HUP9904595A2 - Advanced diagnostic and therapeutic preparations - Google Patents

Abstract

Targetable diagnostic and/or therapeutic agent comprises a suspension, in an aqueous carrier liquid, of a reporter which comprises gas-filled microbubbles stabilised by monolayers of film-forming surfactant. The agent also comprises at least one vector.

Description

DANUBIA Patent and Trademark Office Ltd.

Budapest

Advanced diagnostic and therapeutic products

The invention relates to diagnostic and/or therapeutically effective compositions, more particularly to diagnostic and/or therapeutically effective agents which comprise moieties which interact or have affinity with sites and/or structures within the body and thus allow for enhanced diagnostic imaging and/or treatment of specific sites within the body. Of particular importance are the use of diagnostic agents in ultrasound imaging, these compositions being hereinafter referred to as targeted ultrasound contrast agents.

Ultrasound imaging is known to be a potentially valuable diagnostic tool, for example in the study of the vascular system, especially in cardiography, and in the examination of microvascular tissues. Various contrast agents have been proposed to enhance the resulting acoustic images, including suspensions of solid particles, emulsified liquid droplets, gas bubbles, and encapsulated gases or liquids. It is generally accepted that low-density contrast agents that are easily compressible are particularly effective in terms of the acoustic backscatter they generate, and are therefore of particular interest in gas-containing and gas-generating systems.

Gaseous contrast media are also known to be effective in magnetic resonance (MR) imaging, for example as susceptible contrast agents that reduce MR signal intensity.

89454-215 OE/OEM

-2Oxygen-containing contrast agents also represent potentially suitable paramagnetic MR contrast agents.

Furthermore, in X-ray imaging, it has been observed that gases such as carbon dioxide may be suitable as negative oral contrast agents or intravascular contrast agents.

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Radioisotopes of radioactive gases, such as inert gases such as xenon, have also been proposed for use in scintigraphy, such as for imaging blood clots.

Targeted ultrasound contrast agents are those that contain (i) a reporter moiety capable of interacting with ultrasound radiation and generating a detectable signal; (ii) one or more vectors that have affinity for a specific target site and/or structure within the body, such as specific cells or pathological areas; and (iii) one or more linkers that connect said reporter moiety and the vectors when they are not directly linked.

The molecules and/or structures to which the agent is to be attached are referred to as targets. In order to achieve specific imaging or therapeutic effects in a selected region or structure of the body, the target must be present and accessible within that region/structure. Ideally, it is expressed only within the desired area, but it is usually present in other parts of the body as well, potentially causing background problems. The target may be a specific molecular species (i.e. a target molecule) or an unknown molecule or rather a complex structure (i.e. target structure) that is present in the region to be imaged and/or treated and is capable of specifically or selectively binding to the given vector molecule.

-3The vector is linked or bound to the reporter moiety in order to bind these moieties to the region/structure to be imaged and/or manipulated. The vector may bind specifically to a selected target or may bind only selectively and with affinity to a limited number of other molecules/structures, again causing potential background problems.

W Only a few targeted ultrasound contrast agents are known in the prior art. For example, US-A-5 531 980 describes a system in which the signal carrier is an aqueous suspension of air or gas microbubbles stabilized with one or more film-forming surfactants, which are at least partially lamellar or layered, and these surfactants are bound to one or more vectors, which are bioactive substances designed for specific targeting tasks. It is claimed that the microbubbles are not directly encapsulated with the surfactant, but rather are incorporated into liquid-filled liposomes that stabilize the microbubbles. It is clear that the lamellar or plate-like surfactants present in such liposomes, such as phospholipids, must necessarily be present in the form of one or more lipid bilayers, which have lipophilic ends (back-to-back) and hydrophilic heads on both the outside and the inside (Schneider M., Liposomes as drug carriers: 10 years of research, Drug targeting, Nyon, Switzerland, 3-5 October 1984, Buri P. and Gumma A., Elsevier Amsterdam 1984).

EP-A-0727225 describes targeted ultrasound contrast agents in which the signal carrier is a chemical substance having a sufficient vapor pressure to be present in part as a gas at body temperature. This chemical substance is associated with a surfactant or an albumin.

-4 carrier, which includes a protein, peptide or carbohydrate-based cell adhesion molecule ligand as a vector. The signal carrier portion in such contrast agents corresponds to phase-shift colloidal systems, such as those described in WO-A-9416739; It has now been recognized that the administration of such phase-shift colloids can lead to the formation of microbubbles, which grow uncontrollably, possibly to such an extent that they pose a potential embolism risk, for example in the myocardial vasculature and the brain (Schwarz, Advances in Echo-Contrast [1994(3)], 48-49).

WO-A-9320802 proposes that tissue-specific ultrasound imaging can be enhanced by conjugating an acoustically reflective oligolamellar liposome to tissue-specific ligands, such as antibodies, peptides, lectins, etc. The liposomes are deliberately chosen to be gas-free and thus do not have the beneficial echogenic properties of gas-based ultrasound contrast agents. Further references to this technology, such as targeting fibrin, thrombus and atherosclerotic areas, can be found, for example, in the following publications: Alkanonyuksel, H. et al., J. Phann. Sci. (1996) 85(5), 486-490; J. Am. Coll. Cardiol. (1996) 27(2) Suppl A, 298A; Circulation, 68 Sci. Sessions, Anaheim 1995 Nov. 1316.

There are several publications on ultrasound contrast agents that refer to the possible use of monoclonal antibodies as vectors, without providing practical details, and also refer to signal carriers that contain substances that can be taken up by the reticuloendothelial system and thus enable the imaging of organs such as the liver.

-5 improvements (WO-A-9300933, WO-A-9401140, WO-A-9408627, WO-A9428874, US-A-5088499, US-A-5348016 and US-A-5469854).

Our invention is based on the recognition that gas-filled microbubbles stabilized by a monolayer of film-forming surfactants are particularly suitable as signal carriers in targeted diagnostic and/or therapeutic agents. For example, the flexibility and deformability of such thin, monolayer membranes significantly enhances the echogenicity of the signal carriers compared to liposome systems containing lipid bilayers or multiple such bilayers. This allows the use of very small amounts of signal carriers to achieve high ultrasound contrast and this has safety advantages.

Accordingly, the invention relates to a targeted diagnostic and/or therapeutically effective agent, for example an ultrasound contrast agent, which comprises a signal carrier suspended in a carrier material, for example an injectable carrier liquid, comprising gas-filled microbubbles stabilized by a monolayer of a film-forming surfactant, and the agent further comprises at least one vector.

The term monolayer means that the amphiphilic surfactant forms single-layer films or membranes similar to the so-called Langmuir-Blodgett films at gas-liquid interfaces, where the lipophilic part of the amphiphilic substance is oriented towards the gas phase and the hydrophilic parts interact with water.

As is apparent from WO-A-9729783, electrostatic repulsion between charged phospholipid membranes allows the formation of stable and stabilizing monolayers at the microbubble-carrier fluid interface. It is believed that the flexibility of such thin layers,

-6deformability enhances the echogenicity of the product compared to gas-filled liposomes containing one or more lipid bilayers. The amount of phospholipids used to stabilize such microbubble-containing aqueous suspensions is so small that it is only necessary to form a single layer of surfactant around the gas microbubbles, which provides a film-like structure and stabilizes the microbubbles against collapse. It is believed that microbubbles with liposome-like surfactant bilayers cannot be produced at such low phospholipid concentrations.

A preferred embodiment of the invention is based on the further recognition that limited adhesion to the target is a very useful property of diagnostic and/or therapeutic agents, which property can be achieved by using vectors that provide transient retention at the target rather than fixed binding. Thus, such agents, rather than remaining fixedly bound at a specific site, exhibit a delayed flow along the vascular endothelium through their transient interaction with endothelial cells. Such agents are thus concentrated on the walls of blood vessels, providing them with an increased echogenicity in the case of ultrasound contrast agents, relative to the entire blood flow, which is free of anatomical features. They thus enable better imaging of the capillary system, including the microvasculature, and thus help to distinguish between normal and poorly perfused tissues, for example in the heart, and may also be suitable for imaging structures, such as Kupffer cells, for visualizing thrombi and atherosclerotic lesions, or for visualizing neovascularized and inflamed tissue areas. The solution according to the invention is particularly

-It is suitable for imaging changes that occur in normal blood vessels in dead tissue.

In a further embodiment of the invention, one or more vectors are linked to or incorporated into the carrier in such a way that such vectors do not readily reach the target or target receptors. Thus, increased tissue specificity can be achieved by employing an additional method for the detection of the vectors, for example by subjecting the agent to an external ultrasound treatment after administration, thereby modifying the diffusivity of the vector-containing portions.

Any biocompatible gas may be present in the signal carrier, the term gas being understood to mean any substance (including mixtures) that is essentially or entirely gaseous (including vapor) at normal human body temperature of 37°C. Thus, the gas may be, for example, air, nitrogen, oxygen, carbon dioxide, hydrogen, an inert gas such as helium, xenon, argon or krypton, sulfur fluoride such as sulfur hexafluoride, disulfur decafluoride or trifluoromethylsulfur pentafluoride, selenium hexafluoride, optionally halogenated silane such as methylsilane or dimethylsilane, a low molecular weight hydrocarbon (for example up to 7 carbon atoms), for example an alkane such as methane, ethane, propane, butane or pentane, a cycloalkane such as cyclopropane, cyclobutane or cyclopentane, an alkene such as ethylene, propene, propadiene or butene or an alkyne such as acetylene or propyne, and may also be an ether such as dimethyl ether, ketone, ester, halogenated low molecular weight hydrocarbon (up to 7 carbon atoms) or a mixture thereof. In halogenated gases, at least some of the halogen atoms are fluorine atoms; Thus, the biocompatible hydrocarbon gas may be, for example, one of the following compounds: bromochlorodifluoromethane, chlorodifluoromethane,

-8dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoronethan, chloropentafluoroethane, dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene, ethyl fluoride, 1,1-difluoroethane and perfluorocarbons, such as perfluoroalkanes, perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes (such as perfluoro-n-butane, optionally mixed with one of the following isomers: perfluoroisobutane), perfluoropentanes, perfluorohexanes and perfluoroheptanes; perfluoroalkenes, such as perfluoropropene, perfluorobutenes (such as perfluorobut-2-yne) and perfluorobutadiene; perfluoroalkynes, such as perfluorobut-2-yne; and perfluorocycloalkanes, such as perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutanes, perfluorotrimethylcyclobutanes, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentanes, perfluorocyclohexane, perfluoromethylcyclohexane and perfluorocycloheptane. Other halogenated gases that can be used include, for example, methyl chloride, fluorinated (e.g. perfluorinated) ketones, such as perfluoroacetone and fluorinated (e.g. perfluorinated) ethers, such as perfluorodiethyl ether. The use of perfluorinated gases, such as sulfur hexafluoride and perfluorocarbons, such as perfluoropropane, perfluorobutane and perfluoropentane, is particularly advantageous due to the recognized high stability of microbubbles containing such gases in the bloodstream.

The gas may also comprise any of the following substances: butane, cyclobutane, n-pentane, isopentane, neopentane, cyclopentane, perfluoropentane, perfluorocyclopentane, perfluorohexane or a mixture of one or more of these substances, which are liquid at the processing and handling temperature but gaseous at body temperature, such as are described for example in WO-A-9416739, since the film-forming surfactant monolayer in the signal carrier unit stabilizes the resulting microbubbles against uncontrolled growth.

-9In principle, any film-forming surfactant can be used to form gas-encapsulating monolayers, including, for example, the following: non-polymeric and non-polymerizable wall-forming surfactants, such as those described in WO-A-9521631; polymeric surfactants, such as those described in WO-A-9506518; and phospholipids, such as those described in WO-A-9211873, WO-A-9217212, WO-A9222247, WO-A-9428780, WO-A-9503835 or WO-A-972978. Preferably, 75%, particularly preferably the entire amount, of the film-forming surfactants in the compositions according to the invention is present in the monolayer at the gas-liquid interface.

Examples of phospholipids that can be used include lecithins (e.g. phosphatidylcholines), such as natural lecithins such as egg white lecithin or soybean lecithin, and synthetic or semi-synthetic lecithins such as dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine; phosphatidic acids, phosphatidylethanolamines, phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, cardiolipins, sphingomyelins, fluorinated analogues thereof, and mixtures of any of these or mixtures thereof with other lipids such as cholesterol.

We have found that it is particularly advantageous to use phospholipids which predominantly (at least 75%) contain molecules each of which has a different average charge, this is particularly advantageous if they constitute essentially the only amphiphilic component of the signal carrier and this provides particularly valuable advantages, for example with regard to the stability and acoustic properties of the product. Without being bound by any theory,

-10it is believed that the electrostatic repulsion between charged phospholipid membranes allows for the formation of stable monolayers at gas-liquid interfaces, and as mentioned above, the flexibility and deformability of such thin membranes enhances the echogenicity of the carriers of the invention compared to gas-filled liposomes containing one or more lipid bilayers.

The use of charged phospholipids also provides carriers with advantageous properties such as stability, dispersibility and resistance to collapse without the addition of additional additives such as surfactants and/or viscosity enhancers, thus allowing the number of contrast agent components injected into the patient to be kept to a minimum. For example, the charged surface of the microbubbles can minimize or prevent aggregation by electrostatic repulsion.

Preferably at least 75%, more preferably the entire amount of the phospholipid materials used in the signal carrier according to the invention have an average charge under the conditions of preparation and/or use, which charge may be positive, more preferably negative. Positively charged phospholipids include phosphatidic acid esters, such as palmitolephosphatidic acid or distearoylphosphatidic acid, and amino alcohols, such as the hydroxyethylenediamine ester. Negatively charged phospholipids include, for example, naturally occurring materials (e.g., those derived from soybeans or egg whites), semi-synthetic (e.g., partially or fully hydrogenated) and synthetic phosphatidylserine, phosphatidylglycerols, phosphatidylinositols, phosphatidic acids, and

-11cardiolipins. The fatty acyl groups of such phospholipids generally contain 14-22 carbon atoms, for example a palmitoyl or stearoyl group. Lyso-forms of such charged phospholipids can also be used in the solution according to the invention, the term lyso-termining phospholipids which contain only one fatty acyl group and this is preferably linked to the 1-position carbon atom of the glyceryl moiety by an ester bond. Lyso-forms of phospholipids having such a charge are preferably used in admixture with charged phospholipids containing two fatty acyl groups.

The use of phosphatidylserines in the materials of the invention is particularly preferred among the phospholipids and is particularly preferred when their amount is at least 80%, in particular 85-92%. While not wishing to be bound by any theory, it is possible that the ionic bridge formed between the carboxyl and amino groups of adjacent serine molecules contributes to the stability of such signaling systems. Preferred phosphatidylserines include, for example, saturated (e.g. hydrogenated or synthetic) naturally occurring phosphatidylserine, as well as synthetic distearoylphosphatidylserine, dipalmitoylphosphatidylserine and diarachidoylphosphatidylserine.

Other lipids that can be used include, for example, phosphatidylethanolamines, optionally mixed with one or more lipids such as stearic acid, palmitic acid, stearylamine, palmitylamine, cholesterol, bisalkyl glycerides, sphingoglycolipids, synthetic lipids such as N,N-dimethyloctadecyl-1-octadecaneammonium chloride or bromide (DOD AC, DOD AB) and/or maleic acid bisalkyl esters.

Other lipids that can be used in the preparation of gaseous contrast agents include, for example, the following fatty acids: stearic acid, palmitic acid, 2-n-hexadecylstearic acid, oleic acid, and

-12other other acid-containing lipids. Such lipids can be linked by amide linkage to amino acids containing one or more amino groups; the resulting lipid-modified amino acids (e.g. dipalmitoylysine or distearoyl-2,3-diaminopropionic acid) can be suitable precursors for the attachment of functionalized spacer elements containing coupling sites for conjugation of one or more vector molecules.

Other suitable stabilizers are lipopeptides which contain a lipid linked to the peptide linker moiety which is suitably functionalized for the purpose of coupling one or more vector molecules. Particularly preferred is a positively charged peptide linker element (e.g. containing two or more lysine residues) which is capable of binding to signal-carrying microbubbles via electrostatic interaction, which microbubbles are stabilized by a negatively charged phospholipid or other surface membrane.

Another embodiment of the invention relates to functionalized microbubbles that contain one or more reactive groups for non-specific reaction with receptor molecules located on cell surfaces. For example, microbubbles containing thiol groups can be linked to cell surface receptors via a disulfide exchange reaction. The reversible nature of such reactions means that the flow of the microbubbles can be controlled by changing the redox environment. Similarly, functionalized microbubbles can be used that are provided with membranes containing activated esters, such as N-hydroxysuccinimide esters, for reaction with amino groups found on cell surface molecules.

-13 Previously proposed phospholipid-based microbubble-containing contrast agents (WO-A-9409829) are prepared by contacting a powdered surfactant, such as freeze-dried, preformed liposomes or freeze-dried or spray-dried phospholipid solutions, with air or other gas, followed by an aqueous carrier, followed by mixing to form a microbubble suspension which is to be administered shortly after preparation. However, the disadvantage of such methods is that considerable mixing energy must be applied to form the desired dispersion and the size and size distribution of the microbubbles is dependent on the amount of energy applied and thus cannot be controlled in practice.

The signal carriers or materials according to the invention are however preferably prepared by obtaining the gas microbubble dispersion in an aqueous medium containing a suitable surfactant (e.g. phospholipid), which has, if necessary, been previously autoclaved or otherwise sterilized, and then, preferably after washing and/or size fractionation of the resulting microbubbles, the resulting dispersion is lyophilized, for example in the presence of one or more cryoprotectants/lyoprotectants, to obtain a dried product which can be easily reconstituted into a ready-to-use material in water/aqueous solution and thus a consistently reproducible microbubble dispersion is obtained. This process is described in more detail in WO-A-9729783, the content of which is hereby incorporated by reference; The possibility of removing bubbles of unwanted size and excess surfactants makes this method significantly more advantageous than, for example, the method described in the aforementioned WO-A-9409829 or the prior art WO-A-9608234 (where the bubbles are produced on site after injection,

-14 by shaking a suspension containing various phospholipids and viscosity enhancers, such as propylene glycol and glycerin).

The above process can be used to produce carrier microbubbles with a very narrow size distribution, wherein more than 90% (at least 95%, preferably at least 98%) of the microbubbles have a volume average diameter of 1-7 pm or less and less than 5% (e.g., no more than 3%, preferably no more than 2%) of the microbubbles have a volume average diameter of greater than 7 pm. A washing step can be used to ensure that the carrier is substantially free of unwanted components, such as excess lipids or viscosity enhancers. Carrier-containing materials prepared in this manner have the following advantages over prior art contrast agents:

The dose-dependent echogenicity is greatly increased, since essentially all of the surfactant is involved in stabilizing the microbubbles as a monolayer. In vitro ultrasound studies in dogs have shown that the ultrasound contrast agents prepared in the above manner exhibit a 15 dB increase in backscatter intensity in the myocardium after intravenous injection at a dose of 0.1 µl microbubbles/kg body weight.

The in vivo safety is increased for the same reasons, since the administered dose for such substances is 0.1-10 pg/kg body weight per phospholipid, such as 1-5 pg/kg. The use of such low levels of surfactant clearly has a significant advantage in terms of minimizing possible toxic side effects.

Furthermore, a high efficacy/dose ratio is particularly advantageous for targeted application, since it is generally accepted that a relatively small amount of signal carrier accumulates at the desired site if such a

-15 products are used, which contain vectors with affinity for such sites. These preferred signal carriers according to the invention thus significantly increase the contrast at the desired site, compared to known targetable ultrasound contrast agents. Their high efficiency allows the microbubbles to be seen individually by ultrasound and thus a sensitivity as good as, if not better than, scintigraphy, which is currently the most widely used method for targeted imaging, although the resolution of scintigraphic images is not the best.

A particular advantage of phosphatidylserine-based agents is their biocompatibility; thus, no acute toxic effects, such as changes in blood pressure or heart rate, were observed in animal experiments in dogs when dogs were administered intravenously with phosphatidylserine-based contrast agent boluses prepared as above and the dose used ranged up to 10 times the normal imaging dose.

The use of charged phospholipids may also be advantageous in that they contain functional groups, such as carboxyl or amino groups, which allow for easy coupling of the vectors, optionally using linkers. It is noted, however, that other functional groups may be incorporated into such systems by mixing the lipid containing the desired functional group with the film-forming surfactant prior to microbubble formation.

It is generally not necessary to use additives such as emulsifiers and/or viscosity enhancers, which are commonly used in many existing contrast agent formulations. As mentioned above, the advantage of this is that the number of components can be kept to a minimum, ensuring the lowest possible concentration of the agent.

-16 viscosity. Since the preparation of the agents generally includes a freeze-drying step, it may be advantageous to add cryoprotective/lyoprotective or bulking agents, for example the following can be used for this purpose: alcohols, such as aliphatic alcohols, such as tert-butanol, polyols, such as glycerol, carbohydrates, such as sugar or sucrose, mannitol, prehalose, or cyclodextrin or polysaccharides, such as dextran, or polyglycols, such as polyethylene glycol. The use of physiologically well-tolerated sugars, such as sucrose, is particularly advantageous.

The lyophilized products prepared as described above are particularly easy to reconstitute with water, requiring only minimal agitation, such as gentle hand shaking for a few seconds. The size of the resulting microbubbles is consistently reproducible and independent of the amount of agitation energy applied, in practice the size being determined by the size of the microbubbles in the initial microbubble dispersion; surprisingly this size is substantially maintained in the lyophilized and reconstituted product. Thus, since the microbubble size in the initial dispersion can be easily controlled by process parameters such as the type, speed and duration of agitation, the size of the final microbubbles can be easily controlled.

The lyophilized dried product has been shown to be stable for at least several months under ambient conditions. The resulting microbubble dispersion when mixed with water is stable for at least 8 hours, which provides considerable flexibility when converting the dried product into a ready-to-use form prior to injection.

-17The high efficacy of these preferred signal carriers allows the use of smaller than usual bubbles, as they produce ultrasound contrast effects well above the minimum detection level of currently used ultrasound imaging equipment. Potential advantages of such small bubbles include reduced vascular occlusion, longer circulation time, increased ability to reach the target, and reduced accumulation in the lung or other non-target organs, and their use and compositions containing them are further aspects of the invention.

It is also possible to use these small bubbles to enhance the increased ultrasound contrast effect of bubble clusters. It is known from theory that the ultrasound contrast effect of a given number of bubbles, which is the total volume of the bubbles V, in a dilute dispersion increases if the bubbles aggregate to form a larger gas phase, which has a total volume similar to V. In this way, it is possible to use small bubbles that do not provide substantially ultrasound contrast until they accumulate (this can occur in the target areas, more advantageously than in non-target areas, which contain a low density of target molecules). Small bubbles can also be made to coalesce, for example, by interaction with the target, and thus increase the contrast in the target area. The interconnection between the bubbles and the subsequent accumulation can also be influenced if the signal carrier - in addition to containing the vector ensuring binding to the specific site - also contains unreacted parts that are able to react with the functional groups of the other bubbles.

In accordance with the invention, the signal carrier unit generally remains associated with the vectors. However, in one type of targeting method, sometimes referred to as pretargeting, the vector (often a

-18 monoclonal antibody) is administered alone; the carrier is then administered linked to a moiety that is capable of specifically binding to the pre-targeting vector molecule (if the pre-vector is an antibody, the carrier can be linked to an immunoglobulin binding molecule, such as a protein A or an anti-immunoglobulin antibody). The advantage of this method is that there is sufficient time to eliminate vector molecules that do not bind to their target, thus substantially reducing the background problems associated with excess carrier-vector conjugate. In one embodiment of the invention, pre-targeting is performed with a specific vector, and then the carrier units are administered linked to another vector and a moiety that binds to the first vector.

In a further embodiment of the invention, for example, in determining the degree of blood perfusion in target areas, such as the myocardium, it is important to measure the rate at which contrast agents bind to, displace or are removed from the target. This can be done in a controlled manner by administering an additional vector and/or substance capable of displacing or releasing the contrast agent from the target.

Imaging modalities that can be used in accordance with the invention include two- and three-dimensional imaging methods, such as B-mode imaging (e.g., using a time-varying amplitude of the signal envelope, which envelope is generated from the fundamental frequency of the emitted ultrasound pulses, their sub- or higher harmonics, or the sum or difference of the frequencies of the emitted pulses or such harmonics, images generated from the fundamental frequency or its second harmonics being preferred), color Doppler imaging, Doppler amplitude imaging, and the latter two with any of the aforementioned modalities.

-19 is a combination of. Surprisingly, excellent second harmonic signals are obtained from the targeted monolayer-stabilized microbubbles of the invention. To reduce the effect of motion, successive images of tissues, such as the heart or kidney, can be collected using a suitable synchronization technique (e.g., filtering out those resulting from ECG or respiratory motion). Measurement of changes in resonance frequency or frequency absorption, which changes accompany fixed or delayed microbubbles, can also be used to detect contrast agent.

The invention provides a means for delivering therapeutic agents to a desired site in combination with vector-mediated targeting. A therapeutic agent is a substance that has a beneficial effect on a specific disease in a living human or animal organism. Although the combination of drugs and ultrasound contrast agents has been proposed, for example, in WO-A-9428873 and WO-A-9507072, these products do not contain vectors that have affinity for specific sites and thus have a comparatively much lower specific retention at the desired site prior to or during the release of the active agent.

In the present invention, the therapeutic compounds may be encapsulated within or attached to the microbubbles or incorporated into the stabilizing membranes. Thus, the therapeutic agent may be attached to a portion of the membrane, for example by a covalent or ionic bond, or may be physically incorporated into the stabilizing material, particularly if the active agent has a similar polarity or solubility to the membrane material, thereby preventing leakage from the product before it can exert its effect in the body. The release of the active agent may be initiated by contact with the blood or other external or

-20 by internal action, for example by an enzyme-catalyzed dissolution process or by the use of ultrasound. The disruption of gas-containing microparticles by external ultrasound is a known phenomenon with ultrasonic contrast agents, as described, for example, in WO-A-9325241; the release rate of the active substance may vary depending on the type of therapeutic administration, using a specific amount of ultrasonic energy from the transducer.

The drug substance may be covalently attached to the surface of the encapsulating membrane using a suitable coupling agent, such as those described herein. For example, a phospholipid or lipopeptide derivative may be prepared to which the drug substance is attached by a biodegradable bond or linker, and this derivative may then be incorporated into the material used to prepare the signal carrier, as described above.

Suitable therapeutic agents for use in the drug delivery compositions of the invention include any known therapeutic agent or active analog thereof that contains a thiol group that is capable of binding to a thiol-containing microbubble under oxidative conditions to form a disulfide bond. In combination with a vector or vectors, such drug/vector-modified microbubbles can accumulate in the target tissue; the addition of a reducing agent, such as reduced glutathione, can then dissociate the drug molecule from the microbubble in the target cell environment, thereby increasing the drug concentration and therapeutic effect. Alternatively, the composition can be prepared without the therapeutic agent, which is then coupled to or coated on a microbubble immediately prior to use; for example, the therapeutic agent can be administered in an aqueous solution.

-21 to a microbubble suspension prepared in the medium, and then shaken to bind the therapeutic agent to the microbubbles.

Other drug delivery systems include vector-modified phospholipid membranes doped with lipopeptide structures containing a poly-L-lysine or poly-D-lysine chain in combination with the targeting vector. For use in gene therapy/antisense technologies, especially receptor-mediated drug delivery, the microbubble carrier is condensed with DNA or RNA by electrostatic interaction with cationic polylysine. The advantage of this method is that the vector or vectors used for targeted delivery are not directly linked to the polylysine carrier molecule. The polylysine chain is also tightly bound to the microbubble membrane due to the presence of lipid chains. The use of ultrasound to increase the efficiency of delivery is advantageous.

In another method, free polylysine chains are first modified with drug or vector molecules and then condensed onto the negative surface of targeted microbubbles.

Among the active ingredients that can be used in the solution according to the invention, the following are mentioned, for example, without any intention of limitation: antineoplastics, such as vincristine, vinblastine, vindesine, busulfan, chlorambucil, spiroplatin, cisplatin, carboplatin, methotrexate, adriamycin, mitomycin, bleomycin, cytosine arabinoside, arabinosyl adenine, mercaptopurine, mitotane, procarbazine, dactinomycin (antinomycin D), daunorubicin, doxorubicin hydrochloride, taxol, plicamycin, aminoglutethimide, estramustine, flutamide, leuprolide, megestrol acetate, tamoxifen, testolactone, trilostane, amsacrine (mAMSA), asparaginase (L asparaginase), etoposide, interferon a-2a and 2b, blood products, such as hematoporphyrins or derivatives thereof;

-22biological response modifiers, such as muramyl peptides, antifungal agents, such as ketoconazole, nystatin, griseofulvin, flucytosine, miconazole or amphotericin B, hormones or hormone analogues, such as growth hormone, melanocyte stimulating hormone, estradiol, beclomethasone dipropionate, betamethasone, cortisone acetate, dexamethasone, flunisolide, hydrocortisone, methylprednisolone, paramethasone acetate, prednisolone, prednisone, triamcinolone or fludrocortisone acetate; vitamins, such as cyanocobalamin or retinoids; enzymes, such as alkaline phosphatase or manganese superoxide dismutase; antiallergic agents, such as amelexanox; tissue factor inhibitors, such as monoclonal antibodies and Fab fragments thereof, synthetic peptides, non-peptides and compounds that regulate tissue factor expression; platelet inhibitors, such as GPIa, GPIb and GPIIb-IIIa, ADP receptors, thrombin receptors, von Willebrand factor, prostaglandins, aspirin, ticlopidine, clopidogrel and reopro; inhibitors of coagulation protein targets, such as Flla, FVa, FVIIa, FVIIIA, FIXa, FXa, tissue factor, heparins, hirudin, hirulog, argatroban, DEGR-rFVIIa and annexin V; fibrin formation inhibitors and fibrinolysis promoters, such as t-PA, urokinase, plasmin, streptokinase, rt-plasminogen activator and rstaphylokinase; anti-angiogenic factors, such as medroxyprogesterone, pentosan polysulfate, suramin, taxol, thalidomide, angiostatin, interferon-alpha, metalloproteinase inhibitors, platelet factor 4, somatostatin, thrombospondin; blood circulation promoting agents, such as propranolol; metabolic potentiators such as glutathione, anti-tuberculosis drugs such as p-aminosalicylic acid, isoniazid, capreomycin sulfate, cyclohexene, ethambutol, ethionamide, pyrazinamide, rifampin, or streptomycin sulfate; antiviral agents such as acyclovir, amantadine, azidothymidine, ribavirin, or vidarabine; vasodilators such as diltiazem, nifedipine, verapamil, erythritol tetranitrate, isosorbide dinitrate,

-23-nitroglycerin or pentaerythritol tetranitrate; antibiotics such as dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil, cephalexin, ceffadine, erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin, nafcillin, penicillin, polymyxin or tetracycline; anti-inflammatory agents such as diflunisal, ibuprofen, indomethacin, meclefenamate, mefenic acid, naproxen, phenylbutazone, piroxicam, tolmetin, aspirin or salicylates; antiprotozoals such as chloroquine, metronidazole, quinine or meglumine antimonate; antirheumatic agents such as penicillamine; narcotics such as paregorin; opiates such as codeine, morphine or opium; cardiac glycosides such as deslaneside, digitoxin, difoxin, digitalin or digitalis; neuromuscular blocking agents such as atracurine mesylate, gallamine triethiodide, hexafluorenium bromide, metocurarine iodide, pancuronium bromide, succinylcholine chloride, tubocurarine chloride or vecuronium bromide; sedatives such as amobarbital, amobarbital sodium, apropbarbital, butabarbital sodium, chloral hydrate, ethchlorvynol, ethinamate, flurazepam hydrochloride, glutethimide, methotrimeprazine hydrochloride, methyprilon, midazolam hydrochloride, paraldehyde, pentobarbital, secobarbital sodium, talbutal, temazepam or triazolam; local anesthetics such as bupivacaine, chloroprocaine, etidocaine, lidocaine, mepivacaine, procaine or tetracaine; general anesthetics, droperidol, etomidate, fentanyl citrate droperidol, ketamine hydrochloride, methohexital sodium or thiopental and their pharmaceutically acceptable salts (e.g. acid addition salts, such as hydrochloride or hydrobromide or basic salts, such as sodium, calcium or magnesium salts) or derivatives thereof (e.g. acetates). Therapeutically active substances that can be used also include genetic materials, such as nucleic acids, and RNA and DNA of natural or synthetic origin, including recombinant RNA and DNA. Certain

DNA encoding -24 proteins can be used to treat a variety of diseases. For example, tumor necrosis factor or interleukin-2 genes can be used to treat advanced cancers; thymidine kinase genes can be used to treat breast cancer or brain tumors; interleukin-2 genes can be used to treat neuroblastoma, malignant melanoma or kidney cancer; interleukin-4 genes can be used to treat cancers.

