invention. Confirmation of the microparticulate nature of the products is performed using microscopy as described in WO-A-9607434. Ultrasonic transmission measurements can be made using a wideband transducer to indicate product suspensions which provides increased sound attenuation compared to a standard. The flow cytometric analysis of the products can be used to confirm the binding of the antibodies to them. The ability of targeted agents to specifically bind to cells expressing the target can be studied by means of microscopy and / or the use of a flow chamber containing immobilized cells, for example, using a population of cells expressing the target structure and an additional population of cells that do not express the target. Streptavidin / radioactive, fluorescent or enzyme-labeled avidin can be used to analyze binding to biotin.

Example 1 - Preparation and biological evaluation of microbubbles containing multiple specific gas of DSPS "added" with a lipopeptide consisting of a peptide that binds to heparin sulfate (KRKR) and a peptide of fibronectin (WOPPRARI).

This example is directed to the preparation of targeted microbubbles comprising multiple peptide vectors arranged in a linear sequence. a) Synthesis of a lipopeptide consisting of a peptide that binds to heparin sulfate (KRKR) and a fibrolectin peptide (WOPPRARI).

The lipopeptide is synthesized in an ABI 433A automatic peptide synthesizer starting with an Fmoc-Ileng resin (Novabiochem) at the 0.1 mmol scale using 1 mmol ammonia cartridges. All amino acids and palmitic acid were preactivated using HBTU before coupling. The simultaneous removal of the peptide from the resin and the side chain protecting groups in TFA containing 5% phenol, 5% EDT, 5% anisole and 5% H20 was carried out for two hours which provides an untreated product yield of 150 mg. Purification was carried out by preparative CLAP (Vydac column 218TP1022) of an aliquot of 40 mg of untreated material using a gradient of 70 to 80% B for 40 min. (A = 0.1% TFA / water and B = MeOH) at a flow rate of 9 ml / min. After lyophilization 16 mg of pure material are obtained (analytical CLAP, gradient, 71-100% of B, where B = MeOH, A = TFA 0.01% / water: column -Vydac 218TP54: detection - UV 260 and fluorescence, EX2a0r Em350 - product retention time = 19.44 min). Further characterization of the product is carried out using MALDI mass spectrometry; expected, M + H at 2198, found at 2199. b) Preparation of microbubbles containing "added" DSPS gas with a multiple specific lipopeptide consisting of a heparin sulfate binding peptide (KRKR) and a fibronectin peptide (WOPPRARI).

DSPS (Avanti, 4.5 mg) and lipopeptide of a) (0.5 mg) are weighed in each of two flasks, and 0.8 ml of a 1.4% propylene glycol / 2.4% glycerol solution are added to each flask. The mixture is heated at 80 ° C for 5 min (2 flasks are stirred during heating). The samples are cooled to room temperature and the free space is purged with perfluorobutane gas. The flasks are shaken in a lid mixer for 45 s and the microbubbles are rotated overnight. The bubbles are washed several times with deionized water and analyzed by Coulter counter (size: 1.3 micrometers (87%), 3-5 micrometers (11.5%)) and acoustic attenuation (maximum frequency reached: 3.5 MHz). The bubbles are stable at 120 mmHg. MALDI mass spectrum analysis was used to confirm incorporation into DSPS microbubbles as follows; AC. 0.05-0.1 ml of microbubble suspension is transferred to a clean bottle and 0.05-0.1 ml of methanol is added. The suspension is sonicated for 30 s and the solution is analyzed by EM MALDI. The positive mode provides M + H at 2200, expected for the lipopeptide, 2198. c) In vitro study of microbubbles containing "added" DSPS gas with a multiple specific lipopeptide consisting of a peptide that binds to heparin sulfate (KRKR) and a fibronectin peptide (WOPPRARI): binding to endothelial cells under conditions flow The human endothelial cell line ECV 304, derived from normal umbilical cord (ATCC CRL-1998) was cultured in 260 ml of Nunc culture flasks (chutney 153732) in RPMI 1640 medium (Bio Whittaker) to which L-glutamine 200 was added. mM, penicillin / streptomycin (10,000 U / ml and 10,000 mcg / ml) and 10% fetal bovine serum (Hyclone Lot No. AFE 5183). Cells were subcultured with a division ratio of 1: 5 to 1: 7 when they reached confluence. 22 mm diameter coverslips were sterilized (BDH, Cat. No. 406/0189/40) and placed in the bottom of 12-well culture plates (Costar) before they were added in the upper cells, in 0.5 ml of complete medium with serum. When the cells reached confluence, the coverslips were placed in a flow chamber manufactured for the purpose. The chamber consists of a slot engraved on a glass plate on which the coverslips are placed with the cells with the cells facing the groove forming a flow channel. Microbubbles for ultrasound of section b) are passed from a reservoir that is at 37 ° C through the flow chamber and back to the reservoir using a peristaltic pump. The flow velocity was adjusted to simulate relevant physiological cutting speeds. The flow chamber was placed under a microscope and the interaction between the microspheres and the cells was directly observed. A microscope mounted camera was connected for color video printing and a monitor. There is a gradual accumulation of microbubbles on the cells, which is dependent on the flow velocity. By increasing the flow rate, the cells begin to detach from the coverslip, the microbubbles are still attached to the cells. Control bubbles that do not present the vector do not adhere to endothelial cells and disappear from the cells under conditions of minimal flow. d) Experiment in vivo in dog Case 1) A 22 kg common dog (mongrel) is anesthetized with pentobarbital and mechanically ventilated. The thorax is opened by sternotomy in the midline, the anterior pericardium is removed, and 30 mm gelled silicone rubber spacers are inserted between the heart and a P5-3 transducer of an ATL HDI-3000 ultrasound scanner. The scanner for intermittent short axis image formation is adjusted once at each end of systole by delayed activation of EGC. A net volume of 2 ml microbubbles of b) was injected as a rapid intravenous bolus. 3 seconds later, the right ventricle is observed in the form of an image which contains contrast material, another 3 seconds later, the lventricle is also filled, and a shadow of transitory attenuation is observed that obscures the view of the posterior parts of the lventricle. There is also a substantial increase in the brightness of the myocardium, also in the portions of the heart distal to the lventricle when the attenuation shadow subsists. After the initial bolus step, the ultrasound scanner is adjusted for high-output high-speed image formation of continuous frames, which method is known to cause destruction of ultrasound contrast agent bubbles in the tissue regions of the cells. which images are formed. After a few seconds, the scanner is adjusted again to its initial setting. The myocardium then appears darker and closer to the baseline value. Moving the divided image to a new position results in the reappearance of the contrast effects, moving the slide back to the initial position results again in a brightness of tissue that is close to the baseline.

Case 2) Tcomparativol A net volume of 2 ml of microbubbles prepared in a manner identical to b) above is injected, with the exception that lipopeptide is not included in the preparation, using the same imaging procedure as in the above. The improvement of the myocardial echo was much less intense and of shorter duration compared to that observed in case 1. At the end of the left ventricular attenuation phase, there was also an almost complete loss of myocardial contrast effects, and it was not observed an increase in myocardial echo in the posterior part of the left ventricle as in case 1.

Example 2 - microbubbles containing multiple specific gas of DSPS "added" with RGCD-Mal-PEG or -DSPE and a lipopeptide consisting of a peptide that binds to heparin sulfate (KRKR) and a fibronectin peptide (WOPPRARI).

This Example is directed to the preparation of directed microbubbles comprising multiple peptide vectors. a) Synthesis of 3-maleimidopropionylamido-PEG2000-acildiestearoyl phosphatidylethanolamine (PE-PEG-MAL) A mixture of distearoyl phosphatidylethanolamine (DSPE), (37.40 mg, 0.005 mmol), N-hydroxysuccinimido-PEG2000-p? Aleimide, NHS-PEG-MAL, (100 mg, 0.25 mmol) and triethylamine is stirred at room temperature for 24 hours. (35 μl, 0.25 mmol) in a chloroform / methanol (3: 1) solution. After evaporation of the solvents under reduced pressure, the residue is purified by flash chromatography (chloroform / methanol, 8: 2). The product is obtained as a white wax, 92 mg (66%) and the structure is verified by NMR and MALDI MS. b) Summary of RGDC The RGDC peptide is synthesized in an ABI 433A automated peptide synthesizer (scale 0.25 mmoles, Fmoc-Cys (Trt) -Wang resin, (Novabioche) All amino acids were activated using HBTU, the untreated peptide is removed from the resin and it is simultaneously deprotected in TFA containing 5% EDT, 5% phenol and 5% water.After evaporation of the excess separation solution, the peptide is precipitated and triturated several times with diethyl ether before air drying. the untreated peptide by preparative CLAP and the fractions containing the pure product are combined and freeze-dried.The final characterization is performed using analytical CLAP and MALDI EM. c) Preparation of microbubbles filled with multiple specific gas encapsulated by phosphatidylserine and "added" with RGCD-Mal-PEG? 10t, -DSPE and a lipopeptide comprising a heparin-binding hepatic sulfate (KRKR) and a fibronectin peptide (WOPPRARI) Weigh into a clean vial DSPS (Avanti, 5.0 mg), lipopeptide (0.5 mg) from example 1 a) and PE-PEG-MAL (0.5 mg) from section a), and add 1.0 ml of a propylene glycol solution 1.4% / glycerol 2.4%. The mixture is sonicated for 3-5 min, heated at 80 ° C for 5 min and then filtered through a 4.5 micrometer filter. The mixture is cooled to room temperature and the free space is purged with perfluorobutane gas. The flasks are shaken in a lid mixer for 45 s and the microbubbles are centrifuged at 1000 rpm for 3 min. The infranatant is exchanged with 1 ml of PBS containing 1 mg of the RGDC peptide and the pH is adjusted to 8. The conjugation reaction is allowed to proceed for 2 h. The bubbles are washed in PBS and then with water until all the unreacted RGDC from the infranatant has been removed, when observed by EM MALDI. The microbubbles are further analyzed by Coulter counter (98% between 1 and 7 micrometers). d) In vitro binding assay Binding of microbubbles to endothelial cells is carried out under flow conditions using the in vitro assay described in example 1 c). There is a gradual accumulation of microbubbles on the cells, which is dependent on the flow velocity. Control bubbles that do not have vectors do not adhere to the endothelial cells detaching from the cells under conditions of minimal flow.

Example 3) Preparation of microbubbles containing multiple specific gas encapsulated with DSPS and typed with anti-CD62-Mal-PEG2000-PE and typed with anti-ICAM-l-Mal-PEG, 000-PE This example is directed to the preparation of microbubbles comprising multiple vectors of antibodies for directed ultrasound. a) Preparation of microbubbles containing gas encapsulated with DSPS and PE-PEG2000-MAL Weigh into a limpip DSPS bottle (Avanti, 4.5 mg) and PE-PEG20oo ~ Maleimi, * y of example 2 a) (0.5 mg) and add 1 ml of a 1.4% propylene glycol / glycerol 2.4% solution. The mixture is heated at 80 ° C for 5 min and then filtered through a 4.5 micrometer filter. The sample is cooled to room temperature and the free space is purged with perfluorobutane gas. The flasks are shaken in a lid mixer for 45 s and the microbubbles are washed three times with distilled water. b) Thiolation of antibodies against CD62 and against ICAM-1 To 0.3 mg of each of antibodies against CD62 and against ICAM-1 dissolved in PBS buffer (pH 7, 0.5 ml) is added Traut's reagent and the solutions are stirred at room temperature for 1 h. Excess reagent is separated from the modified protein on a NAP-5 column (Pharmacia). c) Conjugation of antibodies typed against CD62 and against ICAM-1 to microbubbles containing gas encapsulated with DSPS and PSPS-PEG2000-MAL 0.5 ml of mixed thiolated antibody preparation from b) is added to an aliquot of microbubbles of a) and the conjugation reaction is allowed to proceed for 30 min on a rotary table. After centrifugation at 2000 rpm for 5 min, the infranatant is removed. The microbubbles are washed three additional times with water. You can also vary the length of the PEG separadpr to include larger chains, for example PEG3400 and PEG5000 p more certains, for example PEG600 or PEG800. The addition of a third antibody such as thiolated anti CD34 is also considered.

