US 20040141922A1 (19) United States (2) Patent Application Publication (10) Pub. No.: US 2004/0141922 A1 Klaveness et al. (43) Pub. Date: Jul. 22, 2004

(54) DIAGNOSTIC/THERAPEUTICAGENTS tion-in-part of application No. 08/958,993, filed on Oct. 28, 1997, now Pat. No. 6,264,917. (75) Inventors: Jo Klaveness, Oslo (NO); Pal Rongved, Oslo (NO); Anders Hogset, (60) Provisional application No. 60/049,264, filed on Jun. Oslo (NO); Helge Tolleshaug, Oslo 6, 1997. Provisional application No. 60/049,265, filed (NO); Anne Naevestad, Oslo (NO); on Jun. 6, 1997. Provisional application No. 60/049, Halldis Hellebust, Oslo (NO); Lars 268, filed on Jun. 7, 1997. Hoff, Oslo (NO); Alan Cuthbertson, Oslo (NO); Dagfinn Lovhaug, Oslo (30) Foreign Application Priority Data (NO); Magne Solbakken, Oslo (NO) Oct. 28, 1996 (GB)...... 962.2366.4 Correspondence Address: Oct. 28, 1996 (GB)...... 9622.367.2 Li CAI Oct. 28, 1996 (GB)...... 962.2368.0 Amersham Health, Inc. Jan. 15, 1997 (GB)...... 9700699.3 101 Carnegie Center Apr. 24, 1997 (GB)...... 9708265.5 Princeton, NJ 08540-6231 (US) Jun. 6, 1997 (GB)...... 97.11842.6 Jun. 6, 1997 (GB)...... 97.11846.7 (73) Assignee: NYCOMED IMAGING AS Publication Classification (21) Appl. No.: 10722,075 (51) Int. Cl." ...... A61K 49/00; A61K 38/00 (22) Filed: Nov. 26, 2003 (52) U.S. Cl. …...... …... 424/9.52; 514/2 Related U.S. Application Data (57) ABSTRACT Targetable diagnostic and/or therapeutically active agents, (63) Continuation of application No. 09/765,614, filed on e.g. ultrasound contrast agents, having reporters comprising Jan. 22, 2001, now abandoned, which is a continua gas-filled microbubbles stabilised by monolayers of film tion of application No. 08/960,054, filed on Oct. 29, forming surfactants, the reporter being coupled or linked to 1997, now Pat. No. 6,261,537, which is a continua at least one vector. Patent Application Publication Jul. 22, 2004 Sheet 1 of 2 US 2004/0141922 A1

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i Patent Application Publication Jul. 22, 2004 Sheet 2 of 2 US 2004/0141922 A1

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DIAGNOSTIC/THERAPEUTIC AGENTS [0009] The vector is attached or linked to the reporter moiety in order to bind these moieties to the region/structure [0001] This application claims benefit under 35 U.S.C. to be imaged and/or treated. The vector may bind specifi 119(e) of provisional applications serial Nos. 60/049,264 cally to a chosen target, or it may bind only selectively, and 60/049,265 both filed 6 Jun. 1997; and No. 60/049,268 having affinty also for a limited number of other molecules/ filed 7 Jun. 1997. structures, again creating possible background problems. [0002] This invention relates to diagnostic and/or thera peutically active agents, more particularly to diagnostic [0010] There is a limited body of prior art relating to and/or therapeutically active agents incorporating moieties targeted ultrasound contrast agents. Thus, for example, U.S. which interact with or have affinity for sites and/or structures Pat. No. 5,531,980 is directed to systems in which the within the body so that diagnostic imaging and/or therapy of reporter comprises an aqueous suspension of air or gas particular locations within the body may be enhanced. Of microbubbles stabilised by one or more film-forming sur particular interest are diagnostic agents for use in ultrasound factants present at least partially in lamellar or laminar form, imaging, which are hereinafter referred to as targeted ultra said surfactant(s) being bound to one or more vectors sound contrast agents. comprising “bioactive species designed for specific target ing purposes”. It is stated that the microbubbles are not [0003] It is well known that ultrasound imaging comprises directly encapsulated by surfactant material but rather that a potentially valuable diagnostic tool, for example in studies this is incorporated in liquid-filled liposomes which stabilise of the vascular system, particularly in cardiography, and of the microbubbles. It will be appreciated that lamellar or tissue microvasculature. A variety of contrast agents has laminar surfactant material such as phospholipids present in been proposed to enhance the acoustic images so obtained, such liposomes will inevitably be present in the form of one including suspensions of solid particles, emulsified liquid or more lipid bilayers with the lipophilic tails “back-to droplets, gas bubbles and encapsulated gases or liquids. It is back” and the hydrophilic heads both inside and outside (see generally accepted that low density contrast agents which e.g. Schneider, M. on “Liposomes as drug carriers: 10 years are easily compressible are particularly efficient in terms of of research” in Drug targeting, Nyon, Switzerland, 3-5 Oct. the acoustic backscatter they generate, and considerable interest has therefore been shown in the preparation of 1984, Buri, P and Gumma, A. (Ed), Elsevier, Amsterdam gas-containing and gas-generating systems. 