The lipophilic derivative of the drug, which is bound to the microbubble membrane by hydrophobic interactions, can exert its therapeutic effect as part of the microbubble or after release from the microbubble, for example after application of ultrasound. If the drug does not have the desired physical properties, a lipophilic group can be introduced to link the drug to the membrane. Preferably, the lipophilic group should be introduced in a way that does not affect the in vivo activity of the molecule or the lipophilic group can be cleaved and the drug released. The introduction of lipophilic groups can be carried out by various chemical means, depending on the functional groups present in the drug. The covalent bond can be carried out using functional groups present in the drug molecule that are capable of reacting with appropriately functionalized lipophilic compounds. Such lipophilic moieties include branched or unbranched alkyl chains, cyclic compounds, aromatic groups, and fused aromatic and non-aromatic ring systems. In some cases, the lipophilic moiety comprises a suitably functionalized steroid, such as cholesterol or a similar compound. Functional groups particularly suitable for derivatization include nucleophilic groups, such as amino, hydroxy and sulfhydryl groups. A suitable method for lipophilic derivatization is the addition of sulfhydryl groups to active ingredients, such as captopril.

-25, direct alkylation, for example by reaction with an alkyl halide under basic conditions, and thiol ester formation by reaction with an activated carboxylic acid. Active agents containing carboxylic acid functional groups, such as atenolol or chlorambucil, can be derivatized by amide or ester formation by coupling the amines with alcohols having suitable physical properties. In a preferred embodiment, cholesterol is coupled to the therapeutic compound, thus forming a degradable ester bond.

The invention is preferably used, for example, in angiogenesis, which is the formation of new blood vessels by branching out from existing blood vessels. The primary stimulus for this process is inadequate nutrient and oxygen supply to cells in tissues (hypoxia). The cells respond by secreting angiogenic factors, which can be very diverse, one of which is vascular endothelial growth factor. These factors trigger the secretion of proteolytic enzymes that degrade basement membrane proteins, as well as inhibitors that block the action of these potentially harmful enzymes. The combined effect of reduced signaling from receptors that bind angiogenic factors and their association with them, induces endothelial cell migration, proliferation, and rearrangement, and ultimately basement membrane synthesis around the new blood vessels.

Tumors must initiate angiogenesis when they reach a millimeter size in order to sustain their growth. Because angiogenesis is accompanied by characteristic changes in endothelial cells and their proliferation, this process is a promising target for therapeutic intervention. The changes that accompany angiogenesis are also very promising for diagnosis, as a

-26Malignant disease is a prime example, but this concept also holds promise for inflammatory and various inflammation-related diseases. These factors are also involved in the revascularization of damaged parts of the myocardium, which occurs when the stenosis is relieved within a short period of time.

The following tables list several known receptors/targets that are involved in hematopoiesis. Using the targeting principles described herein, angiogenesis can be detected by most imaging modalities used in medicine. Contrast-enhanced ultrasound imaging has the added advantage that the contrast medium consists of microspheres that are confined to the interior of blood vessels. Even though the target antigens are found on a variety of cell types, the microspheres will bind exclusively to endothelial cells.

So-called prodrugs can also be used with the substances of the invention. These active ingredients can be derivatized to alter their physicochemical properties and make them suitable for incorporation into the signal carrier; such derivatized active ingredients can be considered prodrugs and are generally inactive until the derivatizing group is cleaved off and the active form of the active ingredient is formed.

By directing gas-filled bubbles containing prodrug-activating enzymes to pathological sites, enzyme targeting can be imaged, allowing visualization of when the microbubbles properly reach pathological sites and simultaneously disappear from non-target areas. This can help determine the optimal timing of prodrug injection for individual patients.

Another solution is to encapsulate the prodrug, the prodrug-activating enzyme, and the vector in the same microbubble in a system where the prodrug is only released by certain external stimuli.

-27. Such a stimulus could be, for example, a tumor-specific protease, as described above, or the bursting of microbubbles by external ultrasound after they have reached the desired target.

With the solution according to the invention, various active ingredients can be easily delivered to diseased or dead areas, such as the heart, the general vasculature, as well as the liver, spleen, kidneys and other areas, such as the lymphatic system, body cavities or the gastrointestinal system.

The products of the invention can be used for targeted drug delivery in vivo or in vitro. In the latter case, the products can be used in in vitro systems, such as in kits for diagnosing various diseases or for characterizing various components of blood and tissue samples. Methods such as those used to bind certain blood components or cells to polymeric particles (e.g. monodisperse magnetic particles) and separate them from a sample in vitro can also be used in the solution of the invention, in which the low density of the signal-carrying units is used to separate the gaseous material by flotation and repeated washing.

The coupling of the signal carrier unit to the desired vector (and/or therapeutic agent) can be carried out by covalent or non-covalent bonding, generally by bringing into interaction the functional groups attached to the receptor and/or vector and/or any intermediate linker group/spacer element. Chemically reactive groups that can be used for this purpose include, for example, amino, hydroxyl, sulfhydryl, carboxyl and carbonyl groups, as well as carbohydrate groups, vicinal diols, thioethers, 2-amino

-28alcohols, 2-aminothiols, guanidyl, imidazolyl and phenol groups.

Covalent coupling of the signal carrier and the vector can thus be accomplished with a coupling agent containing reactive moieties capable of reacting with such functional groups. Reactive groups capable of reacting with sulfhydryl groups include α-haloacetyl compounds of the formula X-CH ₂ CO- (wherein X=Br, Cl or J), which are particularly reactive with sulfhydryl groups, but can also be used to modify imidazolyl, thioetherphenol and amine groups, as described in Gurd, FRN, Methods Enzymol. (1967) 11, 532. N-Maleimide derivatives are also selective for sulfhydryl groups, but may also be useful for coupling amino groups under certain circumstances. N-Maleimides can be incorporated into coupling systems for carrier-vector conjugation, as described by Kitagawa et al. (Kitagawa, T. et al., Chem. Pharm. Bull. (1981) 29, 1130), or they can be used as polymer crosslinkers to stabilize vesicles (Kovacic, P. et al. in J. Am. Chem. Soc. (1959) 81, 1887). Reagents such as 2-iminothiolane (Traut, R. et al., Biochemistry (1973) 12, 3266), which introduce the thiol group via conversion of the amino group, can also be considered sulfhydryl reagents if the coupling is effected by disulfide bridge formation. Reagents that introduce reactive disulfide bonds into either the carrier or the vector may also be useful, as they can create linkage by disulfide exchange between the vector and carrier, such reagents include, for example, Ellman's reagent (DTNB), 4,4'-dithiodipyridine, methyl-3-nitro-2-pyridyl disulfide, and methyl-2-pyridyl disulfide (Kimura, T. et al., Analyt. Biochem. (1982) 122, 271).

-29Reactive molecular moieties capable of reacting with amino groups include alkylating and acylating agents. Examples of alkylating agents include the following:

i) α-haloacetyl compounds which are specific for amino groups without the presence of reactive thiol groups and which are described by the general formula -X-CH2-CO, where X=Cl, Br or J (e.g. Wong, Y-H.H., Biochemistry (1979) 24, 5337);

ii) N-maleimide derivatives which react with the amino groups either by a Michael type reaction or are linked to the ring carbonyl group by acylation (Smyth, D. G. et al., J. Am. Chem. Soc. (1960) 82, 4600 and Biochem. J. (1964) 91, 589);

iii) aryl halides, such as reactive nitrohalogen aromatic compounds;

iv) alkyl halides (Me Kenzie, J.A. et al., J. Protein Chem. (1988) 7, 581);

v) aldehydes and ketones that are capable of forming Schiff bases with amino groups, the resulting adduct is usually stabilized by reduction to form a stable amine;

vi) epoxide derivatives, such as epichlorohydrin and bisoxyranes, which are capable of reacting with amino, sulfhydryl or phenolic hydroxyl groups;

vii) chlorine-containing derivatives of s-triazines, which are highly reactive towards nucleophiles such as amino, sulfhydryl and hydroxyl groups;

viii) aziridines containing s-triazine compounds (Ross, W.C.J., Adv. Cancer Res. (1954) 2), which are capable of reacting with nucleophiles, such as amino groups, by ring opening;

ix) squaric acid diethyl esters (Tietze, L.F., Chem. Ber. (1991) 124, 1215); and

x) α-haloalkyl ethers, which are more reactive with alkylating agents than normal alkyl halides due to the activating effect of the ether oxygen atoms (Benneche, T. et al., Eur. J. Med. Chem. (1993) 28,463).

Examples of acylating agents reactive with the amino group include:

i) isocyanates and isothiocyanates, especially aromatic derivatives, which form stable urea and thiourea derivatives and which are used for crosslinking proteins (Schick, A.F. et al., J. Biol. Chem. (1961) 236, 2477);

ii) sulfonyl chlorides (Herzig, D.J. et al., Biopolymers (1964) 2, 349), which are suitable for introducing fluorescent signal-carrying groups into the linker;

(iii) acid halides;

iv) active esters such as nitrophenyl esters or N-hydroxysuccinimidyl esters;

v) acid anhydrides, such as mixed, symmetrical or N-carboxyanhydrides;

vi) other suitable reagents for forming amide bonds (Bodansky, M. et al., Principles of Peptide Synthesis (1984) Springer-Verlag);

vii) acyl azides, for example in which the azide group is liberated from preformed hydrazine derivatives with sodium nitrite (e.g. Wetz, K. et al., Anal. Biochem. (1974) 58, 347);

viii) azlactones which are linked to polymers such as bisacrylamide (e.g. Rasmussen, J.K., Reactive Polymers (1991) 16, 199);

ix) imidoesters which form stable amidines upon reaction with amino groups (Hunter, M.J. and Ludwig, M.L., J.Am. Chem. Soc. (1962) 84, 3491).

Carbonyl groups, such as aldehyde groups, can be reacted with weak protein bases at a pH such that the nucleophilic protein side chain functional groups are protonated. Weak bases include 1,2-aminothiols, for example, those found on the N-terminal cysteine residues, which selectively form stable 5-membered thiazolidine rings with aldehyde groups (Ratner, S. et al., J. Am. Chem. Soc. (1937) 59, 200). Other weak bases include, for example, phenylhydrazones (Heitzman, H. et al., Proc. Natl. Acad. Sci. USA (1974) 71, 3537).

Aldehydes and ketones can also be reacted with amines to form Schiff bases, which can be advantageously stabilized by reductive amination. Alkoxylamino groups readily react with ketones and aldehydes to form stable alkoxyamines (Webb, R. et al., Bioconjugate Chem. 2 (1990) 1, 96).

Reactive groups capable of reacting with carboxyl groups include diazo compounds, such as diazoacetate esters and diazoacetamides, which are converted to ester groups with high specificity (Herriot R.M., Adv. Protein.Chem. (1947) 3,169). Carboxylic acid modifying reagents, such as carbodiimides, which react via O-acyl urea formation followed by amide bond formation, may also be used; coupling may be facilitated by addition of an amine or the coupling may result in direct vector-receptor binding. Suitable water-soluble carbodiimides include 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide (CMC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Zot, H.G. and Puett, D., J. Biol. Chem. (1989) 264,15552). Other suitable carboxylic acid modifying reagents

-32reagents include isoxazolium derivatives, such as Woodwards reagent K, 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 useful reactive materials include vicinal diones, such as p-phenylenediglyoxal, which can also be used to react with guanidyl groups (Wagner et al., Nucleic acid Res. (1978) 5, 4065); and diazonium salts, which undergo electrophilic substitution reactions (Ishizaka T., J. Immunol. (1960) 85, 163). Bisdiazonium compounds can be readily prepared by reacting aryldiamines with sodium nitrite in acidic solution. It will be appreciated that the functional groups on the carrier and/or vector may be converted to other functional groups prior to the reaction, for example to provide additional reactivity or selectivity. Suitable methods for this purpose include, for example, the conversion of amines to carboxylic acids using dicarboxylic anhydrides; conversion of amines to thiols, for example using N-acetylhomocysteine thiolactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane or thiol-containing succinimidyl derivatives; conversion of thiols to carboxylic acids, for example using α-haloacetates; conversion of thiols to amines, for example using ethyleneimine or 2-bromoethylamine; conversion of carboxylic acids to amines, for example using carbodiimides and then diamines; and conversion of alcohols to thiols, for example using tosyl chloride, followed by transesterification with thioacetate, followed by hydrolysis to obtain thiols using sodium acetate.

-33A vector-signal carrier coupling can also be performed using enzymes as zero-length coupling agents; for example, transglutaminase, peroxidase, and xanthine oxidase can be used. Reverse proteolysis can also be used for coupling via amide bond formation.

Non-covalent vector-carrier coupling can be achieved, for example, by electrostatic charge interactions, such as between a polylysinyl-functionalized carrier and a polyglutamyl-functionalized vector, by chelation to form stable metal complexes, or by high affinity binding interactions, such as avidin/biotin binding. Polylysine, non-covalently applied to a negatively charged membrane surface, can also non-specifically increase the affinity of microbubbles for a cell through charge interactions.

Alternatively, the vector may be linked to a protein known to bind phospholipids. In many cases, a single phospholipid molecule may be linked to the protein, such as the translocase, while other proteins are linked to surfaces that contain predominantly phospholipid head groups and are therefore useful for linking vectors to phospholipid microspheres; such proteins include, for example, β2-glycoprotein I (Chonn, A., Semple, S.C. and Cullis, P.R., Journal of Biological Chemistry (1995) 270, 25845-25849). Phosphatidylserine-binding proteins are described, for example, by Igarashi et al. (Igarashi, K. et al., Journal of Biological Chemistry 270(49), 29075-29078); Thus, a conjugate consisting of a vector and such a phosphatidylserine binding protein can be used to attach the vector to phosphatidylserine-encapsulated microbubbles. If the amino acid sequence of the binding protein is known, the phospholipid binding portion can be synthesized or isolated and

-34 is used for conjugation with the vector, thus bypassing the biological activity that is located elsewhere in the molecule.

Molecules that specifically bind to the surface (or membrane) of microspheres can also be obtained by directly screening molecular libraries for microsphere-binding molecules. For example, phage libraries containing small peptides can be used for this screening. For example, the screening is performed by simply mixing the microspheres and the phage containing the library and then eluting the phage that bind to the floating microspheres. If necessary, the screening can also be performed under physiological conditions (e.g. in blood) to eliminate peptides that cross-react with blood components. The advantage of this type of screening is that only binding molecules that do not destabilize the microspheres are selected, since only molecules bound to intact floating spheres rise to the surface. It is also possible to apply some kind of stress during the selection process (e.g., by applying pressure) to ensure that destabilizing binding molecules are not selected. Furthermore, the selection can be performed under shear conditions, for example, by first allowing the phages to react with the microspheres and then allowing the microspheres to pass through a surface coated with anti-phage antibodies under flow conditions. In this way, it is possible to select binding molecules that are resistant to the shear conditions that exist in vivo. Binding molecules identified in this way can be linked (by chemical linkage, or peptide synthesis, or at the DNA level in the case of recombinant vectors) to vector molecules, providing a general method for linking any vector to microspheres.

-35 A vector that is linked to or includes a peptide lipo-oligosaccharide or lipopeptide linker that contains a membrane insertion-mediating element may also be useful. An example of this is described in Leenhouts, J.M. et al., Febs Letters (1995) 370(3), 189-192. Non-bioactive molecules that contain known membrane insertion anchor/signal groups may also be useful for certain applications, such as the hydrophobic segment H1 from the Na, K-ATPase α-subunit, described in Xie, Y. and Morimoto, T. in J. Biol. Chem. (1995) 270(20) 11985-1199. The anchor group may also be a fatty acid or cholesterol.

The coupling can also be carried out using avidin or streptavidin, which contain four high-affinity binding sites for biotin. Avidin is therefore suitable for conjugating vectors to signal carriers when both the vector and the signal carrier are biotinylated. Examples of this are described in the following literature: Bayer, E.A. and Wilchek, M., Methods Biochem. Anal. (1980) 26, 1. This method can also be extended to the coupling of signal carrier to signal carrier, a process that promotes the coalescence of the bubbles and consequently a potential increase in echogenicity. Avidin or streptavidin can also be directly coupled to the surface of the signal carrier microparticles.

Non-covalent binding can also take advantage of the bifunctional nature of bispecific immunoglobulins. These molecules are capable of specifically binding to two antigens and thus linking them. For example, bispecific IgG or chemically prepared bispecific F(ab)'2 fragments can be used as linkers. Heterobifunctional bispecific antibodies have also been described as being capable of linking two different antigens (Bode, C. et al., J.

-36Biol. Chem. (1989) 264, 944 and Staerz U.D. et al., Proc. Natl. Acad. Sci. USA (1986) 83, 1453). Similarly, any carrier and/or vector containing two or more antigenic determinants (Chen Aa et al., Am. J. Pathol. (1988) 130, 216) can be linked to antibody molecules to form a multi-bubble, cross-linked system with potentially increased echogenicity.

The coupling agents used in the solution according to the invention generally create a certain specificity of coupling between vector and signal carrier or signal carrier and signal carrier, and these coupling agents can also be used to couple one or more therapeutically active substances.

In some cases, it is advantageous to use a PEG component as a stabilizer, together with the vector or vectors or directly with the signal carrier in the same molecule, if the PEG does not serve as a spacer.

So-called zero-length linkers can also be used if desired according to the invention, these induce a direct bond between two reactive chemical groups without introducing an additional linking agent (e.g., carbodiimides or enzymatically generated amide bond), such as biotin/avidin systems, which induce non-covalent signal carrier-vector binding, as well as materials that induce hydrophobic or electrostatic interactions.

Most often, however, the linker comprises, for example, two or more reactive moieties as described above, which are connected by a spacer element. The presence of such a spacer allows the bifunctional linkers to react with specific functional groups within the molecule or between two different molecules, resulting in a bond between the two components and introducing an external, linker-like

-37bound material into the signal carrier-vector conjugate. The reactive groups in the coupling agent can be the same (homobifunctional agents) or different (heterobifunctional agents) or if several different reactive groups are present (heteromultifunctional agents), thus different potential reagents can be formed that create a covalent bond between any chemical substances, either intramolecularly or intermolecularly.

The nature of the external material introduced by the coupling agent can have a critical impact on the targeting capability and overall stability of the final product. Thus, it may be necessary to introduce labile linkages, for example, those that are biodegradable or have a spacer containing chemically sensitive or enzymatically cleavable sites. The spacer may also contain polymeric components, for example, those that act as surfactants or that enhance bubble stability. The spacer may also contain reactive groups, for example, those mentioned above, to enhance surface crosslinking, or may contain tracer elements, such as a fluorescent label, a spin label, or a radioactive material.

The contrast agents of the invention are thus suitable for use in any imaging method, since contrast elements, such as X-ray contrast agents, light imaging patterns, spin labels or radioactive moieties, can all be easily incorporated or linked to the signal carrier moieties.

Spacer elements usually contain aliphatic chains that effectively separate the reactive moieties from each other by a distance of between 5 and 30 Å. They can also contain macromolecular structures, such as PEGs, which are of particular importance in biotechnical and biomedical applications (Milton Harris, J. Poly(ethylene glycol) chemistry, biotechnical and biomedical

-38applications Plenum Press, New York, 1992 ). PEGs are soluble in most solvents, including water, and are highly hydrated in aqueous environments, with two or three water molecules bound to each ethylene glycol segment; this has the effect of preventing the adsorption of other polymers or proteins to PEG-modified surfaces. PEGs are known to be nontoxic, nondestructive to active proteins or cells, while covalently bound PEGs are known to be nonimmunogenic and nonantigenic. Furthermore, PEGs can be readily modified and linked to other molecules without significantly affecting their chemical properties. Their advantageous solubility and biological properties are evident in the many uses known for PEGs and their copolymers, including block copolymers such as PEG-polyurethanes and PEG-polypropylenes.

Suitable molecular weights for the PEG spacers used according to the invention are generally between 120 Daltons and 20 kDaltons.

The main mechanism by which particles are taken up by cells of the reticuloendothelial system (RES) is opsonization by plasma proteins in the blood; these mark foreign particles which are then taken up by the RES. The biological properties of the PEG spacer elements used in the invention may increase the circulation time of contrast agents in a manner similar to that observed with PEGylated liposomes (Klibanov, A.L. et al., FEBS Letters (1990) 268, 235-237 and Blume, G. and Cevc, G., Biochim. Biophys. Acta 35 (1990) 1029, 91-97). Increased coupling efficiency to the desired region can also be achieved by using antibodies that are attached to the ends of the PEG spacers (Maruyama, K. et al., Biochim. Biophys. Acta (1995) 1234, 74-80 and Hansen, C.B. et al., Biochim. Biophys. Acta (1995) 1239, 133-144).

-39In certain cases, it may be advantageous to use the PEG component as a stabilizer together with the vector or vectors or directly with the signal carrier in the same molecule, where the PEG does not serve as a spacer.

Further examples of 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 starches, glycogen, dextran and aminodextrans; polyamino acids and their methyl and ethyl esters, such as those found in homo- and copolymers of lysine, glutamic acid and aspartic acid; polypeptides, oligosaccharides and oligonucleotides, which may or may not contain enzyme cleavage sites.

In general, spacer elements may contain cleavable groups, such as vicinal glycol, azo, sulfone, ester, thioester or disulfide groups. Spacers containing biodegradable methylene diester or diamide groups of the following general formula may also be used:

-(Z) _- .YXC(R'R ² ).XY(Z) _n - in the formula X and Z are -O-, -S- and -NR-, where R is hydrogen or an organic group; Y is carbonyl, thiocarbonyl, sulfonic, phosphoryl or other similar acid-forming group; m and n are 0 or 1; R and R are hydrogen, an organic group or a group of the formula -XY(Z) _m - or together form a divalent organic group -; these groups, as described for example in the publication WO-A-9217436, are easily biodegradable in the presence of esterases, for example in vivo, but are stable in such

-40 in the absence of enzymes. These can thus be advantageously linked to therapeutic agents to enable their slow release.

Poly[N-(2-hydroxyethyl)methacrylamides] are potentially suitable spacer materials due to their low affinity for cells and tissues (Volfová, L, Ríhová, B. and V.R. and Vetvicka, P., J. Bioact. Comp. Polymers (1992) 7, 175-190). Experiments with similar polymers, mainly containing the very similar 2-hydroxylpropyl derivative, have shown that they are only minimally endocytosed by the mononuclear phagocyte system (Goddard, P., Williamson, I., Bron, J., Hutchkinson, L.E., Nicholls, J. and Petrák, K., J. Bioct. Compat. Polym. (1991) 6, 4-24.).

Other potentially suitable polymer spacer materials include, for example:

i) methyl methacrylate and methacrylic acid copolymers; these may be erodible (Lee, P.I., Pharm. Res. (1993) 10, 980) and are more swellable than neutral polymers due to the carboxylate substituent;

ii) block copolymers of polymethacrylates and biodegradable polyesters (San Roman, J. and Guillen-Garcia, P., Biomaterials (1991) 12, 236-241);

iii) cyanoacrylates are polymers of 2-cyanoacrylic acid esters, which are biodegradable and can be used in the form of nanoparticles as selective drug carriers (Forestier, F., Gerrier, P., Chaumard, C., Quero, A.M., Couvreur, P. and Labarre, C., J. Antimicrob. Chemoter. (1992) 30, 173-179);

iv) polyvinyl alcohols, which are water-soluble and generally biocompatible (Langer, R., J. Control. Release (1991) 16, 53-60);

v) vinyl methyl ether and maleic anhydride copolymers, which are described as being bioerodible (Finné, U., Hannus, M. and Urtti, A., Int. J. Pharm. (1992) 78, 237-241);

vi) polyvinylpyrrolidones, for example those having a molecular weight of less than 25,000 and which are rapidly excreted by the kidneys (Hespe, W., Meier, A. M. and Blankwater, Y. M., Arzeim.-Forsch./Drug Res. (1977) 27, 1158-1162);

vii) polymers and copolymers of short chain aliphatic hydroxy acids such as glycolic acid, lactic acid, butyric acid, valeric acid and caproic acid (Carli, F., Chim. Ind. (Milano) (1993) 75,494-9), including copolymers containing aromatic hydroxy acids to increase the degradation rate (Imasaki, K., Yoshida, M., Fukuzaki, H., Asano, M., Kumakura, M., Mashimo, T., Yamanaka, H. and Nagai. T, Int. J. Pharm. (1992) 81, 31-38);

viii) polyesters containing alternating units of ethylene glycol and terephthalic acid, such as Dacron®, which are non-degradable but highly biocompatible;

ix) block copolymers containing segments of aliphatic hydroxy acid polymers (Younes, H., Nataf, P.R., Cohn, D., Appelbaum, Y.J., Pizov, G. and Uretzky, G., Biomater.Artif.Cells Artif.Organs (1988) 16,705-719), e.g. together with polyurethanes (Kobayashi, H., Hyon, S.H. and Ikada, Y., Water-curable and biodegradable prepolymers ” - J. Biomed.Mater.Res.(1991) 25,1481-1494);

x) polyurethanes, which are known to be well tolerated in implants and which can be combined with flexible soft segments, such as polytetramethylene glycol, polypropylene glycol or polyethylene glycol, and aromatic hard segments, such as 4,4'-methylenebis(phenylene isocyanate) (Ratner, B.D., Johnston, A.B. and Lenk, T.J, J. Biomed. Mater. Res:

-42Applied Biomaterials (1987) 21,59-90; Sa Da Costa,V.et al., J.Coll.Interface Sci. (1981) 80,445-452 and Affrossman,S.et al., Clinical Materials (1991) 8,25-31);

xi) poly(1,4-dioxan-2-one) derivatives, which are considered biodegradable due to their hydrolyzable ester linkages (Song,C.X., Cui,X.M. and Schindler,A., Med.Biol. Eng. Comput.(1993) 31, 5147-150) and which may contain glycol linkages to enhance their absorbability (Bezwada,R.S., Shalaby,S.W. and Newman,H.D.J., Agricultural and synthetic polymers: Biodegradability and utilization (1990) (Glass,J.E. and Swift,G.), 167174 - ACS symposium Series, #433, Washington DC., U.S.A. American Chemical Society);

xii) polyanhydrides, such as copolymers of sebacic acid (octanedioic acid) and bis(4-carboxyphenoxy)propane, which have been shown in rabbit experiments (Brem, H., Kader, A., Epstein, J.I., Tamargo, R.J., Domb, A., Langer, R. and Leong, K.W., Sci. Cancer Ther. (1989) 0 5, 55-65) and rat experiments (Tamargo, R.J., Epstein, J.I., Reinhard, C.S., Chasin, M. and Brem, H., J. IS Biomed. Mater. Res. (1989) 23, 253-266) to be suitable for the controlled release of active ingredients in the brain without any detectable toxic effects;

xiii) biodegradable polymers containing ortho-ester groups, which have been used for controlled release in vivo (Maa, Y.F. and Heller, J., J. Control. Release (1990) 14, 21-28); and xiv) polyphosphazenes, which are inorganic polymers containing alternating phosphorus and nitrogen atoms (Crommen, J.H., Vandorpe, J. and Schacht, E.H., J. Control. Release (1993) 24, 167-180).

The following tables summarize coupling agents and protein modifying agents that can be used in the preparation of targeted agents of the invention.

-43Heterobifunctional coupling agents

Couplings Reactivity 1 Reactivity 2 Note ABH carbohydrate photoreactive ANB-NOS -nh ₂ photoreactive ADP(l) -SH photoreactive iodizable disulfide linker APG -nh ₂ photoreactive Reacts selectively with Arg between pH=7-8 ASIB(l) -SH photoreactive iodizable ASBA(1) -COOH photoreactive iodizable EDC -nh ₂ -COOH zero-length linker GMBS -nh ₂ -SH sulfo-GMBS -nh ₂ -SH water soluble HSAB -nh ₂ photoreactive sulfo-HSAB -nh ₂ photoreactive water soluble MBS -nh ₂ -SH sulfom-MBS -nh ₂ -SH water soluble m ₂ c ₂ h carbohydrate -SH MPBH carbohydrate -SH NHS-ASA(1) -nh ₂ photoreactive iodizable sulfo-NHSASA(l) -nh ₂ photoreactive water soluble iodinated sulfo-NHS-LCASA(l) -nh ₂ photoreactive water-soluble iodinated PDPH carbohydrate -SH disulfide linker PNP-DTP -nh ₂ photoreactive SADP -nh ₂ photoreactive disulfide linker sulfo-SADP -nh ₂ photoreactive water-soluble disulfide linker SAED -nh ₂ photoreactive disulfide linker SAND -nh ₂ photoreactive water-soluble disulfide linker SANPAH -nh ₂ photoreactive sulfo-SANPAH -nh ₂ photoreactive water soluble

Couplings Reactivity 1 Reactivity 2 Note SASD(l) -NH ₂ photoreactive water-soluble iodinated disulfide linker SIAB -nh ₂ -SH sulfo-SIAB -nh ₂ -SH water soluble SMCC -nh ₂ -SH sulfo-SMCC -nh ₂ -SH water soluble SMBP -nh ₂ -SH sulfo-SMPB -nh ₂ -SH water soluble SMPT -nh ₂ -SH sulfo-LC-SMPT -nh ₂ -SH water soluble SPDP -nh ₂ -SH sulfo-SPDP -nh ₂ -SH water soluble sulfo-LC-SPDP -nh ₂ -SH water soluble sulfo-SAMCA(2) -nh ₂ photoreactive sulfo-SAPB -nh ₂ photoreactive water soluble

Note: (1) = iodinated; (2)= fluorescent

Homobifunctional coupling agents

Couplings Reactivity Note BS -nh ₂ BMH -SH BASED(l) photoreactive iodizable disulfide linker BSCOES -nh ₂ sulfo-BSCOES -nh ₂ water soluble DFBN -nh ₂ DMA -nh ₂ DMP -nh ₂ DMS -nh ₂ DPDPB -SH disulfide linker DSG -nh ₂ DSP -nh ₂ disulfide linker

Coupling agents Reactivity Note DSS -NH ₂ DST -nh ₂ sulfo-DST -nh ₂ water soluble DTBP -nh ₂ disulfide linker DTSSP -nh ₂ disulfide linker EGS -nh ₂ sulfo-EGS -nh ₂ water soluble SPBP -nh ₂

Biotinylating agents

Couplings Reactivity Note biotin-BMCC -SH biotin-DPPE* production of biotinylated liposomes biotin-LC-DPPE* production of biotinylated liposomes biotin-HPDP -SH disulfide linker biotin hydrazide carbohydrate biotin-LC-hydrazide carbohydrate iodoacetyl-LC-biotin -nh ₂ NHS-iminobiotin -nh ₂ reduced affinity for avidin NHS-SS-biotin -nh ₂ disulfide linker photoreactivatable biotin nucleic acids sulfo-NHS-biotin -nh ₂ water soluble sulfo-NHS-LC-biotin -nh ₂

Note: DPPE=dipalmitoylphosphatidylethanolamine; LC=long chain

-46Tools for protein modification

I like Reactivity Function Ellman's reagent -SH quantitation/detection/protection DTT -S.S- reduction 2-mercaptoethanol -S.S- reduction 2-mercaptylamine -S.S- reduction Traut's reagent -nh ₂ -SH introduction SATA -nh ₂ protected -SH introduction AMCA-NHS -nh ₂ fluorescence signal AMCA hydrazide carbohydrate fluorescence signal AMCA-HPDP -S.S- fluorescence signal SBF chloride -S.S- -SH fluorescence detection N-ethylmaleimide -S.S- -SH blocking NHS acetate -NH ₂ -NH ₂ blocking and acetylation citric acid -nh ₂ reversible blocking and negative charge introduction DTPA -nh ₂ chelating agent intake BNPS box tryptophan tryptophan group cleavage Bolton-Hunter -NH ₂ introduction of an iodinated group

Other potentially useful protein modifications include: partial or complete deglycosidation with neuraminidase, endoglycosidases or periodates, since deglycosidation often results in reduced uptake in the liver, spleen, macrophages, etc., while neo-glycosylation of proteins often results in increased uptake in the liver and macrophages; production of truncated forms by proteolytic cleavage, which leads to reduced size and shorter half-life in the circulatory system; cationization, for example, according to methods described in the following references: Kumagi et al., J. Biol. Chem. (1987) 262, 15214-15219; Triguero et al., Proc. Natl. Acad. Sci. USA (1989)

86, 4761-4765; Pardridge et al., J. Pharmacol. Exp. Therap. (1989) 251, 821-826 and Pardridge and Boado, Febs Lett. (1991) 288, 30-32.