Example 4) Multiple targeted gas-containing microbubbles directed from DSPS coated non-covalently with polylysine and a fusion peptide comprising a PS-binding component and a fibronectin peptide sequence NH2F.N.F.R.L.K.A.G.O.K.I.R.F.G.G.G.G.W.O.P.R.A.I.OH. a) Synthesis of the fragmentp fusion peptide that binds to PS-fibronectin NHoF.N.F.R.L.K.A.G.O.K.I.R.F.G.G.G.G.W.O.P.R.R.A.I.OH.

The peptide is synthesized in an automated ABI 433A peptide synthesizer starting with Fmoc-Ile-Wang resin (Novabiochem) at 0.1 mmol scale using 1 mmol ammo cartridges. All amino acids are preactivated using HBTU before coupling.

The simultaneous removal of the peptide from the resin and side chain prptectcres lbs is carried out in TFA, which contains 5% fencl, 5% EDT and 5% H20 for 2 hpras, which yields an untreated prpductc yield of 302 mg. The purification per preparative CLAP (Vydac 218TP1022 column) of an aliquot of 25 mg of untreated material is carried out using a gradient of 20 to 40% B for 40 min (A = 0.1% TFA / water and B = 0.1 TFA) % / acetonitrile) at a flow velocity of 9 ml / min. After lipfilization, 10 mg of purp material (analytical CLAP, gradient, 20 to 50% B, where B = 0.1% TFA / acetonitrile, A = 0.1% TFA / water: vdadac column 218TP54: UV detection 214) and 260 nm - product retention time = 12.4 min). An additional characterization of the product is carried out using MALDI mass spectrometry; expected, M + H 2856, found, at 2866. b) Preparation of non-covalently coated DSPS microbubbles with polylysine and a fusion peptide of the PS / fibronectin binding fragment NH2F.N.F.R.L.K.A.G.O.K.I.R.F.G.G.G.G.W.O.P.R.A.I.OH.

DSPS (5 mg, Avanti) is weighed into a clean vial together with poly-L-lysine (Sigma, 0.2 mg) and peptide from a) above (0.2 mg). To the bottle add 1.0 ml of a 1.4% propylene glycol / glycerol 2.4% solution. The mixture is heated at 80 ° C for 5 min. The mixture is cooled to room temperature and the free space is purged with perfluorobutane gas. The flasks are shaken in a lid mixer for 45 s and the microbubbles are centrifuged at 1000 rpm for 3 min. After extensive washing with water, PBS and water, the final solution is examined to detect polylysine and peptide content using MALDI EM. No polypeptide material is observed in the final wash solution. Then acetonitrile (0.5 ml) is added and the bubbles are destroyed by sonication. Analysis of the resulting solution for polylysine and fusion peptide that binds to PS-fibronectin is then carried out using MALDI MS. The results are as follows: The spacer element contained within the fusion peptide that binds to PS-fibronectin (-GGG-) can also be substituted with other spacers such as PEG20oo-11 $ -polyalanine (-AAA-). It is also considered that a pre-directed form may be used, wherein the fusion peptide of the fragment binds to DSPS / fibronectin first allowed to associate with cells via the binding of the peptide to fibronectin. This is followed by the administration of PS microbubbles which then bind to the PS binding peptide.

Example 5 - Microbubbles containing multiple specific aas encapsulated with fpsphatidylserine and biotin-PEG300-alanyl-cholesterol and functionalized with streptavidin / biotinyl-endotelyl-1 peptide (biotin-D-Trp-Leu-Asp-Ile-Trp.OH) and antipolimentary biotinyl-fibrin (biotin- GPRPPERHOS.NH ,, * This example is directed to the preparation of directed ultrasound microbubbles where streptavidin is used as a linker between indicators and biptinadps vectcres. a) Synthesis of biPtin-PEG3.100-ß-alanine cholester To a hydrochloride solution of c-clesteryl-ß-alanine (15 mg, 0.03 m.sup.-1) in 3 ml of chloroform / wet methanol (2.6: 1), triethylamine (42 ml, 0.30 mmol) was added. The mixture is stirred for 10 min at room temperature and a solution of biotin-PEG3400-NHS (100 mg, 0.03 mmol) in 1,4-dioxane (1 ml) is added dropwise. After stirring at room temperature for 3 h, the mixture is evaporated to dryness and the residue is purified by flash chromatography to provide white crystals, yield; 102 mg (89%). The structure is verified by EM MALDI and NMR. b) Synthesis of peptide endotelin-1 bjptinadp (biotin-D-Trp-Leu-Asp-Ile-Trp.OH) The peptide is synthesized in an ABI 433A peptide automate synthesizer starting with Fmoc-Trp (Boc) -Wang (Novabiochem) resin at 0.1 mmol scale using 1 mmol ammo cartridges. All amino acids are preactivated using HBTU before coupling. The simultaneous removal of the peptide from the resin and the side chain protecting groups in TFA containing 5% anizol and 5% H20 for 2 hours is carried out which provides an untreated product yield of 75 mg. Purification by preparative CLAP (column Vydac 218TP1022) of an aliquot of 20 mg of crude material is carried out using a gradient of 30 to 80% B for 40 min (A = 0.1% TFA / water and B = 0.1% TFA). / acetonitrile) and a flow rate of 9 ml / min. After lyophilization of the pure fractions, 2 mg of pure material are obtained (analytical CLAP, gradient, 30-80% of B where B = 0.1% TFA / acetonitrile, A = 0.01% TFA / water: column - Vydac 218TP54: detection - UV 214 nm - product retention time = 12.6 min). An additional product characterization is carried out using MALDI mass spectrometry, expected, M + H at 1077, found 1077. c) Synthesis of biotinyl-fibrin-anti-polymerizing peptide (Biotin-GPRPPERHOS.NH) This peptide is synthesized and purified using protocols similar to those described in section b) above. The pure product is characterized by CLAP and EM MALDI. d) Preparation of microbubbles filled with multiple specific gas encapsulated with phosphatidylserine and biotin-PEG3100-β-alanine cholesterol Weigh into a DSPS bottle (Avanti, 4.5 mg) and biotin-PEG3400-β-alanine cholesterol from section a) (0.5 mg) and add 0.8 ml of a 1.4% propylene glycol / glycerol 2.4% solution. The mixture is heated at 80 ° C for 5 min (2 flasks are stirred during heating). The sample is cooled to room temperature and then the free space is purged with perfluorobutane gas. The bottle is shaken in a mixer with a lid for 45 s and the microbubbles are rolled up overnight. The microbubble suspension is washed several times with deionized water and analyzed by Coulter counter and acoustic attenuation. e) Conjugation with labeled streptavidin with fluorescein and biotinylated peptides in section b) and Q)To the microbubble preparation of d) streptavidin was added with fluorescein (0.2 mg) dissolved in PBS (1 ml). The bubbles are cycled on a rotating table for 3 h at room temperature. After extensive washing with water and analysis by microcosm of fluorescence, the microbubbles are incubated in 1 ml of PBS containing peptide biotinil-endotelil-1 (0.5 mg) and peptide biotinil-fibrin-anti-polymeriser (0.5 mg) of sections b ) and c) respectively, for 2 h. Extensive washing of the microbubbles is performed to remove the conjugated peptide.

Example 6 - Multiple specific gas filled microbubbles encapsulated with phosphatirylserine and a biotinylated lipopeptide used to prepare an "interposition" of streptavidin with a biotinyl-endothelin-1 peptide mixture (biotin-D-Trp-Leu-Asp-Ile-Trp. OH) and biotinyl-fibrin-antipolymer peptide (biotin-GPRPPERHOS.NH) a) Synthesis of lipopeptide dipalmitoyl-lisinyl-tryptophanyl-lysinyl-lysinyl-lisinyl (biotinyl) -glycine The lipopeptide is synthesized in an automatic ABI 433A peptide synthesizer from Fmoc-Gly-Wang resin (Novabiochem) at 0.1 mmol scale using 1 mmol ammo cartridges. All amino acids and palmitic acid are preactivated using HBTU before coupling. The simultaneous removal of the peptide from the resin and the side chain protecting groups in TFA containing 5% phenol, 5% EDT, 5% anisole and 5% H20 for 2 h is carried out, which provides a yield of untreated product of 150 mg. Purification by preparative CLAP (Vydac column 218TP1022) from an aliquot of 40 mg of untreated material is carried out using a gradient of 70 to 100% B for 40 min (A = 0.1% TFA / water and B = MeOH) at a flow rate of 9 ml / min. After lyophilization 14 mg of pure material are obtained (analytical CLAP, gradient, 70-100% of B where B = MeOH, A = 0.1% TFA / water: column - Vydac 218TP54: detection - UV 260 and fluorescence, extinction 280 , emission 350 - product retention time = 22 min). Further characterization of the product is carried out using MALDI mass spectrometry; expected M + H at 1478, found 1471. b) Preparation of microbubbles containing "added" DSPS gas with the biotinylated lipopeptide sequence of section a) They were weighed in each of two DSPS bottles (Avanti, 4. 5 mg) and lipopeptide from a) (0.5 mg) and 0.8 ml of a 1.4% propylene glycol / glycerol 2.4% solution were added to each bottle. The mixture is heated at 80 ° C for 5 min (the flasks are stirred during heating). The samples are cooled to room temperature and the free space is purged with perfluorobutane gas. The bottles are shaken in a lid mixer for 45 s and the microbubbles are formed by spinning at night. The microbubbles are washed several times with deionized water and analyzed by Coulter counter and acoustic attenuation. The MALDI mass spectrum analysis is used to confirm the incorporation into the microbubbles of DSPS as described in example 1 b). c) Preparation of microbubbles filled with multiple specific gas encapsulated with phosphatidylserine and a biotinylated lipopeptide functionalized with streptavidin / biotinyl-endothelin-1 peptide (biotin-D-Trp-Leu-Asp-Ile-Trp.OH) and biotinyl-fibrin peptide -antipoli ester (biotin-GPRPPERHOS.H2) The microbubble preparation of b) is treated analogously to that described in example 5, section e).

Example 7 - Multiple specific gas filled microbubbles encapsulated with phosphatidylserine and biotin-DPPE used to prepare an "interpolation" of streptavidin with a biotinyl-endothelin-1 peptide mixture (biotin-D-Trp-Leu-Asp-Ile-Ile- Trp.OH) and biotinyl-fibrin-antipolimentary peptide (biotin- GPRPPERHOS.NHo) a) Preparation of microbubbles containing biotin To a mixture of phosphatidylserine (5 mg, Avanti) and biotin-DPPE (0.6 mg, Pierce) in a clean bottle add propylene glycol-glycerol 5% in water (1 ml). The dispersion is heated at 80 ° C for 5 min and then cooled to room temperature. The free space is then purged with perfluorobutane and the flask is shaken in a mixer with a lid for 45 s. After centrifugation, the infranatant is removed and the microbubbles formed are extensively washed with water. b) Conjugation of microbubbles filled with capsules with phosphatidylserine and biotin-DPPE with streptavidin and a mixture of biotinyl-endothelin-1 (biotin-D-Trp-Leu-Asp-Ile-Ile Trp. OH) and biotinyl-fibrin peptide antipolimentary (biotin- GPRPPERHOS.NH) The procedure detailed in example 5 section e) is followed.

Example 8 - Multiple specific gas-filled microbubbles encapsulated with phosphatidylserine, streptavidin-Succ-PEG-DSPE and a mixture of biotinylated human endothelium IgG antibody and biotinylated transferrin a) Synthesis of Succ-PEG3 0-DSPE NH2-PEG3400-DSPE is carboxylated using succinic anhydride, for example, by a method similar to that described by Nayar, R. and Schroit, A.J. in Biochemistry (1985) 24, 5967-71. b) Preparation of gas-filled microbubbles encapsulated with phosphatidylserine and Succ-PEG3400-DSPE To a mixture (5 mg) of phosphatidylserine (90-99.9 mmole%) and Succ-PEG3400-DSPE (10-0.1 mmole%) is added propylene glycol-glycerol 5% in water (1 ml). The dispersion is heated to not more than 80 ° C for 5 min and then cooled to room temperature. The dispersion (0.8 ml) is transferred to a bottle (1 ml) and the purge-free space with perfluorobutane. The bottle is shaken in a mixer with lid for 45 s, then the sample is placed on a rotating table. After centrifugation the infranatant is exchanged with water and the washing is repeated. c) Coupling of streptavidin to gas-filled microbubbles encapsulated with phosphatidylserine and Succ-PEG3400-DSPE Streptavidin is covalently linked to Succ-PEG300- DSPE in the membranes by standard coupling methods using water-soluble carbodiimide. The sample is placed on a rotating table during the reaction. After centrifugation in infranatant, it is exchanged with water and the washing is repeated. The functionality of bound streptavidin is analyzed by binding, for example, to fluorescently labeled biotin, biotinylated antibodies (detected by a fluorescently labeled secondary antibody) or biotinylated and fluorescently or radioactively labeled oligonucleotides. The analysis is performed by fluorescence microscopy or scintillation counting. d) Preparation of multiple specific gas-filled microbubbles encapsulated with phosphatidylserine and Succ-PEG3400-DSPE-12? f-non-covalently functionalized with biotinylated human transferrin and human endothelium IgG antibody The microbubbles of section c) are incubated in a solution containing human transferrin and biotinylated human endothelium IgG antibody using the method described by Bayer et al., Meth. Enzymol., 62, 308. The vector-coated microbubbles are washed as described above.