1984). [0011] EP-A-0727225 describes targeted ultrasound con [0004] Gas-containing contrast media are also known to trast agents in which the reporter comprises a chemical be effective in magnetic resonance (MR) imaging, e.g. as having a sufficient vapour pressure such that a proportion of susceptibility contrast agents which will act to reduce MR it is a gas at the body temperature of the subject. This signal intensity. Oxygen-containing contrast media also rep chemical is associated with a surfactant or albumin carrier resent potentially useful paramagnetic MR contrast agents. which includes a protein-, peptide- or carbohydrate-based [0005] Furthermore, in the field of X-ray imaging it has cell adhesion molecule ligand as vector. The reporter moi been observed that gases such as carbon dioxide may be eties in such contrast agents correspond to the phase shift used as negative oral contrast agents or intravascular con colloid systems described in WO-A-9416739; it is now trast agents. recognised that administration of such phase-shift colloids may lead to generation of microbubbles which grow uncon [0006] The use of radioactive gases, e.g. radioactive iso trollably, possibly to the extent where they cause potentially topes of inert gases such as xenon, has also been proposed dangerous embolisation of, for example, the myocardial in scintigraphy, for example for blood pool imaging. vasculature and brain (see e.g. Schwarz, Advances in Echo [0007] Targeted ultrasound contrast agents may be Contrast [1994(3)], pp. 48-49). regarded as comprising (i) a reporter moiety capable of [0012] WO-A-9320802 proposes that tissue-specific ultra interacting with ultrasound irradiation to generate a detect sonic image enhancement may be achieved using acousti able signal; (ii) one or more vectors having affinity for cally reflective oligolamellar liposomes conjugated to tis particular target sites and/or structures within the body, e.g. sue-specific ligands such as antibodies, peptides, lectins etc. for specific cells or areas of pathology; and (iii) one or more The liposomes are deliberately chosen to be devoid of gas linkers connecting said reporter and vector(s), in the event and so will not have the advantageous echogenic properties that these are not directly joined. of gas-based ultrasound contrast agents. Further references to this technology, e.g. in targeting to fibrin, thrombi and [0008] The molecules and/or structure to which the agent atherosclerotic areas are found in publications by is intended to bind will hereinafter be referred to as the Alkanonyuksel, H. et al. in J. Pharm. Sci. (1996) 85(5), target. In order to obtain specific imaging of or a therapeutic 486-490; J. Am. Coll. Cardiol. (1996) 27(2) Suppl A, 298A; effect at a selected region/structure in the body the target and Circulation, 68 Sci. Sessions, Anaheim 13-16 November must be present and available in this region/structure. Ide 1995. ally it will be expressed only in the region of interest, but usually will also be present at other locations in the body, [0013] There is also a number of publications concerning creating possible background problems. The target may ultrasound contrast agents which refer in passing to possible either be a defined molecular species (i.e. a target molecule) use of monoclonal antibodies as vectors without giving or an unknown molecule or more complex structure (i.e. a significant practical detail and/or to reporters comprising target structure) which is present in the area to be imaged materials which may be taken up by the reticuloendothelial and/or treated, and is able to bind specifically or selectively system and thereby permit image enhancement of organs to a given vector molecule. such as the liver—see, for example WO-A-9300933, WO