Vectors useful for the targetable agents of the invention include, for example, the following:

i) antibodies which can be used as vectors for a very wide range of targets and which have the advantageous properties of very high specificity, high affinity (if necessary), the ability to be modified if necessary, etc. Whether an antibody is bioactive or not depends on the given vector/target combination. Both conventional and genetically produced antibodies can be used, the latter allowing the production of antibodies with special properties, e.g. affinity and specificity. It is advantageous to use human antibodies in order to avoid possible immune reactions against the vector molecules. A further suitable group of antibodies are the so-called bi- and multi-specific antibodies, i.e. antibodies which are specific for two or more different antigens in one antibody molecule. Such antibodies may be suitable, for example, for promoting the formation of bleb clusters and may be used for various therapeutic purposes, e.g. for the delivery of toxic substances to the target organ. Various aspects of bispecific antibodies are described in the following literature references: McGuinness B.T. et al., Nat. Biotechnol. (1996) 14, 1149-1154; George A.J. et al., J. Immunol. (1994) 152, 1802-1811; Bonardi et al., Cancer Res. (1993) 53, 3015-3021; French R.R. et al., Cancer Res. (1991) 51, 2353-2361.

ii) Cell adhesion molecules, their receptors, cytokines, growth factors, peptide hormones and fragments thereof. Such vectors are based on interactions between normal biological protein-protein and target molecule receptors and thus in many cases trigger a biological response upon binding to the target and thus

-48bioactive; this is relatively insignificant for vectors that target proteoglycans.

iii) Non-peptide agonists/antagonists or non-bioactive binders for receptors of cell adhesion molecules, cytokines, growth factors and peptide hormones. This category may also include non-bioactive vectors that are neither agonists nor antagonists, but which still have valuable targeting capabilities.

iv) Oligonucleotides and modified oligonucleotides that bind DNA or RNA by Watson-Crick or other types of base pairing. DNA is generally only present in extracellular sites as a consequence of cell damage, so that such nucleotides, which are generally non-bioactive, may be useful, for example, in delivery to necrotic sites, which exhibit a wide variety of pathological symptoms. Oligonucleotides may also be designed to bind to specific DNA or RNA binding proteins, such as transcription factors, which are often overexpressed or activated in tumor cells or in activated immune or endothelial cells. Combinatorial libraries may be used to select oligonucleotides that specifically bind to any potential target molecule and which may thus be used as targeting vectors.

v) DNA-binding agents, which behave similarly to oligonucleotides, but which may be biologically active and/or toxic when taken up by cells.

vi) Protease substrates/inhibitors. Proteases are involved in a variety of pathological conditions. Many substrates/inhibitors are non-peptides, but at least in the case of inhibitors, they are often bioactive.

vii) Vector molecules can be derived from combinatorial libraries without necessarily knowing the exact

-49molecular target, functionally selecting (in vitro, ex vivo or in vivo) molecules that bind to the imagined region/structure.

viii) Various small molecules, including bioactive molecules, known to bind to different types of biological receptors. Such vectors or their targets can be used to generate non-bioactive compounds that bind to the same target.

ix) Proteins or peptides that bind to glycosaminoglycan side chains, such as heparan sulfate, including glycosaminoglycan-binding sites of larger molecules, since binding to glycosaminoglycans does not result in a biological response. Proteoglycans are absent from red blood cells, eliminating unwanted absorption into these cells.

Other peptide vectors and their lipopeptides that are of particular interest in targeted ultrasound imaging include: atherosclerotic plaque-binding peptides such as YRALVDTLK, YAKFRETLEDTRDRMY and RALVDTEFKVKQEAGAK; thrombus-binding peptides such as NDGDFEEIPEEYLQ and GPRG; platelet-binding peptides such as PLYKKIIKKLLES; and cholecystokinin, α-melanocyte-stimulating hormone, heat-stable enterotoxin 1, vasoactive intestinal peptide, synthetic α-M2 peptide from the third heavy chain complement-determining region and analogs thereof for tumor targets.

The following tables summarize various vectors that can be directed to different types of targets and indicate the area where targeted diagnostic and/or therapeutic agents comprising such vectors according to the invention can be used.

-50Protein and peptide vectors-antibodies

Vector type Destination Notes/Areas of use Ref. antibodies in general CD34 vascular diseases in general, normal vascular wall (e.g. myocardium), activated endothelium, immune cells 1 II ICAM-1 II 1 II ICAM-2 II 1 II ICAM-3 II 1 II E-selectin II 1 II P-selectin II 1 II I am a II 1 II Integrins, e.g. VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, βία?, β1<Χ8, βιΑ _ν , LFA-1, Mac-1, CD41a, etc. II 1 II GlyCAM blood vessel walls in lymph nodes (very specific to lymph nodes) 3 II MadCam II 3 II fibrin thrombi 4 II tissue factor activated endothelium, tumors 5 II myosin necrosis, myocardial infarction 6 II CEA (carcinoembryonic antigen) tumors 7 II mucins tumors 8 II multiple drug resistant protein tumors 9 II prostate specific antigen prostate cancer

II Cathepsin B tumors (different types, more or less often specifically overexpressed in different tumors - Cathepsin B is one such protease) 10 II transferrin receptor tumors, vascular wall 11 MoAb 9.2.27 tumors antigen upregulated on cell growth 12 VAP-1 adhesion molecule 13 Lane 3 protein upregulated during phagocytic activity antibodies CD34 (sialomucin) endothelial cells antibodies CD31 (PECAM-1) endothelial cells antibodies intermediate fibers necrotic cells/tissue antibodies CD44 tumor cells the antibodies P2-microglobulin usually b antibodies MHC Class 1 usually b antibodies integrin ανβ3 tumors; angiogenesis c

References

a) Heider, K. Η., Μ. Sproll, S. Susani, E. Patzelt, P. Beaumier, E.

Ostermann, H. Ahom, and G.R. Adolf. 1996. Characterization of a high-affinity monoclonal antibody specific for C D44v6 as a candidate for immunotherapy of squamous cell carcinomas. Cancer Immunology Immunotherapy 43: 245-253.

b) I. Roitt, J. Brostoff, and D. Male. 1985. Immunology, London: Gower Medical Publishing, 4.7 c) Stromblad, S., and DA Cheresh. 1996. Integrins angiogenesis and vascular cell survival. Chemistry & Biology 3: 881-885.

-52Protein and peptide vectors - cell adhesion molecules, etc.

Vector type Destination Note/Featured area Ref L-selectin CD34 MadCAM1 GlyCam 1 vascular diseases in general, normal vascular wall (e.g. myocardium), activated endothelium, lymph nodes 3 Other selectins carbohydrate ligands (sialyl Lewis x) heparan sulfate vascular diseases in general, normal vascular wall (e.g. myocardium), activated endothelium, lymph nodes 14 RGD peptides integrins II 2 PECAM peptides PECAM and others endothelium, cells in the nervous system 15 Integrins, e.g. VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, βια ₇ , βια ₈ , βιΑ _ν , LFA-1, Mac-1, CD41a, etc. laminin, collagen, fibronectin, VCAM-1 thrombuspondin, vitronectin, etc. endothelium, vascular wall, etc. 16 Integrin receptors, such as laminin, collagen, fibronectin, VCAM-1 thrombuspondin, vitronectin, etc. Integrins, e.g. VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, βια ₇ , βια ₈ , βιΑ _ν , LFA-1, Mac-1, CD41a, etc. cells in the immune system, blood vessel walls, etc. 17 18 Neural cell adhesion molecules (N-CAM) intergin ανβ3 RGD peptides proteoglycans N-CAM (homophile) CD31 (PECAM-1) integrins endothelial cells angiogenesis 19 c

-53- ^{í :} r J. ^s .. ^:

Vectors containing cytokines/growth factors/peptide hormones and fragments thereof

Vector type Destination Comment/Area of use Ref Epidermal growth factor EGF receptor or similar receptors tumors 20 Nerve growth factor NGF receptor tumors 21 Somatostatin ST receptor tumors 22 Endothelin endothelin receptor blood vessel wall Interleukin-1 IL-1 receptor different types of inflammation, activated cells 23 Interleukin-2 IL-2 receptor II 24 Chemokines (about 20 different cytokines in part vectors) chemokine receptors, proteoglycans inflammations 25 Tumor necrosis factor TNF receptors inflammation Parathyroid hormone PTH receptors bone diseases kidney diseases Bone morphogenetic protein BMR receptors bone diseases Calcium CT receptors bone diseases Colony stimulating factors (G-CSF, GM-CSF, M-CSF, IL-3) appropriate specific receptors, proteoglycans endothelium 26 Insulin-like growth factor I IGF-I receptor tumors, other growth tissues Atrial natriuretic factor ANF receptors kidney, blood vessel wall Vasopressin Vasopressin receptor kidney, blood vessel wall VEGF VEGF receptor endothelium, vascularization areas

-54* » 4* » ·· • ·· v · ♦· * » • < · · · «·· ' ’·· · · · · * *

Fibroblast growth factors FGF receptors, proteoglycans endothelium angiogenesis 27 Schwann cell growth factor proteoglycan specific receptors 28

Other proteins and peptide vectors

Vector type Destination Area of observation/use Ref Streptavidin kidney kidney diseases 29 Bacterial fibronectin-binding proteins fibronectin blood vessel wall 30 Antibody Fc portion Fc receptors monocytes macrophages liver 31 Transferrin transferrin receptor tumors vascular walls 11 Streptokinase/ tissue plasminogen activator thrombi thrombi Plasminogen, plasmin fibrin thrombi, tumors 32 Mast cell proteinases proteoglycans 33 Elastase proteoglycans 34 Lipoprotein lipase proteoglycans 35 Coagulation and enzymes Coagulation enzymes 36 Extracellular superoxide dismutase proteoglycans 37 Heparin cofactor II proteoglycans 38 Retinal survival factor proteoglycan specific receptors 39 Heparin-binding brain mitogen proteoglycan specific receptors 40 Apolipoproteins, for example proteoglycans specific receptors, 41

Apolipoprotein B (e.g. LDL receptor) Apolipoprotein E LDL receptor proteoglycans 42 Adhesion-promoting proteins, such as purpurin proteoglycans 43 Viral coat proteins, e.g. HIV, Herpes proteoglycans 44 Microbial adhesins, such as Antigen 85 mycobacteria complex fibronectin, collagen, fibrinogen, vitronectin, heparin sulfate 45 β-amyloid precursor proteoglycans β-amyloid accumulates in Alzheimer's disease 46 Tenascin, such as Tenascin C heparin sulfate, integrins 47

Vectors containing non-peptide agonists/antagonists or non-bioactive binding agents of cytokines/growth factors/peptide hormones/cell adhesion molecules

Vector type Destination Comment/Area of use Ref different agonists/antagonists are known for such factors, which act through G-protein-coupled receptors 48 49 Endothelin antagonist Endothelin receptor blood vessel wall Desmopressin (vasopressin analogue) vasopressin receptor kidney vessel wall Demoxytocin (oxytocin analogue) oxytocin receptor reproductive organs, mammary glands, brain Angiotensin II receptor antagonists Angiotensin II receptors blood vessel wall. brain adrenal glands

CV-11974 TCV-116 Non-peptide RGD- -analogs integrins cells in the immune system, blood vessel walls, etc. 50

Vectors containing antifactors

Vector type Destination Comment/Area of use Ref Angiostatin tumor EC plasminogen fragment K Charge-derived inhibitor tumor EC blood vessel wall J β-cyclodextrin tetradecasulfate tumors, inflammations C Fumagillin and its analogues tumors, inflammations This Interferon-a tumor EC K Interferon-γ tumor EC This Interleukin-12 tumor EC This Linomid tumors, inflammations The Medroxyprogesterone tumor EC K Metalloproteinase inhibitors tumor EC K Pentosan polysulfate tumor EC K Platelet factor 4 tumor EC M Somatostatin tumor EC K Sure amine tumor EC K Taxol tumor EC K Thalidomide tumor EC K Thrombospondin tumor EC K

vectors containing factors

Vector type Destination Note/ area of use Ref Acidic fibroblast growth factor tumor EC K Adenosine tumor EC K

Angiogenin tumor EC K Angiotensin II tumor EC K Basic membrane components tumors e.g. tenascin, collagen IV M Basic fibroblast growth factor tumor EC K Bradykinin tumor EC K Calcitonin gene-derived peptide tumor EC K Epidermal growth factor tumor EC K Fibrin tumors K Fibrinogen tumors K Heparin tumor EC K Histamine tumor EC K Hyaluronic acid or its fragments tumor EC K Interleukin-1a tumor EC K Laminin, laminin fragments tumor EC K Nicotinamide tumor EC K Platelet activating factor tumor EC K Platelet-derived endothelial growth factor tumor EC K Prostaglandin E1, E2 tumor EC K Spermine tumor EC K Spermine tumor EC K Material P tumor EC K Transforming Growth Factor-a tumor EC K Transforming growth factor-β tumor EC K Tumor necrosis factor-α tumor EC K Vascular endothelial growth factor/vascular permeability factor tumor EC K Vitronectin tumor EC The

-58Vector molecules or other recognized angiogenic factors with known affinity for angiogenesis-related receptors

Vector type Destination Note/ area of use Ref Angiopoietin tumors, inflammation B az-antiplasmin tumors, inflammation Combination libraries, compounds from which are listed tumors, inflammation for example, compounds that bind to the basement membrane after degradation Endoglycan tumors, inflammation D Endosialin tumors, inflammation D Endostatin (collagen fragment) tumors, inflammation M Factor VII related antigen tumors, inflammation D Fibrinopeptides tumors, inflammation ZC Fibroblast growth factor, basic tumors, inflammation This Hepatocyte growth factor tumors, inflammation I Insulin-like growth factor tumors, inflammation R Interleukins tumors, inflammation I Leukemia inhibitory factor tumors, inflammation The Metalloproteinase inhibitors tumors, inflammation This

Monoclonal antibodies tumors, inflammation for example: against angiogenic factors or their receptors or components of the fibrinolytic system Peptides such as cyclic RGD _d FV tumors, inflammation B,Q Placental growth factor tumors, inflammation J Placental proliferin-related protein tumors, inflammation This Plasminogen tumors, inflammation M Plasminogen activator tumors, inflammation D Plasminogen activator inhibitors tumors, inflammation U,V Platelet activating factor antagonists tumors, inflammation angiogenesis inhibitors The Platelet-derived growth factor tumors, inflammation KE Pleiotropin tumors, inflammation KZA Proliferin tumors, inflammation KE Proliferin-related protein tumors, inflammation This Selections tumors, inflammation for example E-selectin D SPARC tumors, inflammation M Snake venoms (RGD-containing) tumors, inflammation Q Tissue inhibitor of metalloproteinases tumors, inflammation for example TIMP-2 u Thrombin tumors, inflammation H Thrombin receptor activating tetradecapeptide tumors, inflammation H

Thymidine phosphorylase tumors, inflammation D Tumor growth factor tumors, inflammation ZA

Receptors/Targets Associated with Angiogenesis

Vector type Destination Note/ area of use Ref Biglikan tumors, inflammation dermatan sulfate proteoglycan X CD34 tumors, inflammation L CD44 tumors, inflammation F Collagen type I, IV, VI, VIII tumors, inflammation The Decorative tumors, inflammation dermatan sulfate proteoglycan Y Dermatan sulfate tumors, inflammation X Endothelin tumors, inflammation G Endothelin receptors tumors, inflammation G Fibronectin tumors P Flk-1/KDR, Flt-4 tumors, inflammation VEGF receptor D FLT-1 (fms-like tyrosine kinase) tumors, inflammation VEGF-A receptor 0 Heparan sulfate tumors, inflammation P Hepatocyte growth factor receptor (c-met) tumors, inflammation I Insulin-like growth factor/mannose-6-phosphate receptor tumors, inflammation R

Integrins: β3 and β ₅ , integrin α _ν β3, integrin αββι, integrins αβ, integrins βι, integrin α ₂ βι, integrin α _ν β3, integrin and integrin α _ν β5 fibrin receptors tumors, inflammation laminin receptor fibronectin receptor subunit D P Intercellular adhesion molecule-1 and -2 tumors, inflammation P Jagged gene product tumors, inflammation T Ly-6 tumors, inflammation lymphocyte activation protein N Matrix metalloproteinase tumors, inflammation D MHC class II tumors, inflammation Notch gene product tumors, inflammation T Osteopontin tumors Z PECAM tumors, inflammation CD31 P Plasminogen activator receptor tumors, inflammation ZC Platelet-derived growth factor receptors tumors, inflammation This Selectins: E-, P- tumors, inflammation D Sialyl Lewis-X tumors, inflammation blood group antigen M Stress proteins: glucose-regulated heat shock family and others tumors, inflammation molecular chaperones Syndecan tumors, inflammation T Thrombospondin tumors, inflammation M

TIE receptors tumors, inflammation tyrosine kinase with Ig and EGF-like domains This Tissue factor tumors, inflammation Z Tissue metalloproteinase inhibitor tumors, inflammation for example TIMP-2 U Transforming growth factor receptor tumors, inflammation This Urokinase-type plasminogen activator receptor tumors, inflammation D Vascular cellular adhesion molecule tumors, inflammation D Vascular endothelial growth factor-related protein tumors, inflammation Vascular endothelial growth factor-A receptor tumors, inflammation K von Willebrand factor-related antigen tumors, inflammation L

Oligonucleotide vectors

Vector type Destination Note/ area of use Ref Oligonucleotides that are complementary to repetitive sequences, such as ribosomal RNA, genes with Alu sequences DNA accessible by necrosis tumors myocardial infarction other other diseases that involve necrosis 51 Oligonucleotides that are complementary to disease-specific mutations (e.g. mutated oncogenes) DNA that becomes accessible through necrosis at a stage of the relevant disease tumors 51 Oligonucleotides that are complementary to the DNA of the infectious agent the DNA of the infectious agent viral or bacterial infections 51

Triple or quadruple helix-forming oligonucleotides as in the examples above as in the examples above 51 Oligonucleotides containing sequences that recognize DNA or RNA binding proteins DNA-binding protein e.g. transcription factors (often overexpressed/activated in tumors or endothelial/immune cells) Tumors Activated endothelium Activated immune cells

Moc recommended oligonucleotide vectors Vector type Destination Note/ area of use Ref Phosphorthioate oligos as with unmodified oligos as with unmodified oligos 51 2'-O-methyl-substituted oligos you II 51 Circular oligos 11 II 51 Oligos containing a hairpin structure to reduce degradation II II 51 Oligos with terminal phosphorothioate II II 51 2'-fluoro-oligo II II 51 2'-amino-oligo 11 II 51 DNA-binding agents conjugated to oligos (for example, see below) II increased binding affinity compared to pure oligos 52 Peptide nucleic acids (PNAs, oligonucleotides with peptide backbones) II increased binding affinity and stability compared to standard oligos 53

-64Nucleosides and nucleotide vectors

Vector type Destination Note/ area of use Ref Adenosine or its analogues adenosine receptors vascular wall heart 54 ADP, UDP, UTP and more different nucleotide receptors many tissues, such as brain, spinal cord, kidney, spleen 55

Receptors that contain DNA-binding proteins

Vector type Destination Note/ area of use Ref Acridine derivatives distamycin netropsin actinomycin D echinomycin bleomycin DNA made accessible by necrosis tumors, myocardial infarction and other diseases in which necrosis or other processes that release DNA from the cell occur

Receptors containing protease substrates

Vector type Destination Note/ area of use Ref Peptide or non-peptide substrates Cathepsin B tumors, several of which may overexpress different types of proteases, such as Cathepsin B, more or less specifically 10

-65Protease inhibitor-containing receptors

Vector type Destination Note/ area of use Ref Peptide or non-peptide inhibitors, such as N-acetyl-Leu-Leu-norleucinal Cathepsin B tumors, several of which may overexpress different types of proteases, such as Cathepsin B, more or less specifically 10 Bestatin-([(2 S ,3 R)-3 -amino-2-hydroxy-4-phenylbutanoyl]-L-leucine hydrochloride) aminopeptidases tumors, for example on cell surfaces Pefablok (4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) serine proteases tumors, blood vessel walls, etc. Commercially available inhibitors such as captopril enalapril ricinoleate angiotensin converting enzyme endothelial cells Non-peptide compounds with low specificity coagulation factors vascular wall injuries, tumors, etc. Protease nexins (extracellular protease inhibitors) proteoglycans 56 Antithrombin proteoglycans, coagulation factors 57

-66Vectors from combinatorial libraries

Vector type Destination Note/ area of use Ref Antibodies whose structure has been determined over generations any of the above targets, or it may be unknown, we perform functional selection on the vector, which binds to the diseased structures to select it any disease or normal structure, such as thrombi, tumors, or myocardial vessel walls 58, 59, 60 Peptides whose sequences have been determined over generations II II 58, 59, 60 Oligonucleotides whose sequence has been determined over generations II II 58, 59, 60 Modifications of the oligos as above II II 58, 59, 60 Other chemicals whose structures have been determined over generations II II 58, 59, 60

Carbohydrate vectors

Vector type Destination Note/ area of use Ref Neoglycoproteins macrophages general activation/inflammation Oligosaccharides containing galactose at the terminal end asialoglycoprotein receptor liver 61 Hyaluronan aggrecan (a proteoglycan) binding proteins cell surface receptors: CD44 62

Mannose blood brain barrier, brain tumors and other diseases that cause changes in the BBB 63 Bacterial glycopeptides II 64

(Glyco)lipid vectors

Vector type Destination Note/ area of use Ref GM1 gangliosides cholera bacteria in the gastrointestinal tract diagnosis/treatment of cholera Platelet activating factor (PAF) antagonists PAF receptors diagnosis of inflammation Prostaglandin antagonists for inflammation prostaglandin receptors diagnosis of inflammation Thromboxane antagonists of inflammation leukotriene receptors diagnosis of inflammation

Small molecule vectors

Vector type Destination Note/ area of use Ref Adrenaline appropriate receptors beta blockers adrenergic beta- receptors myocardium for beta-1 blockers Alpha-blockers adrenergic alpha- receptors blood vessel wall B enzodiazepines Serotonin analogues serotonin receptors Antihistamines histamine receptors blood vessel wall Acetylcholine receptor antagonists ACh receptors

Verapamil Ca channel blocker myocardium Nifedipine Ca ^z+ channel blocker myocardium Amiloride So*/!!* exchange blocks this exchange in the kidney and is usually upregulated in cells stimulated by growth factors Digitalis glucosides Na ⁺ /K ⁺ -ATPases myocardium peripheral vasculature central nervous system Thromboxane/prostaglandin receptor antagonists or agonists thromboxane/prostaglandin receptors vascular wall, endothelium Glutathione glutathione receptors leukotriene receptors lung brain Biotin biotin transport protein on the cell surface 65 Folate folate transport protein on the cell surface tumors 66 Riboflavin riboflavin transport protein on the cell surface 67

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The following non-limiting examples illustrate the invention in more detail. The microparticulate nature of the product was confirmed by microscopic examination as described in WO-A-9607434. Ultrasonic transmission measurements were performed using a broadband transducer to detect microbubble suspensions that resulted in increased sound attenuation compared to the standard. Flow cytometric analysis of the products was used to confirm the binding of macromolecules. The ability of the targeted microbubbles to specifically bind to cells expressing a target was examined in vitro by microscopy and/or using a flow chamber containing immobilized cells, e.g. the target

We used cells expressing the -Ί4 structure, as well as cell populations that do not express the target. Radioactive, fluorescent or enzyme-labeled streptavidin/avidin systems are used for the analysis of biotin binding.

Example 1

Adhesion of poly-L-lysine-coated phosphatidylserine-encapsulated microbubbles to endothelial cells mg poly-L-lysine with a molecular weight of 115 kDa was dissolved in 400 μΐ water. 40 μΐ perfluorobutane encapsulated (encapsulated) in phosphatidylserine was incubated either in 400 μΐ water or in the poly-L-lysine solution for 15 minutes at room temperature. Zeta potential measurements confirmed that poly-L-lysine-coated microbubbles were positively charged, while uncoated bubbles were negatively charged. The microbubbles described above were tested for cell adhesion, using human endothelial cell growth on a culture plate, with uncoated microbubbles as a control. After incubation, microscopic examination of endothelial cells showed that poly-L-lysine-coated microbubbles adhered to endothelial cells in much greater numbers compared to non-coated microbubbles.

Example 2

Gas-filled microbubbles containing phosphatidylserine and RGDC-Mal-PEH ₃ 4 _O o-DSPE

a) Preparation of BOC-NH-PEG3400-DSPE (tert-butylcarbamate-polyethyleneglycol-distearoylphosphatidylethanolamine) g DSPE (distearoylphosphatidylethanolamine) was added to 150 mg Boc-NH-PEG ₃₄₀ o-SC (tert-butylcarbamate-polyethyleneglycol-succinimidylcarbonate) dissolved in 2 ml chloroform, then 33 μΐ triethylamine was added. The resulting mixture turned into a clear solution after 10 minutes,

After stirring at -7541°C. The solvent is then removed in a rotary evaporator, the residue is dissolved in 5 ml of acetonitrile. The resulting dispersion is cooled to 4°C, centrifuged, the solution is separated from the undissolved material and evaporated to dryness. The structure of the resulting product is confirmed by NMR analysis.

b) Preparation of H2N-PEG3400-DSPE (amino-polyethylene glycol-distearoylphosphatidylethanolamine)

167 mg of Boc-NH-PEG34oo-DSPE was stirred in 5 ml of 4 M hydrochloric acid/dioxane solution for 2.5 hours at ambient temperature. The solvent was then removed in a rotary evaporator, the residue was dissolved in 1.5 ml of chloroform and washed with 2x1.5 ml of water. The organic phase was removed in a rotary evaporator. The obtained material was analyzed by TLC (chloroform/methanol/water=1 3/5/0.8), the obtained product had an Rf=0.6; the structure of the product, which was ninhydrin positive, was confirmed by NMR analysis.

c) Preparation of Mal-PEG34oo-DSPE (3-maleimidopropionate-polyethylene glycol-distearoylphosphatidylethanolamine)

5.6 mg (0.018 mol) of N-succinimidyl-3-maleimidopropionate were dissolved in 0.2 ml of tetrahydrofuran, 65 mg (0.012 mmol) of H ₂ N-PEG34oo-DSEP dissolved in 1 ml of tetrahydrofuran were added, followed by 2 ml of 0.1 M sodium phosphate buffer. The resulting mixture was heated to 30°C, the reaction was monitored by TLC, and the solvent was evaporated.

d) Preparation of RGDC-Mal-PEG34oo-DSPE

0.010 mmol Mal-PEG3400-DSPE and 0.1 M sodium phosphate buffer (pH=7.5) were mixed and added to 0.010 mmol RGDC peptide. The mixture was then warmed to 37°C, if necessary, and the reaction was monitored by TLC, then the solvent was removed.

e) Preparation of gas-filled microbubbles entrapped in phosphatidylserine and RGDC-Mal-PEG ₃₄ oo-DSPE A mixture of mg phosphatidylserine (90-99.9 mol%) and Mal-PEG _34O o. -DSPE (10-0.1 mol%) is added to 1 ml of 5% propylene glycol/glycerol aqueous solution. The resulting dispersion is heated at no more than 80°C for 5 minutes and then cooled to room temperature. The resulting 0.8 ml dispersion is transferred to a 1 ml vial and the supernatant is rinsed with perfluorobutane. The vial is then shaken for 45 seconds using a stirrer and the sample is transferred to a cylindrical table. After centrifugation, the supernatant is replaced with 0.1 M sodium phosphate buffer (pH=7.5). Then, RGDC peptide dissolved in 0.1 M phosphate buffer (pH=7.5) is added to the washed microbubbles, which are placed on a roller table. The washing process is then repeated.

f) Another method for the preparation of gas-filled microbubbles enclosed in phosphatidylserine and RGDC-Mal-PEG _34O o-DSPE mg of phosphatidylserine is mixed with 1 ml of 5% propylene glycol/glycerol aqueous solution. The dispersion is heated at no more than 80°C for 5 minutes and then cooled to room temperature. The resulting 0.8 ml of dispersion is then transferred to a 1 ml vial, the part above the dispersion is rinsed with perfluorobutane. The vial is then shaken with a mixer for 45 minutes and the sample is then transferred to a roller table. After centrifugation, the supernatant is replaced with 0.1 M sodium phosphate (pH=7.5). Then, RGDC-Mal-PEG ₃₄ oo-DSPE dissolved in 0.1 M sodium phosphate buffer (pH=7.5) is added to the washed microbubbles and placed on a roller table. The washing process is repeated after the RGDC-Mal-PEG ₃₄ oo-DSPE microbubbles are incorporated into the membranes.

-ΊΊ -

Example 3

Gas-filled microbubbles enclosed in a phosphatidylserine, phosphatidylcholine and biotin-amidocaproate-PEG3400-Ala-cholesterol system

a) Preparation of Z-Ala-cholesterol (3-0-(carbobenzyloxy-L-alanyl)cholesterol) 1 mmol cholesterol, 5 mmol Z-alanine and 4 mmol dimethylaminopyridine are dissolved in a dimethylformamide/tetrahydrofuran mixture (20 ml + 5 ml) and dicyclohexylcarbodiimide is added. The reaction mixture is stirred at ambient temperature overnight. The dicyclohexylurea is filtered off and the solvent is removed in a rotary evaporator, the residue is dissolved in chloroform, the undissolved dicyclohexylurea is separated by filtration and the solvent is removed in a rotary evaporator. The residue is applied to a silica gel column and the Z-Ala-cholesterol is eluted with toluene/petroleum ether=20/2 and then toluene/diethyl ether=20/2 mixtures. The fractions containing the title compound were combined, the solvent was removed in a rotary evaporator, and the structure of the product was confirmed by NMR.

b) Production of Ala-cholesterol (3-O-(L-alanyl)-cholesterol)

0.48 ml of Z-Ala-cholesterol in 20 ml of tetrahydrofuran and 3 ml of glacial acetic acid was hydrogenated in the presence of 5% palladium on carbon for 2 hours. The mixture was then filtered and concentrated in vacuo.

c) Preparation of Boc-NH-PEG ₃ 4oo-Ala-cholesterol

Ala-cholesterol was added to Boc-NH-PEG3400-Sc (tert-butylcarbamate-polyethylene glycol succinimidyl carbonate) dissolved in chloroform, followed by the addition of triethylamine. The resulting suspension was stirred at 45°C for 10 minutes. The crude product was purified by chromatography.

Production of HiN-PE G3400-Ala-cholesterol

-78Boc-NH-PEG ₃ 4oo-Ala-cholesterol is stirred in a 4 M hydrochloric acid/dioxane solution for 2.5 hours at ambient temperature. The solvent is removed in a rotary evaporator, the residue is taken up in chloroform and then washed with water. The organic phase is evaporated to dryness in a rotary evaporator. The crude product is purified by chromatography.

e) Preparation of biotinamidocaproate-PEG ₃ 4oo-Ala-cholesterol

Biotinamide caproate-N-hydroxysuccinimide ester is added to H2N-PEG ₃ 4oo-Ala-cholesterol dissolved in tetrahydrofuran, which also contains 2 ml of 0.1 M sodium phosphate buffer (pH=7.5). The mixture is heated to 30°C, the reaction is monitored by TLC, and the solvent is evaporated.

f) Gas-filled microbubbles enclosed in a phosphatidylserine, phosphatidylcholine and biotinamidocaproate-PEG ₃ 4oo-Ala-cholesterol system mg of a mixture containing phosphatidylserine and phosphatidylcholine (total 90-99.9 mol%) and biotinamidocaproate-PEG ₃ 4oo-Ala-cholesterol (10-0.1 mol%) are added to 1 ml of 5% propylene glycol-glycerol aqueous solution. The resulting dispersion is heated at no more than 80°C for 5 minutes and then cooled to ambient temperature. 0.8 ml of the resulting dispersion is then transferred to a 1 ml vial, the upper part is rinsed with perfluorobutane, the vial is shaken for 45 s using a stirrer that can be closed with a cap, and the sample is then transferred to a roller table. After centrifugation, the supernatant is replaced with water and the washing operation is repeated.

g) Another method for the preparation of gas-filled microbubbles enclosed in a phosphatidylserine, phosphatidylcholine and biotinamidocaproate-PEG34oo-Ala-cholesterol system: A mixture containing 100 mg of phosphatidylserine and phosphatidylcholine is added to 1 ml of a 5% propylene glycol-glycerol aqueous solution. The mixture is

-79 dispersion is heated to no more than 80°C for 5 minutes and then cooled to ambient temperature. 0.8 ml of the dispersion is then transferred to a 1 ml vial, the headspace is rinsed with perfluorobutane. The vial is shaken for 45 seconds with a capped stirrer, and the sample is then transferred to a roller table. After centrifugation, the supernatant is replaced with water. Biotinamidocaproate-PEG34oo-Ala-cholesterol is then added to the washed microbubbles and then transferred to a roller table for several hours. The washing operation is repeated after the biotinamidocaproate-PEG34oo-Ala-cholesterol has been incorporated into the microbubbles membranes.