Example 9 - Multiple specific gas filled microbubbles encapsulated with phosphatidylserine / streptavidin-Succ-PEG-DSPE and oligonucleotides biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and biotin-GGCGCTGATGATGTTGTTGATTCTT a) Synthesis of Succ-PEG3 0-DSPE Described in example 8 a) b) Preparation of gas-filled microbubbles encapsulated with phosphatidylserine and Succ-PEG3400-DSPE Described in example 8 b) c) Coupling of estraptividin to gas-filled microbubbles encapsulated with phosphatidylserine and Succ-PEG3400-DSPE Described in example 8 c). d) Preparation of gas-filled microbubbles encapsulated with phosphatidylserine / streptavidin-Succ-PEG-DS E and the oligonucleotides biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and biotin-GGCGCTGATGATGTTGTTGATTCTT The microbubbles of section c) are incubated in a solution containing a mixture of biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and biotin-GGCGCTGATGATGTTGTTGATTCTT. The microbubbles coated with oligonucleotide are washed as described above. The binding of the oligonucleotide to bubbles is detected, for example, by using a fluorescent labeled oligonucleotide for binding to bubbles, or by hybridizing the oligonucleotide bound to a labeled complementary oligonucleotide (fluorescence or radioactivity). The functionality of the microbubbles that present oligonucleotide is analyzed, for example, by hybridizing the bubbles with sequences containing immobilized DNA complementary to the attached oligonucleotide. Other useful examples include an oligonucleotide complementary to ribosomal DNA (of which there are many copies per haploid genome) and an oligonucleotide complementary to an oncogene is used (for example ras, of which one copy exists per haploid genome).

Example 10 - Multiple specific gas filled microbubbles encapsulated with phosphatidylserine and phosphatidylethanolamine covalently functionalized with the fibronectin and transferrin proteins a) Preparation of microbubbles Weigh in a clean flask DSPS (Avanti, 4.5 mg) and DSPE (Avanti, 1.0 mg) and add 1 ml of a 1.4% propylene glycol / glycerol 2.4% solution. The mixture is heated at 80 ° C for 5 min and then filtered through a 4.5 micrometer filter. The sample is cooled to room temperature and the free space is purged with perfluorobutane gas. The bottle is shaken in a blender with a lid for 45 s and the microbubbles are washed twice with distilled water and then resuspended in 0.1 M sodium borate buffer, pH 9. b) Modification of fibronectin / transferrin Fibronectin (0.5 mg) and transferrin are mixed (1.3 mg) in PBS and a solution containing NHS-fluorescein in DMSO is added. The mixture is stirred at room temperature for 1 h and then purified by proteins in a column Superdex 200. The mixture of fluorescein-labeled protein is lyophilized in phosphate buffer pH 7.5 c) Modification of microbubbles The lyophilized product of b) is dissolved again in 0.5 ml of water and to the mixture of fibronectin labeled with fluorescein / transferrin 0.1 mmols of SDBP crosslinker (Pierce) are added. The solution is incubated on ice for 2 h, loaded onto a NAP-5 column and eluted with PBS. To this is added 1 ml of the microbubble suspension of a) and the incubation is allowed to proceed for 2 h at room temperature on a rotary table. The material that has not reacted is removed by allowing the microbubbles to float and then replacing the buffer with water, this process is repeated three times.

Example 11) Preparation of multiple specific hollow polymer particles incorporate avidin in the polymeric wall conjugated to the oligonucleotide biotin-GGCGCTGATGATGTTGTTGATTCTT and the peptide endotelin-1 biotin-D-Trp-Leu-Asp-Ile-Ile-Trp. OH This example is directed to the preparation of polymeric ultrasound contrast agents comprising multiple vectors linked to non-surfactant for targeting / therapeutic applications. a) Preparation of polymer particles that incorporate avidin in the polymer wall Hollow polymer particles of P73 (as described in WO 96/07434) containing avidin are prepared by a process involving the lyophilization of an oil-in-water emulsion using the following procedure: an oily solution is prepared by dissolving 0.25 g of the biodegradable polymer P73 [poly (ethylidenebis (16-hydroxyhexadecanoate) -co (adipic acid)] in 5 ml of camphene at 60 [deg.] C. To 0.2 ml of the oil solution 2 mg of avidin are added. aqueous solution by dissolving 0.4 g of the polymer, a- (16-hexadecanoyloxyhexadecanoyl) -w-methoxypolyoxyethylene ester, in 20 ml of water at 60 ° C. The oil solution (0.2 ml) is then mixed with the aqueous solution (0.8 ml) in a vibromezclador (Capmix) for 15 s to form the emulsion of oil in water.The emulsion is frozen in dry ice and methanol, and then dried at a pressure of 200 mTorr for 24 h to remove excess solvent.The powder is rebuilt as a susp Hollow particles are added by adding 1.0 ml of water. The resulting ultrasound contrast agent is confirmed by microscopic observation, size distribution in Coulter, acoustic attenuation and resistance to external pressure. - 13S - b) Synthesis of biotin-D-Trp-Leu-Aspp-Ile-Ile-Trp.OH Described in example 5 b). c) Conjugation of polymer particles that incorporate avidin The particles of a) are centrifuged and the supernatant is replaced with 1 ml of PBS buffer pH 7.5 containing 0.2 mg of biotin-GGCGCTGATGATGTTGTTGATTCTT and 0.2 mg of biotin-D-Trp-Leu-Asp-Ile-Ile-Trp.OH of b) previous. After incubation for 24 h the particles are washed extensively with PBS and water.

Example 12 - Functionalization of microspheres of gas-filled albumins (GAM) with biotin for multiple specific addressing a) Preparation of biotinylated albumin microbubbles A homogeneous suspension of GAM (6 x 108 particles / ml) is used in 5 mg / ml of albumin, where all manipulations are performed at room temperature. Two 10 ml aliquots (170 xg, 5 min) are centrifuged to promote the flotation of the microspheres and 8 ml of the underlying infranatant is removed by careful suction and replaced by an equal volume of buffered saline with air-saturated phosphate, the Preparations are rotated for 15-20 min to resuspend the microspheres. The procedure is repeated twice, after which only negligible amounts of free albumin not associated with microspheres are supposed to remain. 50 μl of NHS-biotin (10 mM in dimethyl sulfoxide) is added to one of the aliquots (final concentration, 50 μM); the other aliquot (control) receives 50 μl of dimethyl sulfoxide. The tubes containing the samples are rotated for 1 h after which 20 μl portions of 50% aqueous glutaraldehyde are added to each tube to crosslink the microspheres. After rotation for another hour, the tubes are placed vertically overnight to allow the flotation of the microspheres. The next day, the suspensions are washed twice with phosphate buffered saline containing 1 mg / ml human serum albumin (PBS / HSA) and resuspended in PBS / HSA after the last centrifugation. In order to determine the presence of biotin associated with microspheres, horseradish peroxidase conjugated streptavidin (strep-HRP) is added to both suspensions and the tubes are rotated for 1 h to allow the reaction to take place. The microspheres are then washed three times, resuspended in 100 mM citrate-phosphate buffer (pH 5) containing 0.1 mg / ml of phenylenediamine dihydrochloride and 0.01% hydrogen peroxide, and rotated for 10 min. The development of a yellow-green color is indicative of the presence of enzyme. The following results are obtained: This confirms that GAM was biotinated. b) Microparticles containing multiple specific gas Subsequently, the biotinylated microspheres are used to prepare multiple specific target products in a manner analogous to that exemplified in examples 5), 6) and 7).

Example 13 - Microbubbles containing multiple specific gas of DSPS functionalized with peptide sue binds to heparin sulfate / fibronectin peptide / RGD peptide and fluorescein. a) Synthesis of a lipopeptide sue contains the sequence RGD and a fluorescein indicator group: dipalmoyl Lvs-Lys-Lys-Lys racetil-Ara-Gly-Asp-Lvs (Fluorescein) 1 Gly. OH The lipopeptide is synthesized as described in example 1) using commercially available amino acids and polymers. The lipopeptide is separated from the resin in TFA containing 5% water, 5% phenol, 5% EDT for 2 h. After evaporation in vacuo, the crude product is precipitated and triturated with diethyl ether. Purification by preparative CLAP (Vydac 218TP1022 column) from an aliquot of 40 mg of untreated material is carried out using a gradient of 60 to 100% B for 40 min (A = 0.1% TFA / water and B = TFA 0.1% / acetonitrile) at a flow rate of 9 ml / min. After lyophilization of 10 mg of pure material (analytical CLAP, gradient, 60-100% of B where B = 0.1% TFA / acetonitrile), A = 0.01% TFA / water: column - vydac 218TP54: Detection - UV 260 - product retention time - 20-22 minutes). The additional characterization of the product was carried out using MALDI mass spectrometry; expected, M + H to 1922, found in 1920. b) Synthesis of a lipopeptide containing a heparin sulfate binding sequence and a fibronectin peptide Synthesis and purification as described in example la). c) Preparation of microburbuids containing multiple specific gas of DSPS functionalized with a peptide that binds to heparin sulfate and a peptide of fibronectin, an acetyl-RSD peptide and fluorescein Weighed in each of two bottles DSPS (Avanti, 4 mg) and lipopeptide from a) (0.5 mg, 0.2 mmol) and lipopeptide from b) (0.5 mg), and 0.8 ml of a propylene glycol solution was added to each bottle at 1.4% / glycerol at 2.4%. The mixture was heated at 80 ° C for 5 minutes (the bottles were shaken during heating). The samples were cooled to room temperature and the free space was purged with perfluorobutane gas. The flasks were shaken in a lid mixer for 45 seconds and the microbubbles formed were rotated overnight. The microbubbles were washed several times with deionized water and analyzed by MALDI mass spectrometry as described in Example I b). It is observed that the microbubbles after analysis by microscopy consist of a size range between 1 and 5 micrometers. In addition, the microbubbles were fluorescent.

Example 14. Microbubbles containing multiple specific gas of DSPS covalently modified with receptor antibody against transferrin labeled with FITC CD71, receptor antibody and "added" with a lipopeptide with affinity for endothelial cells This example is directed to the preparation of ultrasound agents that are directed against multiple vectors. a) Synthesis of a lipopeptide that binds to endothelial cells: 2-n-hexadecilestearyl-Lys-Leu-Ala-Leu-L? s-Leu-Ala-Leu-Lys-Ala-Leu-Lys-Ala-Al-Leu -Lys-Leu-Ala-NH2.

The lipopeptide shown below is synthesized on an automatic ABI 433A peptide synthesizer starting with a Rink amide resin on a 0.1 mmol scale using 1 mmol ammo cartridges.