US 2004/0141922 A1 Jul. 22, 2004 tanes, perfluorocyclohexane, perfluoromethylcyclohexane number of components administered to the body of a subject and perfluorocycloheptane. Other halogenated gases include upon injection of the contrast agents is kept to a minimum. methyl chloride, fluorinated (e.g. perfluorinated) ketones Thus, for example, the charged surfaces of the microbubbles such as perfluoroacetone and fluorinated (e.g. perfluori may minimise or prevent their aggregation as a result of nated) ethers such as perfluorodiethyl ether. The use of electrostatic repulsion. perfluorinated gases, for example sulphur hexafluoride and [0028] Desirably at least 75%, preferably substantially all perfluorocarbons such as perfluoropropane, perfluorobu of phospholipid material used in reporters in agents of the tanes and perfluoropentanes, may be particularly advanta invention consists of molecules bearing a net overall charge geous in view of the recognised high stability in the blood under conditions of preparation and/or use, which charge stream of microbubbles containing such gases. may be positive or, more preferably, negative. Representa [0023] The gas may comprise a substance such as butane, tive positively charged phospholipids include esters of phos cyclobutane, n-pentane, isopentane, neopentane, cyclopen phatidic acids such as dipalmitoylphosphatidic acid or dis tane, perfluoropentane, perfluorocyclopentane, perfluoro tearoylphosphatidic acid with aminoalcohols such as hexane or a mixture containing one or more such gases hydroxyethylethylenediamine. Examples of negatively which is liquid at handling or processing temperatures but charged phospholipids include naturally occurring (e.g. soya gaseous at body temperature, e.g. as described in the afore bean or egg yolk derived), semisynthetic (e.g. partially or mentioned WO-A-9416739, since the film-forming surfac fully hydrogenated) and synthetic phosphatidylserines, tant monolayers in reporter units according to the invention phosphatidylglycerols, phosphatidylinositols, phosphatidic may stabilise the resulting microbubbles against uncontrol acids and cardiolipins. The fatty acyl groups of such phos lable growth. pholipids will typically each contain about 14-22 carbon atoms, for example as in palmitoyl and stearoyl groups. Lyso [0024] In principle, any appropriate film-forming surfac forms of such charged phospholipids are also useful in tant may be employed to form the gas-encapsulating mono accordance with the invention, the term “lyso’ denoting layers, including non-polymeric and non-polymerisable phospholipids containing only one fatty acyl-group, this wall-forming surfactant materials, e.g. as described in WO preferably being ester-linked to the 1-position carbon atom A-9521631; polymer surfactant material, e.g. as described in of the glyceryl moiety. Such lyso forms of charged phos WO-A-9506518; and phospholipids, e.g. as described in pholipids may advantageously be used in admixture with WO-A-921.1873, WO-A-921.7212, WO-A-9222247, WO-A charged phospholipids containing two fatty acyl groups. 9428780, WO-A-9503835 or WO-A-9729783. Advanta geously 75%, preferably substantially all, of the film-form [0029] Phosphatidylserines represent particularly pre ing surfactant present in agents according to the invention is ferred phospholipids of use in agents according to the incorporated into monolayers at the gas-liquid interfaces. invention and preferably constitute a substantial part, e.g. at [0025] Representative examples of useful phospholipids least 80% of the phospholipid content thereof, for example include lecithins (i.e. phosphatidylcholines), for example 85-92%. While we do not wish to be bound by theoretical natural lecithins such as egg yolk lecithin or soya bean considerations, it may be that ionic bridging between the lecithin and synthetic or semisynthetic lecithins such as carboxyl and amino groups of adjacent serine moieties dimyristoylphosphatidylcholine, dipalmitoylphosphatidyl contributes to the stability of such reporter systems. Pre choline or distearoylphosphatidylcholine; phosphatidic ferred phosphatidylserines include saturated (e.g. hydroge acids; phosphatidylethanolamines; phosphatidylserines; nated or synthetic) natural phosphatidylserine and synthetic phosphatidylglycerols; phosphatidylinositols; cardiolipins; distearoylphosphatidylserine, dipalmitoylphosphati sphingomyelins; fluorinated analogues of any of the fore dylserine and diarachidoylphosphatidylserine. going; mixtures of any of the foregoing and mixtures with [0030] Other potentially useful lipids include phosphati other lipids