Example 4

Gas-filled microbubbles containing phosphatidylserine, phosphatidylcholine, biothionamidocaproate-PEG ₃₄ oo-Ala-cholesterol system and active ingredient-cholesterol system

a) Preparation of active ingredient cholesterol 1 mmol cholesterol, 4 mmol of an active ingredient containing an acidic group and dimethylaminopyridine are dissolved in a dimethylformamide/tetrahydrofuran mixture (20 ml + 5 ml), then dicyclohexylcarbodiimide is added thereto. The mixture is then stirred at room temperature for 1 night, then the dicyclohexylurea is filtered off, the solvent is removed and the title compound is purified by chromatography.

b) Preparation of gas-filled microbubbles enclosed in a system of phosphatidylserine, phosphatidylcholine, biothionamidocaproate-PEG ₃₄₀ o-Ala-cholesterol and active ingredient cholesterol A mixture containing mg of phosphatidylserine and phosphatidylcholine (total 90-99.9 mol%) and biothinamidocaproate-PEG ₃ 4oo-Ala-cholesterol (prepared according to Example 3) (total 10-0.1 mol%) is added to 1 ml of 5% propylene glycol-glycerol aqueous solution. The resulting dispersion

-80 is heated at not more than 80°C for 5 minutes and then cooled to room temperature. The resulting 0.8 ml dispersion is transferred to a 1 ml vial, the upper part is rinsed with perfluorobutane, the vial is shaken with a cap stirrer for 45 seconds, and then the sample is transferred to a roller table. After centrifugation, the supernatant is replaced with water and the washing procedure is repeated.

Example 5

Gas-filled microbubbles encapsulated in phosphatidylserine and thiolated anti-CD34-MAL-PE _34O o-DSPE

a) Production of thiolated anti-CD34 antibodies

Thiolation of anti-CD34 antibodies was performed according to the method of Hansen et al. (Hansen, C.B. et al. (1995) Biochim. Biophys. Acta 1239, 133-144).

b) Preparation of gas-filled microbubbles enclosed in phosphatidylserine and thiolated anti-CD34-MaI-PEG ₃₄₀₀ -DSPE To a mixture of mg phosphatidylserine (90-99.9 mol%) and Mal-PEG ₃₄₀ o-DSPE (10-0.1 mol%, prepared according to Example 2) 1 ml of 5% propylene glycol-glycerol aqueous solution is added. The dispersion is heated at no more than 80°C for 5 minutes and then cooled to room temperature. 0.8 ml of the dispersion thus obtained is transferred to a 1 ml vial, the upper part is rinsed with perfluorobutane. The vial is then shaken with a cap stirrer for 45 seconds, and the sample is then transferred to a roller table. After centrifugation, the supernatant is replaced with the appropriate buffer and the thiolated antibody is coupled to the microbubbles, for example according to the method described in the following literature references: Goundalkar, A., Ghose, T. and Mezei, M., J. Pharm. Pharmacol. (1984) 36 465-66 or Hansen, CB et al. (1995) Biochim. Biophys. Acta 1239 133-144. The microbubbles are then placed on a roller table for several hours and washed. The resulting microbubbles

-81 flow cytometric analysis (using fluorescently labeled secondary antibodies) is used to verify the binding of anti-CD34 antibodies to the bubbles. The ability of the bubbles to specifically bind to CD34-expressing cells is studied microscopically using a population of CD34-expressing cells and a population of cells that are not CD34-expressing.

Example 6

Biotin attached to gas-filled microbubbles

Biotin can be coupled to the microbubbles by various methods, for example, according to the method of Corley P. and Loughrey H.C. (Corley, P. and Loughrey, H.C., (1994) Biochim. Biophys. Acta 1195, 149-156). The resulting bubbles are then analyzed by flow cytometry, for example, using fluorescent streptavidin to detect the coupling of biotin to the bubble. In another method, a radioactive or enzyme-labeled streptavidin/avidin system is used to analyze biotin coupling.

Example 7

Gas-filled microbubbles enclosed in a distearoylphosphatidylserine and biotin-DPPE system

To 22.6 mg of distearoylphosphatidylserine (DSPS) is added 4 ml of aqueous 4% propylene glycol-glycerol solution. The dispersion is heated at no more than 80°C for 5 minutes and then cooled to ambient temperature. Then 1.5 mg of biotin-DPPE is dispersed in 1 ml of 4% aqueous propylene glycol-glycerol and the sample is placed on a roller table for 1-2 hours. The suspension is then filled into vials, the upper part is rinsed with perfluorobutane. The vials are shaken for 45 minutes and then placed on a roller table. After centrifugation for 7 minutes, the supernatant is replaced with water and the washing procedure is repeated twice. Evaporative

Normal phase HPLC equipped with a -82Light Scattering Detector confirms that the microbubble membrane contains 4 mol% biotin-DPPE. The average particle diameter of the microbubbles is 4 pm as determined by a Coulter Counter. Ultrasound transmission measurements using a 3.5 MHz broadband transducer showed that the attenuation of the sound beam for < 2 mg/ml particle dispersion is greater than 5 dB/cm.

Example 8

Non-covalent binding of gas-filled microbubbles enclosed in a phosphatidylserine/biotinylated antibody system to streptavidin-Succ-P EG ₃ 4 _O o-DSPE

a) Preparation of Succ-PEG ₃ 4oo-DSPE

NH2-PEG3400-DSPE (prepared according to Example 2) is carboxylated with succinic anhydride, for example according to the method described in Nayar, R. and Schroit, A.J., Biochemistry (1985) 24, 5967-71.

b) Preparation of gas-filled microbubbles encapsulated in the phosphatidylserine/Succ-PEG ₃ 4oo-DSPE system A mixture containing mg of phosphatidylserine (90-99.9 mol%) and Succ-PEG ₃ 4oo-DSPE (10-0.1 mol%) is added to 1 ml of an aqueous, 5% propylene glycol-glycerol solution. The resulting dispersion is heated at no more than 80°C for 5 minutes and then cooled to room temperature. 0.8 ml of the resulting dispersion is transferred to a 1 ml vial, the upper part is rinsed with perfluorobutane. The vial is shaken with a cap-type stirrer for 45 seconds, then the sample is transferred to a roller table. After centrifugation, the supernatant is replaced with water and the washing procedure is repeated. The microbubble can also be produced according to example 2f).

c) Coupling of streptavidin to gas-filled microbubbles enclosed in phosphatidylserine/Succ-PEG ₃ 4oo-DSPE

-83A streptavidin is covalently linked to Succ-PEG ₃ 4oo-DSPE in microbubble membranes using a standard coupling method using water-soluble carbodiimide. The sample is placed on a roller table during the reaction. After centrifugation, the supernatant is replaced with water and the washing procedure is repeated. The functionality of the coupled streptavidin is analyzed by binding, for example, to fluorescently labeled biotin, biotinylated antibodies (detected with fluorescently labeled secondary antibodies) or biotinylated and fluorescently or radioactively labeled oligonucleotides. The analysis was performed by fluorescence microscopy or scintillation counting.

d) Preparation of gas-filled microbubbles enclosed in phosphatidylserine, in which biotin is non-covalently linked to streptavidin-Succ-PEG _34O o-DSPE

The microbubbles prepared according to Example 8c) are incubated in a solution containing biotinylated vectors, e.g. biotinylated antibodies. The vector-coated microbubbles are washed as described above.

Example 9

Gas-filled microbubbles enclosed in a phosphatidylserine system, in which the biotinylated oligonucleotide is non-covalently linked to streptavidin-Succ-PEG _34O o-DSPE

a) Preparation of Succ-PEG ₃₄ oo-DSPE

NH2-PEG ₃₄ ooDSPE (prepared according to Example 2) is carboxylated with succinic anhydride, for example, according to the method described in Nayar, R. and Schroit, AJ, Biochemistry (1985) 24, 5967-71.

b) Gas-filled microbubbles enclosed in a Phosphatidylserine/Succ-PEG ₃₄ oo-DSPE system

-A mixture containing 845 mg of phosphatidylserine (90-99.9 mol%) and Succ-PEG _34O o-DSPE (10-0.1 mol%) is added to 1 ml of an aqueous 5% propylene glycol-glycerol solution, the resulting dispersion is heated at no more than 80°C for 5 minutes and then cooled to ambient temperature. 0.8 ml of such dispersion is then transferred to a 1 ml vial, the adjacent part is rinsed with perfluorobutane, the vial is shaken with a cap stirrer for 45 seconds and then transferred to a roller table. After centrifugation, the supernatant is replaced with water and the washing is repeated. The microbubbles can also be prepared as described in example 2f).

c) Coupling of streptavidin to gas-filled microbubbles enclosed in a phosphatidylserine/Succ-PEG ₃ 4oo-DSPE system Streptavidin is covalently coupled to Succ-PEG ₃₄ oo-DSPE in the membranes of the microbubbles using a standard coupling method using water-soluble carbodiimides. The sample is placed on a roller table during the reaction. After centrifugation, the supernatant is replaced with water and the washing is repeated. The functionality of the coupled streptavidin is examined by binding, for example, to fluorescently labeled biotin, biotinylated antibodies (detected with 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 gas-filled microbubbles entrapped in phosphatidylserine and non-covalent binding of biotinylated oligonucleotide to streptavidin Succ-PE G _34O o-DSPE

Microbubbles prepared according to Example 9c) are incubated in a solution containing a biotinylated oligonucleotide. The oligonucleotide-coated bubbles are washed as above. The

The binding of oligonucleotides to the bubbles can be detected, for example, by using fluorescently labeled oligos to bind to the bubbles or by hybridizing the bound oligonucleotides with a labeled (fluorescent or radioactive) complementary oligonucleotide. The functionality of the oligonucleotide-carrying microbubbles is analyzed, for example, by hybridizing the bubbles with immobilized DNA-containing sequences that are complementary to the linked oligonucleotide. For example, an oligonucleotide that is complementary to ribosomal DNA (which is present in many copies in a haploid genome) and an oligonucleotide that is complementary to an oncogene (for example, where ras is present in one copy in a haploid genome) can be used.

Example 10

Gas-filled microbubbles encased in phosphatidylserine/folate-PEG-Succ-DSPE

a) Preparation of Folate-PEG-Succ-DSPE

Folate-PEG-Succ-DSPE was prepared as described in Lee, R.J. and Low, P.S. in (1995) Biochimica. Biophysica. Acta 1233, 134-144.

b) Preparation of gas-filled microbubbles enclosed in phosphatidylserine/folate-PEG-Succ-DSPE mg of a mixture containing phosphatidylserine (90-99.9 mol%) and folate-PEG-DSPE (10-0.1 mol%) is added to 1 ml of an aqueous, 5% propylene glycol-glycerol solution. The resulting dispersion is heated at no more than 80°C for 5 minutes and then cooled to room temperature. Then 0.8 ml of the resulting dispersion is transferred to a 1 ml vial, the supernatant is rinsed with perfluorobutane, the vial is shaken with a cap-type stirrer for 45 minutes, and the sample is placed on a roller table. After centrifugation, the supernatant is replaced with water and the wash is repeated.

-86 is repeated. The microbubbles can also be prepared as described in Example 2e) or 2f). Folate binding is examined microscopically by examining the binding of folate-containing microbubbles to cells expressing different levels of folate receptors.

Example 11

Gas-filled microbubbles entrapped in phosphatidylserine/thiolated anti-CD34-Mal-PEG _34O o-DSPE, thiolated anti-ICAM-1-Mal-PEG ₃ 4oo-DSPE and thiolated anti-E-selectin-Mal-PEG ₃ 4oo-DSPE systems

a) Production of thiolated anti-CD34 antibodies

Anti-CD34 antibodies are prepared, for example, according to the method described in Hansen, C.B. et al. (1995). Biochim. Biophys. Acta 1239, 133-144.

b) Production of thiolated anti-ICAM-1 antibodies

Thiolation of anti-ICAM-1 antibodies is performed, for example, according to the method described in Hansen, C.B. et al. (1995) Biochim Biophys. Acta 1239, 133-144.

c) Production of thiolated anti-E-selectin antibodies

Thiolation of anti-E-selectin antibodies is carried out, for example, according to the method described in Hansen, C.B. et al. (1995) Biochim Biophys. Acta 1239, 133-144.

d) Gas-filled microbubbles enclosed in a system of phosphatidylserine/thiolated anti-CD34-Mal-PEG34oo-DSPE, thiolated anti-ICAM-1-Mal-PEG _34oo -DSPE and thiolated anti-E-selectin-Mal-PEG ₃ 4oo-DSPE mg of a mixture containing phosphatidylserine (90-99.9 mol%) and Mal-PEG _34O o-DSPE (10.01 mol%, prepared according to Example 2) are added to 1 ml of an aqueous, 5% propylene glycol-glycerol solution. The resulting dispersion is heated at no more than 80°C for 5 minutes, then

-87 is cooled to ambient temperature. 0.8 ml of the dispersion thus obtained is added to a 1 ml vial, the upper part is rinsed with perfluorobutane, the vial is shaken with a cap stirrer for 45 minutes, then the sample is transferred to a roller table. After centrifugation, the supernatant is replaced with the appropriate buffer and the antibodies according to examples 1 la-c) are coupled to the microbubbles, for example according to the method described in the following literature: Goundalkar, A., Ghose, T. and Mezei, M., J. Pharm. Pharmacol. (1984) 36,465- 466 or Hansen, C.B. et al., (1995) Biochim. Biophys. Acta 1239, 133-144. The microbubbles are transferred to the roller table for several hours and then washed.

Example 12

Coupling of FNFRLKAGQKIRFGAAAWEPPRARI peptide to phosphatidylserine-encapsulated gas-filled microbubbles The FNFRLKAGQKIRFGAAAWEPPRARI peptide, which contains phosphatidylserine-binding and heparin-binding regions, is prepared. The peptide is added to preformed phosphatidylserine-encapsulated perfluorobutane microbubbles and mixed thoroughly.

Example 13

Fibronectin covalently linked to gas-filled microbubbles enclosed in phosphatidylserine and phosphatidylethanolamine

a) Preparation of microbubbles 100 mg DSPS and 5 mg DSPE are weighed into a clean vial and 5 ml of 1.4% propylene glycol/2.4% glycerol solution is added. The mixture is heated at 80°C for 5 minutes, then cooled to room temperature, the upper part is flushed with perfluorobutane gas. The vials are shaken with a cap stirrer for 45 seconds, then the microbubbles are washed with double distilled water and resuspended in 0.1 M sodium borate buffer, pH=9.

b) Fibronectin modification

PUBLICATION COPY/ÍT.|A j. :J í ^9994595 mg of fibronectin in 5 ml of 0.01 M Hepes buffer (pH=8) is added to 0.1 mmol of the crosslinking agent SDBP. The resulting mixture is incubated on ice for 2 hours.

c) Microbubble modification

The protein solution from b) is added to the microbubble suspension from a) and incubated for 2 hours at room temperature on a roller table. The unreacted material is removed by allowing the microbubbles to float, then the buffer is replaced with 0.1 ml of sodium borate buffer (pH=9). This operation is repeated three times.

d) In vitro analysis

The resulting microbubbles are tested in vitro as described in Example 21. A gradual accumulation of microbubbles to the cells can be observed.

Example 14

Gas-filled microbubbles enclosed in a phosphatidylserine/3 β-[N-(N',Ν’-dimethylaminoethane)-carbamoyl] cholesterol system

a) Preparation of 3p-[N-(N’,N’-dimethylaminoethane)carbamoyl]cholesterol (DC-chol) (Farhood, H., Gao, X., Barsoum, J. and Hunag, L., Anal. Biochem. 225, 89-93 (1995))

19.40 mg (24:1, 0.22 mmol) of 2-dimethylaminoethylamine and 310 μΐ (2.23 mmol) of triethylamine in 3 ml of dichloromethane were stirred at room temperature and 100 mg (0.22 mmol) of cholesteryl chloroformate in 1,4-dioxane were slowly added. When the reaction was complete, the mixture was evaporated to dryness and the residue was purified by flash chromatography (CHCl ₃ /MeOH, 4:1) to give a white solid, yield 105 mg (95%). The structure was confirmed by NMR and MALDI studies.

b) Preparation of microbubble dispersion

Perfluorobutane-encapsulated microbubbles were prepared from 90% phosphatidylserine and 10% DC-chol by weighing 4.5 mg DSPS and 0.5 mg DC-chol into a 2 ml vial and adding 0.8 ml of 4% aqueous propylene glycol/glycerol solution. The solution was heated at 80°C for 5 min and shaken. The solution was then cooled to room temperature, the supernatant was rinsed with perfluorobutane, the vial was shaken with a cap-type shaker at 4450 oscillations/min for 45 s, and then placed on a roller table. The samples were washed by centrifugation at 2000 rpm for 5 min. The supernatant is removed using a syringe and replaced with an equal volume of distilled water. The supernatant is rinsed again with perfluorobutane and the sample is left on a roller table until a homogeneous appearance is achieved. The washing operation is repeated.

Example 15

Gas-filled microbubbles enclosed in a phosphatidylserine/WEPPRAI-PE system

Phosphatidylethanolamine (PE) is reacted with an equimolar amount of crosslinking agent N-hydroxysuccinimidyl-2,3-dibromopropionate in a 1:1 mixture of dioxane and 0.02 M Hepes buffer (pH=8). It is then incubated on ice for 2 hours, then an equimolar amount of heparin-binding peptide WEPPRARI is added, the pH is adjusted to pH=9 by adding 0.2 M disodium tetraborate and the incubation is continued for another 2 hours at room temperature. The resulting product is purified by chromatography. Monolayer-enclosed microbubbles containing perfluorobutane are prepared from 80-95% phosphatidylserine (PS) and 5-20% peptide-substituted PE.

Example 16

Gas-filled microbubbles enclosed in a phosphatidylserine/inactivated human thrombin-Succ-PEG ₃₄ oo-DSPE system

a) Inactivation of human thrombin

Human thrombin was inactivated by incubation with a 20% molar excess of D-Phe-L-Pro-L-Arg-chloromethyl ketone in 0.05 M Hepes buffer, pH 8, at 37°C for 30 minutes.

b) Phosphatidylserine/Succ-PEG ₃ 4oo-DSPE-encapsulated gas-filled microbubbles A mixture of mg of phosphatidylserine (90-99.9 mol%) and Succ-PEG _34O o-DSPE (10-0.1 mol%, prepared as described in Example 9a) is added to 1 ml of 5% propylene glycol-glycerol aqueous solution. The resulting dispersion is heated at no more than 80°C for 5 minutes and then cooled to ambient temperature. 0.8 ml of the dispersion thus obtained is transferred to a 1 ml vial, the upper part is rinsed with perfluorobutane, the vial is shaken with a cap-type stirrer for 45 minutes, and the sample is then transferred to a roller table. After centrifugation, the supernatant is replaced with water and the washing operation is repeated. The microbubbles can also be produced as described in Example 2f).

c) Preparation of gas-filled microbubbles encased in phosphatidylserine/inactivated human thrombin Succ-PEG ₃₄₀₀ -DSPE

Inactivated human thrombin is covalently bound to Succ-PEG ₃₄₀₀ -DSPE in microbubbles according to Example 16b) using a standard coupling method using water-soluble carbodiimide. The sample is placed on a roller table during the reaction. After centrifugation, the supernatant is replaced with water and the washing is repeated.

Example 17

Gas-filled microbubbles with methotrexate and a prodrug-activating enzyme attached to them

a) Coupling of methotrexate via a peptide linker to gas-filled microbubbles

The coupling of amino acids to the anticancer drug methotrexate (MTX) is well known in the literature (Huennekens, F.M. (1994), TIBTECH 12, 234-239 and references therein). Instead of a single amino acid, a peptide can also be coupled to MTX using similar technology. Such a peptide may contain a linker for coupling MTX to the surface of microbubbles. One such group of linkers includes peptides having the general formula (MTX)-F-K/R-X-R-Z-C, where X is any amino acid and Z is a hydrophobic amino acid. A specific representative of such linkers is (MTX)-F-K-L-R-C. The SH group present in the Cys group is used to couple the MTX peptide to microbubbles (which microbubbles contain, for example, phosphatidylserine and Mal-PEG-DSPE). This is done using standard technology, for example, as described in Example 2. This type of linker is expected to be cleaved by the enzyme cathepsin B, which is often selectively overexpressed outside of tumor cells and on the surface of tumor cells (Panchal, R.G. et al. (1996), Nat. Biotechnoi. 14, 852-856). Thus, the potential prodrug (MTX)-F-K/R-X-R can be selectively released in tumors. This prodrug can be further activated and converted to the active drug MTX using carboxypeptidases, which are endogenously present in the tumor or delivered to the tumor, for example, by tumor-associated antibodies (see below).

b) Prodrug-activating enzyme covalently attached to the surface of gas-filled microbubbles

An example of a prodrug activating enzyme is carboxypeptidase A (CPA), which can be conjugated to the surface of microbubbles, which are, for example, a mixture of phosphatidylserine and phosphatidylethanolamine.

-92, for example, a polyethylene glycol chain with a molecular weight of 3400 Da, carrying an N-hydroxysuccinimide group at both ends (Perron, M. J. and Page, M., Br. J. Cancer 73, 281-287); the microbubbles can be prepared by standard methods. The CPA-containing microbubbles can be directed to pathological sites by incorporating suitable targeting vectors into the CPA-containing bubbles. Alternatively, the CPA can be directly linked to the vector (e.g., antibody), for example, as described above. In the latter case, the CPA-vector conjugate will be linked to the surface of the microbubbles (Hansen, C.B. et al. (1995) Biochim. Biophys. Acta 1239 133-144). Examples of several possible prodrug-enzyme pairs are described, for example, in the following publication: Huennekens, F.M. (1994) TIBTECH 12, 234-239.

Example 18

Gas-filled microbubbles encased in a system of phosphatidylserine, thiolated anti-CEA-Mal-PEG _34O o-DSPE and anticancer prodrug 3',5'-0-dipalmitoyl-5-fluoro-2'-deoxyuridine

a) Production of thiolated anti-CEA antibodies

Thiolation of anti-CEA antibodies was performed according to the method described in Hansen, C.B. et al. (1995) Biochim. Biophys. Acta 1239, 133-144.

b) Gas-filled microbubbles enclosed in a system of phosphatidylserine, thiolated anti-CEA-Mal-PEG ₃₄₀₀ -DSPE and anticancer prodrug 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine mg phosphatidylserine (90-99.9 mol%), Mal-PEG ₃₄₀₀ -DSPE (10-0.1 mol%, prepared according to example 2) and 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine anticancer prodrug (Móri, A. et al. (1995) Cancer Chemother. Pharmacol. 35, 447-456) are added to a mixture of 1 ml of 5% propylene glycol-glycerol solution. The resulting dispersion is not

-93heated at more than 80°C for 5 minutes and then cooled to ambient temperature. 0.8 ml of the resulting dispersion is transferred to a 1 ml vial and the supernatant is rinsed with perfluorobutane. The vial is shaken with a cap stirrer for 45 seconds and the sample is then transferred to a roller table. After centrifugation, the supernatant is replaced with a suitable buffer * and the antibody is coupled to the formed microbubbles, for example by the methods described in the following publications: Goundalkar, A., Ghose, T. and Mezei, M., J. Pharm. Pharmacol. (1984) 36465-466, or Hansen, C.B. et al., (1995) Biochim. Biophys. Acta 1239 133-144. The microbubbles are placed on a roller table for several hours and then washed.

Example 19

Gas-filled microbubbles enclosed in a phosphatidylserine/thiolated anti-CEA-Mal-PEG ₃₄ oo-DSPE and N-trifluoroacetyladriamycin-14-valerate anticancer prodrug system

a) Production of thiolated anti-CEA antibodies

Thiolation of anti-CEA antibodies was performed as described in Hansen, C.B. et al. (1995) Biochim. Biophys. Acta 1239 133-144.

b) Gas-filled microbubbles enclosed in a phosphatidylserine/thiolated anti-CEA-Mal-PEG ₃ 4 _O o-DSPE and N-trifluoroacetyl-adriamycin-14-valerate anticancer prodrug system To a mixture containing mg of phosphatidylserine (90-99.9 mol%), Mal-PEG _34O o-DSPE (10-0.1 mol%, prepared according to Example 2) and N-trifluoroacetyl-adriamycin-14-valerate (Móri, A. et al. (1993) Pharm. Res. 10, 507-514) of an anticancer prodrug, 1 ml of an aqueous 5% propylene glycol-glycerol solution is added. The resulting dispersion is heated at no more than 80°C for 5 minutes and then cooled to ambient temperature. 0.8 ml of the resulting dispersion was then transferred to a 1 ml vial

-94 and rinse the upper part with perfluorobutane. The vial is closed with a cap-type stirrer for 45 seconds and the sample is then placed on a roller table. After centrifugation, the supernatant is replaced with the appropriate buffer and the antibody is coupled to the microbubbles, for example as described in the following references: Goundalkar, A., Ghose, T. and Mezei, M. in J. Pharm. Pharmacol. (1984) 36 465-66, or Hansen, C.B. et al., (1995) Biochim. Biophys. Acta 1239133-144. The microbubbles are placed on a roller table for several hours and then washed.

Example 20

Application

A material containing microbubbles encapsulated in phosphatidylserine, containing inactivated human thrombin-Succ-Mal-PEG34oo-DSPE in the encapsulating membrane, is lyophilized from 0.01 M phosphate buffer pH=7.4. The resulting product is redispersed in sterile water and then injected intravenously into patients at risk of venous thrombosis in the leg vein. The leg is examined by standard ultrasound. The thrombi are located with increased contrast compared to the surrounding tissues.

Example 21

Preparation and biological evaluation of DSPS gas-filled microbubbles doped with a lipopeptide containing a heparin sulfate binding peptide (KRKR) and a fibronectin peptide (WOPPRARI)

In the example, we demonstrate the production of targeted microbubbles containing multiple peptide vectors arranged in a linear sequence.

a) Preparation of a lipopeptide containing heparin sulfate binding peptide (KRKR) and fibronectin peptide (WOPPRARI)

I

The lipopeptide was synthesized on an ABI 433A automated peptide synthesizer starting from Fmoc-Ile-Wang resin using 0.1 mmol steps of 1 mmol amino acid loading. Each amino acid and palmitic acid were preactivated with HBTU before coupling. The cleavage of the peptides from the resin and the removal of the side chain protecting groups were simultaneously performed in TFA containing 5% phenol, 5% EDT, 5% anisole and 5% H2O, the reaction time was 2 h, yielding 150 mg of crude product. The product was purified by preparative HPLC on a 40 mg aliquot of crude starting material, the gradient was 70 -> 100% B over 40 min (A = 0.1% TFA/water and B = MeOH) at a flow rate of 90 ml/min. After lyophilization, 16 mg of pure material was obtained (analytical HPLC, gradient 70-100% B, where B=MeOH, A=0.01% TFA/water: detection UV 260 and fluorescence, Exzeo, Em ₃ 5o, product retention time 19.44 min). Further product characterization was performed by MALDI mass spectrometry: expected M+H 2198, measured 2199.

b) Preparation of gas-filled DSPS microbubbles doped with a multispecific lipopeptide containing heparin sulfate binding peptide (KRKR) and fibronectin peptide (WOPPRARI)

4.5 mg DSPS and 0.5 mg lipopeptide according to point a) are weighed into two vials and 0.8 ml of a solution containing 1.4% propylene glycol/2.4% glycerin is added, the mixture is heated at 80°C for 5 minutes (a

-96 while shaking the vials). The samples are then cooled to room temperature, the supernatant is flushed with perfluorobutane gas. The vials are then shaken with a cap stirrer for 45 seconds and treated on a roller table overnight. The resulting microbubbles are washed several times with deionized water and analyzed with a Coulter Counter (size: 1-3 μ, 87%, 3-5 μ, 11.5%), and then tested for acoustic attenuation (frequency at maximum attenuation 3.5 MHz). The microbubbles are stable at 120 mmHg. MALDI mass spectroscopic analysis is used to verify the incorporation of the lipopeptide into the DSPS bubbles: approximately 0.05-0.1 ml of the microbubble suspension is transferred to a clean vial and 0.05-0.1 ml of methanol is added. The resulting suspension was sonicated for 30 seconds and the solution was analyzed by MALDI MS. In positive mode, the M+H value was 2200 (expected value for the lipopeptide 2198).

c) In vitro study of gas-filled DSPS microbubbles containing a multispecific lipopeptide containing heparin sulfate binding peptide (KRKR) and fibronectin peptide (WOPPRARI): binding to endothelial cells under flow conditions

ECV 304 human endothelial cell line derived from normal umbilical cord (ATCC CRL-1998) was cultured in 260 ml Nunc culture flasks (chutney 153732) in RPMI 1640 medium supplemented with L-glutamine (200 mM), penicillin/streptomycin (10,000 U/ml and 10,000 pg/ml), and 10% fetal bovine serum. The cells were subcultured at a split ratio of 1:5 to 1:7 when confluent. 22 mm diameter coverslips were sterilized and placed at the bottom of 12-well culture plates, and the cells were plated in complete medium containing 0.5 ml of serum. When the cells reached confluence,

-97 confluence, the coverslips were placed in a custom-made flow chamber, which contained notches cut into the glass slide, onto which the coverslips containing the cells were placed so that the cells faced the notch, thus forming a flow channel. The microbubbles produced in part b) were flowed through the flow chamber, these microbubbles were introduced from a reservoir maintained at 37°C and then returned to the reservoir using a peristaltic pump. The flow was adjusted to simulate the physiological shear rate. The flow chamber was placed under a microscope and the interaction between the microbubbles and the cells was directly observed. The camera mounted on the microscope was connected to a color video printer and a monitor. The gradual accumulation of microbubbles on the cells occurred in a flow rate-dependent manner. By further increasing the flow rate, the cells began to detach from the coverslip, but the microbubbles remained attached to the cells. Control bubbles without vector did not adhere to endothelial cells and disappeared from the chamber even under minimal flow conditions.

d) In vivo experiment with dogs

Case 1 A mixed breed dog weighing 100 kg was anesthetized with pentobarbital and mechanically ventilated. The chest was opened by a midline sternal incision, the anterior pericardium was removed, and a 30 mm gelled silicone rubber spacer was inserted between the heart and the P5-3 transducer of the ATL HDI-3000 ultrasound scanner. The scanner was then set to intermittent short axis imaging with delayed EGC at each end-systole. A 2 ml volume of pure microbubble as described in b) was injected as a rapid intravenous bolus; 3 seconds later, the imaged right

-98 ventricle was visible to contain contrast agent and a further 3 seconds later the left ventricle also filled and a transient attenuation shadow became visible which obscured the image of the posterior part of the left ventricle. A significant increase in brightness was observed in the myocardium as the attenuation shadow disappeared in the heart regions adjacent to the left ventricle. After the initial bolus was delivered, the ultrasound scanner was switched to a continuous, high-resolution, high-output mode which is known to disrupt microbubbles of ultrasound contrast agents in the imaged tissue sections. After a few seconds the scanner was returned to its original setting. The myocardium then became darker and approached the baseline. By moving the imaging aperture to a new position the contrast effect reappeared, by returning the aperture to its original position tissue brightening occurred again in the immediate vicinity of the baseline.

Case 2 (comparative) ml of microbubbles prepared according to point b), which did not contain lipopeptide, were used as injections and otherwise everything was done as above. The myocardial echo enhancement was much less intense and lasted for a shorter time than in case 1. After the end of the left ventricular attenuation phase, the myocardial contrast effect almost completely disappeared and the myocardial echo enhancement in the posterior part of the left ventricle, as noted in case 1, was not observable.

Example 22

Preparation of gas-filled microbubbles enclosed in a DSPS/thiolated anti-CD34-Mal-PEG2ooo-PE system

a) Gas-filled microbubbles encapsulated in DSPS/PE-PEGiooo-Mal

-994.5 mg (2.9 mmol) DSPS and 0.5 mg PE-PEG _2O oo-Mal from Example 50 were weighed into a clean vial and 1 ml of a 1.4% propylene glycol/2.4% glycerol solution was added. The resulting mixture was heated at 80°C for 5 seconds and then filtered through a 4.5 µm filter. The resulting sample was cooled to room temperature, the headspace was rinsed with <perfluorobutane, the vials were shaken with a cap shaker for 45 minutes, and the resulting microbubbles were washed three times with distilled water.

b) Thiolation of anti-CD34 antibodies

0.3 mg of anti-CD34 antibody was dissolved in 0.5 ml of phosphate buffered saline (PBS, pH=7) and 0.3 mg of Traut's reagent was added and the solution was shaken on silica gel at room temperature for 1 hour. The excess reagent was separated from the modified protein on a NAP-5 column.

c) Conjugation of thiolated anti-CD34 antibody to gas-filled microbubbles enclosed in DSPS/DSPE-PEG ₂ ooo-Mal system

0.5 ml of the thiolated antibody preparation according to b) above is added to the aliquot of the microbubbles according to a) and the conjugation reaction is carried out for 30 minutes on a roller table. Centrifugation is then carried out at 2000 rpm for 5 minutes, and the supernatant is then removed. The microbubbles are washed three times with water.

d) Detection of antibodies enclosed in microbubbles with FITC-conjugated secondary antibody

To the microbubble suspension from point c) 0.025 ml of FITC-conjugated goat anti-mouse antibody is added. The mixture is incubated in a dark room at room temperature for 30 minutes on a roller table and then centrifuged at 2000 rpm for 5 minutes. The supernatant is removed and the microbubbles are washed three more times with water.

-100 washes. Flow cytometric analysis shows that 98% of the population in the microbubble suspension is fluorescent.