All amino acids and 2-n-hexadecyl stearic acid were preactivated using HBTU before coupling. The simultaneous removal of the peptide from the resin and the side chain protecting groups was carried out in TFA containing 5% EDT and 5% H20 for 2 hours which provides an untreated product with a yield of 150 mg. Purification by preparative CLAP (column Vydac 218TP1022) from an aliquot of 40 mg of untreated material was carried out using a gradient of 90 to 100% B for 5O minutes (A = 0.1% TFA / water and B = MeOH ) at a flow rate of 9 ml / min. After lyophilization 10 mg of pure material are obtained (analytical CLAP, gradient, 90-100 B% where B = MeOH, A = TFA 0.01% / water: column - Vydac 218TP54: Detection = UV 214 nm - retention time of product - 23 minutes). The additional product characterization was carried out using MALDI mass spectrometry; expected, M + H at 2369, found at 2373. b) Preparation of microbubbles containing "added" DSPS gas with lipopeptide that binds to endothelial cells and PE-PEG200-MAL They were weighed in a clean DSPS bottle (Avanti, 4. 5 mg) and lipopeptide from A (0.5 mg) together with PE-PEG2000-maleimide from example 2 (0.5 mg) and 1 ml of a 1.4% propylene glycol / glycerol 2.4% solution was added. The mixture was heated at 80 ° C for 5 minutes and then filtered through a 4.5 micrometer filter. The mixture was cooled to room temperature and the free space was purged with perfluorobutane gas. The flasks were shaken in a lid mixer for 45 s and the microbubbles were washed three times with distilled water. c) Thiolation of receptor antibody against transferrin labeled with FITC The Ab antibody against transferrin CD71 labeled with FITC (100 mg / ml, Becton, Dickinson), 0.7 ml in PBS was modified with Traut's reagent (0.9 mg, Pierce) at room temperature for 1 h. The excess reagent was separated from the modified protein on a NAP-5 column (Pharmacia). d) Conjugation of the receptor antibody against transferrin labeled with thiolated FITC to microbubbles containing "added" DSPS gas with lipopeptide that binds to endothelial cells and DSPE-PEG-, nnQ-MAL An aliquot of 0.5 ml of the protein fraction (2 ml in total) was added from c) to the microbubbles of b) and the conjugation reaction was allowed to proceed for 10 min on a rotary table. After centrifugation at 1000 rpm for 3 min, the protein solution was removed and the conjugation was further repeated twice with 1 ml aliquots and 0.5 ml of protein solution, respectively. The bubbles were then washed four times in distilled water and a sample was analyzed for the presence of antibody by flow cytometry and microscopy. A fluorescent population of > 92 ' rpoog Cytometric comparison of negative control microbubbles flow of DSPS (left curve) with bubbles conjugated with antibody against transferrin labeled with FITC CD71 (black curve, right) showing 92% fluorescence in the population.

The incorporation into the microbubbles of the lipopeptide was confirmed by MALDI mass spectrometry as described in example I b).

Example 15: Preparation of gas-filled microbubbles coated with multiple specific transferrin / avidin for directed ultrasound imaging This example is directed to the preparation of microbubbles containing multiple protein vectors for ultrasound / therapy a) Synthesis of thiol-functionalized lipid molecule: Dipalmitoyl-Lys-Lys-Lys-Aca-Cvs .OH The lxpido structure shown above was synthesized on an ABI 433A automatic peptide synthesizer starting with Fmoc-Cys (Trt) -Wang (Novabiochem) resin at 0.25 ml scale using 1 mmol amino acid cartridges. All amino acids and palmitic acid were preactivated using HBTU coupling chemistry. The simultaneous removal of the peptide from the resin and the deprotection of the side chain protecting groups was carried out in TFA containing 5% EDT and H205% for 2 hours which gives an untreated product yield of 250 mg. Purification is carried out by preparative CLAP (Vydac 218TP1022 column) of an aliquot of 40 mg of untreated material using a gradient of 90 to 100 B% for 50 min (A = 0.1% TFA / water and B = MeOH) a a flow rate of 9 ml / min. After lyophilization, 24 mg of pure material are obtained (analytical CLAP, gradient, 70-100 B% where B = 0.1% TFA / acetonitrile, A =% TFA 0.01% / water: column -vydac 218TP54: Detection - UV 214 nm - product retention time - 23 min). Additional product characterization was carried out using MALDI mass spectrometry; expected, M + H at 1096, found at 1099. b) Preparation of microbubbles containing "added" DSPS gas with a thiol-containing lipid structure; Weigh in a clean DSPS bottle (Avanti, 4.5 mg) and the lipid structure of a) before (0.5 mg), and add 0.8 ml of a solution containing 1.4% propylene glycol / 2.4% glycerol in water. The mixture was heated at 80 ° C for 5 minutes (the bottles were stirred during heating) and filtered while still hot through a 40 micron filter. The samples were cooled to room temperature and the free space was purged with perfluorobutane gas. The flasks were shaken in a lid mixer for 45 s and the microbubbles were placed on a rotating table overnight. The bubbles were washed several times with deionized water and analyzed for thiol group incorporation using Ellmans reagent. c) Modification of transferrin v avidin with fluorescein-NHS v sulfo-SMPB To a mixture of 2 mg of transferrin (Holo, human, Alpha Therapeutic Corp) and 2 mg of avidin (Sigma) in PBS (1 ml) is added 0.5 ml of a solution of DMSO containing 1 mg of sulfo-SMPB (Pierce ) and 0.5 mg of fluorescein-NHS (Pierce). The mixture is stirred for 45 minutes at room temperature and then passed through a Shepadex 200 column using PBS as eluent. The protein fraction is collected and stored at 4 ° C before use. d) Conjugation of microbubbles with modified transferrin-avidin To the thiol-containing microbubbles of b) is added 1 ml of solution c) of transferrin protein / modified avidin. After adjusting the pH of the solution to 9, allow the conjugation reaction to proceed for 2 h at room temperature. After extensive washing with deionized water the microbubbles are analyzed by a Coulter counter (81% between 1 and 7 micrometers) and fluorescence microscopy. Highly fluorescent microbubbles are observed.

Example 16: Preparation of gas-filled microbubbles functionalized for directed ultrasound imaging This example is directed to the preparation of microbubbles having a reactive group on the surface for non-specific targeting, mainly using disulfide exchange reactions to carry out binding to a multiplicity of cell targets. Weigh in a clean DSPS bottle (Avanti, 5.0 mg) and the thiol containing the lipid structure of example 15 a) (1.0 mg) and add 0.8 ml of a solution containing 1.4% propylene glycol / 2.4% glycerol. The mixture is heated at 80 ° C for 5 minutes (the flasks are stirred during heating) and filtered while still hot through a 40 micron filter. The samples are cooled to room temperature and the free space is purged with perfluorobutane gas. The bottles are shaken in a mixer with a lid for 45 s and the microbubbles are placed on a rotating table overnight. The bubbles are washed several times with deionized water and analyzed for thiol group incorporation using the Ellmans reagent.

Example 17 - Microburbu containing gas, specific multiple of DSPS sue comprise a lipopeptide which targets endothelial cells and a molecule containing captopril This example is directed to the preparation of ultrasonic agents for combined targeting and therapeutic applications. a) Synthesis of a functionalized lipopeptide with captopril The structure shown below is synthesized using a manual nitrogen sparger apparatus starting with Rink Amida MBHA resin protected with Fmoc (Novabiochem) at a scale of 0.125 mmoles. All amino acids were purchased from Novabiochem and palmitic acid from Fluka. The coupling was carried out using standard TBTU / HOBt / DIEA protocols. Bromoacetic acid was coupled through the side chain of Lys as a symmetric anhydride using pre-activation with DIC. Captopril (Sigma) was dissolved in DMF and introduced into the solid phase using DBU as a base. The simultaneous removal of the peptide from the resin and the deprotection of the side chain protecting groups was carried out in TFA containing 5% EDT, 5% water and 5% ethylmethyl sulfide for 2 h. A 10 mg aliquot of the untreated material was purified by preparative liquid chromatography (Vydac 218TP1022 column) using a gradient of 70 to 100% B for 60 min (A = 0.1% TFA / water and B = 0.1% TFA / acetonitrile) at a flow rate of 10 ml / min. After lyophilization a yield of 2 mg of pure material is obtained (analytical CLAP: gradient 70-100% B for 20 min, A = 0.1% TFA / water and B = 0.1% TFA / acetonitrile / flow rate 1 ml / min; Vydac column 218TP54, UV detection 214 nm, retention time 26 min). Further characterization is carried out using MALDI mass spectrometry, which provides M + H at 1265 as expected. b) Synthesis of a lipopeptide with affinity for endothelial cells: dipalmitoyl-Lys-Lys-Lys-Aca-Ile-Arg-Arg-Val-Ala-Arg-Pro-Pro-Leu-NH-, The lipopeptide is synthesized on an ABI 433A automatic peptide synthesizer starting with Rink amide resin (Novabiochem) at 0.1 mmol scale using 1 mmol amine cartridges. All amino acids and palmitic acid were preactivated using HBTU before of the coupling. Simultaneous removal of the peptide from the resin and side chain protecting groups was carried out in TFA containing 5% phenol 5% EDT and H205% for 2 hours which provides an untreated product yield of 160 mg. Purification by preparative CLAP (Vydac 218TP1022 'column) from an untreated 35 mg aliquot is carried out using a gradient of 70 to 100% B for 40 min (A = 0.1% TFA / water and B = MeOH) a a flow rate of 9 ml / min. After lyophilization, 20 mg of pure material are obtained (analytical CLAP; gradient 70-100% of B, where B = MeOH, A = TFA 0.1% / water: column - vydac 218TP54: Detection - UV 214 and 260 nm - product retention time = 16 min). Further characterization of the product was carried out using MALDI mass spectrometry; expected, M + H at 2050, found at 2055. c) Preparation of bubbles containing DSPS gas comprising a lipopeptide for targeting endothelial cells and a molecule containing captopril for delivery of a medicament.

Weigh into a DSPS bottle (Avanti, 4.5 mg), product of a) (0.5 mg) and product of b) (0.5 mg) and add 1.0 ml of a 1.4% propylene glycol / glycerol 2.4% solution to each bottle. The mixture is heated at 80 ° C for 5 minutes (the flasks are stirred during heating). The samples are collected at room temperature and the free space is purged with perfluorobutane gas. The flasks are first shaken in a lid mixer for 45 s and then rotated for 1 h followed by extensive washing with deionized water. No detectable concentrations of initial material are found in the final wash solution, as evidenced by EM MALDI. The MALDI mass spectral analysis was used to confirm the incorporation of the products of section a) and b) into the microbubbles as described in Example I b). d) In vitro study of microbubbles containing DSPS gas comprising a lipopeptide for targeting endothelial cells and a sleep molecule containing captopril for therapeutic applications.

The in vitro assay described in example 1 c) was used to examine the binding of cells under flowing conditions. There is a gradual accumulation of the microbubbles on the cells, which is dependent on the flow velocity. As the flow rate increased, the cells began to detach from the coverslip, the microbubbles still remained attached to the cells. Control bubbles that do not present the vector do not adhere to endothelial cells and disappear from cells under conditions of minimal flow.

Example 18 - Preparation of microburbules containing multiple specific gas of DSPS loaded with a lipopeptide containing a helical peptide with affinity for cell membranes and the peptide antibiotic polymyxin B sulfate.

This example is directed to the preparation of targeted microbubbles comprising multiple peptide vectors having a combined targeting and a therapeutic application. a) Synthesis of a lipopeptide comprising a helical peptide with affinity for cell membranes: hexadecyl-stearyl-Lys-Leu-Ala-Leu-Lys-Leu-Ala-Leu-Lys-Ala-Leu-ys-Ala-Ala-leu -Lys-Leu-Ala-NH-,.

Described in example 14 (a) b) Preparation of microbubbles containing multiple specific gas Weigh into a clean DSPS bottle (Avanti, 5.0 mg), lipopeptide from a) (0.3 mg) and polymyxin B sulfate (Sigma, 0.5 mg) and add 1.0 ml of a 1.4% propylene glycol / glycerol / 2.4% solution. . The mixture is sonicated for 3-5 min, heated at 80 ° C for 5 minutes and then filtered through a 4.5 micron filter.The mixture is cooled to room temperature and the free space is purged with perfluorobutane gas. The bottle is shaken in a lid mixer for 45 seconds and the microbubbles are centrifuged at 1000 rpm for 3 minutes.The microbubbles are washed in water until no polymyxima B sulphate or lipopeptide can be detected in the infranatant by EM MALDI. shows that the size distribution of the bubble population is between 1-8 micrometers, as desired.To the washed bubbles (ca. 0.2 ml) methanol (0.5 ml) is added and the mixture is placed in a sonic bath during 2 min It is found that the resulting clear solution, after analysis by MALDI-MS, contains both lipopeptide and polymyxime B sulphate (expected 1203, found 12.07).