such as cholesterol. dylethanolamines optionally admixed with one or more [0026] It has been found that the use of phospholipids lipids such as stearic acid, palmitic acid, stearylamine, predominantly (e.g. at least 75%) comprising molecules palmitylamine, cholesterol, bisalkyl glycerols, sphingogly individually bearing net overall charge may be particularly colipids, synthetic lipids such as N,N-dimethyl-N-octade advantageous, especially when used as essentially the sole cyl-1-octadecanammonium chloride or bromide (DODAC, amphiphilic component of the reporter, and may convey DODAB), and/or maleic acid bisalkylesters. valuable benefits in terms of parameters such as product [0031] Additional exemplary lipids which may be used to stability and acoustic properties. Without wishing to be prepare gas-containing contrast agents include fatty acids, bound by theoretical considerations, it is believed that stearic acid, palmitic acid, 2-n-hexadecylstearic acid, oleic electrostatic repulsion between charged phospholipid mem acid and other acid-containing lipid structures. Such lipid branes may encourage the formation of stable monolayers at structures may be coupled by amide bond formation to the gas-liquid interfaces; as noted above, the flexibility and amino acids containing one or more amino groups; the deformability of such thin membranes will enhance the resulting lipid-modified amino acids (e.g. dipalmitoyllysine echogenicity of reporters used in accordance with the inven or distearoyl-2,3-diaminopropionic acid) may be useful pre tion relative to gas-filled liposomes comprising one or more cursors for the attachment of functionalised spacer elements lipid bilayers. having coupling sites for conjugation of one or more vector [0027] The use of charged phospholipids may also provide molecules. reporters with advantageous properties regarding, for [0032] Further useful stabilisers include lipopeptides com example, stability, dispersibility and resistance to coales prising a lipid attached to a peptide linker portion which is cence without recourse to additives such as further surfac suitably functionalised for coupling to one or more vector tants and/or viscosity enhancers, thereby ensuring that the molecules. A particular preference is the inclusion of a US 2004/0141922 A1 Jul. 22, 2004 positively charged peptide linker element (e.g. comprising [0037] Echogenicity per dose may be greatly enhanced two or more lysine residues) capable of anchoring through since substantially all of the surfactant material participate in electrostatic interaction with reporter microbubbles stabi stabilisation of the microbubbles as monolayers. In vivo lised by negatively charged phospholipid or other surfactant ultrasound tests in dogs have shown that ultrasound contrast membranes. agents prepared as above may produce an increase in [0033] Another embodiment of the invention comprises backscattered signal intensity from the myocardium of 15 functionalised microbubbles carrying one or more reactive dB following intravenous injection of doses as low as 0.1 ul groups for non-specific reaction with receptor molecules microbubbles/kg body weight. located on cell surfaces. Microbubbles comprising a thiol [0038] Safety in vivo is improved for the same reasons, moiety, for example, may bind to cell surface receptors via since such agents may, for example, be administered in disulphide exchange reactions. The reversible nature of such doses such that the amount of phospholipid injected is as low reactions means that microbubble flow may be controlled by as 0.1-10 ug/kg body weight, e.g. 1-5 ug/kg. The use of such altering the redox environment. Similarly, functionalised low levels of surfactant may clearly be of substantial advan microbubbles with membranes comprising activated esters tage in minimising possible toxic side effects. such as N-hydroxysuccinimide esters may be used to react with amino groups found on a multiplicity of cell surface [0039] The high efficacy/dose ratio is also particularly molecules. advantageous in targeting applications, since it is generally understood that rather low amounts of reporter will accu [0034] Previously, proposed microbubble-containing con mulate at sites of interest when using products comprising trast agents based on phospholipids, for example as vectors having affinity for such sites. These preferred report described in WO-A-9409829, are typically prepared by ers according to the invention may