Example 23

DSPS/thiolated anti-CD62-Mal-PEG _2O oo-PE-encapsulated gas-filled microbubbles

The procedure for preparing anti-CD62 antibodies is as described in Example 22.

Example 24

DSPS/thiolated anti-ICAM-l-Mal-PEG _2OO o-PE-encapsulated gas-filled microbubbles

The procedure described in Example 22 was followed to prepare microbubbles containing anti-ICAM-1 antibodies.

Example 25

DSPS/thiolated anti-CD62-Mal-PEG ₂ ooo-PE thiolated anti-ICAM-1-MAl-PEG ₂ ooo-PE-encapsulated gas-filled microbubbles

In the example, we demonstrate the production of microbubbles containing multiple antibody vectors for targeted ultrasound imaging.

a) Production of gas-filled microbubbles enclosed in DSPS/PE-PEG _2O oo-Mal

4.5 mg DSPS and 0.5 mg PE-PEG ₂ ooo-Mal according to example 2a) are weighed into a clean vial, 1 ml of 1.4% propylene glycol/2.4% glycerol solution is added, the resulting mixture is heated at 80°C for 5 minutes, then filtered through a 4.5 pm filter. The sample is cooled to room temperature, the upper part is flushed with perfluorobutane gas, then the vials are shaken with a cap stirrer for 45 seconds and the resulting microbubbles are washed three times with distilled water.

b) Thiolation of anti-CD62 and anti-ICAM antibodies

-101 0.3-0.3 mg of anti-CD62 and anti-ICAM-1 antibodies are dissolved in PBS buffer (pH=7, 0.5 ml), Traut's reagent is added and the resulting solution is stirred at room temperature for 1 hour. The excess reagent is separated from the modified protein on a NAP-5 column.

c) Conjugation of thiolated anti-CD62 and anti-ICAM-1 antibodies to gas-filled microbubbles enclosed in DSPS/DSPE-PEGzooo-Mal

0.5 ml of the mixture of b) and thiolated antibody preparation is added to the microbubble aliquot of a) and the conjugation reaction is allowed to proceed for 30 minutes on a roller table. The material is then centrifuged at 2000 rpm for 5 minutes and the supernatant is removed. The microbubbles are washed three more times with water.

The PEG spacer length is varied by using longer (PEG3400 and PEG _5O oo) or shorter (e.g. PEG _6O o or PEGgoo) chains. It is also possible to add a third antibody to the thus thiolated anti-CD34.

Example 26

Targeted gas-filled microbubbles containing polylysine non-covalently linked to DSPS and a PS-binding component, as well as a fibronectin peptide sequence FNFRLKAGOKIRFGGGGWOPPRAI

a) Preparation of PS-binding/FNFRLKAGOKIRFGGGGGWOPPRAI fibronectin fragment fusion peptide

The peptide was synthesized using an ABI 433A automated peptide synthesizer starting from Fmoc-Ile-Wang resin in 0.1 mmol steps using 1 mmol of amino acid loading. All amino acids were preactivated using HBTU before coupling. Simultaneous removal of peptides and side chain protecting groups from the resin into TFA was performed,

- 102 which also contained 5% phenol, 5% EDT and 5% H2O, the operation was carried out for 2 hours, thus 302 mg of crude product was obtained. After purification by preparative HPLC, a 25 mg aliquot of crude material was obtained, the gradient was 20 -> 40% B over 40 minutes (A=0.1% TFA/water and B=0.1% TFA/acetonitrile), flow rate 9 ml/min. After lyophilization, 10 mg of pure material was obtained (analytical HPLC, gradient 20 -> 50% B, where B=0.1% TFA/acetonitrile, A=0.01% TFA/water, detection UC 214 and 260 nm, product retention time 12.4 minutes). Further product characterization was performed by MALDI mass spectroscopy: expected M+H 2856, measured 2866.

b) Preparation of gas-filled microbubbles containing DSPS, non-covalently bound polylysine and PS-binding/fibronectin fragment fusion peptide (FNFRLKAGOKIRFGGGGWOPPRAI) mg DSPS is weighed into a clean vial, together with 0.2 mg poly-L-lysine and 0.2 mg of the peptide according to point a) above. 1 ml of 1.4% propylene glycol/2.4% glycerol is also weighed into the vial. The resulting mixture is heated at 80°C for 5 minutes, then cooled to room temperature, the upper part is flushed with perfluorobutane gas, and the vials are shaken with a cap stirrer for 45 minutes, then the resulting microbubbles are centrifuged at 100 rpm. The material was then washed vigorously with water, PBS buffer, and then water, and the final solution was assayed for polylysine and peptide content using MALDI MS. No polypeptide material was detected in the final wash solution. 0.5 ml of acetonitrile was then added to the microbubbles and the bubbles were disrupted by sonication. The resulting solution was then assayed for polylysine and PS-binding/fibronectin fusion peptide using MALDI MS. The results obtained are as follows:

- 103 MALDI expected

MALDI measured

Poly-L-lysine

DSPS-binding peptide

786, 914 1042,1170

2856

790, 919

1048, 1177

2866

The spacer element (-GGG-) in the PS-binding/fibronectin fusion peptide can be replaced with another spacer, such as PEG ₂ -chain polyalanine (-AAA-). Pre-targeting can also be used, in which the DSPS-binding/fibronectin fragment fusion peptide is first allowed to associate with cells via fibronectin peptide linkage, and then PS microbubbles are added, which then attach to the PS-binding peptide.

Example 27

Gas-filled microbubbles encapsulated in phosphatidylserine/biotin-PEG ₃ 4oo-al anyl cholesterol and functionalized with streptavidin/biotinyl endothelin-1 peptide (biotin-D-Trp-Asp-Ile-Ile-Trp.OH) and biotinyl fibrin antipolymer peptide (biotin-GPRPPERHOS.NH ₂ )

In this example, we demonstrate the production of steerable, targetable ultrasonic microbubbles using streptavidin as a linker between biotinylated signal carrier(s) and vector(s).

a) Preparation of biotin-PEG ₃ 4oo-alanine-cholesterol mg (0.03 mmol) of cholesteryl-b-alanine hydrochloride from Example 59 was mixed with 3 ml of chloroform/wet methanol (2.6:1) and 42 ml (0.30 mmol) of triethylamine was added. The resulting mixture was stirred for 10 minutes at room temperature, then a solution of 100 mg (0.03 mmol) of biotin-PEG ₃₄₀ o-NHS in 1 ml of 1,4-dioxane was added dropwise. The resulting material was stirred at room temperature for 3 hours, then the mixture was evaporated to dryness and the residue was flash-dried.

- 104 chromatography to give a white crystalline solid, yield 102 mg, 89%. Its structure was confirmed by MALDI-MS and NMR studies.

b) Preparation of biotinylated endothelin-1 peptide (biotin-D-Trp-Leu-Asp-Ile-Trp.OH)

The peptide was synthesized on an ABI 433A automated peptide synthesizer starting from Fmoc-Trp(Boc)-Wang resin in 0.1 mmol steps using 1 mmol of amino acid loading. Each amino acid was preactivated with HBTU before coupling. Simultaneous deprotection of the peptide and side chains was performed with 5% anisole and 5% H ₂ O in TFA for 2 h to yield 75 mg of crude product. A 20 mg aliquot was purified by preparative HPLC, gradient 30 -> 80% B, 40 min (A=0.01% TFA/water and B=0.1% TFA/acetonitrile, flow rate 9 ml/min. After lyophilization of the pure fractions, 2 mg of pure material was obtained (analytical HPLC, gradient 30-80% B, where B=0.1% TFA/acetonitrile, A=0.01% TFA/water, detection UV 214 n, product retention time 12.6 min). Further product characterization was performed by MALDI mass spectroscopy: expected M+H 1077, measured 1077.

c) Preparation of biotinyl fibrin antipolymer peptide (biotin-GPRPPERHOS.NH ₂ )

The peptide was prepared and purified according to point b), and the obtained pure product was characterized by HPLC and MALDI MS.

d) Multi-specific gas-filled microbubbles enclosed in phosphatidyl/biton-P EG34oo-b-alanine-cholesterol

4.5 mg DSPS and 0.5 mg biotin-PEG ₃ 4oo-b-alanine-cholesterol according to point a) are weighed into a vial and 0.8 ml of 1.4% propylene glycol/2.4% glycerol solution is added. The resulting mixture is heated at 80°C for 5 minutes (the vials are shaken during heating). The sample

- 105 then cooled to room temperature, the headspace flushed with perfluorobutane gas, the vials shaken with a cap shaker for 45 minutes, then incubated overnight on a roller table. The microbubble suspensions were then washed several times with deionized water and examined with a Coulter Counter and for acoustic attenuation.

e) Conjugation with fluorescein-labeled streptavidin and biotinylated peptides according to points b) and c)

To the microbubble preparation according to point d) 0.2 mg of fluorescein-conjugated streptavidin is added dissolved in 1 ml of PBS. The bubbles are placed on a roller table for 3 hours at room temperature. They are then washed vigorously with water and examined by fluorescence microscopy, the microbubbles are incubated for 2 hours in 1 ml of PBS, which also contains 5 mg of biotinyl endothelin peptide and 0.5 mg of biotinyl fibrin anti-polymerant peptide according to points b) and c). The microbubble is then washed vigorously to remove the conjugated peptides.

Example 28

Phosphatidylserine and biotin-DPPE gas-filled microbubbles for the production of streptovidin “sandwich” using a mixture of biotinyl-endothelin-1 peptide (biotin-D-Trp-Leu-Asp-Ile-Ile-Trp.OH) and biotinyl-fibrin-anti-polymerant peptide (biotin-GPRPPERHOS.NH ₂ )

a) Production of biotin-containing microbubbles

A mixture of 0.5 mg of phosphatidylserine and 0.6 mg of biotin-DPPE is weighed into a vial, 1 ml of 5% propylene glycol-glycerol aqueous solution is added, the resulting dispersion is heated at 80°C for 5 minutes, then cooled to ambient temperature. The supernatant is rinsed with perfluorobutane, and the vial is shaken with the cap-mounted stirrer for 45 minutes.

-106 After centrifugation, the supernatant is removed and the microbubbles are washed vigorously with water.

b) Conjugation of gas-filled microbubbles enclosed in phosphatidylserine and biotin-DPPE with streptovidin and biotinyl-

- with a mixture of endothelin-1 peptide (biotin-D-Trp-Leu-Asp-Ile-Ile-Trp.OH) and biotinyl fibrin anti-polymerant peptide (biotin-GPRPPERHOS.NH ₂ )

The preparation is carried out as described in Example 27.

Example 29

Functionalization of microbubbles enclosed in PFB gas-containing DSPS with heparin sulfate binding peptide/fibronectin peptide/RGD peptide and fluorescein

a) Preparation of the lipopeptide containing the RGD sequence and the fluorescein signaling group: Dipalmitoyl-Lys-Lys-Lys-Lys[acetyl-Arg-Gly-Asp-Lys(fluorescein)]Gly.OH

The lipopeptide was prepared from commercially available amino acids and polymers as described in Example 21a). The lipopeptide was cleaved from the resin with TFA containing 5% water, 5% phenol, 5% EDT for 2 hours. After concentration in vacuo, the crude product was precipitated and taken up in diethyl ether. After purification by HPLC

-107 40 mg aliquot of crude material was obtained, gradient 60 -> 100% B, over 40 min (A= 0.1% TFA/water and B=0.1% TFA/acetonitrile), flow rate 9 ml/min. After lyophilization, 10 mg of crude product was obtained (analytical HPLC gradient 60-100% B, where B=0.1% TFA/acetonitrile, A=0.01% TFA/water, detection UV 260, product retention time 20-22 min). Further product characterization was performed by MALDI mass spectrometry: expected M+H 1922, measured 1920.

b) Lipopeptide containing a heparin sulfate binding sequence and a fibronectin peptide

The preparation and purification are carried out as described in Example 21a), c) Multispecific gas-filled DSPS microbubbles functionalized with heparin sulfate binding peptide, fibronectin peptide, acetyl-RGD peptide and fluorescein 4 mg (3.9 mmol) DSPS, 0.5 mg (0.2 mmol) lipopeptide according to point a) and 0.5 mg lipopeptide according to point b) are weighed into 2-2 vials and 0.8 ml of 1.4% propylene glycol/2.4% glycerol solution is added. The mixtures are heated at 80°C for 5 minutes (the vials are shaken during heating). The samples are then cooled to room temperature, the supernatant is flushed with perfluorobutane gas, the vials are shaken with a cap stirrer for 45 minutes, and then kept on a roller table overnight. The resulting microbubbles are then washed several times with deionized water and analyzed by MALDI mass spectrometry as described in Example 21b). The microbubbles are examined microscopically and found to be between 1 and 5 pm in size and to exhibit fluorescence.

Example 30

Gas-filled microbubbles containing DSPS covalently bound to CD71 FITC-labeled anti-transferrin receptor

-108 are modified with antibodies and doped with a lipopeptide with affinity for endothelial cells. In the example, we demonstrate the production of targeted ultrasound materials containing multiple vectors.

a) Production of endothelial cell-binding lipopeptide: 2-n-

-hexadecylstearyl-Lys-Leu-Ala-Leu-Lys-Leu-ALa-Leu-Lys-Ala-Leu-Lys-Ala-Ala-Leu-Lys-Leu-Ala-NH ₂

The following lipopeptide was prepared using an ABI 433A automated peptide synthesizer starting from Rink Amid resin in a 0.1 mmol scale using 1 mmol of amino acid loading.

Each amino acid and 2-n-hexadecylstearic acid were preactivated with HBTU before coupling. Simultaneous removal of the peptide and side chain protecting groups from the resin was carried out for 2 h with TFA containing 5% EDT and 5% H ₂ O to give 50 mg of crude product. A 40 mg aliquot of crude product was purified by preparative HPLC, gradient 90 -> 100% B, 50 min (A = 0.1% TFA/water and B = MeOH), flow rate 9 ml/min. After lyophilization, 10 mg of crude product was obtained (analytical HPLC, gradient 90 -> 100% B, B = MeOH, A = 0.01% TFA/water, detection UV 214 nm, product retention time 23 min). Further product characterization was performed by MALDI mass spectrometry: expected M+H 2369, measured 2373.

- 109 -

b) Preparation of gas-filled microbubbles in which DSPS is doped with endothelial cell-binding lipopeptide and PEGiooo-Mal

4.5 mg DSPS and 0.5 mg lipopeptide according to point a) are weighed into a vial together with 0.5 mg PE-PEG ₂ ooo-Mal according to example 50 and 1 ml of 1.4% ethylene glycol/2.4% glycerol solution is added. The resulting mixture is heated at 80°C for 5 minutes and then filtered through a 4.5 pm filter. The sample is then cooled to room temperature, the upper part is flushed with perfluorobutane gas, the vial is shaken with a cap stirrer for 45 seconds and the resulting microbubbles are washed three times with distilled water.

c) Thiolation of FITC-labeled anti-transferrin receptor antibody

FITC-labeled CD71 anti-transferrin receptor Ab (100 mg/ml in PBS, 0.7 ml) was reacted with 0.9 mg of Traut's reagent at room temperature for 1 hour. The excess reagent was then separated from the modified protein on a NAP-5 column.

d) Conjugation of thiolated FITC-labeled anti-transferrin receptor antibody to DSPS gas-filled microbubbles doped with endothelial cell-binding lipopeptide and DSPE-PEGiooo-Mal

A 0.5 ml aliquot of the protein fraction from point c) above (total 2 ml) is added to the microbubbles from point b) and the conjugation reaction is carried out for 10 minutes on a roller table. After centrifugation at 1000 rpm, the protein solution is removed and the conjugation is repeated twice more with 1 ml and 0.5 ml aliquots of the protein solution, respectively. The bubbles are then washed four times in distilled water, the sample is analyzed for antibodies by flow cytometry and microscopy. The fluorescent population is >92%.

-110 The attached Figure 1 shows the results of the flow cytometry study compared with negative control DSPS microbubbles (left curve), the bubbles are conjugated with DC71 FTIC-labeled anti-transferrin antibody (solid curve on the right), they show 92% fluorescence.

The incorporation of lipopeptides into microbubbles is confirmed by MALDI mass spectrometry as described in Example 21b).

Example 31

Gas-filled microbubbles containing DSPS, a lipopeptide for targeting to endothelial cells, and a captopril-containing molecule

The example demonstrates the production of ultrasonic materials, which contain a therapeutic agent in addition to the guiding materials.

a) Preparation of captopril-functionalized lipopeptide

The above structure was prepared using a manual nitrogen sparge apparatus starting from Fmoc-protected Rink Amide BMHA resin in a 0.125 mmol scale. The coupling was performed according to standard TBTU/HOBt/DIEA protocols. The bromoacetic acid was coupled via the Lys side chain as a symmetrical anhydride using DIC preactivation. Captorpil was dissolved in DMF and introduced into the solid phase using DBU as a base. Simultaneous removal of the resin and side chain protecting groups from the resin was achieved within 2 hours.

-Illa was performed with TFA, which also contained 5% EDT, 5% water and 5% ethyl methyl sulfide. A 10 mg aliquot was purified by preparative liquid chromatography, gradient 70 -> 100%, 60 min (A=0.1% TFA/water and B=0.1% TFA/acetonitrile), flow rate 10 ml/min. After lyophilization, 2 mg of pure material was obtained (analytical HPLC gradient 70 -> 100% B 20 min, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow rate 1 ml/min, detection UV 214 nm, retention time 26 min). Further characterization was performed by MALDI mass spectrometry: M+H 1265.

b) Preparation of a lipopeptide with affinity for endothelial cells: dipalmitoyl-Lys-Lys-Lys-Aca-Ile-Arg-ARg-Val-Ala-Arg-Arg-Pro-Pro-Leu-NH ₂

The lipopeptide was synthesized on an ABI433A automated peptide synthesizer starting from Rink Amid resin using 1 mmol amino acid loadings in a 0.1 mmol scale. The amino acids and palmitic acid were preactivated with HBTU before coupling. Simultaneous removal of the peptide and side chain protecting groups from the resin was performed with TFA for 2 h, which also contained 5% phenol, 5% EDT and 5% water, yielding 160 mg of crude product. Purification by preparative HPLC yielded 35 mg of aliquot of crude material, gradient 70 -> 100% B in 40 min (A=0.1% TFA/water, B=MeOH), flow rate 9 ml/min. Lyophilization yielded 20 mg of crude material (analytical

-112 HPLC, gradient 70 -> 100% B, B=MeOH, A=0.1% TFA/water, detection UV 214 and 260 nm, product retention time 16 min). Further product characterization was performed by MALDI mass spectrometry: expected M+H 2050, measured 2055.

c) Production of DSPS-containing gas-filled microbubbles containing lipopeptide for targeting to endothelial cells and captopril-containing molecule for drug delivery

4.5 mg DSPS, 0.5 mg product from a) and 0.5 mg product from b) are weighed into a vial and 1 ml of 1.4% propylene glycol/2.4% glycerol solution is added. The resulting mixture is heated at 80°C for 5 minutes (with shaking). The sample is then cooled to room temperature, the headspace is flushed with perfluorobutane gas. The vial is first shaken with a cap stirrer for 45 seconds, then treated on a roller table for 1 hour, after which the contents are intensively washed with deionized water. There is no detectable starting material in the final wash solution, as confirmed by MALDI MS analysis. MALDI mass spectrometry was used to confirm that products a) and b) were incorporated into the microbubbles, as described in Example 21b).

d) In vitro study of gas-filled microbubbles containing DSPS, a molecule containing lipopeptide for delivery to endothelial cells and captopril for therapeutic purposes The in vitro study according to Example 21c) was used to study cell attachment under flow conditions. A gradual accumulation of microspheres occurs on the cells, depending on the flow rate. By further increasing the flow rate, the cells began to detach from the coverslip, but the microbubbles remained attached to the cells. The vector-free

-113 control microbubbles did not adhere to endothelial cells and disappeared from the chamber under minimal flow conditions.

Example 32

Gas-filled microbubbles containing DSPS to which is attached a lipopeptide containing a helical peptide that has affinity for cell membranes and the peptide antibiotic polymyxin B sulfate

In the example, we demonstrate the production of targeted microbubbles containing multiple peptide vectors that can be used in combination for targeting and therapeutic use.

a) Preparation of a lipopeptide containing a helical peptide with affinity for cell membranes: hexadecylstearyl-Lys-Leu-Ala-Leu-Lys-Leu-Ala-Leu-Lys-Ala-Leu-Lys-Ala-Leu-lys-Ala-Ala-Leu-Lys— -Leu-Ala-NH ₂

This is prepared as described in Example 30a).

b) Preparation of multi-specific gas-filled microbubbles mg DSPS, 0.3 mg lipopeptide according to point a) and 0.1 mg polymyxin B sulfate are weighed into a clean vial and 1 ml of 1.4% propylene glycol/2.4% glycerol solution is added. The resulting mixture is sonicated for 3-5 minutes, heated at 80°C for 5 minutes, and finally filtered through a 4.5 pm filter. The mixture is cooled to room temperature and the upper part is flushed with perfluorobutane gas. The vial is then shaken with a cap mixer for 45 seconds, and the resulting microbubbles are centrifuged at 1000 rpm for 3 minutes. The microbubbles are then washed with water until polymyxin B sulfate or lipopeptide can be detected in the supernatant by MALDI-MS. Microscopic examination showed that the size distribution of the bubbles was in the range of 1-8 pm. For the washed bubbles (approximately 0.2 ml), 0.5 ml of methanol was added.

-114 is added, then the mixture is placed in an ultrasonic bath for 2 minutes. According to the MALDI-MS analysis, the resulting clear solution contains both lipopeptide and polymyxin B sulfate (expected value 1203, measured 1207).

Example 33

Gas-filled microbubbles containing DSPS doped with a lipopeptide containing an IL-1 receptor-binding sequence and modified with a branched structure containing the active ingredient methotrexate

In the example, we demonstrate the production of targeted microbubbles containing multiple vectors for targeting/therapeutic purposes.

a) Preparation of a lipopeptide containing an interleukin-1 receptor binding peptide: dipalmitoyl-Lys-Gly-Asp-Trp-Asp-Gln-Phe-Gly-Leu-T rp-Arg-Gly-Ala-AIa.OH

The lipopeptide was synthesized on an ABI433A automated peptide synthesizer starting from Fmoc-Ala-Wang resin in a 0.1 mmol scale using 1 mmol of amino acid loading. The amino acids and palmitic acid were preactivated with HBTU before coupling. Simultaneous removal of the lipopeptide and side chain protecting groups from the resin was carried out for 2 h with TFA containing 5% H ₂ O, 5% anisole, 5% phenol and 5% EDT, yielding 150 mg of crude product. Purification by preparative HPLC yielded a 30 mg aliquot of crude material, gradient 90 —>

-115 100% B 40 min (Α=0.1% TFA/water, B=MeOH), flow rate 9 ml/min. After lyophilization, 4 mg of crude material was obtained (analytical HPLC, gradient 90 -> 100% B over 20 min, B=MeOH, A=0.01% TFA/water, detection UV 218 nm, product retention time 23 min). Further product characterization was performed by MALDI spectrometry: expected M+H 2083, measured 2088.

b) Preparation of a branched methotrexate core structure containing a thiol group

The methotrexate structure was prepared on an ABI433A automated peptide synthesizer starting from Fmoc-Cys(Trt) Tentagel resin in a 0.1 mmol scale. The product and the protecting groups were simultaneously removed from the resin for 2 hours with TFA containing 5% EDT and 5% H ₂ O, yielding 160 mg of crude product. After purification by preparative HPLC, a 30 mg aliquot of crude material was obtained, gradient 10 -> 30% B 40 min (A=0.1% TFA/water, B=0.1% TFA/acetonitrile), flow rate 9 ml/min. After lyophilization of the pure fractions, 9 mg of pure material was obtained (analytical HPLC, gradient 5 -> 50% B= 0.1% TFA/acetonitrile, A=0.01% TFA/water, detection UV 214 nm, product retention time 9.5 min). The product was also characterized by MALDI mass spectrometry: expected M+H 1523, measured 1523.

- 116-

c) Production of microbubbles with multiple specific gas fillings

4.5 mg DSPS, 0.5 mg thiol-containing lipopeptide according to Example 64a) and 0.2 mg lipopeptide according to point a) are placed in a clean vial and 1 ml of 1.4% propylene glycol/2.4% glycerol solution is added. The resulting mixture is ultrasonically stirred for 3-5 minutes, then heated at 80°C for 5 minutes, then filtered through a 4.5 pm filter. The mixture is then cooled to room temperature, the upper part is flushed with perfluorobutane gas. The vial is then shaken for 45 seconds in a cap mixer, the resulting microbubbles are centrifuged at 1000 rpm for 3 minutes, and the supernatant is discarded.

d) Conjugation of methotrexate branched structure to thiolated microbubbles

0.5 mg of methotrexate as described in d) above is dissolved in PBS, pH=8. The solution is then added to the thiol-containing microbubbles as described in c) and left to stand for 16 hours to allow disulfide bonds to form. It is then washed vigorously with PBS and water and examined microscopically and by MALDI MS.

The disulfide bond linking the methotrexate structure to the microbubbles can be reduced in vivo to release the free drug molecule, so that the microbubbles together with the tumor-specific vector form the drug delivery system. A physiologically acceptable reducing agent, such as glutathione, can be used to release the drug.

Example 34

Gas-filled microbubbles coated with poly-L-lysine complexed with a fluorescein-labeled DNA fragment from plasmid pBR322

- Example 117A demonstrates the production of microbubbles suitable for gene therapy/antisense use. Specific targeting can be achieved by further doping of the microbubble membranes with vector modified lipid structures according to Example 21.

a) Gas-filled microbubbles enclosed in DSPS

4.5 mg of DSPS was weighed into a clean vial, 1 ml of 1.4% propylene glycol/2.4% glycerol solution was added, the mixture was sonicated for 2 min, and then heated at 80°C for 5 min. Immediately after heating, the solution was filtered through a 4 pm filter, cooled to room temperature, and the supernatant was flushed with perfluorobutane gas. The vials were then shaken on a cap shaker for 45 s, the resulting microbubbles were then washed with deionized water, and the supernatant was discarded. The microbubbles were then suspended in 0.5 ml of water.

b) Preparation and application of poly-L-lysine/DNA complex to microbubbles enclosed in DSPS mg poly-L-lysine (70-150 kD) is weighed into a clean vial and 0.1 ml of fluorescein-labeled pBR322 plasmid dissolved in TE buffer (10 mM Tris HCl, pH 8) is added. The solution is made up to 0.6 ml with water and the pH is adjusted to pH=8. The complexation is carried out for approx. 1 hour, then 0.05 ml of polylysine-DNA solution is added to the microbubble suspension according to point a) above. After 1 hour, it is determined microscopically that the bubbles are fluorescent, confirming the presence of DNA.

Example 35

Preparation of gas-filled microbubbles containing a dabsylated atherosclerotic plaque-binding sequence and a branched core peptide containing RGDS

-118 In this example, we demonstrate the production of microbubbles that contain a thiol group on their surface for modification with thiol-containing vectors for targeting/drug delivery and drug release purposes.

a) preparation of the branched peptide dabsyl-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Lys(HN ₂ -Arg-Gly-Asp-Ser)-Gly-Cys.OH

The peptide was synthesized on an ABI433A automated peptide synthesizer starting from Fmoc-Cys(Trpt)-Tentagel resin using 1 mmol of amino acid in 0.1 mmol steps. The amino acids were preactivated with HBTU before coupling. Simultaneous removal of the peptide and side chain protecting groups from the resin was carried out for 2 h with TFA containing 5% phenol, 5% EDT and 5% water, yielding 160 mg of crude product. Purification by preparative HPLC yielded a 30 mg aliquot of crude material, gradient 10 -> 60% B over 40 min (A=0.1% TFA/water, B=acetonitrile), flow rate 9 ml/min. After lyophilization, 2.5 mg of crude material was obtained (analytical HPLC, gradient 10 -> 50% B over 20 min, B=0.1% TFA/acetonitrile, A=0.01% TFA/water, detection UV 214 and 435 nm, product retention time 25 min). The product was further characterized by MALDI mass spectrometry: expected M+H 2070, measured 2073.

-119 -

b) Production of thiol-containing gas-filled microbubbles

This is done as described in example 64a).

c) Oxidative coupling of thiolated microbubbles with multiple specific peptides via disulfide bonds

The sub-float of the microbubbles prepared according to the previous point b) is discarded and replaced with 1 mg of the dabsyl peptide according to point a) in 0.7 ml of dilute ammonia solution (pH=8). Then 0.2 ml of a stock solution containing 6 mg of potassium ferricyanate dissolved in 2 ml of water is added to this. The vial is then placed on a roller table and the thiol oxidation is continued for 2 hours. The bubbles are then washed vigorously with water until the sub-float is free of dabsyl peptide, as confirmed by HPLC and MALDI MS studies. The peptides bound to the microbubbles are detected by reduction of the disulfide bonds, for which tris-(2-carboxyethyl)phosphine is used as a water-soluble reducing agent. After the reduction, the sub-float is free of dabsyl peptide, as confirmed by HPLC and MALDI MS studies.

Other physiologically acceptable reducing agents, such as reduced glutathione, may also be used to initiate release.

Example 36

Microbubbles enclosed in gas-filled DSPS and biotin-PEG34oo-acyl-phosphatidylethanolamine functionalized with streptavidin, oligonucleotide biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and biotinylated fibrin antipolymer peptide (biotin-GPRPPERHOS.NHi)

a) Preparation of biotin-PEG34oo-acyl-phosphatidylethanolamine A mixture of 100 mg (0.03 mmol) dipalmitoyl-phosphatidylethanolamine, 100 mg (0.03 mmol) biotin-PEG-COj-NHS and 42 μΐ (0.30 mmol) triethylamine was stirred in chloroform/methanol, a mixture (3:1) at room temperature for 2

- Stir for 120 hours, then evaporate the solvent under reduced pressure, purify the residue by flash chromatography (methylene chloride/methanol/water=40/80/l). The product obtained is a yellowish, gummy substance, amount 112 mg (94%), its structure was confirmed by NMR and MALDI MS studies.

b) Coupling of fluorescein-conjugated streptavidin to gas-filled microbubbles

Gas-filled microbubbles were prepared as described in the previous examples by mixing DSPS and biotin-PEG3 ₄ oo-acylphosphatidylethanolamine. The microbubble suspension was divided into 0.2 ml aliquots and fluorescein-conjugated streptavidin was added as indicated in the table below. The samples were incubated on a roller table for 15 or 30 minutes at ambient temperature, and excess protein was removed by washing with PBS. The samples were analyzed by flow cytometry and a Coulter Counter. The results obtained are summarized in the table below.

Results:

Aliquot number Added Streptavidin (mg/200 ml sample) Incubation time (room temperature) % fluorescent particles Average particle diameter (microns) 1 0 2.0 - 2 0 - 12 (foam) 3 0.2 (3xl0' ⁹ mmol) 30 minutes 7.8 3.9 4 2 (3x10' ⁸ mmol) 30 minutes 26.2 4.2 5 10 (1.5xl0' ⁷ mmol) 15 minutes 30.5 so 6 20 (3x10 ⁷ mmol) 30 minutes 97.9 5.2 7 40 (6xl0' ⁸ mmol) 15 minutes 96.7 5.1

-121 -

8DSPS control 20 (3x10 ⁷ mmol) 15 minutes 0.6 3.7

c) Conjugation of streptavidin-coated microbubbles to biotin GAAAGGTAGTGGGGTCGTGTGCCGG oligonucleotide and biotinylated fibrin-anti-polymerase biotin-GPRPPERHQS peptide

Aliquot number 6 of the above table is centrifuged, the supernatant is replaced with 1 ml of PBS buffer (pH=7.5) containing 0.2 mg of biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and 0.2 mg of biotin-GPRPPERHQS (prepared according to examples 27b) and c). After 24 hours of incubation, the particles are washed vigorously with PBS and water.

As described above, other biotinylated vectors or therapeutic agents can also be attached to streptavidin- or avidin-coated microbubbles.