Example 19) - Preparation of microbubbles containing multiple specific gas of DSPS "added" with a lipopeptide comprising an IL-1 receptor binding sequence and modified with a branched structure containing the drug methotrexate This example is directed to the preparation of targeted microbubbles comprising multiple vectors for targeting / therapeutic / drug-release applications. a) Synthesis of a polypeptide comprising a peptide that binds to interleukin-1 receptor (dipalmitoyl-Lys-Gly-Asp-Trp-Asp-Gln-Phe-Gly-Leu-Trp-Arg-Gly-Ala-Ala. OH The lipopeptide is synthesized in an ABI 433A automatic peptide synthesizer starting with Fmoc-Ala-Wang resin (Novabiochem) at the 0.1 mmol scale using 1 mmol ammo cartridges. All amino acids and palmitic acid are preactivated using HBTU before coupling. The simultaneous removal of lipopeptide from the resin and from the side chain protecting groups is carried out in TFA containing 5% H20, 5% anisole, 5% phenol and 5% EDT for 2 hours which provides a product yield untreated 150 mg. Purification by preparative CLAP (vydac column 218TP1022) from an aliquot of 30 mg of untreated material is carried out using a gradient of 90 to 100% B for 40 min ((A = 0.1% TFA / water and B = MeOH ) at a flow rate of 9 ml / min After lyophilization, 4 mg of pure material are obtained (analytical CLAP, gradient 90-100% of B during 20 min, where B = MeOH, A = 0.1% TFA / water: column - vydac 218TP54: Detection - UV 214 nm - product retention time = 23 min.) Further characterization of the product was carried out using MALDI mass spectrometry, expected, M + H at 2083, found at 2088. b) Synthesis of a branched methotrexate core structure containing a thiol portion The methotrexate structure is synthesized in an automatic ABI 433A peptide synthesizer starting with Fmoc-Cys (Trt) Tentagel resin in a 0.1 mmol scale. The simultaneous removal of product from the resin and the deprotection of the protecting groups is carried out in TFA containing 5% EDT and 5% H20 for 2 hours which provides an untreated product yield of 160 mg. Purification is carried out by preparative CLAP (vydac column 218TP1022) of an aliquot of 30 mg of untreated material using a gradient of 10 to 30% B for 40 min (A = 0.1% TFA / water and B = TFA 0.1% / acetonitrile) and at a flow rate of 9 ml / min. After lyophilization of the pure fractions, 9 mg of pure material are obtained (analytical CLAP, gradient 5-50% of B, where B = 0.1% TFA / acetonitrile, A = 0.01% TFA / water: vydac column 218TP54: Detection - UV 214 nm - product retention time = 9.5 min). Further characterization of the product was carried out using MALDI mass spectrometry; expected, M + H to 1523, found at 1523. c) Preparation of microbubbles sue contain multiple specific gas Weigh in a clean flask DSPS (Avanti, 4.5 mg), and thiol containing lipopeptide from example 15 a) (0.5 mg) and lipopeptide from a) (0.2 mg) above, and add 1.0 ml of a propylene glycol 1.4 solution % / glycerol 2.4%. The mixture is sonicated for 3-5 min, heated at 80 ° C for 5 minutes and then filtered through a 4.5 micrometer filter. The mixture is cooled to room temperature and the free space is purged with perfluorobutane gas. The flasks are shaken in a lid mixer for 45 s and the microbubbles are centrifuged at 1000 rpm for 3 minutes after which they are discarded in infranatant. d) Conjugation of a branched structure of methotrexate with tipped microbubbles The methotrexate structure of b) above (0.5 mg) is dissolved in PBS, pH 8.0. The solution is then added to the thiol containing bubbles of c) and the formation of the disulfide bond is allowed to proceed for 16 h. After extensive washing with PBS and water, the bubbles are analyzed by microscopy and EM MALDI. It is also considered pertinent that the binding of the disulfide bond of the methotrexate structure to the microbubbles can be reduced by releasing the free drug molecule in vivo. This, in combination with a tumor-specific vector, is a drug delivery system. A physiologically relevant reducing agent such as glutathione can be used to carry out the release of the medicament.

Example 20) Preparation of microbubbles re-coated with poly-L-lißin complexing with fluorescein-labeled DNA fragments of plasmid pBR322 This example is directed to the preparation of microbubbles for gene therapy / antisense applications. It is considered that specific targeting can be obtained by additional addition of microbubble membranes with lipid structures modified with vector as described in example 1. a) Preparation of microbubbles containing DSPS gas Weigh into a clean DSPS bottle (Avanti, 4.5 mg). 1.5 ml of a 1.4% propylene glycol / glycerol 2.4% solution is added and the mixture is sonicated for 2 min and then heated at 80 ° C for 5 minutes. Immediately after heating, the solution is filtered through a 4 micron filter. The sample is cooled to room temperature and the free space is purged with perfluorobutane gas. The bottle is shaken in a mixer with a lid for 45 s. Then the bubbles are washed once with deionized water and the infranatant is discarded. Subsequently the microbubbles are resuspended in 0.5 ml of water. b) Preparation of the poly-L-lysine / DNA complex and loaded with DSPS microbubbles To 1 mg of poly-L-lysine (70-150 kD) in a clean bottle is added 0.1 ml of fluorescein-labeled digested plasmid pBR322 (Biorad) dissolved in TE buffer (10 mM Tris-HCl, pH 8). The solution is made up to a total of 0.6 ml by addition of water and the pH is adjusted to 8. The complex formation is allowed to take place for 1 h and then 0.5 ml of polylysine-DNA solution is added to the microbubble suspension. from a) previous. After 1 h microscopy is used to show that the bubbles are fluorescent confirming the presence of DNA.

Example 21: Preparation of microbubbles filled with multiple specific gas containing a branched core peptide comprising a sequence that binds to the atherosclerotic plaque Dabsilada and RGDS This example is directed to the preparation of microbubbles having a thiol group on the surface for modification with thiol-containing vectors for drug delivery / delivery and drug delivery. a) Synthesis of the branched peptide Dabsil-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Lys (NH2-Arg-Glv-Asp-Ser) -Gly-Cys.OH The peptide is synthesized in an automatic ABI 433A peptide synthesizer starting with Fmoc-Cys (Trt) Tentagel resin at the 0.1 mmol scale using 1 mmole amino acid cartridges. All amino acids were preactivated using HBTU before coupling. The simultaneous removal of the peptide from the resin and the side chain protecting groups in TFA containing 5% phenol 5% EDT and 5% H20 was carried out for 2 hours which gives a yield of crude product of 160 mg. Purification by preparative CLAP (vydac column 218 TP1022) from an aliquot of 30 mg of crude material was carried out using a gradient of 10 to 60% B for 40 min (where (A = 0.1% TFA / water and B) = acetonitrile) at a flow rate of 9 ml / min After freeze drying, 2.5 mg of pure material are obtained (analytical CLAP, gradient 10-50% B for 20 min, where B = 0.1% TFA / acetonitrile and A = TFA 0.01% / water: column - vydac 218TP54: Detection - UV 214 and 435 nm - product retention time = 21 min.) Further characterization of the product was carried out using MALDI mass spectrometry, expected, M + H a 2070, found at 2073. b) Preparation of thiol-containing gas-filled microbubbles As described in example 15 a) and b). c) Oxidative coupling of microbubbles typed with multiple specific peptide via disulfide bond formation.

The microbubble infranatant of subsection b) above was discarded and replaced with a solution of dabsyl-peptide from a) (1 mg) in 0.7 ml of diluted ammonia solution (pH 8). To this 0.2 ml of a concentrated solution containing 6 mg of potassium ferricianate dissolved in 2 ml of water was added. The bottle was placed on a rotating table and oxidation of the thiol was allowed to proceed for 2 h. The bubbles were then extensively washed with water until the infranatant was free of the dabsyl-peptide as evidenced by CLAP and EM MALDI. The detection of peptide bound to microbubbles was carried out by reduction of the disulfide bond using a water soluble reducing agent Tris- (2-carboxyethyl) -phosphine. After the reduction, the infranatant was found to contain free dabsyl-peptide as evidenced by CLAP and EM MALDI. Other relevant physiological reducing agents such as reduced glutathione were also considered useful for initiating the release.

Example 22 - Microparticles containing gas comprising bis (l-hydroxyhexadecanoate) polymer of ethylidene and adipoyl chloride and biotin-amidocaprate-Ala covalently bound to the polymer a) Synthesis of Z-Ala-polymer (3L0- (carbobenzyloxy-L- alanyl) -polymer) The polymer is prepared from ethylidene bis (16-hydroxyhexadecanoate) and adipoyl chloride as described in WO-A-9607434, and a 10,000 molecular weight polymer fraction is purified using gel permeation chromatography (GPC). . 10 g of the material (corresponding to 1 mmol of OH groups), Z-alanine, (5 mmol) and dimethylaminopyridine (4 mmol) are dissolved in dry dimethylformamide / tetrahydrofuran and then dicyclohexylcarbodiimide is added. The reaction mixture is stirred at room temperature overnight. Dicyclourea is filtered off and the solvent is removed using rotary evaporation. The product is purified by chromatography, the fractions containing the title compound are combined and the solvent is removed using rotary evaporation. The structure of the product is confirmed by NMR. b) Synthesis of Ala-polymer (3-0- (L-alanyl) -polymer) Z-Ala-polymer (0.1 mmol) is stirred in toluene / tetrahydrofuran and glacial acetic acid (15% of the total volume) and hydrogenated in the presence of 5% palladium in carbon for 2 hours. The reaction mixture is filtered and concentrated in vacuo. c) Synthesis of biotinamidocaproate-Ala-polymer A solution of N-hydroxysuccinimide biotinamidocaproate ester in tetrahydrofuran is added to H2N-Ala-polymer dissolved in * a mixture of tetrahydrofuran and dimethylformamide and 0.1M sodium phosphate buffer having a pH of 7.5. The reaction mixture is heated to 30 ° C and stirred vigorously; the reaction is monitored by CCD until its completion. The solvent is evaporated and the untreated product is used without further purification. d) Particles containing gas comprising biotin-amidocaproate-Ala-polymer and PEG 10000 methylether 16-hexadecanoyloxyhexadecanoate Add 10 ml of a 5% w / w solution of biotin-amidocaproate-Ala-polymer to (-) - camphene maintained at 60 ° C, to 30 ml of a 1% w / w aqueous solution of PEG 10000 methylether 16- hexadecanoyloxyhexadecanoate (prepared as described in WO-A-9607434) at the same temperature. The mixture is emulsified using a rotor stator mixer (Ultra 'Turax1 ^ T25) for several minutes, and then frozen in a dry ice / methanol bath and lyophilized for 48 hours, which gives the title product as a powder White. e) Acoustic characterization and microscopy of the product Confirmation is made of the "microparticulate nature of the product using optical microscopy as described in WO-A-9607434. Ultrasonic transmission measurements using a 3.5 MHz wideband transducer indicate that a <2 particle suspension. mg / ml provides an attenuation of the sound beam of at least 5 dB / cm. f) Multiple specific microparticles Subsequently, the biotinylated microspheres are used to prepare multiple specific target products similar to those exemplified in examples 5), 6) and 7).

Example 23) Preparation of microbubbles containing multiple specific hasps encapsulated with DSPS and biotin-PEG ^ nn-acyl-phosphatidylethanolamine and fused with streptavidin, oli q onu c 1 eot gone biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and f ibrin peptide- • ant i - Biotinylated polymerizer (Biotin-GPRPPERHOS .NH) a) Synthesis of biotin-PEG ^ nn-acyl-phosphatidylethanolamine A mixture of dipalmitoylphosphatidylethanolamine (21.00 mg, 0.03 mmol), biotin-PEG-C02-NHS (100 mg, 0.03 mmol) and triethylamine (42 μl, 0.30 mmol) in a chloroform / methanol solution is stirred for 2 hours at room temperature. (3: 1) After evaporation of the solvents under reduced pressure, the residue is subjected to flash chromatography (methylene chloride / methanol / water 40: 8: 1). The product is obtained as a yellow gum, 112 mg (94%) and the structure is verified by NMR and MALDI MS. b) Streptavidin binding to fluorescein conjugated to gas-filled microbubbles Microbubbles containing gas were prepared by mixing DSPS and biotin-PEG3400-acyl-phosphatidylethanolamine as described in example 5 a). The suspension of microbubbles is divided into 0.2 ml aliquots and streptavidin conjugated with fluorescein is added, as shown in the table below. The samples were incubated on a rotary table for 15 or 30 minutes at room temperature before removal of excess protein by washing in PBS.