therefore considerably contacting powdered surfactant, e.g. freeze-dried preformed improve contrast at sites of interest compared to known liposomes or freeze-dried or spray-dried phospholipid solu targetable ultrasound contrast agents. Their high efficacy tions, with air or other gas and then with aqueous carrier, may effectively make it possible to “see” single agitating to generate a microbubble suspension which must microbubbles using ultrasound, giving a sensitivity close to then be administered shortly after its preparation. Such or potentially even higher than that of scintigraphy, which processes, however, suffer the disadvantages that substantial currently is probably the most useful technique in targeting, agitational energy must be imparted to generate the required although the resolution in scintigraphic pictures is not dispersion and that the size and size distribution of the impressive. microbubbles are dependent on the amount of energy applied and so cannot in practice be controlled. [0040] A particular advantage of phosphatidylserine [0035] The reporters or agents according to the present based agents is their biocompatibility; thus no acute toxic invention, on the other hand, may advantageously be pre effects such as changes in blood pressure or heart rate have pared by generating a gas microbubble dispersion in an been observed in animal tests on dogs injected with intra appropriate surfactant (e.g. phospholipid)-containing aque venous boluses of phosphatidylserine-based contrast agents ous medium, which may if desired previously have been prepared as described above at doses of up to ten times a autoclaved or otherwise sterilised, and then, preferably after normal imaging dose. washing and/or size fractionation of the thus-formed [0041] The use of charged phospholipids may also be of microbubbles, subjecting the dispersion to lyophilisation, advantage in that they will contain functional groups such as e.g. in the presence of one or more cryoprotectants/lyopro carboxyl or amino which permit ready linking of vectors, if tectants, to yield a dried product which is readily reconsti desired by way of linking units. It should be noted that other tutable in water/aqueous solutions to generate consistently functional groups may also be incorporated into such sys reproducible microbubble dispersions. This process is tems by mixing a lipid containing a desired functional group described in greater detail in WO-A-9729783, the contents with the film-forming surfactant prior to microbubble gen of which are incorporated herein by reference; the ability to eration. remove bubbles of unwanted size and excess surfactant material render this process of substantial advantage over [0042] It is generally unnecessary to incorporate additives processes such as those described in the aforementioned such as emulsifying agents and/or viscosity enhancers such WO-A-9409829 and in prior art such as WO-A-9608234 as are commonly employed in many existing contrast agent (where bubbles are generated on site prior to injection by formulations into agents of the invention. As noted above, shaking a suspension of different phospholipids and viscos this is of advantage in keeping to a minimum the number of ity enhancers such as propylene glycol and glycerol). components administered to the body of a subject and ensuring that the viscosity of the agents is as low as possible. [0036] The above-described process may be used to gen Since preparation of the agents typically involves a freeze erate reporter microbubbles with a very narrow size distri drying step as discussed above, it may however be advan bution, e.g. such that over 90% (e.g. at least 95%, preferably tageous to include a cryoprotectant/lyoprotectant or bulking at least 98%) of the microbubbles have volume mean agent, for example an alcohol, e.g. an aliphatic alcohol such diameter in the range 1-7 um and less than 5% (e.g. not more as t-butanol; a polyol such as glycerol; a carbohydrate, e.g. than 3%, preferably not more than 2%) of the microbubbles a sugar such as sucrose, mannitol, trehalose or a cyclodex have volume mean diameter above 7 um. The washing step trin, or a polysaccharide such as dextran; or a polyglycol may be used to ensure that the reporter is substantially free such as polyethylene glycol. The use of physiologically of unwanted components such as excess lipids or viscosity enhancers. Agents containing reporters prepared in this way well-tolerated