Example 37

Gas-filled microbubbles encased in DSPS functionalized with a thrombus-carrying lipopeptide and production of the thrombolytic enzyme tissue plasminogen activator

The example demonstrates the production of ultrasound contrast agents that can be delivered to thrombi and contain a therapeutic thrombolytic agent.

a) Preparation of a lipopeptide with affinity for thrombi (dipalmitoyl-Lys-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln.NH ₂ )

-122\

The lipopeptide was synthesized on an ABI 433A automated peptide synthesizer starting from Rink Amid resin using 1 mmol amino acid loadings in 0.1 mmol increments. The amino acid and palmitic acid were preactivated with HBTU before coupling. The peptides and side chain protecting groups were simultaneously removed from the resin using TFA containing 5% phenol, 5% EDT, 5% anisole and 5% _H2O for 2 h to yield 80 mg of crude product. A 20 mg aliquot of the resulting material was purified by preparative HPLC. After lyophilization, 6 mg of pure material was obtained. The product was identified by MALDI mass spectrometry and analytical HPLC.

b) Tissue plasminogen activator modification with Sulpho-SMPB

0.1 mM ammonium carbonate buffer containing 0.1 mg t-PA was made up to 0.2 ml by adding water. To the resulting solution was added 0.4 mg Sulpho-SMPB dissolved in 0.05 ml DMSO. The protein solution was left to stand at room temperature for 45 min and then purified on a Superdex 200 column. The product was eluted with PBS and the modified protein fraction was collected.

c) Preparation of DSPS/thrombus-binding lipopeptide and thiol-containing lipopeptide-entrapped gas-filled microbubbles and conjugation of modified tissue plasminogen activator mg DSPS is weighed into a clean vial together with 0.5 mg of lipopeptide according to point a) and 0.5 mg of thiol-containing lipopeptide according to example 64a). Then 1 ml of 1.4% propylene glycol/2.4% glycerol is added

- 123 solution and the resulting mixture are sonicated for 2 minutes and then heated at 80°C for 5 minutes. Immediately after heating, the solution is filtered through a 4 pm filter, the sample is cooled to room temperature and the upper part is flushed with perfluorobutane gas. The vial is shaken for 45 seconds in a cap-type mixer, then the resulting microbubbles are washed twice with deionized water. The supernatant is discarded and replaced with 1 ml of the protein solution according to point b) above. The conjugation reaction is carried out for 1 hour. The microbubbles are then centrifuged, the supernatant is replaced again with 1 ml of the protein solution. The incubation step is repeated until all the protein solution has been used. The microbubbles are then washed vigorously with water and analyzed with a Coulter Counter. The microbubbles were tested in a flow chamber as described in Example 21c). The protein-modified microbubbles were found to bind in greater numbers than the lipopeptide/DSPS or DSPS-only microbubbles.

The targeting/therapeutic/ultrasound activity of microbubbles was investigated in vitro and in vivo in thrombogenesis.

Example 38

Production of gas-filled microbubbles enclosed in lipopeptide-containing DSPS containing a helical peptide with affinity for cell membranes

In this example, we demonstrate the production of microbubbles containing peptide vectors that can be targeted to setjmembrane structures.

a) Production of a lipopeptide containing a helical peptide with affinity for cell membranes

-124 -

The lipopeptide was prepared on an ABI 433A automatic peptide synthesizer starting from Rink Amid resin using 1 mmol amino acid loadings in 0.2 mmol steps. The amino acids and 2-n-hexyl stearic acid were preactivated with HBTU before coupling. The lipopeptides and side chain protecting groups were simultaneously removed from the resin with TFA containing 5% H ₂ O for 2 h, yielding 520 mg of crude product. A 30 mg aliquot of the resulting product was purified by preparative HPLC, gradient 90 -> 100% B in 40 min (A=0.1% TFA/water, B=MeOH), flow rate 9 ml/min. After lyophilization, 10 mg of crude material was obtained (analytical HPLC, gradient 90 -> 100% B over 20 min, B=MeOH, A—0.01 Λ TFA/water, detection UV 214 nm, product retention time 23 min). Further characterization of the product was performed by MALDI mass spectrometry: expected M+H 2369, measured 2375.

b) Production of gas-filled microbubbles

4.5 mg DSPS and 0.5 mg lipopeptide from point a) are weighed into a clean vial and 1 ml of 1.4% propylene glycol/2.4% glycerol solution is added. The resulting mixture is sonicated for 3-5 minutes, heated at 80°C for 5 minutes, then filtered through a 4.5 mm filter. The mixture is cooled to room temperature and the upper part is flushed with perfluorobutane gas. The vials are shaken with a cap stirrer for 45 seconds, the resulting microbubbles are stirred at 1000 rpm

- 125 Centrifuge for 3 minutes. The microbubbles are then washed until the lipopeptide is no longer detectable by MALDI-MS. The resulting product is subjected to Coulter Counter, acoustic attenuation and pressure stability tests. 0.5 ml of methanol is added to the washed microbubbles (approximately 0.2 ml) and the mixture is sonicated for 2 cycles. The resulting clear solution contains the lipopeptide when analyzed by MALDI-MS.

c) In vitro and in vivo studies

The in vitro and in vivo characteristics of the obtained microbubbles are similar to those of the microbubbles of Example 21.

Example 39

Microbubbles enclosed in phosphatidylserine and biotinylated lipopeptide

a) Preparation of dipalmitoyl-lysinyl-tryptophanyl-lysinyl-lysinyl-lysinyl(biotinyl)-glycine lipopeptide

The lipopeptide was synthesized on an ABI433A automated peptide synthesizer starting from Fmoc-Gly-Wang resin using 1 mmol amino acid loadings in 0.1 mmol steps. The amino acids and palmitic acid were preactivated with HBTU before coupling. The peptide and side chain protecting groups were simultaneously removed from the resin with TFA containing 5% phenol, 5% EDT, 5% anisole and 5% H ₂ O for 2 h to give 150 mg of crude product. 40 mg of the resulting product was purified by preparative HPLC, gradient 70 -> 100% B in 40 min (A=0.1% TFA/water, B=MeOH), flow rate 9 ml/min. After lyophilization, 14 mg of crude product was obtained (analytical HPLC, gradient 70 -> 100% B, B=NaOH, A=0.01% TFA/water, detection UV 260 nm and fluorescence Ex280, Em350, product retention time 22 min). Further product characterization was performed by MALDI mass spectrometry: expected M+H 1478, measured 1471.

-126 -

b) Gas-filled microbubbles containing DSPS doped with the biotinylated lipopeptide sequence according to point a)

4.5 mg DSPS and 0.5 mg (0.2 mmol) of the lipopeptide from point a) are weighed into two vials each, 0.8 ml of 1.4% propylene glycol/2.4% glycerol solution is added and the resulting mixture is heated for 80 min with shaking for 5 min. The samples are then cooled to room temperature, the upper part is flushed with perfluorobutane gas and the vials are shaken with a cap stirrer for 45 s and then kept on a roller table overnight. The resulting microbubbles are washed several times with deionized water and then subjected to a Coulter Counter and acoustic attenuation test. MALDI mass spectrometry analysis confirmed the incorporation of the lipopeptide into the DSPS microbubbles: approx. 50-100 ml of microbubbles are transferred to a clean vial and 50-100 ml of water is added. The mixture was sonicated for 30 seconds and then dropped onto a clean sample plate (1 ml + 0.5 ml ACH matrix). Positive mode analysis showed M+H 1474, expected value 1478.

Example 40

Preparation of multispecific gas-filled microbubbles entrapped in DSPS containing lipopeptide containing non-bioactive interleukin-1 receptor-binding peptide

The example demonstrates the production of targeted microbubbles containing a non-bioactive peptide vector directed to the IL-1 receptor that does not induce signal transduction or inhibit IL-1 binding.

a) Preparation of lipopeptide containing non-bioactive interleukin-1 receptor-binding peptides

- 127 The lipopeptide was prepared on an ABI 433A automated peptide synthesizer starting from Fmoc-Ala-Wang resin using 0.1 mM steps of 1 mM amino acid loadings. The amino acids and palmitic acid were preactivated with HBTU before coupling. Simultaneous removal of the lipopeptide and side chain protecting groups from the resin was carried out with TFA containing 5% H ₂ O, 5% anisole, 5% phenol and 5% EDT for 2 h to yield 150 mg of crude product. A 30 mg aliquot of the product was purified by preparative HPLC, gradient 90 -> 100% B over 40 min (A=0.1% TFA/water, B=MeOH), flow rate 9 ml/min. After lyophilization, 4 mg of crude product was obtained (analytical HPLC, gradient 90 -> 100% B over 20 min, B=MeOH, A=0.01% TFA/water, detection UV 214 nm, product retention time 23 min). Further product characterization was performed by MALDI mass spectrometry: expected M+H 2083, measured 2088.

b) Production of gas-filled microbubbles

4.5 mg DSPS and 0.5 mg lipopeptide from point a) are weighed into a clean vial and 1 ml of 1.4% propylene glycol/2.4% glycerol solution is added. The resulting mixture is stirred for 3-5 minutes, heated at 80°C for 5 minutes, then filtered through a 4.5 pm filter. The mixture is cooled to room temperature, the upper part is flushed with perfluorobutane gas, the vials are shaken in a cap mixer for 45 minutes, then the resulting microbubbles are centrifuged at 1000 rpm for 3 minutes. The microbubbles are then washed with water until lipopeptide can no longer be detected by MALDI MS analysis. 0.5 ml of methanol is added to the washed microbubbles (approximately 0.2 ml) and the mixture is treated in an ultrasonic bath for 2 minutes. The resulting clear solution contains the lipopeptide according to MALDI MS analysis (expected value 2083, measured 2088).

- 128 -

Example 41

Perfluoropropane-filled microbubbles containing DSPC, DSPS, and endothelial cell-binding lipopeptide for targeted ultrasound imaging

0.8 ml of DSPC:DPSP (3:1) (5 mg/ml) solution in propylene glycol/glycerol solution (4% in water) was added to 0.5 mg of the lipopeptide as described in Example 31b). The resulting mixture was heated at 80°C for 5 minutes and shaken. The solution was then cooled to ambient temperature and the supernatant was flushed with perfluorobutane gas. The vials were shaken for 85 seconds with a cap stirrer and placed on a roller table for 5 minutes. The samples were then centrifuged at 2000 rpm for 5 minutes, the supernatant was removed and replaced with distilled water. The supernatant was flushed again with perfluorobutane gas and the sample was kept on a roller table until it appeared homogeneous. The washing operation is repeated, the resulting ultrasound contrast agent is tested for Coulter Counter analysis, acoustic attenuation measurement and resistance to external pressure. The microbubbles are subjected to an in vitro test as described in Example 21. A gradual accumulation of microbubbles bound to cells is observed.

Example 42

Sulfur hexafluoride-containing microbubbles containing DSPC, DSPS, and endothelial cell-binding lipopeptide for targeted ultrasound imaging

0.8 ml of a solution of DSPC:DSPS (3:1) (5 mg/ml) in propylene glycol/glycerol solution (4% in water) was added to 0.5 mg of the lipopeptide as described in Example 31b). The resulting mixture was heated at 80°C for 5 minutes and shaken, then the solution was cooled to ambient temperature and the supernatant was purged with sulfur hexafluoride gas.

-129. The vials are then shaken for 45 seconds on a cap shaker and then treated on a roller table for 5 minutes. The samples are then centrifuged at 2000 rpm for 5 minutes, the supernatant is removed and replaced with distilled water. The supernatant is rinsed again with sulfur hexafluoride and the samples are kept on a roller table until they appear homogeneous. The washing operation is repeated.

The resulting ultrasound contrast material is tested for resistance to external pressure using a Coulter Counter, acoustic attenuation method, and other methods.

Example 43

Preparation of gas-filled microbubbles containing DSPG and endothelial cell-binding lipopeptide for targeted ultrasound imaging

0.8 ml of a solution containing DSPG (5 mg/ml) in propylene glycol/glycerol solution (4% in water) is added to 0.5 mg of the lipopeptide as described in Example 31b). The mixture is heated at 80°C for 5 minutes and shaken. The solution is then cooled to ambient temperature, the supernatant is flushed with perfluorobutane gas. The vials are shaken for 45 seconds on a cap shaker and treated on a roller table for 5 minutes. The samples are then centrifuged at 2000 rpm for 5 minutes, the supernatant is removed and replaced with distilled water. The supernatant is flushed again with perfluorobutane gas and the sample is treated on the roller table until it appears homogeneous. The washing operation is repeated. The resulting ultrasound contrast agent is tested for resistance to external pressure by Coulter Counter analysis, acoustic attenuation measurement and external pressure. The microbubbles are tested in vitro as described in Example 21: a gradual accumulation of microbubbles bound to cells is observed.

-130 -

Example 44

Preparation of perfluoropropane-filled microbubbles containing DSPG and endothelial cell-binding lipopeptide for targeted ultrasound imaging

0.8 ml of a solution containing DSPG (5 mg/ml) in propylene glycol/glycerol solution (4% in water) was added to 0.5 mg of the lipopeptide as described in Example 31b). The mixture was heated at 80°C for 5 minutes and shaken. The solution was then cooled to room temperature, the supernatant was flushed with perfluoropropane, the vials were shaken for 45 seconds on a cap shaker and treated on a roller table for 5 minutes. The samples were then centrifuged at 2000 rpm for 5 minutes, the supernatant was removed and replaced with distilled water. The supernatant was flushed again with perfluorobutane gas and the sample was treated on the roller table until homogeneous. The washing operation was repeated. The resulting ultrasound contrast agent is tested for resistance to external pressure by Coulter Counter analysis, acoustic attenuation measurement and external pressure. The microbubbles are tested in vitro as described in Example 21: a gradual accumulation of microbubbles bound to cells is observed.

Example 45

Preparation of sulfur hexafluoride-containing microbubbles containing DSPG and endothelial cell-binding lipopeptide for targeted ultrasound imaging

To 0.8 ml of a solution containing DSPG (5 mg/ml) in propylene glycol/glycerol solution (4% in water) was added 0.5 mg of the lipopeptide described in Example 31b). The mixture was heated and shaken at 80°C for 5 minutes, the solution was cooled to ambient temperature and the supernatant was flushed with sulfur hexafluoride gas. The vial was shaken for 45 seconds on a cap shaker and then on a roller table for 5 minutes.

-131. The sample is then centrifuged at 2000 rpm for 5 minutes, the supernatant is removed and replaced with distilled water. The supernatant is rinsed again with sulfur hexafluoride and the sample is kept on the roller table until it appears homogeneous. The washing operation is repeated. The resulting ultrasound contrast agent is tested for resistance to external pressure by Coulter Counter analysis, acoustic attenuation measurement and external pressure.

Example 46

Gas-filled microbubbles containing DSPS non-covalently bound to polylysine mg DSPS were weighed into a clean vial together with 0.2 mg poly-L-lysine and 1 ml of 1.4% propylene glycol/2.4% glycerol solution was added. The resulting mixture was heated at 80°C for 5 min, cooled to room temperature and the supernatant was flushed with perfluorobutane gas. The vials were then shaken for 45 s with a cap stirrer and the resulting microbubbles were centrifuged at 1000 rpm for 3 min. They were then washed vigorously with water, PBS buffer and then water and the polylysine content was assayed by MALDI MS. No polypeptide material was detected in the final wash. Then 0.5 ml of acetonitrile was added to the microbubbles and sonicated until all microbubbles were disrupted. The resulting solution was re-analyzed for polylysine using MALDI MS. The results obtained were as follows:

MALDI expected MALDI measured poly-L-lysine 786, 914, 1042, 1170 790, 919, 1048, 1177

Example 47

Fabrication of functionalized gas-filled microbubbles for targeted ultrasound imaging

-132 In this example, we demonstrate the production of microbubbles containing a reactive group on their surface for non-specific targeting, in principle using disulfide exchange reactions, through which they bind multiple times to cellular targets.

a) Preparation of thiol-functionalized lipid molecule

The above lipid structure was prepared using an ABI 433A automatic peptide synthesizer starting from Fmoc-Cys(Trt)-Wang resin using 0.25 mM steps of 1 mM amino acid loadings. The amino acids and palmitic acid were preactivated with HBTU. The peptide and side chain protecting groups were simultaneously removed from the resin with TFA containing 5% EDT and 5% H ₂ O for 2 h, yielding 250 mg of crude product. 40 mg of the resulting product was purified by preparative HPLC with a gradient of 90 -> 100% B in 50 min (A=0.1% TFA/water, B=MeOH), flow rate 9 ml/min. After lyophilization, 24 mg of pure product was obtained (analytical HPLC, gradient 70 -» 100% B, t, B=0.1% TFA/acetonitrile, A=0.01% TFA/water, detection UV 214 nm, product retention time 23 min). The product was also characterized by MALDI mass spectrometry: expected M+H 1096, measured 1099.

b) Preparation of gas-filled microbubbles containing DSPS doped with thiol-containing lipid structure

4.5 mg DSPS and 0.5 mg (0.4 mmol) of the lipid from point a) above are weighed into a clean vial and 0.8 ml of 1.4% propylene glycol/2.4% glycerol aqueous solution is added and the resulting mixture is heated at 80°C for 5 minutes with shaking, then filtered hot 40

-133 mm filter. The sample is cooled to room temperature, the upper part is flushed with perfluorobutane gas, the vials are shaken in a cap mixer for 45 seconds, then treated on a roller table overnight. The resulting microbubbles are washed several times with deionized water and the incorporation of thiol groups is analyzed using Ellmans reagent

Example 48

Preparation of gas-filled microbubbles containing DSPS doped with thrombus-binding lipopeptides

a) Preparation of a lipopeptide with affinity for thrombi (dipalmitoyl-LyskAsn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln.NH ₂ )

......

The lipopeptide was synthesized on a Rink Amid resin using an ABI 4433A automated peptide synthesizer using 1 mmol amino acid loadings in a 0.1 mmol scale. The amino acids and palmitic acid were preactivated with HBTU before coupling. Simultaneous removal of the peptide and side chain protecting groups from the resin was carried out for 2 h with TFA containing 5% phenol, 5% EDT, 5% anisole and 5% H ₂ O to yield 80 mg of crude product. 20 mg of this was purified by preparative HPLC. After lyophilization, 6 mg of pure product was obtained. The product was characterized by MALDI mass spectrometry and analytical HPLC.

-134 -

b) Generation of ultrasonic microbubbles targeted to thrombi

4.5 mg DSPS and 1 mg lipopeptide from point a) are weighed into a vial and 0.8 ml of 1.4% propylene glycol/2.4% glycerol solution is added. The mixture is heated at 80°C for 5 minutes, then filtered through a 4 pm filter, then cooled to room temperature and the upper part is flushed with perfluorobutane gas. The vials are shaken with a cap stirrer for 45 seconds, then the resulting microbubbles are washed well with deionized water. The microbubbles are characterized microscopically and by Coulter Counter analysis. The MALDI MS method is used to confirm the presence of lipopeptides as described in the previous examples.

Example 49

Production of transferrin-containing gas-filled microbubbles for targeted ultrasound imaging

a) Preparation of thiol-functionalized lipid molecule o

NH,

The above lipid structure was prepared using an ABI 433A automated peptide synthesizer starting from Fmoc-Cys(Trt)-Wang resin using 0.25 mM steps with 1 mM amino acid loadings. The amino acids and palmitic acid were preactivated with HBTU before coupling. The peptide and side chain protecting groups were simultaneously removed from the resin with TFA containing 5% EDT and 5% H ₂ O for 2 h. This yielded 250 mg of crude product. 40 mg of this was purified by preparative HPLC gradient 90 -> 100% B 50 min

- 135 (A-0.1% TFA/water, B=MeOH), flow rate 9 ml/min. After lyophilization, 24 mg of pure material was obtained (analytical HPLC, gradient 70 -» 100% B, B=0.1% TFA/acetonitrile, A=0.01% TFA/water, detection UV 214 nm, product retention time 23 min). Further product characterization was performed by MALDI mass spectrometry: expected M+H 1096, measured 1099.

b) Microbubbles containing DSPS doped with thiol-containing lipid

4.5 mg DSPS and 0.5 mg (0.4 mmol) of the lipid from point a) above are weighed into a clean vial and 0.8 ml of a 1.4% propylene glycol/2.4% glycerol solution is added. The resulting mixture is heated at 80°C for 5 minutes and then filtered while still hot through a 40 mm filter. The samples are then cooled to room temperature, the upper part is flushed with perfluorobutane gas, the vials are shaken in a cap mixer for 45 seconds and then treated on a roller table overnight. The resulting microbubbles are washed several times with deionized water and tested for the incorporation of thiol groups with Ellmans reagent.

c) Transferrin modification using fluorescein-NHS and Sulpho-SMPB To mg transferrin (Holo, human) in 1 ml PBS, 0.5 ml of a DMSO solution containing 1 mg Sulpho-SMPB and 0.5 mg fluorescein-MHS is added. The resulting mixture is stirred for 45 minutes at room temperature and then passed through a Sephadex 200 column, eluting with PBS. The protein fractions are collected and stored at 4°C until use.

d) Conjugation of microbubbles with transferrin

To the thiol-containing microbubbles according to point b) 1 ml of the modified transferrin protein solution according to point c) is added. The pH is adjusted to pH=9 and the conjugation reaction is carried out for 2 hours at room temperature. The microbubbles are then washed thoroughly

-136 is examined with deionized water and Coulter Counter analysis (97% between 1 and 5 mm) and fluorescence microscopy (strongly fluorescent microbubbles).

Example 50

Preparation of gas-filled microbubbles containing DSPS containing PE-PEG ₂ ooo-Mal conjugated to thiolated trypsin fluorescein

a) Preparation of Boc-H-PEG ₂ oooDSPE (tert-butylcarbamate-polyethyleneglycol-distearoylphosphatidylethanolamine) mg DSPE is added to 150 mg Boc-NH-PEG ₂ ooo-SC dissolved in 2 ml chloroform, followed by the addition of 33 μΐ triethylamine. The resulting mixture is stirred at 41°C for 10 minutes until the starting material dissolves. The solvent is then removed in a rotary evaporator, the residue is taken up in 5 ml acetonitrile and the resulting dispersion is cooled to 4°C and centrifuged. The solution is then filtered and evaporated to dryness. The structure of the resulting material is confirmed by NMR analysis.

b) Preparation of H ₂ N-PEG ₂ ooo-DSPE (amino-polyethylene glycol-distearoylphosphatidylethanolamine

167 mg of Boc-NH-PEG ₂ ooo ^_ DSPE in 5 ml of 4 M hydrochloric acid/dioxane solution was stirred for 2.5 hours at ambient temperature, then the solvent was removed in a rotary evaporator, the residue was dissolved in 1.5 ml of chloroform and washed twice with 1.5 ml of water. The organic phase was concentrated in vacuo. According to TLC analysis (chloroform/methanol/water=1 3/5/0.8) the material contained a single ninhydrin positive spot, Rf=0.6; the structure was confirmed by NMR analysis.

c) Preparation of MAl-PEG ₂ ooo-DSPE (3-maleimidopropionate-polyethylene glycol-distearoylphosphatidylethanolamine

-1375.6 mg (0.018 mmol) of N-succinimidyl-3-maleimidopropionate were dissolved in 0.2 ml of tetrahydrofuran and added to 65 mg (0.012 mmol) of H ₂ N-PEG ₂ ooo-DSPE dissolved in 1 ml of tetrahydrofuran and 2 ml of 0.1 M sodium phosphate buffer (pH=7.5). The resulting mixture was heated to 30°C and the reaction was monitored by TLC, after which the solvent was removed in vacuo. The title compound was purified by flash chromatography on silica gel (80/20=chloroform/methanol). The structure of the crude product was confirmed by NMR analysis and mass spectrometry.

d) Preparation of gas-filled microbubbles containing ** DSPS doped with PE-PEG2ooo~Mal

4.5 mg DSPS and 0.5 mg PE-PEG ₂ ooo-Mal from point c) are weighed into a clean vial and 1 ml of 1.4% propylene glycol/2.4% glycerol solution is added. The resulting mixture is heated at 80°C for 5 minutes and then filtered through a 4.5 mm filter. The sample is cooled to room temperature, the upper part is flushed with perfluorobutane gas, the vials are shaken for 45 seconds in a cap shaker and the resulting microbubbles are washed with multi-distilled water.

e) Preparation of fluorescein-labeled trypsin To 1 mg of trypsin in 1 ml of PBS, add 0.2 ml of a DMSO solution containing 1 mg of fluorescein-NHS. The resulting mixture is stirred for 45 minutes at room temperature, then applied to a Sephadex 2000 column and the product is eluted with PBS, flow rate 1 ml/min. Collect 5 ml of the protein fraction and store at 4°C.

f) Preparation of thiolated, fluorescein-labeled trypsin

To the protein fraction from point e) 1 mg of Traut's reagent is added and the mixture is stirred at room temperature for 12 hours. Then 4 ml of the product modified with Traut's reagent is applied to a Sephadex 2000 column and the product is eluted with PBS. The maximum

Protein fractions showing a fluorescence intensity of -138 are collected, their total volume is 6 ml.

g) Conjugation of microbubbles with thiolated fluorescein-labeled trypsin

The microbubbles prepared according to point d) are incubated on a roller table in 1 ml of the protein solution according to point f). The conjugation is carried out for 10 minutes at pH=7.3 - 7.8, then centrifuged and the supernatant is removed. The operation is repeated three more times, after which the bubbles are washed four times with water to remove unconjugated protein.

D. Bubbles containing active enzyme were confirmed by cleavage of Arg-pNA derivative in PBS.

E. Bubbles were analyzed by Coulter Counter analysis and echogenicity measurement.

PEK-022-015 Bubbles are stable under pressure Total concentration 0.83 Diameter 1-3 mm Diameter 3-5 mm 40 Diameter 5-7 mm 28 Maximum attenuation frequency 3.3 Attenuation at 2 MHz 4.9 Attenuation at 3.5 MHz 7.8 Attenuation at 5.0 MHz 7.1

Figure 2 shows the results of the flow cytometry assay compared to the negative control bubbles (left curve). 98% of the bubbles are fluorescent.

-139 -

Example 51

Preparation of gas-filled microbubbles containing DSPS and captopril-containing molecules for diagnostic and therapeutic purposes a) Preparation of lipopeptide functionalized with captopril o

The above lipopeptide was prepared by manual bubbling method starting from Fmoc-protected Rink Amid BMHA resin in 0.125 mmol scale. Coupling was performed according to standard DBTU/HOBt/DIEA protocol. Bromoacetic acid was coupled via the side chain of Lys as a symmetrical anhydride using DIC preactivation. Captopril dissolved in DMF was introduced into the solid phase using DBU as base. Simultaneous removal of peptide and side chain protecting groups was performed from the resin using TFA containing 5 Λ EDT, 5% water and 5% ethyl methyl sulfide for 2 h. 10 mg of the obtained crude product was purified by preparative liquid chromatography gradient 70 -> 100% B over 60 min (A=0.1% TFA/water, B=0.1% TFA/acetonitrile), flow rate 10 ml/min. After lyophilization, 2 mg of pure material was obtained (analytical HPLC, gradient 70 -> 100% B over 20 min, A=0.01% TFA/water, B=0.1% TFA/acetonitrile, flow rate 1 ml/min, detection UV 214 nm, product retention time 26 min). Further characterization was performed by MALDI mass spectrometry: M+H= 1265, identical to the expected value.

-140 -

b) Gas-filled microbubbles containing DSPS and a captopril-containing compound

4.5 mg DSPS and 0.5 mg of the product from point a) are weighed into a vial and 1 ml of a 1.4% propylene glycol/2.4% glycerol solution is added. The mixture is sonicated for 5 minutes and then heated at 80°C for 5 minutes with shaking. The vials are then cooled, the supernatant is flushed with perfluorobutane gas and the vials are shaken for 45 seconds in a cap-type mixer, and the resulting microbubbles are then thoroughly washed with deionized water. According to the MALDI spectrometric measurement performed, no compound from point a) can be detected in the final wash liquid. The incorporation of the captorpil-containing lipopeptide into the microbubbles was confirmed by MALDI-MS analysis: approximately 50 μΐ of microbubbles are placed in a clean vial containing 100 μΐ of 90% methanol. The mixture is sonicated for 30 seconds and analyzed by MALDI mass spectrometry, which shows an M+H peak corresponding to the lipopeptide of point a).

Example 52

Preparation of gas-filled microbubbles containing a vector and DSPS with affinity for adrenergic receptors for diagnostic and therapeutic purposes

a) Preparation of a protected atenolol derivative suitable for solid-phase coupling

i) Preparation of 4-[(2,3-Epoxy)propoxy]phenylacetate

A mixture of 4.98 g (0.030 mol) of methyl-4-hydroxyphenylacetate, 23.5 ml (0.30 mol) of epichlorohydrin and 121 μΐ (1.5 mmol) of pyridine was stirred at 85°C for 2 h, then cooled and the excess epichlorohydrin was distilled off. The residue was dissolved in ethyl acetate, washed with brine and dried over Na ₂ SO ₄ . The solution was filtered, concentrated and the remaining dark material was chromatographed (silica gel, hexane/ethyl acetate=7/3), yielding 2.25 g

-141 (34%) colorless oil is obtained. 'H (300 MHz) and ¹³ C NMR (75 MHz) studies confirm the structure.

ii) Preparation of methyl 4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetate A mixture of g (9 mmol) methyl 4-[(2,3-epoxy)propoxy]phenylacetate, 23 ml (0.27 mmol) isopropylamine and 1.35 ml (74.7 mmol) water was stirred at room temperature overnight. The mixture was then concentrated, the oily residue was dissolved in chloroform and dried over Na ₂ SO 4 , then filtered and concentrated to give a yellow oil in quantitative yield, which was used in the next step without further purification. The structure was confirmed by ¹ H and ¹³ C NMR analysis.

iii) Preparation of 4-[2-Hydroxy-3-[(1-methylethyl)amino)propoxy]phenylacetic acid hydrochloride

563 mg (2 mmol) of methyl-4-[2-hydroxy-3-[(1-methylethyl)amino)propoxy]phenylacetate were heated in 15 ml of 6 M hydrochloric acid at 100°C for 4 h, then the mixture was concentrated, the residue was dissolved in water and lyophilized. 'H and ¹³ C NMR studies confirmed the structure, MALDI mass spectrometry showed M+H 268, identical to the expected value.

arc) Preparation of N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy)phenylacetic acid mmol 4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetic acid hydrochloride is dissolved in 2 ml of water and added to a solution of 1.60 g (7.2 mmol) of sodium bicarbonate dissolved in 15 ml of water/dioxane=2/l. Then 0.48 g (2.2 mmol) of di-tert-butyl dicarbonate dissolved in 5 ml of dioxane is added. The course of the reaction is followed by TLC analysis (silica gel, CHC13/MeOH/AcOH=85/10/5), the di-tert-butyl dicarbonate is added in small portions until the conversion is complete. The

The reaction mixture was then poured into water saturated with potassium hydrogen sulfate and the server phase was extracted with ethyl acetate. The server phase was washed with water and then brine, dried over Na ₂ SO< and filtered to give 0.6 g of crude product. This was purified by chromatography (silica gel, CHCl ₃ /MeOH/AcOH=85/10/5). The solution was concentrated, the residue was dissolved in glacial acetic acid and lyophilized to give 415 mg (56%) of product as a white solid. The structure was confirmed by ¹ H and ¹³ C NMR analysis.

b) Preparation of atenolol functionalized lipopeptide

The above structure was synthesized by manual bubbling method starting from Fmoc-protected Rink Amid BMHA resin using compound from point a) in 0.125 mmol scale. Coupling was performed according to standard TBTU/HOBt/DIEA protocols. Simultaneous removal of peptide and side chain protecting groups from the resin was performed for 2 hours with TFA containing 5% EDT and 5% water. The crude product was precipitated from ether and purified by preparative liquid chromatography, gradient 70 -» 100% B 60 min (A=0.1% TFA/water, B=0.1% TFA/acetonitrile), flow rate 10 ml/min. After lyophilization, 38 mg of pure product was obtained (analytical HPLC, gradient 70 -> 100% B over 20 minutes, A=0.01% TFA/water, B=0.1% TFA/acetonitrile, flow rate 1 ml/min, detection UV 214 nm, product retention time 25

- 143 min). Further characterization is performed by MALDI mass spectrometry (ACH matrix), measured value M+H 1258, expected value 1257.

c) Preparation of gas-filled microbubbles containing DSPS and lipopeptide-containing atenolol

To a mixture of 4.5 mg DSPS and 0.5 mg of the product from point b) is added 1 ml of a 1.4% propylene glycol/2.4% glycerol solution in a vial. The resulting mixture is sonicated for 5 minutes, heated at 80°C for 5 minutes with shaking, and then cooled. The supernatant is flushed with perfluorobutane gas and the vials are shaken for 45 seconds on a cap mixer, then the contents of the vials are thoroughly washed with deionized water. By MALDI spectrometry, the compound from point b) cannot be detected in the final wash liquid. The incorporation of the atenolol-containing lipopeptide into the microbubble is confirmed by MALDI MS spectrometry: approximately 50 μΐ of microbubbles are added to a clean vial containing 100 μΐ of 90% methanol. The mixture was sonicated for 30 seconds and analyzed by MALDI MS (ACH-matrix), according to which the M+H peak value 1259 corresponds to the lipopeptide according to point b).

d) In vitro analysis

The microbubbles were tested in vitro as described in Example 21. A gradual accumulation of microbubbles bound to cells was observed.