Results Samples were analyzed by flow cytometry and Coulter counter. The results are summarized in the table above. c) Conjugation of streptavidin-coated microbubbles with the oligonucleotide biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and biotin-biotin fibrin-anti-polymer peptide biotin-GPRPPERHOS The particles of the above aliquot number 6 were centrifuged and the supernatant was replaced with 1 ml of PBS buffer, pH 7.5 containing 0.2 mg of biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and 0.2 mg of biotin-GPRPPERHOS (example 5 c). After incubation for 24 h the particles were washed extensively with PBS and water. It is considered that other biotinylated vectors or therapeutic agents can be conjugated to streptavidin or avidin-coated microbubbles using this method.

Example 24) Preparation of microbubbles encapsulated with DSPS and functionalized with thrombus-directed lipopeptide and tissue plasminogen activator thrombolytic enzyme This example is directed to the preparation of US agents directed to thrombi comprising a therapeutic thrombolytic agent. a) Synthesis of a lipopeptide with affinity for thrombi (dipalmitoyl-Lys-Asn-Asp-Gly-As-Phe-Glu-Glu-lie-Pro-Glu- The lipopeptide is synthesized in an ABI 433 A automatic peptide synthesizer starting with Rink amide resin (Novabiochem) at 0.1 mmol scale using 1 mmole amino acid cartridges. All amino acids and palmitic acids were preactivated using HBTU before coupling.

The simultaneous removal of the peptide from the resin and the side chain protecting groups was carried out in TFA containing 5% phenol, 5% EDT, 5% anisole and 5% H20 for 2 h, which provides a product yield without try 80 mg. Purification was carried out by preparative CLAP (vydac column 218TP1022) of an aliquot of 20 mg of the crude material. After lyophilization 6 mg of the pure material are obtained. The product is characterized by analytical MALDI and CLAP mass spectrometry. b) Modification of tissue plasminogen activator with sulfo-SMPB A solution of 0.1 ml of ammonium carbonate buffer containing 0.1 mg of t-PA (Sigma is brought up to 0.2 ml by the addition of water) 0.4 ml of sulfo-SMPB (Pierce) dissolved in 0.05 ml is added to this solution. of DMSO. The protein solution is allowed to stand at room temperature for 45 minutes and then the purification is carried out on a Superdex 200 column. The product is eluted in PBS and the modified protein fraction is collected. c) Preparation of microbubbles encapsulated with DSPS / lipopeptide that binds thrombi and thiol-containing lipopeptide, and conjugation to modified tissue plasminogen activator It is weighed in a DSPS glass bottle (Avanti, . 0 mg), together with 0.5 mg of lipopeptide of a) and 0.5 mg of the thiol-containing lipopeptide of example 15 a). To this is added 1.0 ml of a 1.4% propylene glycol / glycerol 2.4% solution and the mixture is sonicated for 2 min and then heated at 80 ° C for 5 minutes. Immediately after heating the solution is filtered through a 4 micron filter. The sample is cooled to room temperature and the free space is purged with perfluorobutane gas. The bottles are shaken in a lid mixer for 45 s and the microbubbles are washed twice with deionized water. The infranatant and replaced with a 1 ml aliquot of the protein solution of b) above. It is allowed to process the conjugation reaction for 1 h. The bubbles are centrifuged and the infranatant is exchanged with an additional 1 ml of protein solution. The incubation step is repeated until all the protein solution is used. Then the microbubbles are washed extensively with water and analyzed by Coulter counter. The microbubbles are tested in a flow chamber test described in example 1 c). I know that protein-modified microbubbles bind in higher numbers compared to those that comprise either lipopeptide / DSPS or DSPS alone. It is considered that the targeting / therapeutic / ultrasound activities of these microbubbles is evaluated in in vitro and in vivo thrombogenesis models.

Example 25 - Microbubbles filled with multiple specific PFB gas encapsulated with DSPS and a lipopeptide comprising a peptide that binds heparin sulfate (KRKR) and a fibronectin peptide (WOPPRARI) for targeting and a lipopeptide containing atenolol. for therapeutic applications a) Synthesis of a lipopeptide consisting of a peptide that binds to heparin sulfate (KRKR) and a fibronectin peptide (WOPPRARI) Synthesis and purification described in the example 1 a) b) Synthesis of a protected atenolol derivative suitable for solid phase coupling i) Synthesis of methyl 4- (2,3-hepoxy) propoxyl phenylacetate A mixture of methyl 4-hydroxyphenylacetate (4.98 g, 0.0 mmol), epichlorohydrin (23.5 ml, 0.30 mol) and pyridine (121 μl, 1.5 mmol) was stirred at 85 ° C for 2 h. The reaction mixture is cooled and the excess epichlorohydrin (rotary evaporator) is distilled off. The residue is taken up in ethyl acetate, washed with brine and dried (Na 2 SO 4). The solution is filtered and concentrated. The dark residue is subjected to chromatography (silica, hexane / ethyl acetate 7: 3) to provide 2.25 g (34%) of a colorless oil. The NMR spectra XH (300 MHz) and 13 C NMR (75 MHz) are in agreement with the structure. ii) Synthesis of methyl 4- [2-hydroxy-3- [(1-methylethyl) amino] propoxyl phenylacetate] A mixture of methyl 4- ((2,3-epoxy) propoxy] phenylacetate (2.00 g, 9.00 mmol), isopropylamine (23 mL, 0.27 mol) and water (1.35 mL, 74.7 mmol) is stirred at room temperature during the night. The reaction mixture is concentrated (rotary evaporator) and the oily residue is dissolved in chloroform and dried (Na 2 SO 4). Filtration and concentration provide a quantitative yield of a yellow oil which is used in the next step without further purification. The structure is verified by XH and 13C NMR analysis. iii) Synthesis of 4- [2-hydroxy-3-? (1- methylethyl) aminol propoxyl phenylacetic A solution of methyl 4 - [2-hydroxy -3 - [(1-methylethyl) amino] propoxy] phenylacetate (563 mg, 2. 00 mmoles) in 6 M hydrochloric acid (15 ml) is heated at 100 ° C for 4 h. The reaction mixture is concentrated (rotary evaporator) and the residue is taken up in water and lyophilized. The 1 H and 13 C NMR spectra are in agreement with the structure and the mass spectrometry MALDI provides an M + H at 268, as expected. iv) Synthesis of N-Boc-4- r 2 -hydroxy-3- (1-methylethyl) amino] propoxyl phenylacetic acid A solution of 4- [2-hydroxy-3- [(1-methylethyl) amino] propoxy] phenylacetic acid hydrochloride (2.0 mmol) in water (2 ml) is added to a solution of sodium bicarbonate (0.60 g, 7.2 mmol) in water / dioxane (2: 1, 15 ml). A solution of di-butyl dicarbonate (0.48 g, 2.2 mmol) in dioxane (5 ml) is added. The progress of the reaction is monitored by CCD analysis (silica, CHCl3 / MeOH / AcOH 85: 10: 5) and portions of di-tert-butyl dicarbonate are added until the conversion is complete. The reaction mixture is poured into water saturated with potassium acid sulfate and the organic material is extracted into ethyl acetate. The organic phase is washed with water and brine, dried (Na2SO4) and filtered to provide 0.6 g of the untreated material. The product is purified by chromatography (silica, CHCl3 / meOH / AcOH 85: 10: 5). The solution is concentrated and the residue is taken up in glacial acetic acid and lyophilized. Yield, 415 mg (56%) of a white solid. The structure is confirmed by 1 H and 13 C NMR analysis. c) Synthesis of a lipopeptide functionalized with atenolol The structure shown above is synthesized in a manual nitrogen sparger starting with Rink Amida MBHA resin protected with FMOC (Novabiochem) at a scale of 0.125 mmoles, using amino acids from Novabiochem, palmitic acid from Fluka and the compound of a). The coupling is carried out using TBTU / HOBt / DIEA protocols standard. The simultaneous removal of the peptide from the resin and the deprotection of the side chain protecting groups is carried out in TFA containing 5% EDT and 5% water for 2 h. The untreated material is precipitated from the ether and purified by preparative liquid chromatography (vydac 218TP1022 column) using a gradient of 70 to 100% B for 60 min (A = 0.1% TFA / water and B = 0.1% TFA / acetonitrile) at a flow rate of 10 ml / min. After lyophilization a yield of 38 mg of pure material is obtained (analytical CLAP; gradient, 70-100% B for 20 min, A = 0.1% TFA / water and 0.1% TFA / acetonitrile), flow rate 1 ml / min, column -vydac 218TP54: Detection - UV 214 nm - time Hold 25 minutes). Further characterization is carried out using MALDI mass spectrometry; (matrix ACH) that provides, M + H to 1958, expected 1257. d) Preparation of microbubbles filled with DSPS gas comprising a lipopeptide which consists of a peptide that binds to heparin sulfate (KRKR) and a fibronectin peptide (WOPPRARI) and a lipopeptide containing atenolol.

A propylene glycol solution is added 1. 4% / glycerol / 2.4% (1.0 ml) to a mixture of DSPS (Avanti, 5.0 mg), product of a) (0.5 mg) and product of c) (0.5 mg) in a bottle. The mixture is sonicated for 5 min and then heated at 80 ° C for 5 min. The bottles are shaken during heating). The solution is filtered and cooled. The free space is purged with perfluorobutane gas and the flask is shaken in a mixer with a lid for 45 s followed by extensive washing with deionized water. The incorporation of lipopeptide containing atenolol in the bubbles is confirmed by EM MALDI as described in example 1 b). e) In vitro study of microbubbles filled with multiple specific gas An in vitro analysis of the microbubble suspension is carried out as described in example 1 c). There is a gradual accumulation of the misbubbles on the cells, which is dependent on the flow velocity. By increasing the flow rate, the cells begin to detach from the coverslip, the microbubbles are still attached to the cells. Control bubbles that do not present the vector do not adhere to the endothelial cells and disappear from the cells under conditions of minimal flow.

Example 26 - Microbubbles filled with DSP PFB gas containing cholesteryl or chlorambucil ester for diagnostic and therapeutic applications This example is directed to a non-specific modification of a multiplicity of cellular receptors on endothelial cells a) Synthesis of cholesteryl 4-T4-ibis (2-chloroethyl) aminol phenyl] butanoate DIC (170 μl, 1.10 mmol) is added to a solution of chlorambucil (Sigma, 669 mg, 2.20 mmol) in dry dichloromethane (15 ml). The mixture is stirred at room temperature for 0.5 h and added to a solution of cholesterol (Aldrich, 387 mg, 1.00 mmol) and DMAP (122 mg, 1.00 mmol) in dichloromethane (10 ml). The reaction mixture is stirred overnight and then poured onto 5% sodium bicarbonate. The phases are separated and the organic phase is washed with brine and dried (MgSO4). The solution is filtered and concentrated and the product is purified by column chromatography (silica, chloroform) to provide 560 mg (83%) yield of a colorless oil. The product is characterized by MALDI mass spectrometry, which provides M + H to 674, as expected. Further characterization is carried out using XH and 13C NMR analysis, which provides spectra according to the structure. b) Preparation of microbubbles containing DSPS gas comprising a chlorambucil cholesteryl ester for diagnostic and / or therapeutic applications A propylene glycol solution is added 1. 4% / glycerol 2.4% (1.0 ml) to a mixture of DSPS (Avanti, 4.5 mg) and the product of a) (0.5 mg) in a bottle. The mixture is sonicated for 5 min and then heated at 80 ° C for 5 min (the flask is stirred during heating) and cooled. The free space is purged with perfluorobutane gas and the flask is shaken in a mixer with a lid for 45 s followed by extensive washing with deionized water. MALDI mass spectrometry does not show a detectable level of the compound of a) in the final wash solution. The incorporation of chlorambucil cholesteryl ether into the bubbles is confirmed by ME MALDI as follows: ca. 50 μl of microbubbles to a clean bottle containing 100 μl of 90% methanol. The mixture is sonicated for 30 s and analyzed by EM MALDI which provides a peak M + H at 668 corresponding to the structure of a). In combination with a tumor specific vector, it is considered that these microbubbles are useful as agents for delivery of a targeted medication.