Example 53

Gas-filled microbubbles containing DSPS and a lipopeptide containing a heparin sulfate binding peptide (KRKR) and a fibronectin peptide (WOPPRARI) for targeting and a lipopeptide containing atenolol for therapeutic purposes

a) Production of a lipopeptide containing heparin sulfate binding peptide (KRKR) and fibronectin peptide (WOPPRARI)

-144 -

The lipopeptide was synthesized on an ABI 433A automated peptide synthesizer starting from Fmoc-Ile-Wang resin using 1 mmol amino acid loadings in 0.1 mmol steps. The amino acids and palmitic acid were preactivated with HBTU before coupling. The peptide and side chain protecting groups were simultaneously removed from the resin with TFA containing 5% phenol, 5% EDT, 5% anisole and 5% H ₂ O for 2 h. The amount of crude product obtained was 150 mg, of which 40 mg was purified by preparative HPLC, gradient 70 -> 100% B 40 min (A=0.1% TFA/water, B=MeOH), flow rate 9 ml/min. After lyophilization, 16 mg of pure product was obtained (analytical HPLC, gradient 70 -> 100% B, B=MeOH, A=0.01% TFA/water, detection UV 260 nm, fluorescence Ex280, Em350, product retention time 19.44 min). The product was also characterized by MALDi mass spectrometry: expected M+H 2198, measured 2199.

b) Preparation of a protected atenolol derivative suitable for solid-phase coupling

i) Preparation of methyl 4-[(2,3-epoxy)propoxy]phenylacetate ⁴ >98 g (0.030 mol) of 4-hydroxyphenylacetate, 23.5 ml (0.30 mol) of epichlorohydrin and 121 μΐ (1.5 mmol) of pyridine are stirred at 85°C for 2 hours. The mixture is then cooled, the excess epichlorohydrin is distilled off (rotaevapor). The residue is dissolved in ethyl acetate, washed with brine and dried over Na ₂ SO ₄ . The solution is then

- 145 filtered and concentrated. The remaining dark material was chromatographed on silica gel (hexane/ethyl acetate=7/3) to give 2.25 g (34%) of a colorless oil. The 'H (300 MHz) and ¹³ C NMR (75 MHz) spectra confirmed the structure.

ii) Preparation of methyl 4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetate A mixture of g (9 mmol) ethyl 4-[(2,3-epoxy)propoxy]phenylacetate, 23 ml (0.27 mol) isopropylamine and 1.35 ml (74.7 mmol) water was stirred at room temperature overnight, then concentrated (rotaevapor) and the oily residue was dissolved in chloroform and dried over Na ₂ SO ₄ . The material was then filtered, concentrated to give a yellowish oil in quantitative yield, which was used in the next step without further purification. The structure was confirmed by ^] H and ¹³ C NMR analysis.

iii) Preparation of 4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetic acid hydrochloride

563 mg (2 mmol) of methyl-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetate were dissolved in 15 ml of 6 M hydrochloric acid, heated at 100°C for 4 hours, then concentrated (rotaevapor) and the residue was dissolved in water and then lyophilized. The 'H and ¹³ C NMR spectra confirmed the structure, the M+H value of 286 measured by MALDI mass spectrometry corresponded to the expected value.

(iv) Preparation of N-Boc-4-[2-hydroxy-3-[(1-methylethyl)- amino] propoxy]phenylacetic acid mmol 4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetic acid hydrochloride is dissolved in 2 ml of water and added to 0.6 g (7.2 mmol) of sodium hydrogen carbonate dissolved in 15 ml of water/dioxane=2/l. 0.48 g (2.2 mmol) of di-tert-butyl dicarbonate dissolved in 5 ml of dioxane is added. The course of the reaction is monitored by TLC analysis

-146 (silica gel, CHCl ₃ /MeOH/AcOH= 85/10/5), di-tert-butyl dicarbonate was added until the conversion was complete. The reaction mixture was then poured into water saturated with potassium hydrogen sulfate, the organic material was extracted with ethyl acetate, the organic phase was washed with water and brine, dried over Na ₂ SO 4 , filtered to give 0.6 g of crude material. The product was then purified by chromatography (silica gel, CHCl ₃ /MeOH/AcOH=85/10/5). The solution was concentrated, the residue was dissolved in glacial acetic acid and lyophilized to give 415 mg (56%) of a white solid. The structure was confirmed by 'H and ¹³ C NMR analysis.

c) Preparation of atenolol functionalized lipopeptide

The above compound was prepared by manual bubbling method starting from Fmoc-protected Rink Amid BMHA resin in 0.125 mmol scale using the appropriate amino acids and palmitic acid and the compound of point a). The coupling was performed according to standard TBTU/HUBt/DIEA protocols. Simultaneous removal of peptide and side chain protecting groups from the resin was performed with TFA containing 5% EDT, 5% water for 2 hours. The crude material was precipitated from ether and purified by preparative liquid chromatography, gradient 70 -> 100% B in 60 min (A=0.1% TFA/water, B=0.1% TFA/acetonitrile), flow rate 10 ml/min. After lyophilization the yield was 38 mg of pure material (analytical HPLC, gradient 70 -> 100% B

- 147 20 min (A=0.1% TFA/water, B=0.1% TFA/acetonitrile), flow rate 1 ml/min, detection UV 214 nm, retention time 25 min). The product was also characterized by MALDI mass spectrometry (ACH matrix), measured M+H 1258, expected 1257.

d) Preparation of gas-filled DSPS microbubbles containing heparin sulfate binding peptide (KRKR), fibronectin peptide (WOPPRARI) and atenolol-containing lipopeptide ml 1.4% propylene glycol/2.4% glycerol, 5 mg DSPS, 0.5 mg product from point a) and 0.5 mg product from point c) are added to a mixture in a vial. The mixture is sonicated for 5 minutes, then heated at 80°C for 5 minutes with shaking. The solution is then filtered, cooled, the supernatant is flushed with perfluorobutane gas and the vial is shaken for 45 seconds in a cap mixer, then thoroughly washed with deionized water. The incorporation of the atenolol-containing lipopeptide into the microbubbles is confirmed by MALDI MS spectrometry: approx. 50 μΐ of microbubbles are weighed into a clean vial containing 100 μΐ of 90% methanol. The mixture is sonicated for 30 seconds and analyzed by MALDI MS (ACH matrix), two M+H peaks are obtained at 2202 and 1259, corresponding to lipopeptides a) and c), respectively.

e) In vitro analysis

The microbubbles were tested using the in vitro assay described in Example 21. A gradual accumulation of microbubbles bound to cells was observed.

Example 54

Gas-filled microbubbles containing DSPS and a lipophilic atenolol derivative with affinity for adrenergic receptors for diagnostic and therapeutic purposes

-148 -

a) Preparation of N-Hexadecyl-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetamide

i) Preparation of methyl 4-[(2,3-epoxy)propoxy]phenylacetate

A mixture of 4.98 g (0.030 mol) of methyl 4-hydroxyphenylacetate, 23.5 ml (0.30 mol) of epichlorohydrin and 121 μΐ (1.5 mmol) of pyridine was stirred at 25°C for 2 h, then cooled, the excess epichlorohydrin was distilled off, the residue was dissolved in ethyl acetate, washed with brine and dried over Na ₂ SO ₄ . The solution was then filtered and concentrated. The residue was chromatographed on silica gel (hexane/ethyl acetate=7/3) to give 2.25 g (34%) of a colorless oil. The obtained 'H (300 MHz) and ¹³ C NMR (75 MHz) spectra confirmed the structure.

ii) Preparation of methyl-4-[2-hydroxy-3-[(1-methyl-ethyl)amino]propoxy]phenylacetate A mixture of g (9 mmol) methyl-4-[(2,3-epoxy)propoxy]phenylacetate, 23 ml (0.27 mol) isopropylamine and 1.35 ml (74.7 mmol) water was stirred at room temperature overnight, then concentrated (rotaevapor) and the oil residue was dissolved in chloroform and dried over Na ₂ SO ₄ . The material was then filtered and concentrated to give a yellow oil in quantitative yield, which was used in the next step I without further purification. The structure was confirmed by ¹ H and ¹³ C NMR analysis.

iii) Preparation of 4-[2-Hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetic acid

563 mg (2 mmol) of methyl 4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetate were dissolved in 15 ml of 6 M hydrochloric acid and heated at 100°C for 4 h. The mixture was then concentrated (rotaevapor) and the residue was dissolved in water and lyophilized. The 'H and ¹³ C NMR spectra confirmed the structure, and the M+H value of 268 obtained by MALDI mass spectrometry was as expected.

-149 - iv) Preparation of N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetic acid mmol 4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetic acid hydrochloride is dissolved in 2 ml of water and added to 0.60 g (7.2 mmol) of sodium hydrogen carbonate dissolved in 15 ml of water/dioxane=2/l. 0.48 g (2.2 mmol) of ditert-butyl dicarbonate dissolved in 5 ml of dioxane is added. The course of the reaction is monitored by TLC (silica gel, CHCl3/MeOH/AcOH=85/10/5), the ditert-butyl dicarbonate portions being added until the conversion is complete. The reaction mixture was then poured into water saturated with potassium hydrogen sulfate, the organic material was extracted with ethyl acetate, the organic phase was washed with water and brine, dried over Na ₂ SO 4 , filtered to give 0.6 g of crude product. This was chromatographed on silica gel (CHCl 3 /MeOH/AcOH=85/10/5). The solution was then concentrated, the residue was dissolved in glacial acetic acid and lyophilized. This gave 415 mg (56%) of a white solid. The structure was confirmed by 'H and ¹³ C NMR analysis.

v) Preparation of N'-Boc,N-hexadecyl-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetamide mg (0.25 mmol) N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetic acid and 60 mg (0.25 mmol) hexadecylamine were dissolved in 5 ml DMF, cooled to 0°C and 48 mg (0.25 mmol) N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (water-soluble carbodiimide) and 39 mg (0.25 mmol) HOBt were added. The mixture was stirred at 0°C for 1 hour and then at room temperature overnight. The mixture was then poured into 25 ml water containing 2.5 g sodium carbonate and 4 g sodium chloride. The precipitate formed is filtered, washed with water and dissolved in chloroform. The chloroform phase is washed with 5% sodium carbonate and water and dried over Na ₂ SO 4 . The solution is

-150 then filtered, concentrated, thus obtaining 150 mg of organic, white, crude product. This was chromatographed on silica gel (chloroform/methanol=95/5), thus obtaining 118 mg (80%) of white material. Its structure was confirmed by ¹ H (500 MHz) and ¹³ C (125 MHz) NMR analysis. The M+Na peak obtained by MALDI mass spectrometry was 614.

vi) Preparation of N-hexadecyl-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetamide mg N'-Boc-N-hexadecyl-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy)phenylacetamide is dissolved in 9 ml of dichloromethane and 1 ml of trifluoroacetic acid is added. The mixture is stirred for 2 hours at room temperature, the TLC performed (silica gel, chloroform/methanol=95/5) shows the complete conversion of the starting material. The solvents are evaporated, the residual material is dissolved in a water/acetonitrile mixture, lyophilized, thus obtaining a white solid in quantitative yield. The structure is confirmed by 'H (500 MHz) ¹³ C (125 MHz) NMR analysis, the MALDI spectrometry data measured M+H 492 and expected M+Na 514.

b) Gas-filled microbubbles containing DSPS and N-hexadecyl-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetamide for diagnostic and therapeutic purposes ml of a 1.4% propylene glycol/2.4% glycerol solution is added to a mixture containing 4.5 mg of DSPS and 0.5 mg of N-hexadecyl-4-[2-hydroxy-3-[(1-methylethyl)amino)propoxy]phenylacetamide in a vial. The resulting mixture is sonicated for 5 minutes and then heated at 80°C for 5 minutes with shaking. The solution is then filtered, cooled, the supernatant is purged with perfluorobutane gas and the vial is shaken in a cap mixer for 45 seconds and then thoroughly washed with deionized water. The compound according to point a)

-151 incorporation into microbubbles is confirmed by MALDI MS spectrometry: approximately 50 μΐ of microbubbles are weighed into a clean vial containing 100 μΐ of 90% methanol. The mixture is sonicated for 30 seconds and then confirmed by MALDI-MS spectrometry, the measured value M+H 492 corresponds to N-hexadecyl-4-[2-hydroxy-3-[(1methylethyl)amino)propoxy)phenylacetamide.

Example 55

Gas-filled microbubbles containing DSPS and a folic acid-containing compound for diagnostic purposes

a) Production of lipopeptide containing folic acid

The above compound was prepared by manual bubbling method starting from Fmoc-protected Rink Amid BMHA resin in 0.125 mmol scale using the appropriate amino acids, palmitic acid and folic acid. Coupling was performed according to standard TBTU/HOBt/DIEA protocols. Simultaneous removal of peptide and side chain protecting groups from the resin was performed with TFA containing 5% EDT and 5% water for 2 h. The crude material was precipitated from ether and analyzed by MALDI mass spectrometry, the obtained M+H peak value was 1435, expected value 1430. The material was further characterized by analytical HPLC, gradient 70 -> 100% B 20 min (A=0.1% TFA/water, B=0.1% TFA/acetonitrile), flow rate 1 ml/min, retention time 27 min, UV at 368 nm.

-152 -

b) Preparation of gas-filled microbubbles containing DSPS and folic acid-containing lipopeptide ml of 1.4% propylene glycol/2.4% glycerol solution is weighed into a vial to a mixture containing 4.5 mg of DSPS and 0.5 mg of the product according to point a). Then 40 μΐ DMSO and diluted ammonia (to pH=8) are added and the resulting mixture is treated with ultrasound for 5 minutes, then heated at 80°C for 5 minutes with shaking. The solution is then filtered, cooled, the part above the solution is flushed with perfluorobutane gas and the vial is shaken in a cap mixer for 45 seconds, then thoroughly washed with deionized water. The incorporation of the compound according to point a) into the bubbles is confirmed by MLADI MS spectrometry: approx. 50 μΐ of microbubbles are weighed into a clean vial containing 100 μΐ of 90% methanol. The mixture is sonicated for 30 seconds and then analyzed by MALDI MS spectrometry (ACH matrix), the resulting M+H peak is 1238, corresponding to the structure in point a).

c) In vitro analysis

The microbubbles are tested in vitro as described in Example 21. A gradual accumulation of microbubbles bound to cells is observed.

Example 56

Gas-filled microbubbles containing DSPS and chlorambucil cholesteryl ester for diagnostic and therapeutic purposes

a) Preparation of cholesteryl 4-[4-[bis(2-chloroethyl)amino]phenyl]butanoate

170 μΐ (1.10 mmol) of DIC were added to 669 mg (2.20 mmol) of chlorambucil dissolved in 15 ml of anhydrous dichloromethane and the mixture was stirred at room temperature for 0.5 h, then 387 mg (1 mmol) of cholesterol dissolved in 10 ml of dichloromethane and 122 mg (1

- 153 mmol) DMAP. The mixture is stirred overnight, then poured into 5% sodium bicarbonate solution, the phases are separated, the organic phase is washed with brine and dried over MgSO _4. The solution is then filtered, concentrated, the resulting product is chromatographed on silica gel (chloroform), thus 560 mg (83%) of a colorless oil are obtained. The product is confirmed by MALDI mass spectrometry, the obtained M+H value is 674, corresponding to the expected value. Further characterization is also performed by (500 MHz) and ¹³ C (125 MHz) NMR analysis, the spectra obtained are consistent with the structure.

b) Preparation of gas-filled microbubbles containing DSPS and chlorambucil cholesteryl ester for diagnostic and/or therapeutic purposes ml of 1.4% propylene glycol/2.4% glycerol solution is mixed in a vial with a mixture containing 4.5 ml of DSPS and 0.5 mg of the product according to point a), then sonicated for 5 minutes and heated with shaking for 5 minutes, then cooled. The supernatant is flushed with perfluorobutane gas, the vials are shaken in a cap mixer for 45 seconds, then thoroughly washed with deionized water. Compound a) is not present in the final wash liquid in a quantity detectable by MALDI mass spectrometry. The incorporation of chlorambucil cholesteryl ester into the bubbles is confirmed by MALDI MS spectrometry: approx. 50 μΐ of microbubbles are weighed into a clean vial containing 100 μΐ of 90% methanol. The mixture is sonicated for 30 seconds and then analyzed by MALDI MS spectrometry, the resulting M+H peak is 668, corresponding to the structure in point a).

Example 57

Gas-filled microbubbles containing DSPS and a lipopeptide containing atenolol and a chlorambucil-cholesteryl derivative for diagnostic and therapeutic purposes

- 154 -

a) Preparation of a protected atenolol derivative suitable for solid-phase coupling

i) Preparation of methyl-4-[(2,3-epoxy)propoxy]

A mixture of 4.98 g (0.030 mol) of methyl 4-hydroxyphenylacetate, 23.5 ml (0.30 mol) of epichlorohydrin and 121 μΐ (1.1 mmol) of pyridine was stirred at 85°C for 2 h, then cooled, the excess epichlorohydrin was distilled off (rotaevapor), the residue was dissolved in ethyl acetate, washed with brine and dried over Na ₂ SO ₄ . The solution was then filtered, concentrated, and the dark residue was chromatographed on silica gel (hexane/ethyl acetate=7/3) to give 2.25 g (34%) of a colorless oil. The *H (300 MHz) and ¹³ C (75 MHz) NMR spectra were consistent with the structure.

ii) Preparation of methyl 4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetate A mixture of g (9 mmol) methyl 4-[(2,3-epoxy)propoxy]phenylacetate, 23 ml (0.27 mol) isopropylamine and 1.35 ml (74.7 mmol) water was stirred at room temperature overnight, then concentrated, the oily material was dissolved in chloroform and dried over Na ₂ SO ₄ . The material was filtered and concentrated to give a yellow oil in quantitative yield, which was used in the next step without further purification. The structure was confirmed by *H and ¹³ C NMR analysis.

iii) 4-[2-Hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetic acid hydrochloride

563 mg (2 mmol) of methyl-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetate were dissolved in 15 ml of 6 M hydrochloric acid, heated at 100°C for 4 hours, then concentrated, the residue was dissolved in water and lyophilized. The ^] H and ¹³ C NMR spectra confirmed the structure, and the M+H 268 obtained by MALDI mass spectrometry corresponded to the expected value.

iv) Preparation of N-Boc-4-[2-Hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetic acid 1 ml (2 mmol) of 4-[2-hydroxy-3-[(1-methylethyl)amino)propoxy]phenylacetic acid hydrochloride is dissolved in 2 ml of water and added to 0.6 g (7.2 mmol) of sodium hydrogen carbonate dissolved in 15 ml of water/dioxane=2/l. 0.48 g (2.2 mmol) of di-tert-butyl dicarbonate dissolved in 5 ml of dioxane is added. The course of the reaction is monitored by TLC (silica gel, CHCl ₃ /MeOH/AcOH=85/10/5), the di-tert-butyl dicarbonate portions are added until the conversion is complete. The mixture was then poured into water saturated with potassium hydrogen sulfate, the organic phase was extracted with ethyl acetate, the organic phase was washed with water and brine, dried over Na ₂ SO 4 and filtered to give 0.6 g of crude material. The product was chromatographed on silica gel (CHCl ₃ /MeOH/AcOH=85/10/5). The solution was concentrated, the residue was dissolved in glacial acetic acid and lyophilized to give 415 mg (56%) of a white solid. The structure was determined by the 'H and 13

This was confirmed by C NMR analysis.

b) Preparation of atenolol functionalized lipopeptide

The above compound was prepared by manual bubbling method starting from Fmoc-protected Rink Amid BMHA resin in 0.125 mmol scale using the appropriate amino acids, palmitic acid and the compound of point a). The coupling was carried out using the standard DBTU/HOBt/DIEA

-156. The simultaneous removal of peptide and side chain protecting groups from the resin was carried out with TFA containing 5% EDT and 5% water. The crude product was precipitated from ether and purified by preparative liquid chromatography, gradient 70 -> 100% B over 60 min (A=0.1% TFA/water, B=0.1% TFA/acetonitrile), flow rate 10 ml/min. After lyophilization, 38 mg of pure material was obtained (analytical HPLC, gradient 70 -> 100% B over 20 min, A=0.01% TFA/water, B=0.1% TFA/acetonitrile, flow rate 1 ml/min, detection UV 214 nm, product retention time 25 min). The product was also confirmed by MALDI spectrometry (ACH matrix), measured M+H 1258, expected 1257.

c) Preparation of cholesteryl 4-[4-bis(2-chloroethyl)amino]phenyl]butanoate

170 μΐ (1.10 mmol) of DIC were mixed with 669 mg (2.20 mmol) of chlorambucil dissolved in 15 mL of anhydrous dichloromethane. The mixture was stirred at room temperature for 0.5 h, then 387 mg (1 mmol) of cholesterol and 122 mg (1 mmol) of DMAP dissolved in 10 mL of dichloromethane were added. The mixture was stirred overnight and then poured into 5% sodium bicarbonate solution. The phases were separated, the organic phase was washed with brine, dried over MgSO ₄ , filtered, concentrated and chromatographed on silica gel (chloroform) to give 560 mg (83%) of a colorless oil. The product was determined by MALDI mass spectrometry to have an M+H value of 674, which is in accordance with the expected value. H (500 MHz) and ¹³ C (125 MHz) NMR analyses confirm the above structure.

d) Preparation of gas-filled microbubbles containing DSPS and a lipopeptide containing atenolol and a chlorambucil cholesterol ester

- 157 1 ml of 1.4% propylene glycol/2.4% glycerol solution is mixed in a vial with a mixture containing 5 mg DSPS, 0.5 mg of the product from point b) and 0.5 mg of the product from point c), then sonicated for 5 minutes and heated at 80°C for 5 minutes with shaking. The solution is then filtered, then cooled, the upper part is rinsed with perfluorobutane gas and the vial is shaken for 45 seconds, then thoroughly washed with deionized water. The incorporation of compounds b) and c) into the microbubbles is confirmed by MALDI MS spectrometry: approximately 50 μΐ microbubbles are weighed into a clean vial containing 100 μΐ 90% methanol. The mixture is treated with ultrasound for 30 seconds and then analyzed by MALDI MS spectrometry (ACH matrix), the obtained M+H peaks correspond to the lipopeptide according to point b) and the cholesteryl esters according to point c).

e) In vitro analysis

The microbubbles are tested in vitro as described in Example 21. A gradual accumulation of microbubbles bound to cells is observed.

Example 58

Gas-filled microbubbles containing DSPS and lipopeptide containing atenolol for cellular delivery and lipophilic captopril thiol ester for therapeutic purposes

a) Preparation of a protected atenolol derivative suitable for solid-phase coupling

i) Preparation of methyl 4-[(2,3-epoxy)propoxy]phenylacetate

A mixture of 4.98 g (0.030 mol) of methyl 4-hydroxyphenylacetate, 23.5 ml (0.30 mol) of epichlorohydrin and 121 μΐ (1.1 mmol) of pyridine was stirred at 85°C for 2 h, then cooled, the excess epichlorohydrin was distilled off (rotaevapor), the residue was dissolved in ethyl acetate, washed with brine and dried over Na ₂ SO ₄ . The solution was then filtered,

-158 is concentrated, the remaining dark material is chromatographed on silica gel (hexane/ethyl acetate=7/3), thus 2.25 g (34%) of a colorless oil is obtained. The 'H (300 MHz) and ¹³ C (75 MHz) NMR spectra are consistent with the structure.

ii) Preparation of methyl 4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetate A mixture of g (9 mmol) methyl 4-[(2,3-epoxy)propoxy]phenylacetate, 23 ml (0.27 mol) isopropylamine and 1.35 ml (74.7 mmol) water was stirred at room temperature overnight, then concentrated, the oily material was dissolved in chloroform and dried over Na ₂ SO 4 . The material was filtered and concentrated to give a yellow oil in quantitative yield, which was used in the next step without further purification. The structure was confirmed by ^] H and ¹³ C NMR analysis.

iii) 4-[2-Hydroxy-3-[(1-methylethyl)amnio]propoxy]phenylacetic acid hydrochloride

563 mg (2 mmol) of methyl-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetate were dissolved in 15 ml of 6 M hydrochloric acid, heated at 100°C for 4 hours, then concentrated, the residue was dissolved in water and lyophilized. The ^J H and ¹³ C NMR spectra confirmed the structure, the M+H 268 obtained by MALDI mass spectrometry corresponded to the expected value.

(iv) Preparation of N-Boc-4-[2-Hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetic acid 1 ml (2 mmol) of 4-[2-hydroxy-3-[(1-methylethyl)amino)propoxy]phenylacetic acid hydrochloride is dissolved in 2 ml of water and added to 0.6 g (7.2 mmol) of sodium hydrogen carbonate dissolved in 15 ml of water/dioxane=2/l. 0.48 g (2.2 mmol) of di-tert-butyl dicarbonate dissolved in 5 ml of dioxane is added. The course of the reaction is monitored by TLC (silica gel, CHCl ₃ /MeOH/AcOH=85/10/5), the di-tert-butyl-159 -dicarbonate portions are added until the conversion is complete. The mixture was then poured into water saturated with potassium hydrogen sulfate, the organic phase was extracted with ethyl acetate, the organic phase was washed with water and brine, dried over Na ₂ SO ₄ and filtered to give 0.6 g of crude material. The product was chromatographed on silica gel (CHCl ₃ /MeOH/AcOH=85/10/5). The solution was concentrated, the residue was dissolved in glacial acetic acid and lyophilized. This gave 415 mg (56%) of a white solid. The structure was confirmed by ¹ H and ¹³ C NMR analysis.

b) Preparation of atenolol functionalized lipopeptide

The above compound was prepared by manual bubbling method starting from Fmoc-protected Rink Amid BMHA resin in 0.125 mmol scale using the appropriate amino acids and palmitic acid and the compound of point a). The coupling was carried out according to standard TBTU/HUBt/DIEA protocols. Simultaneous removal of peptide and side chain protecting groups from the resin was carried out with TFA containing 5% EDT, 5% water for 2 hours. The crude material was precipitated from ether and purified by preparative liquid chromatography, gradient 70 -> 100% B 60 min (A=0.1% TFA/water, B=0.1% TFA/acetonitrile), flow rate 10 ml/min. After lyophilization, the yield was 38 mg of pure material (analytical HPLC, gradient 70 -> 100% B 20 min (A=0.1% TFA/water, B=0.1% TFA/acetonitrile), flow rate

- 160 1 ml/min, detection UV 214 nm, retention time 25 min). The product was also characterized by MALDI mass spectrometry (ACH matrix), measured M+H 1258, expected 1257.

c) Preparation of captopril cholanic acid thiol ester

A mixture of 361 mg (1 mmol) of 5-p-cholanic acid and 77 μΐ (0.50 mmol) of DIC in 5 ml of dichloromethane was stirred for 10 min and then added to 130 mg (0.600 mmol) of captopril and 180 μΐ (1.20 mmol) of DBU dissolved in 10 ml of dichloromethane. The mixture was stirred overnight, then poured into dilute hydrochloric acid and 30 ml of chloroform were added. The phases were separated, the organic phase was washed with water and brine, and then dried over MgSO<. After filtration and concentration, the crude product was chromatographed (silica gel, chloroform/methanol/acetic acid=95/4/l). The product was lyophilized from acetonitrile/water/ethanol. Thus, 137 mg (49%) of an off-white solid were obtained. The structure is confirmed by *H (500 MHz) and ¹³ C (125 MHz) NMR spectroscopy. MALDI mass spectrometry has been performed and the M+Na peak value in positive mode is m/z 584.

d) Preparation of gas-filled microbubbles containing DSPS and lipopeptide containing atenolol for cell delivery and lyophilic captopril thiol ester for therapeutic purposes ml of 1.4% propylene glycol/2.4% glycerol solution is added to a mixture containing 5 mg DSPS and 0.5 mg of the product according to point b) and 0.5 mg of the product according to point c) in a vial, then sonicated for 5 minutes and heated at 80°C for 5 minutes with shaking, then cooled. The upper part is flushed with perfluorobutane gas and the vials are shaken for 45 seconds, then thoroughly washed with deionized water. No detectable amount of compound b) or c) is present in the final wash liquid by MALDI spectrometry. Compounds b) and c)

- 161 incorporation into microbubbles is confirmed by MALDI MS spectrometry: approximately 50 μΐ of microbubbles are weighed into a clean vial containing 100 μΐ of 90% methanol. The mixture is sonicated for 30 seconds and then analyzed by MALDI MS spectrometry (ACH matrix), the M+H peaks obtained correspond to compounds b) and c).

e) In vitro analysis

The microbubbles are tested in vitro as described in Example 21. A gradual accumulation of microbubbles bound to cells is observed.

Example 59

Gas-filled microbubbles containing phosphatidylserine and biotinamide-PEG-p-Ala-cholesterol and a chlorambucil-cholesterol ester for diagnostic and therapeutic purposes

a) Preparation of cholesterol-N-Boc-P-alaninate

510 μΐ DIC was mixed with 1.25 g (6.60 mmol) Boc-p-Ala-OH in 15 ml dichloromethane under an inert atmosphere, then the resulting mixture was shaken for 30 min and transferred to a flask containing 1.16 g (3 mmol) cholesterol and 367 mg (3 mmol) DMAP dissolved in 15 ml dichloromethane. The mixture was stirred for 2 h and then poured into an aqueous solution of potassium hydrogen sulfate. The phases were separated, the aqueous phase was extracted with chloroform, the organic phases were combined, washed with aqueous potassium hydrogen sulfate solution, then with water and dried over MgSO _4. After filtration and concentration, the resulting crude product was chromatographed on silica gel (chloroform/methanol=9/l), yielding 1.63 g (97%) of a white solid. The structure was confirmed by ^[ H NMR (500 MHz) spectroscopy.

b) Preparation of cholesteryl-p-alaninate hydrochloride

- 162 279 mg (0.500 mmol) of the compound from point a) is dissolved in 5 ml of 1 M hydrochloric acid/1,4-dioxane solution, stirred at room temperature for 4 hours, then concentrated, thus obtaining cholesteryl-β-alaninate hydrochloride in quantitative yield. The structure is confirmed by *H NMR (500 MHz) spectrum and MALDI mass spectrometry, the measured M+Na peak is 482, expected 481.

c) Biotin-PEG34oo-P-Ala-cholesterol mg (0.03 mmol) cholesteryl-p-alaninate hydrochloride was dissolved in 3 ml chloroform/aqueous methanol=2.6/l and 42 μΐ (0.30 mmol) triethylamine was added. The material was stirred for 10 minutes at room temperature, then 100 mg (0.03 mmol) biotin-PEG34oo-NHS dissolved in 1 ml 1,4-dioxane was added dropwise, then stirred at room temperature for 3 hours, then evaporated to dryness and the residue was purified by flash chromatography to give a crystalline material, yield 102 mg, 89%. The structure was confirmed by MALDI MS and NMR analyses.

d) Preparation of cholesteryl 4-[4-bis(2-chloroethyl)amino]phenyl]butanoate

170 μΐ (1.10 mmol) of DIC were mixed with 669 mg (2.20 mmol) of chlorambucil dissolved in 15 ml of anhydrous dichloromethane and stirred at room temperature for 0.5 h, then 387 mg (1 mmol) of cholesterol dissolved in 10 ml of dichloromethane and 122 mg (1 mmol) of DMAP were added. The mixture was stirred overnight and then poured into 5% sodium bicarbonate solution. The phases were separated, the organic phase was washed with brine and dried over MgSO ₄ . The solution was filtered, concentrated, and the resulting product was chromatographed on silica gel (chloroform) to give 560 mg (83%) of the title compound as a colorless oil. The product was analyzed by MALDI mass spectrometry, and the value obtained was M+H 674, corresponding to

-163 as expected. Further analysis was performed using 'H (500 MHz) and ¹³ C (125 MHz) NMR spectra, and the spectra obtained were consistent with the structure.

e) Preparation of gas-filled microbubbles ml of 1.4% propylene glycol/2.4% glycerol solution is added to a mixture containing 5 mg DSPS, 0.5 mg product from point c) and 0.5 mg product from point d) in a vial, then sonicated for 5 minutes, then heated at 80°C for 5 minutes with shaking, then cooled. The supernatant is flushed with perfluorobutane gas, the vials are shaken for 45 seconds, then thoroughly washed with deionized water. No detectable amounts of compound c) or d) are present in the final wash liquid by MALDI spectrometry. The incorporation of compounds c) and d) into the microbubbles is confirmed by MALDI MS spectrometry: approx. 50 μΐ microbubbles are weighed into a clean vial containing 100 μΐ 90% methanol. The mixture was sonicated for 30 seconds and then analyzed by MALDI MS spectrometry (ACH matrix), the M+H peaks obtained corresponded to compounds c) and d).

Example 60

Gas-filled microbubbles containing DSPS and a lipopeptide containing a bestatin derivative for diagnostic and therapeutic purposes

a) Preparation of lipopeptide containing bestatin derivative

-164The above compound was prepared using a manual bubbler starting from Fmoc-protected Rink Amide BMHA resin using the appropriate amino acids and palmitic acid in a 0.125 mmol scale. Coupling was performed according to standard TBTU/HOBt/DIEA protocols. Simultaneous removal of peptide and side chain protecting groups from the resin was performed over 2 hours with TFA containing 5% EDT and 5% water. The crude product was precipitated from ether and purified by preparative liquid chromatography, gradient 70 -» 100% B over 60 minutes (A=0.1% TFA/water and B=0.1% TFA/acetonitrile), flow rate 10 ml/min. After lyophilization, 12 mg of pure material was obtained (analytical HPLC gradient 70 -> 100% B 20 min, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow rate 1 ml/min, detection UV 214 nm, retention time 25 min). The product was further characterized by MALDI spectrometry (ACH matrix), the obtained M+H value was 1315, the expected 1314.

b) Gas-filled microbubbles containing DSPS and a lipopeptide containing a bestatin derivative for diagnostic and therapeutic purposes ml of a 1.4% propylene glycol/2.4% glycerol solution are mixed in a vial with 4.5 mg of DSPS and 0.5 mg of the product according to point a), the resulting mixture is treated with ultrasound for 5 minutes, then heated at 80°C for 5 minutes with shaking, then cooled. The upper part of the material is flushed with perfluorobutane gas, the vial is shaken for 45 seconds in a cap mixer, then washed thoroughly with deionized water. Component b) cannot be detected in the final washing liquid by MALDI spectrometry. The incorporation of the atenolol-containing lipopeptide into the microbubbles is confirmed by MALDI mass spectrometry: approx. 50 μΐ microbubbles are weighed into a clean vial with 100 μΐ 90% methanol, the mixture is sonicated for 30 seconds, then

-165 is analyzed by MALDI mass spectrometry (ACH matrix), the result is M+H peak 1320, expected 1314, corresponding to the lipopeptide according to point a).

c) In vitro analysis

The microbubbles are tested in vitro as described in Example 21. A gradual accumulation of microbubbles bound to cells is observed.

Example 61

Gas-filled microbubbles containing DSPS and chlorambucil-containing lipopeptide for diagnostic and therapeutic purposes

a) Production of chlorambucil-containing lipopeptide

i · - ·

The above compound was prepared using a manual bubbler starting from Fmoc-protected Rink Amide BMHA resin in a 0.125 mmol scale using the appropriate amino acids and palmitic acid. Coupling was performed according to standard TBTU/HOBt/DIEA protocols. Chlorambucil was coupled via the Lys side chain as a symmetrical anhydride using DIC preactivation. Simultaneous removal of peptide and side chain protecting groups from the resin was performed over 2 h with TFA containing 5% EDT, 5% water and 5% ethyl methyl sulfide. A 10 mg aliquot of the crude material was purified by preparative liquid chromatography, gradient 70 -> 100% B over 60 min (A=0.1% TFA/water and B=0.1% TFA/acetonitrile), flow rate 10 ml/min. After lyophilization, 30 mg of pure material was obtained (analytical HPLC gradient 70 100% B 20 min, A=0.1% TFA/water and B=0.1%

TFA/acetonitrile, flow rate 1 ml/min, detection UV 214 nm,

-166 retention time 26.5 min). The product was further characterized by MALDI mass spectrometry, M+H 1295, expected 1298.

b) Gas-filled microbubbles containing DSPS and chlorambucil-containing lipopeptide for diagnostic and therapeutic purposes ml of 1.4% propylene glycol/2.4% glycerol solution are mixed with 4.5 mg of DSPS and 0.5 mg of the product according to point a) in a vial. The resulting mixture is treated with ultrasound for 5 minutes, then heated at 80°C for 5 minutes with shaking, then cooled. The upper part of the material is flushed with perfluorobutane gas and the vial is shaken for 45 seconds in a cap mixer, then the contents are thoroughly washed with deionized water. Compound a) detectable by MALDI mass spectrometry is not present in the last washing liquid. The incorporation of the chlorambucil-containing lipopeptide into the microbubbles is confirmed by MALDI mass spectrometry: approx. 50 μΐ microbubbles are weighed into a clean vial with 100 μΐ 90% methanol, the mixture is sonicated for 30 seconds, then analyzed by MALDI mass spectrometry (ACH matrix): M+H peak 1300, expected 1294, and M+Na peak 1324, expected 1317.

c) In vitro analysis

The microbubbles are tested in vitro as described in Example 21. A gradual accumulation of microbubbles bound to cells is observed.

Example 62

Gas-filled microbubbles containing DSPS, an atenolol-containing lipopeptide, and a lipophilic captopril derivative for diagnostic and therapeutic purposes

a) Preparation of a protected atenolol derivative suitable for solid-phase coupling

i) Preparation of methyl 4-[(2,3-epoxy)propoxy]phenylacetate

-A mixture of 1674.98 g (0.030 mol) of methyl 4-hydroxyphenylacetate, 23.5 ml (0.30 mol) of epichlorohydrin and 121 μΐ (1.1 mmol) of pyridine is stirred at 85°C for 2 hours, then cooled, the excess epichlorohydrin is distilled off (rotaevapor), the residue is dissolved in ethyl acetate, washed with brine and dried over Na ₂ SO 4 . The solution is then filtered, concentrated, the residue is chromatographed on silica gel (hexane/ethyl acetate=7/3), thus 2.25 g (34%) of a colorless oil is obtained. The ¹ H (300 MHz) and ¹³ C (75 MHz) NMR spectra are consistent with the structure.

ii) Preparation of methyl 4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetate A mixture of g (9 mmol) methyl 4-[(2,3-epoxy)propoxy]phenylacetate, 23 ml (0.27 mol) isopropylamine and 1.35 ml (74.7 mmol) water was stirred at room temperature overnight, then concentrated, the oily material was dissolved in chloroform and dried over Na ₂ SO 4 . The material was filtered and concentrated to give a yellow oil in qualitative yield, which was used in the next step without further purification. The structure was confirmed by ¹ H and ¹³ C NMR analysis.

iii) 4-[2-Hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetic acid hydrochloride

563 mg (2 mmol) of methyl-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetate were dissolved in 15 ml of 6 M hydrochloric acid, heated at 100°C for 4 h, then concentrated, the residue was dissolved in water and lyophilized. The Ή and ¹³ C NMR spectra confirmed the structure, and the M+H 268 obtained by MALDI mass spectrometry corresponded to the expected value.

arc) Preparation of N-Boc-4-[2-Hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetic acid

-168 2 ml (2 mmol) of 4-[2-hydroxy-3-[(1-methylethyl)amino)propoxy]phenylacetic acid hydrochloride are dissolved in 2 ml of water and added to 0.6 g (7.2 mmol) of sodium hydrogen carbonate dissolved in 15 ml of water/dioxane=2/l. 0.48 g (2.2 mmol) of di-tert-butyl dicarbonate dissolved in 5 ml of dioxane are added. The reaction was monitored by TLC (silica gel, CHCl ₃ /MeOH/AcOH=85/10/5), di-tert-butyl dicarbonate was added in portions until the conversion was complete. The mixture was then poured into water saturated with potassium hydrogen sulfate, the organic phase was extracted with ethyl acetate, the organic phase was washed with water and brine, dried over Na ₂ SO ₄ and filtered to give 0.6 g of crude material. The product was chromatographed on silica gel (CHCl ₃ /MeOH/AcOH=85/10/5). The solution was concentrated, the residue was dissolved in glacial acetic acid and lyophilized. This gave 415 mg (56%) of a white solid. The structure was confirmed by 'H and ^l3 C NMR analysis.

b) Preparation of atenolol-functionalized lipopeptide

The above compound was prepared by manual bubbling method starting from Fmoc-protected Rink Amid BMHA resin in 0.125 mmol scale using the appropriate amino acids, palmitic acid and the compound of point a). Coupling was performed according to standard DBTU/HOBt/DIEA protocols. Simultaneous removal of peptide and side chain protecting groups from the resin was performed with TFA containing 5% EDT.

169. - - - -. ·· and contains 5% water. The crude product is precipitated from ether, purified by preparative liquid chromatography, gradient 70 -> 100% B over 60 min (A=0.1% TFA/water, B=0.1% TFA/acetonitrile), flow rate 10 ml/min. After lyophilization, 38 mg of pure material is obtained (analytical HPLC, gradient 70 -> 100% B over 20 min, A=0.01% TFA/water, B=0.1% TFA/acetonitrile, flow rate 1 ml/min, detection UV 214 nm, product retention time 25 min). The product was also confirmed by MALDI spectrometry (ACH matrix), measured M+H 1258, expected 1257.

c) Preparation of N-[(S)-3-Hexadecylthio-2-methylpropionyl]proline

188 μΐ (1.10 mmol) of DIEA were added to 170 mg (0.500 mmol) of 1-iodohexadecane, 120 mg (0.550 mmol) of captopril and 165 μΐ (1.10 mmol) of DBU dissolved in 5 ml of tetrahydrofuran. The resulting mixture was heated at 70°C for 2 hours and then concentrated. The residue was poured into water saturated with potassium hydrogen sulfate and the organic material was extracted with chloroform. The organic phase was then washed with water and dried over MgSO ₄ . The resulting product was chromatographed on silica gel (CHCl ₃ /MeOH/AcOH=85/10/5) and lyophilized to give 105 mg (48%) of a white solid. The structure was confirmed by *H (500 MHz) and ¹³ C (125 MHz) NMR spectroscopy and MALDI mass spectrometry was also performed in MH negative mode at m/z 440, consistent with the expected value.

d) Gas-filled microbubbles containing DSPS, an atenolol-containing lipopeptide and a lipophilic captopril derivative for diagnostic and therapeutic purposes ml of 1.4% propylene glycol/2.4% glycerol solution are mixed with 4.5 mg of DSPS and 0.5 mg of the products according to points b) and c) in a vial, the resulting mixture is treated with ultrasound for 5 minutes, then heated at 80°C for 5 minutes with shaking, then cooled. The upper part of the material

-170 perfluorobutane gas and shake the vial for 45 seconds in a cap mixer, then wash the contents thoroughly with deionized water. According to MALDI mass spectrometry, no compounds b) or c) can be detected in the last washing liquid. The incorporation of compounds b) and c) into the microbubbles is confirmed by MALDI mass spectrometry: approx. 50 μΐ microbubbles are weighed into a clean vial with 100 μΐ 90% methanol, the mixture is treated with ultrasound for 30 seconds, then analyzed by MALDI mass spectrometry (ACH matrix): the obtained M+H peaks correspond to compounds b) and c).

e) In vitro analysis

The microbubbles are tested in vitro as described in Example 21. A gradual accumulation of microbubbles bound to cells is observed.

Example 63

Gas-filled microbubbles containing DSPS and an atenolol cholesterol derivative for diagnostic and therapeutic purposes

a) Preparation of methyl 4-[(2,3-epoxy)propoxy]phenylacetate

A mixture of 4.98 g (0.030 mol) of methyl 4-hydroxyphenylacetate, 23.5 ml (0.30 mol) of epichlorohydrin and 121 μΐ (1.1 mmol) of pyridine was stirred at 85°C for 2 h, then cooled, the excess epichlorohydrin was distilled off (rotaevapor), the residue was dissolved in ethyl acetate, washed with brine and dried over Na ₂ SO ₄ . The solution was then filtered, concentrated, and the dark residue was chromatographed on silica gel (hexane/ethyl acetate=7/3) to give 2.25 g (34%) of a colorless oil. The 'H (300 MHz) and ¹³ C (75 MHz) NMR spectra were consistent with the structure.

b) Preparation of methyl 4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy)phenylacetate

- 171 A mixture of 2 g (9 mmol) of methyl 4-[(2,3-epoxy)propoxy]phenylacetate, 23 ml (0.27 mol) of isopropylamine and 1.35 ml (74.7 mmol) of water was stirred at room temperature overnight, then concentrated, the oily material was dissolved in chloroform and dried over Na ₂ SO 4 . The material was filtered and concentrated to give a yellow oil in qualitative yield, which was used in the next step without further purification. The structure was confirmed by ^] Η and ¹³ C NMR analysis.

c) 4-[2-Hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetic acid hydrochloride

563 mg (2 mmol) of methyl-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetate were dissolved in 15 ml of 6 M hydrochloric acid, heated at 100°C for 4 hours, then concentrated, the residue was dissolved in water and lyophilized. The 'H and ¹³ C NMR spectra confirmed the structure, the M+H 268 obtained by MALDI mass spectrometry corresponded to the expected value.

d) Preparation of N-Boc-4-[2-Hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetic acid 1 ml (2 mmol) of 4-[2-hydroxy-3-[(1-methylethyl)amino)propoxy]phenylacetic acid hydrochloride is dissolved in 2 ml of water and added to 0.6 g (7.2 mmol) of sodium hydrogen carbonate dissolved in 15 ml of water/dioxane=2/l. 0.48 g (2.2 mmol) of di-tert-butyl dicarbonate dissolved in 5 ml of dioxane is added. The course of the reaction is monitored by TLC (silica gel, CHCl ₃ /MeOH/AcOH=85/10/5), the di-tert-butyl dicarbonate portions are added until the conversion is complete. The mixture was then poured into water saturated with potassium hydrogen sulfate, the organic phase was extracted with ethyl acetate, the organic phase was washed with water and brine, dried over Na ₂ SÜ4 and filtered to give 0.6 g of crude material. The product was chromatographed on silica gel (CHCl ₃ /MeOH/AcOH=85/10/5). The solution was concentrated, the

-172 The residue was dissolved in glacial acetic acid and lyophilized. This gave 415 mg (56%) of a white solid. The structure was determined by the ’H and 13

This was confirmed by C NMR analysis.

e) Preparation of cholesterol-N-Boc-P-alaninate

510 μΐ DIC was mixed with 1.25 g (6.60 mmol) of Bοο-β-Ala-OH in 15 ml of dichloromethane under an inert atmosphere. The mixture was then shaken for 30 min and transferred to a flask containing 1.16 g (3 mmol) of cholesterol and 367 mg (3 mmol) of DMAP dissolved in 15 ml of dichloromethane. The mixture was stirred for 2 h and then poured into an aqueous solution of potassium hydrogen sulfate. The phases were separated, the aqueous phase was extracted with chloroform, the organic phases were combined, washed with aqueous potassium hydrogen sulfate, then with water and dried over MgSO ₄ . After filtration and concentration, the crude product obtained was chromatographed on silica gel (chloroform/methanol=99/l) to give 1.63 g (97%) of a white solid. The structure was confirmed by NMR (500 MHz) spectroscopy.

f) Preparation of cholesteryl-3-alaninate hydrochloride

279 mg (0.500 mmol) of the compound from part a) was dissolved in 5 ml of 1 M hydrochloric acid/1,4-dioxane and stirred at room temperature for 4 h. The mixture was then concentrated to give cholesteryl-β-alaninate hydrochloride in quantitative yield. The structure was confirmed by Ή NMR (500 MHz) and MALDI mass spectrometry: M+Na peak 482, expected 481.

g) Cholesteryl-N-Boc-4-[2-hydroxy-3-[(l-

Preparation of 100 mg (0.15 mmol) of N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetyl-β-alaninate and 74 mg (0.15 mmol) of cholesteryl-β-alaninate hydrochloride were dissolved in 5 ml of DMF and 26 ml (0.15 mmol) of DIEA was added, followed by 23 mg (0.15 mmol) of

-173HOBt and 29 mg (0.15 mmol) of water-soluble carbodiimide (WSC). The mixture was then stirred at room temperature overnight and then poured into 25 ml of water containing 2.5 g of sodium carbonate and 4 g of sodium chloride. The precipitate formed was extracted with chloroform, the organic phase was washed with water and dried over MgSO _4. It was then filtered, concentrated, and the resulting 132 g of crude material was purified by column chromatography (silica gel, chloroform/methanol/acetic acid=95/4/l). The fractions were combined, concentrated, dissolved in glacial acetic acid, and then lyophilized. Thus, 83 mg (69%) of a yellowish-white solid was obtained. The structure was confirmed by 'H NMR analysis.

h) Cholesteryl-N-4-[2-hydroxy-3-[(l-

Preparation of N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetyl-β-alaninate trifluoroacetate mg (0.05 mmol) was dissolved in 4 ml of anhydrous dichloromethane, 2 ml of trifluoroacetic acid was added and the mixture was stirred for 2 hours and then concentrated. The product was lyophilized from acetonitrile/water to give a whitish-yellow solid in quantitative yield. The product was analyzed by MALDI mass spectrometry: M+H 708, as expected.

i) Gas-filled microbubbles containing DSPS, an atenolol-containing lipopeptide and a lipophilic captopril derivative for diagnostic and therapeutic purposes ml of a 1.4% propylene glycol/2.4% glycerol solution are mixed in a vial with 4.5 mg of DSPS and 0.5 mg of the product according to point h). The mixture is sonicated for 5 minutes, then heated at 80°C for 5 minutes with shaking, then cooled. The supernatant is flushed with perfluorobutane gas, the vial is shaken in a cap shaker for 45 seconds, and the contents are thoroughly washed with deionized water. MALDI

-174 -

There is no detectable amount of product b) in the final wash liquid by mass spectrometry. The incorporation of compound h) into the microbubbles was confirmed by MALDi mass spectrometry.

j) In vitro analysis

The microbubbles are tested in vitro as described in Example 21. A gradual accumulation of microbubbles bound to cells is observed.

Example 64

Multispecific transferrin/avidin-coated gas-filled microbubbles for targeted ultrasound imaging

In this example, we demonstrate the production of vector-containing microbubbles for use in targeted ultrasound/therapeutic procedures.

a) Thiol functionalized lipid molecule: dipalmitoyl-Lys-Lys-

-Lys-Aca-Cys.OH production

The above lipid compound was prepared on an ABI433A automated peptide synthesizer starting from Fmoc-Cys(Trt)-Wang resin using 1 mmol amino acid loadings in a 0.25 mmol scale. The amino acids and palmitic acid were preactivated by HBTU coupling. Simultaneous removal of peptide and side chain protecting groups from the resin was carried out with TFA containing 5% EDT and 5% H ₂ O for 2 h to yield 250 mg of crude product. A 40 mg aliquot of crude product was purified by preparative HPLC, gradient 70 —» 100% B 50

-175 min (A—0.1% TFA/water, B—MeOH), flow rate 9 ml/min. After lyophilization, 24 mg of crude product was obtained (analytical HPLC, gradient 70 -> 100% B, B=0.1% TFA/acetonitrile, A=0.01% TFA/water, detection UV 214 nm, product retention time 23 min). Further product characterization was performed by MALDI mass spectrometry: expected M+H 1096, measured 1099.

b) Production of a gas-filled microbubble doped with a thiol-containing lipid molecule

4.5 mg of DSPS and 0.5 mg of lipid molecule a) as described above are weighed into a clean vial and 0.8 ml of a 1.4% propylene glycol/2.4% glycerol solution is added. The resulting mixture is heated for 80 minutes with shaking for 5 minutes and then filtered while still hot through a 40 pm filter. The sample is then cooled to room temperature, the upper part is flushed with perfluorobutane gas and the vials are shaken with a cap stirrer for 45 seconds and then treated on a roller table overnight. The resulting microbubbles are washed several times with deionized water and analyzed for thiol group incorporation with Ellman's reagent.

c) Transferrin modification using fluorescein-NHS and Sulpho-SMPB A mixture of 1 mg transferrin (Holo, human) and 2 mg avidin in 1 ml PBS was added to 0.5 ml DMSO solution containing 1 mg Sulpho-SMPB and 0.5 mg fluorescein-NHS. The resulting mixture was stirred for 45 min at room temperature and then passed through a Sephadex 200 column, eluting with PBS. The protein fractions were collected and stored at 4°C until use.

d) Conjugation of microbubbles with modified transferrin/avidin

To the thiol-containing microbubbles according to point b) 1 ml of the modified transferrin/avidin protein solution according to point c) is added. The pH

-176 is adjusted to pH=9 and the conjugation reaction is carried out for 2 hours at room temperature. The microbubbles are then washed thoroughly with deionized water and examined by Coulter Counter analysis (81% between 1 and 7 gm) and fluorescence microscopy (highly fluorescent microbubbles can be observed).

Example 65

Gene transfer with gas-filled microbubbles

In this example, we demonstrate the production of targeted microbubbles for gene transfer purposes.

a) Preparation of gas-filled microbubbles containing DSPS and poly-L-lysine-coated lipopeptide

4.5 mg of DSPS and 4.5 mg of the lipopeptide of Example 41 were weighed into two 2 ml vials and 0.8 ml of a 4% aqueous solution of propylene glycol/glycerol was added to each vial. The solutions were heated at 80°C for 4 minutes with shaking, then cooled to room temperature and the supernatant was flushed with perfluorobutane gas. The vials were shaken in a cap shaker at 4450 oscillations/min for 45 seconds and then placed on a roller table for 5 minutes. The contents of the vials were then mixed and the resulting material was centrifuged at 2000 rpm for 5 minutes. The supernatant was removed and replaced with an equal volume of distilled water. The washing procedure was repeated once more. 20.6 mg of poly-L-lysine HBr is dissolved in 2 ml of water and a 0.4 ml aliquot is made up to 2 ml with water. 0.12 ml of DSPS-lipopeptide microbubble suspension is then added to 1.2 ml of the thus diluted poly-L-lysine solution. After incubation, the excess polylysine is removed by vigorous water washing.

b) Transfection of cells

Endothelial cells (ECV 304) were cultured in 6-well plates to homogenize confluent layers. The transfection mixture was prepared

-\ΊΊ prepared, which contains 5 pg DNA (Enhanced Green Fluorescent Protein vector, CLONTECH) and 50 μΐ microbubble suspension according to point a) in RPMI medium, the final volume is 250 μΐ. The mixture is left to stand for 15 minutes at room temperature, then 1 ml complete RPMI medium is added. The medium is removed from the cell culture plate and the DNA-microbubble mixture is added to the cells. The cells are incubated in a cell culture incubator at 30°C.

c) Ultrasonic treatment After a minute of incubation, the selected wells are exposed to continuous ultrasound waves, 1 MHz 0.5 W/cm ² , 30 seconds.

d) Incubation and testing

The cells are incubated in a cell culture incubator at 37°C for approximately 4.5 hours. The medium containing the DNA microbubbles is then removed by aspiration and 2 ml of complete RPMI medium is added. The cells are incubated for 40-70 hours before testing. The majority of the medium is then removed and the cells are examined by fluorescence microscopy. The results obtained are compared to the control in which DNA or DNA-polylysine was added to the cells.

Example 66

Flotation of endothelial cells with microbubbles containing vectors that specifically bind to endothelial cells

The experiment was conducted to demonstrate that the invention can be used to separate cells to which microbubbles were directed. ECV 304 human endothelial cell line, derived from normal umbilical cord (ATCC CRL-1998), was cultured in Nunc culture flasks (chutney 153732) in RPMI 1640 medium,

-178 then 200 mM L-glutamine 10,000 U/ml and 10,000 ug/ml penicillin/streptomycin and 10% fetal calf serum were added. The cells were subcultured after trypsinization, with a split ratio of 1:5 to 1:7 when confluent was reached. 2 million cells from the trypsinized confluent cultures were added to a five-tube centrifuge set. Then control microbubbles or endothelial cell-bound microbubbles (as per Examples 21 and 38) were added at 2, 4, 6, 8 or 10 million bubbles per tube. The cells collected at the bottom of the tubes were counted with a Coulter Counter after centrifugation at 400 g for 5 minutes. It was found that 4 or more microbubbles that bound to the cells brought the cells to the surface of the liquid in the centrifuge tube. All of the microbubbles from Example 38 floated the cells, while only 50% of the microbubbles from Example 21 did.

Example 67

Gas-filled distearoylphosphatidylserine microbubbles containing a vector-containing lipopeptide with affinity for endothelial receptors for targeted ultrasound imaging ^a ) Preparation of 4'-[(3,4-Dimethyl-5-isoxazolyl)-sulfamoyl]succinanilinic acid

267 mg (1 mmol) of sulfisoxazole were dissolved in 10 ml of DMF and added to a mixture containing 1 g (10 mmol) of succinic anhydride and 122 mg (1 mmol) of 4-dimethylaminopyridine. The resulting mixture was stirred at 80°C for 2 hours and then concentrated. The residue was dissolved in 5% aqueous sodium bicarbonate solution, extracted with ethyl acetate, the aqueous solution was acidified with dilute hydrochloric acid, the organic phase was extracted with ethyl acetate, the organic phase was washed with dilute hydrochloric acid, water, then brine, and dried over activated carbon.

-179 and dried over MgSO ₄ . The solution was then filtered and concentrated to give 280 mg (76%) of a white solid. The structure was confirmed by 'H (300 MHz) and ¹³ C (75 MHz) NMR spectroscopy. MALDI mass spectroscopy (ACH matrix) showed the M+Na peak at m/z 390 and the M+K peak at m/z 406, as expected.

b) Preparation of sulfisoxazole-functionalized lipopeptide

The above compound was prepared using a manual nitrogen sparge apparatus starting from Fmoc-protected Rink Amide BMHA resin in a 0.125 mmol scale. Using the appropriate amino acids, palmitic acid and the compound from point a). The coupling was carried out according to standard TBTU/HOBt/DIEA protocols. Simultaneous removal of peptide and side chain protecting groups from the resin was carried out with TFA containing 5% EDT and 5% water. The crude material was precipitated from ether. The product was analyzed by analytical HPLC, gradient 70 -> 100% B, 20 min (A=0.1% TFA/water and B=0.1% TFA/acetonitrile), flow rate 1 ml/min, detection UV 240 nm, retention time 27 min. Further analysis is also performed using MALDI mass spectrometry: M+H at m/z 1359, expected value 1356.

c) production of gas-filled microbubbles containing the product according to point b)

-180 1 ml of 1.4% propylene glycol/2.4% glycerol solution is mixed with 4.5 mg DSPS and 0.5 mg of the product from point b) in a vial, then sonicated for 5 minutes, then heated at 80°C for 5 minutes with shaking, then cooled. The supernatant is flushed with perfluorobutane gas, the vial is shaken in a cap mixer for 45 seconds, then washed thoroughly with deionized water. No detectable compound from point b) is present in the final wash liquid by MALDI mass spectrometry. The incorporation of the isoxazole-containing lipopeptide into the microbubbles is confirmed by MALDI MS mass spectrometry: approx. 50 μΐ microbubbles are weighed into a clean vial containing 100 μΐ 90% methanol, the mixture is sonicated for 30 seconds and then analyzed by MALDI MS spectrometry (ACH matrix), the resulting M+H peak at m/z 1359 corresponds to lipopeptide b).

Claims (37)

-181 Patent claims 1. A targetable diagnostic and/or therapeutic agent comprising a signal carrier suspended in an aqueous carrier liquid, comprising gas-filled microbubbles stabilized by a monolayer of a film-forming surfactant, and further comprising at least one vector. 2. The agent of claim 1, wherein the gas is air, nitrogen, oxygen, carbon dioxide, hydrogen, an inert gas, sulfur fluoride, selenium hexafluoride, a low molecular weight hydrocarbon, ketone, ester, halogenated low molecular weight hydrocarbon, or any mixture thereof. 3. The agent of claim 2, wherein the gas is a perfluorinated ketone, a perfluorinated ether or a perfluorocarbon. 4. The agent of claim 2, wherein the gas is sulfur hexafluoride or perfluoropropane, perfluorobutane or perfluoropentane. 5. An agent according to any one of claims 1-4, wherein the film-forming surfactant is a non-polymeric or non-polymerizable wall-forming surfactant, a polymeric surfactant or a phospholipid. 6. The agent of claim 5, wherein at least 75% of the film-forming surfactant is phospholipid molecules, each of which has an individual average charge. 7. The agent of claim 6, wherein at least 75% of the film-forming surfactant is one or more phospholipids selected from the group consisting of phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, or cardiolipin. 8. The agent of claim 7, wherein at least 80% of the phospholipids are phosphatidylserine. -182 - 9. The agent according to any one of claims 1-8, wherein the film-forming surfactant comprises a lipopeptide. 10. The agent of any one of claims 1-9, wherein the vector is any of the following: a cell adhesion molecule; a cell adhesion molecule receptor; a cytokine; a growth factor; a peptide hormone and fragments thereof; non-peptide agonists/antagonists and non-bioactive binding agents for cell adhesion molecules, cytokines, growth factors and peptide hormone receptors; oligonucleotides and modified oligonucleotides; DNA binding agents, protease substrates/inhibitors; molecules from combinatorial libraries; and small bioactive molecules. 11. An agent according to any one of claims 1-11, wherein the affinity of the vector or vectors for the target organ is such that the agent interacts with it but is not fixedly bound thereto. 12. The agent of claim 11, wherein the vector or vectors are ligands of cell adhesion proteins or cell adhesion proteins for which corresponding ligands are present on endothelial cell surfaces. 13. The agent of any one of claims 1-12, wherein the vector or vectors are positioned such that they are not readily directed to the target. 14. The agent of any one of claims 1-13, wherein the vector is covalently linked or bound to the signal carrier. 15. The agent of any one of claims 1-13, wherein the vector is associated or bound to the signal carrier by electrostatic charge interaction. 16. The agent of any one of claims 1-13, wherein the vector is bound or linked to the signal carrier via an avidin-biotin and/or streptavidin-biotin interaction. - 183 - 17. The agent of any one of claims 1-6, further comprising a moiety that is effective as a radioactive or X-ray contrast agent, a light imaging pattern, or a spin label. 18. The agent according to any one of claims 1-17, further comprising a therapeutically active compound. 19. The agent of claim 18, wherein the therapeutic compound is any of the following: an antitumor agent, a blood product, a biological response modifier, an antifungal agent, a hormone or hormone analog, a vitamin, an enzyme, an antiallergic agent, a tissue factor inhibitor, a platelet inhibitor, a coagulation protein inhibitor, a fibrin formation inhibitor, a fibrinolysis promoter, an antiangiogenic agent, a circulatory agent, a metabolic potentiator, an antituberculosis agent, an antiviral agent, a vasodilator, an antibiotic, an anti-inflammatory agent, an antiprotozoan, an antirheumatic agent, a narcotic, an opiate, a cardiac glycoside, a neuromuscular blocking agent, a sedative, a local anesthetic, a general anesthetic, or a genetic agent. 20. The agent of claim 18 or 19, wherein the therapeutic compound is covalently linked or bound to the signal carrier via disulfide groups. 21. The agent of claim 18 or 19, wherein the lipophilic or lipophilic derivatized therapeutic compound binds to the surfactant monolayer, stabilizing the gas-filled microbubbles of the signal carrier through hydrophobic interactions. 22. Combined preparation containing: i) a first-administerable composition comprising a pre-targeting vector having affinity for the selected target; and -184- ii) a secondly administrable composition which is an agent according to any one of the preceding claims and which comprises a vector which has affinity for said pre-targeting vector. 23. The combination composition of claim 22, wherein the pretargeting vector is a monoclonal antibody. 24. Combined preparation containing: i) a firstly administrable composition comprising an agent according to any one of claims 1 to 21; and ii) a secondly administrable composition comprising a substance capable of displacing or releasing said agent from the target. 25. Combined preparation containing: i) a first administrable composition comprising an agent according to claim 20; and ii) a second administrable composition comprising a reducing agent capable of reductively cleaving disulfide groups linking or linking the therapeutic compound and the signal carrier in said first administrable composition. 26. A method for producing targetable diagnostic and/or therapeutic agents according to claim 1, characterized in that at least one vector is bound or linked to a signal carrier comprising gas-filled microbubbles stabilized by a monolayer of a film-forming surfactant or gas-filled signal carrier microbubbles are produced using a film-forming surfactant to which at least one vector is attached. 27. The method of claim 26, wherein the therapeutic compound is also combined with the signal carrier. 28. The method according to claim 27, characterized in that the coupling of the active ingredient containing a thiol group to the signal carrier, gas-filled -185 The attachment of thiol-containing surfactants to monolayers of microbubbles is carried out under oxidative conditions by forming disulfide bonds. 29. Use of the agent according to any one of claims 1-21 as a targetable ultrasound contrast agent. 30. A method for creating enhanced images in a human or non-human organism, characterized in that the agent according to any one of claims 1-21 is administered to the organism and an ultrasound, magnetic resonance, X-ray, radiographic or light image is created in at least a part of the body. 31. The method according to claim 30, characterized in that i) administering to the body a pre-targeting vector having affinity for the selected target organ, and then ii) administering an agent according to any one of claims 1-21 comprising a vector having affinity for said pre-targeting vector. 32. The method of claim 31, wherein the pretargeting vector is a monoclonal antibody. 33. The method of claim 30, characterized in that i) administering to the body an agent according to any one of claims 1-21, and then ii) administering a substance capable of displacing or releasing said cells from the target organ. 34. The method according to any one of claims 30-33, characterized in that the agent further comprises a compound with a therapeutic effect. 35. The method of claim 34, wherein the therapeutic compound is covalently linked or bound to the signal carrier via disulfide groups and its administration is - 186 is then administered a composition comprising a reducing agent capable of reductively cleaving said disulfide bond. 36. A method for testing in vitro the targeting of an agent according to any one of claims 1-21, characterized in that cells expressing the target are fixed in a flow chamber, the agent suspended in a carrier is passed through the chamber and the binding of said agent to said cells is tested. 37. The method of claim 36, wherein the flow of the carrier fluid in the flow chamber is controlled to simulate shear conditions occurring in vivo. The authorized person: DANUBE Patent and Trademark Office Ltd. Mrs. OlchváryGézáné^) patent attorney