Example 27 - Multiple specific gas-filled microbubbles of DSPS comprising a lipopeptide containing tenolol and a chlorambucil cholesterol derivative for diagnostic and therapeutic applications a) Synthesis of a protected derivative of atenolol suitable for coupling in solid phase As described in example 25 section b). b) Synthesis of a lipopeptide functionality with atenolol As described in example 25 section c). c) Synthesis of 4-ß4- [bis (2-chloroethyl) aminol phenyl] cholesteryl butanoate As described in Example 25 section d). d) Preparation of DSPS microbubbles comprising a lipopeptide containing atenolol and a cholesteryl ester of cloambucil A solution of propylene glycol 1.4% / glycerol 2.4% (1.0 ml) is added to a mixture of DSPS (Avanti, 5.0 mg), the product of b) (0.5 mg) and e) (0.5 mg) in a flask. The mixture is sonicated for 5 minutes and then heated at 80 ° C for 5 min (the flask is shaken during heating). The solution is filtered and cooled. The free space is purged with perfluorobutane gas and the flask is shaken in a mixer with a lid for 45 s followed by extensive washing with deionized water. The incorporation of atenolol containing lipopeptide and chlorambucil analogue into the bubble membrane is confirmed by EM MALDI as described in example 1 e). e) In vitro study of microbubbles containing multiple specific DSPBs of DSPS include a lipopeptide containing atenolol and a chlorambucil cholesterol derivative for diagnostic and therapeutic applications The in vitro assay described in example 1 c) is used to determine the cell binding under flow conditions. There is a gradual accumulation of the microbubbles on the cells, which was dependent on the flow velocity. By increasing the flow rate, the cells began to detach from the coverslip, the microbubbles were still attached to the cells. Control bubbles that do not present the vector do not adhere to the endothelial cells and disappear from the cells under conditions of minimal flow.

Example 28 - Multiple specific gas-filled microbubbles of DSPS comprising a lipopeptide containing atenolol for targeting to cells and lipophilic diol ester of captopril for therapeutic use a) Synthesis of a protected derivative of atenolol suitable for coupling in solid phase As described in example 25, section b) b) Synthesis of a lipopeptide functionalized with atenolol As described in example 25, section c). c) Thiol ester synthesis of captoic acid from captopril A mixture of 5-β-colanic acid (Sigma, 361 mg, 1.00 mmol) and DIC (77 μl, 0.50 mmol) in dichloromethane (5 ml) is stirred for 10 min, and then added to a captopril solution (Sigma). , 130 mg, 0.600 mmol) and DBU (180 μL, 1.20 mmol) in dichloromethane (10 mL). The reaction mixture is stirred overnight and poured onto dilute hydrochloric acid. Chloroform (30 ml) is added. The phases are separated and the organic phase is washed with water and brine, and dried (MgSO4). After filtration and concentration, the untreated material is subjected to chromatography (silica, chloroform / methanol / acetic acid 95: 4: 1). The product is lyophilized from a mixture of acetonitrile / water / ethanol. Yield, 137 mg (49%) of a whitish solid. The structure is verified by NMR (500 MHz) and 13 C (125 MHz) NMR spectroscopy. Further characterization is carried out using MALDI mass spectrometry which provides a M + Na peak in the positive mode at m / z 584. d) Preparation of gas-filled microbubbles of DSPS comprising a lipopeptide containing atenolol for cell targeting and a lipophilic thiol ester of captopril for therapeutic use. A solution of propylene glycol 1.4% / glycerol 2.4% (1.0 ml) is added to a mixture. of DSPS (Avanti, 5.0 mg) and product of b) (0.5 mg) and c) (0.5 mg) in a bottle. The mixture is sonicated for 5 min and then heated at 80 ° C for 5 min (the flask is stirred during heating) and cooled. The free space is purged with perfluorobutane gas and the flask is shaken in a mixer with a lid for 45 s followed by extensive washing with deionized water. MALDI mass spectrometry does not show a detectable level of compound of b) and c) in the final wash solution. The incorporation of the compounds of b) and c) into the bubbles is confirmed by EM MALDI as follows. They transfer ca. 50 μl of microbubbles to a clean bottle containing 100 μl of 90% methanol. The mixture is sonicated for 30 s and analyzed by EM MALDI (ACH-matrix), which provides peaks according to the structures of b) and e), respectively. e) In vitro study of microbubbles containing d) gas The in vitro assay described in example 1 c) is used to determine cell attachment under flow conditions. There is a gradual accumulation of the microbubbles on the cells, which is surprising in the flow velocity. By increasing the flow rate, the cells begin to detach from the coverslip, the microbubbles are still attached to the cells. Control bubbles that do not present the vector do not adhere to endothelial cells and disappear from cells under minimal flow conditions.

Example 29 Phosphatidylserine gas-filled microbubbles comprising biotinamide-PEG-β-Ala-Cholesterol and chlorambucil cholesteryl ester for diagnostic and therapeutic applications a) Synthesis of cholesteryl N-Boc-β-alaninate DIC (510 μl) is added to a solution of Boc-β-Ala-OH (1.25 g, 6.60 mmol) in dichloromethane 815 ml) under an inert atmosphere. The reaction mixture is stirred for 30 min and transferred to a flask containing a solution of cholesterol (1.16 g, 3.00 mmol) and DMAP (367 mg, 3.00 mmol) in dichloromethane (15 ml). The reaction mixture is stirred for 2 h and then mixed with an aqueous solution of potassium acid sulfate. The phases are separated and the aqueous phase is extracted with chloroform. The combined organic phases are washed with aqueous potassium acid sulfate and water, and dried over MgSO4. After filtration and evaporation, the untreated product (silica, chloroform / methanol 99: 1) is chromatographed to give 1.63 g (97%) of a white solid. The structure is confirmed by 1 H NMR (500 MHz). b) Synthesis of cholesteryl β-alaninate hydrochloride A solution of a) compound (279 mg, 0.500 mmol) in 1 M hydrochloric acid and 1,4-dioxane (5 ml) is stirred at room temperature for 4 h. The reaction mixture is concentrated to provide a quantitative yield of cholesteryl β-alaninate hydrochloride. The structure is confirmed by 1 H NMR analysis (500 MHz) and MALDI mass spectrometry, which provides a M + Na peak at 482, expected 481. c) Biotin-PEG, ^ 0 ,, - ß -Ala -cholesterol To a solution of cholesteryl β-alaninate hydrochloride (15 mg, 0.03 mmol) in chloroform / wet methanol (2.6: 1, 3 ml) is added triethylamine (42 μl, 0.30 mmol). The mixture is stirred for 10 minutes at room temperature and a solution of biotin-PEG3400-NHS (100 mg, 0.03 mmol) in 1,4-dioxane (1 ml) is added dropwise. After stirring at room temperature for 3 hours the mixture is evaporated to dryness and the residue is purified by flash chromatography to give white crystals, yield; 102 mg (89%). The structure is verified by EM MALDI and by NMR analysis. d) Synthesis of 4-ß4- [bis (2-chloroethyl) amino] phenyl] butanol ester of cholesteryl DIC (170 μl, 1.10 mmol) is added to a solution of chlorambucil (Sigma, 669 mg, 2.20 mmol) in dry dichloromethane (15 ml). The mixture is stirred at room temperature for 0.5 h and added to a solution of cholesterol (Aldrich, 387 mg, 1.00 mmol) and DMAP (122 mg, 1.00 mmol) in dichloromethane (10 ml). The reaction mixture is stirred overnight and then poured into a 5% sodium bicarbonate solution. The organic phase is washed with brine and dried over MgSO4. The solution is filtered and concentrated and the product is purified by column chromatography (silica, chloroform) to provide 560 ng (83%) of colorless oil yield. The product is characterized by MALDI mass spectrometry, which provides M + H to 674, as expected. Further characterization is carried out using XH (500 MHz) and 13C (125 MHz) NMR analysis which provides spectrum 'according to the structure. e) Preparation of microbubbles filled with gas A solution of propylene glycol 1.4% / glycerol 2.4% (1.0 ml) is added to a mixture of DSPS (Avanti, 5 mg) and the product of c) (0.5 mg) and d) (0.5 mg) in a bottle. The mixture is sonicated for 5 min and then heated at 80 ° C for 5 min (the flask is stirred during heating) and cooled. The free space is purged with perfluorobutane gas and the flask is shaken in a mixer with a lid for 45 s followed by extensive washing with deionized water. MALDI mass spectrometry does not show a detectable level of the compound of c) and d) in the final wash solution. The incorporation of the compounds of c) and d) into the bubbles is confirmed by EM MALDI as described in example 1 b).

Example 30 - Gas-filled microbubbles of DSPS comprising a lipopeptide containing chlorambucil for diagnostic and therapeutic applications This example is directed to the preparation of microbubbles with non-specific affinity by a multiplicity of cell surface molecules. a) Synthesis of a lipopeptide dream contains chlorambucil a manual nitrogen sparger starting with Rink Amida MBHA resin protected with Fmoc (Novabiochem) at a scale of 0.125 mmoles. The standard amino acids of Novabiochem and palmitic acid from Fluka are acquired. The coupling is carried out using the standard TBTU / HOBt / DIEA protocol. Chlorambucil (Sigma) is coupled through the Lys side chain as a symmetric anhydride using pre-activation with DIC. The simultaneous removal of the peptide from the resin and the deprotection of the side chain protecting groups in TFA containing 5% EDT, 5% water and 5% ethylmethyl sulfide for 2 h are carried out. A 10 mg aliquot of the untreated material is purified by preparative liquid chromatography (vydac 218TP1022 column) using a gradient of 70 to 100% B for 60 min (A = 0.1% TFA / water and B = 0.1% TFA / acetonitrile) at a flow rate of 10 ml / min. After lyophilization a yield of 30 mg of pure material is obtained (analytical CLAP, gradient, 70-100% of B during 20 min, A = 0.01% TFA / water: and B = 0.1% TFA / acetonitrile, flow rate 1 ml / min; column - vydac 218TP54: Detection - UV 214 nm - retention time 26.5 minutes). Further characterization is carried out using MALDI mass spectrometry; what it provides, M + H to 1295, expected 1294. b) Preparation of gas-filled microbubbles sue comprises a lipopeptide containing chlorambucil for diagnostic and therapeutic applications A solution of propylene glycol 1.4% / glycerol 2.4% (1.0 ml) is added to a mixture of DSPS (Avanti, 4.5 mg) and the product of a) (0.5 mg) in a flask. The mixture is sonicated for 5 min and then heated at 80 ° C for 5 min (the flask is stirred during heating) and cooled. The free space is purged with perfluorobutane gas and the flask is shaken in a mixer with a lid for 45 s followed by extensive washing with deionized water. MALDI mass spectrometry does not show a detectable level of compound of a) in the final wash solution. The incorporation of chlorambucil containing lipopeptide in the bubbles by EM MALDI is confirmed as follows. They transfer ca. 50 μl of microbubbles to a clean bottle containing ca. 100 μl of 90% methanol. The mixture is sonicated for 30 s and analyzed by EM MALDI (ACH-matrix) which provides a peak M + H at 1300, expected at 1294 and a peak M + Na at 1324, expected 1317. c) In vitro study of microbubbles containing "added" DSPS gas with a lipopeptide containing chlorambucil for diagnostic and therapeutic applications The microbubbles are evaluated using the in vitro flow assay described in example 1 e). A gradual accumulation of the microbubbles takes place which is dependent on the flow velocity. By increasing the flow rate the cells begin to detach from the coverslip, the microbubbles are still bound in the cells. Control bubbles that do not present the vector do not adhere to the endothelial cells and disappear from the cells under conditions of minimal flow.

Example 31 - Full microbubbles of DSPS gas comprising a lipopeptide containing atenolol and a lipophilic derivative of captopril for diagnostic and therapeutic applications a) Synthesis of an appropriate protected atenolol derivative for solid phase coupling As described in example 25 b). b) Synthesis of N- f (S) -3-hexadecylthio-2-methylpropionill proline DIEA (188 μl, 1.10 mmol) is added to a solution of 1-iodohexadecane (176 mg, 0.500 mmol), captopril (120 mg, 0.550 mmol) and DBU (165 μl)., 1.10 mmol) in tetrahydrofuran (5 ml). The mixture is heated at 70 ° C for 2 h and then concentrated. The residue is poured into water saturated with potassium hydrogen sulfate and the organic material is extracted into chloroform. The organic phase is washed with water and dried (MgSO4). The product is purified by chromatography (silica, CHCl3 / MeOH / AcOH 85: 10: 5) and lyophilized to give 105 mg (48%) of a white solid material. The structure is verified by XH (500 MHz) and 13C (125 MHz) analysis and is further characterized by MALDI mass spectrometry, which provides M-H in negative mode at m / z 440, as expected. c) Preparation of microbubbles filled with DSPS gas comprising a lipopeptide containing atenolol and a lipophilic derivative of captopril for diagnostic and therapeutic applications A propylene glycol solution is added 1. 4% / 2.4% glycerol (1.0 ml) to a mixture of DSPS (Avanti, 4.5 mg), product of b) (0.5 mg) and c) in a flask. The mixture is sonicated for 5 min and then heated at 80 ° C for 5 min (the flask is stirred during heating) and then cooled. The free space is purged with perfluorobutane gas and the flask is shaken in a mixer with a lid for 45 s followed by extensive washing with deionized water. MALDI mass spectrometry does not show a detectable level of the compound of b) or c) in the final wash solution. The incorporation of compound b) and c) containing the lipopeptide into bubbles is confirmed by EM MALDI, as described in example 1 b). d) In vitro study of microbubbles containing DSPS gas comprising a lipopeptide containing atenolol and a lipophilic captopril derivative for diagnostic and therapeutic applications Microbubbles were evaluated using the in vitro flow assay described in example 1 e). There is a gradual accumulation of the microbubbles on the cells, which is dependent on the flow velocity. By increasing the flow rate, the cells begin to detach from the coverslip, the microbubbles are still attached to the cells. Control bubbles that do not present the vector do not adhere to the endothelial cells and disappear from the cells under conditions of minimal flow.

EXAMPLE 32 Flotation of Endothelial Cells by DSPS Microbubbles Comprising Multiple Specific Lipopeptide Which Attaches to Endothelial Cells This example is carried out to demonstrate that the invention can also be used for cell separation. The human endothelial cell line ECV 304, derived from the normal umbilical cord (ATCC), was cultured CRL-1998) in Nunc culture flasks (chutney 153732) in RPMI 1640 medium (Bio Whitaker) to which was added 200 mM L-glutamine, Penicillin / streptomycin (10,000 U / ml and 10.00 mcg / ml) and fetal bovine serum 10 % (Hyclone Lot no AFE 5183). The cells were subcultured after tripzinization with a division ratio of 1: 5 to 1: 7 when they reached confluence. Two million cells of tripzinized confluent cultures were added to a set of five centrifuge tubes followed either by DSPS control microbubbles, microbubbles of Example 1 or DSPS microbubbles added with endothelial cell binding lipopeptide of Example 14 a) a a concentration of 2, 4, 6, 8 or 10 million bubbles per tube. The cells at the bottom of the tubes after centrifugation at 400 g for 5 minutes were counted by Coulter counter. It was found that the union of four or more microbubbles to a cell is carried to flotation. In addition, all cells floated by lipopeptide bubbles bound to endothelial cells while approximately 50% floated with the microbubbles of example 1).

Example 33 - Gene transfer by microbubbles filled with PFB gas This example is directed to the preparation of targeted microbubbles for gene transfer. a) Preparation of lipopeptide bubbles DSPS / PFB gas, coated with poly-L-lysine DSPS (4.5 mg) and lipopeptide of 17 b) (0.5 mg) were weighed in 2 ml flasks. To each flask was added 0.8 ml of propylene glycol / glycerol (4%) in water. The solution is heated at 80 ° C for 5 minutes and stirred. The solution is then cooled to room temperature and the free space is purged with perfluorobutane. The flasks are shaken in a Capmix (Espe Capmix, 4450 oscillations / min) for 45 seconds and placed on a rotating table for 5 minutes. The contents of the flasks are mixed and the sample is washed by centrifugation at 2000 rpm for 5 minutes. The infranatant is removed and the same volume of distilled water is added. The washing procedure is repeated once more. Poly-L-lysine hydrobromide (Sigma, 20.6 mg) is dissolved in 2 ml of water and then an aliquot (0.4 ml) is made up to 2 ml of water. To 1.2 ml of the diluted poly-L-lysine solution 0.12 ml of DSPS-lipopeptide bubble suspension is added. After the incubation the excess polylysine is removed by extensive washing with water. b) Transfection of cells Endothelial cells (ECV 304) were grown in 6 plates to a uniform subconfluent layer. A transfection mixture consisting of 5 μg of DNA (an improved green fluorescent protein vector from CLONTECH) and 50 μl of a microbubble suspension of a) in RPMI medium is prepared to a final volume of 250 μl. The mixture is allowed to stand for 15 min at room temperature and then 1 ml of complete RPMI medium is added. The medium is removed from the cell culture vessel and the DNA-microbubble mixture is added to the cells. The cells are incubated in a cell culture incubator (37 ° C). c) Ultrasonic treatment After 15 minutes of incubation, the selected wells are exposed to 1 MHz continuous wave ultrasound, 0.5 W / cm2 for 30 seconds. d) Incubation and examination The cells are further incubated in the cell culture incubator (37 ° C) for about 4.5 hours. The medium containing the DNA microbubbles is then removed by aspiration, and 2 ml of RPMI complete medium is added. The cells are incubated for 40-70 hours before examination. Then most of the medium is removed and the cells are examined by fluorescence microscopy. The results are compared with the results of control experiments where DNA or DNA-polylysine is added to the cells. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (37)

2. Having described the invention as above, the content of the following claims is claimed as property: 1. A dirigible or steerable diagnostic and / or therapeutically active agent, characterized in that it comprises a suspension in an aqueous carrier liquid of an indicator comprising a gas-containing or gas-generating material, the agent is capable of forming at least two types of binding pairs with a target, wherein the gas-containing or gas-generating material is conjugated with at least two vectors at a time. vector capable of joining at least two binding sites. The agent according to claim 1, characterized in that the gas comprises air, nitrogen, oxygen, carbon dioxide, hydrogen, an inert gas, a sulfur fluoride, selenium hexafluoride, a low molecular weight hydrocarbon, a ketone. , an ester, a halogenated hydrocarbon of low molecular weight or a mixture of any of the foregoing.
3. 3. The agent according to claim 2, characterized in that the gas comprises a perfluorinated ketone, a perfluorinated ether or a perfluorocarbon.
4. 4. The agent according to claim 2, characterized in that the gas comprises sulfur hexafluoride or a perfluoropropane, perfluorobutane or perfluoropentane. The agent according to any of the preceding claims, characterized in that it comprises gas microbubbles stabilized by a surface membrane resistant to soalesence, a film-forming protein, a polymeric material, a non-polymeric and non-polymerizable wall-forming material or a surfactant or surfactant. 6. The agent according to claim 5, characterized in that the surfactant comprises at least one phospholipid. The agent according to claim 6, characterized in that at least 75% of the surfactant material comprises phospholipid molecules individually presenting a net total charge. The agent according to claim 7, characterized in that at least 75% of the film-forming surfactant material comprises one or more phospholipids which are selected from phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, phosphatidic acids and cardiolipins. 9. The agent according to claim 8, characterized in that at least 80% of the phospholipids comprise phosphatidylserines. The agent according to any of claims 1 to 9, characterized in that the gas-containing or gas-generating material is conjugated to one or more targeting vectors having specificity for one or more cell surface receptors and further comprising able to bind to a receptor system in a way that induces a therapeutic response. 11. The agent according to any of the preceding claims, characterized in that the vector or vectors are selected from antibodies; cell adhesion molecules; cell adhesion molecule receptors; cytokines; growth factors; peptide hormones and parts thereof; non-bioactive binders of receptors for cell adhesion molecules, cytokines, growth factors and peptide hormones; oligonucleotides and modified oligonucleotides; medicines that bind to DNA; protease substrates / inhibitors; molecules generated from combinational libraries; small bioactive molecules; and proteins and peptides which bind to cell surface proteoglycans. 12. The agent according to any of the preceding claims, characterized in that the vector or vectors have affinity for targets at a level such that the agent interacts but does not bind in a fixed manner to the targets. 13. The agent according to claim 12, characterized in that the vector or vectors are selected from ligands for cell adhesion proteins and cell adhesion proteins which have corresponding ligands on the surface of the endothelial cell. 14. The agent according to any of the preceding claims, characterized in that the vector or vectors are positioned so that they are not easily exposed to the target. The agent according to any of the preceding claims, characterized in that the vector or vectors are coupled or linked to the indicator by means of avidin-biotin and / or streptavidin-biotin interactions. 16. The agent according to any of claims 1 to 14, characterized in that the vector or vectors can be coupled covalently or non-covalently, or can be attached to the indicator. 17. The agent according to any of claims 1 to 14, characterized in that the vector is coupled or linked to an indicator by means of an electrostatic charge interaction. 18. The agent according to any of the preceding claims, characterized in that it also contains portions which are radioactive or reflective as X-ray contrast agents, light imaging probes or spin labels. 19. The agent according to any of the preceding claims, characterized in that it also comprises a therapeutic compound. The agent according to claim 19, characterized in that the therapeutic compound is an antineoplastic agent, a blood product, a biological response modifier, an antifungal agent, a hormone or hormone analog, vitamin, enzyme, antiallergic agent, tissue factor inhibitor, platelet inhibitor, coagulation protein target inhibitor, fibrin formation inhibitor, fibrinolysis promoter, antiangiogenic, circulatory drug, metabolic enhancer, antituberculous, antiviral, vasodilator, antibiotic, antiinflammatory, antiprotozoal, antirheumatic, narcotic, opiate, cardiac glycoside, neuromuscular blocker, sedative, local anesthetic, general anesthetic or genetic material. The agent according to claim 19 or claim 20, characterized in that the therapeutic compound is covalently coupled or linked to the indicator through disulfide groups. 22. The agent according to claim 19 or claim 20, characterized in that a lipophilic or derivatized therapeutic compound (which forms derivatives) lipophilically binds to the indicator through hydrophobic interactions. 23. A combined formulation, characterized in that it comprises: i) a first administrable composition comprising a pre-directed vector having affinity for a selected target; and ii) a second administrable composition comprising an agent according to any of the preceding claims, the agent comprises a vector having affinity for the predicted vector. 24. The combined formulation according to claim 23, characterized in that the pre-directed vector comprises a monoclonal antibody. 2
5. 5. A combined formulation, characterized in that it comprises: i) a first administrable composition comprising an agent according to any of claims 1 to 22, and ii) a second administrable composition comprising a substance capable of displacing or releasing the agent from its objective. 2
6. 6. A combined formulation, characterized in that it comprises: i) a first administrable composition comprising an agent according to claim 21; and ii) a second administrable composition comprising a reducing agent capable of reductively separating the disulfide groups which couple or bind to the therapeutic compound and the indicator in the agent of the first administrable composition. 2
7. 7. A process for the preparation of a steerable diagnostic and / or therapeutically active agent, according to claim 1, characterized in that it comprises coupling or linking at least one vector to an indicator comprising a material that contains gas or generates gas so that the agent is capable of forming at least two types of binding pairs with a target. 2
8. 8. The process according to claim 27, characterized in that the therapeutic compound is also combined with the indicator. 2
9. 9. The use of an agent according to any of claims 1 to 22, characterized in that it is used as a targetable or steerable ultrasound contrast agent. 30. A method for generating improved images of a human or non-human animal body, characterized in that it comprises administering to the body an agent according to any of claims 1 to 22 and generating an image by ultrasound, magnetic resonance, X-rays, an image radiographic or by illumination of at least one part of the body. 31. The method according to claim 30, characterized in that it comprises the steps of: i) administering to the body a pre-directed vector having affinity for a selected target; and subsequently ii) administering an agent according to any of claims 1 to 22, the agent comprises a vector having affinity for the predicted vector. 32. The method according to claim 31, characterized in that the pre-directed vector comprises a monoclonal antibody. The method according to claim 30, characterized in that it comprises the steps of: i) administering to the body an agent according to claims 1 to 22, and subsequently ii) administering a substance capable of displacing or releasing the agent from its objective. 34. The method according to any of claims 30 to 33, characterized in that the agent further comprises a therapeutic compound. 35. The method of conformance with claim 34, characterized in that the therapeutic compound is covalently coupled or linked to the indicator through disulfide groups, and subsequently a composition comprising a reducing agent capable of reductively separating such disulfide groups is administered. 36. A method for in vitro research of targeting by an agent according to any of claims 1 to 22, characterized in that the cells expressing a target are fixedly placed in a flow chamber, a suspension of the agent in a liquid carrier is passed through the chamber, and the binding of the agent to the cells is examined. 37. The method according to claim 36, characterized in that the flow velocity of the carrier liquid is controlled to simulate shear rates found in vivo.