sugars such as sucrose is preferred. may exhibit the following advantages over prior art contrast [0043] Lyophilised dried products prepared as described agent materials: above are especially readily reconstitutable in water, requir US 2004/0141922 A1 Jul. 22, 2004 ing only minimal agitation such as may, for example, be [0048] Again in the context of the present invention, for provided by gentle hand-shaking for a few seconds. The size example in assessment of blood perfusion rates in targeted of the microbubbles so generated is consistently reproduc areas such as the myocardium, it is of interest to measure the ible and is independent of the amount of agitational energy rate at which contrast agents bound to the target are dis applied, in practice being determined by the size of the placed or released therefrom. This may be achieved in a microbubbles formed in the initial microbubble dispersion; controlled manner by administration of an additional vector surprisingly this size parameter is substantially maintained and/or other substance able to displace or release the con in the lyophilised and reconstituted product. Thus, since the trast agent from its target. size of the microbubbles in the initial dispersion may readily [0049] Ultrasound imaging modalities which may be used be controlled by process parameters such as the method, in accordance with the invention include two- and three speed and duration of agitation, the final microbubble size dimensional imaging techniques such as B-mode imaging may readily be controlled. (for example using the time-varying amplitude of the signal [0044] The lyophilised dried products have also proved to envelope generated from the fundamental frequency of the be storage stable for at least several months under ambient emitted ultrasound pulse, from sub-harmonics or higher conditions. The microbubble dispersions generated upon harmonics thereof or from sum or difference frequencies reconstitution in water are stable for at least 8 hours, derived from the emitted pulse and such harmonics, images permitting considerable flexibility as to when the dried generated from the fundamental frequency or the second product is reconstituted prior to injection. harmonic thereof being preferred), colour Doppler imaging [0045] The high efficacy of these preferred reporters may and Doppler amplitude imaging, and combinations of the make it possible to use smaller bubbles than usual while still two latter with any of the above modalities. Surprisingly generating ultrasound contrast effects significantly above the excellent second harmonic signals have been obtained from minimum detection levels of current ultrasound imaging targeted monolayer-stabilised microspheres in accordance equipment. Such smaller bubbles have potential advantages with the present invention. To reduce the effects of move such as reduced clogging of vessels, longer circulation ment, successive images of tissues such as the heart or times, greater ability to reach targets, and lower accumula kidney may be collected with the aid of suitable synchro tion in lungs or other non-target organs, and their use and nisation techniques (e.g. gating to the ECG or respiratory agents containing them constitute further features of the movement of the subject). Measurement of changes in invention. resonance frequency or frequency absorption which accom pany arrested or retarded microbubbles may also usefully be [0046] It may also be possible to use such smaller bubbles made to detect the contrast agent. to exploit the enhanced ultrasound contrast effects of bubble clusters. It is known from theory that the ultrasound contrast [0050] The present invention provides a tool for therapeu effect of a specific number of bubbles with total volume V tic drug delivery in combination with vector-mediated direc in a dilute dispersion increases when the bubbles aggregate tion of the product to the desired site. By “therapeutic” or to form a larger gas phase with the same total volume V. It “drug” is meant an agent having a beneficial effect on a may therefore be possible to use small bubbles which give specific disease in a living human or non-human animal. substantially no ultrasound contrast until they are clustered Whilst combinations of drugs and ultrasound contrast agents (as may occur in target areas in preference to non-target sites have been proposed in, for example, WO-A-9428873 and having low densities of target molecules). Small bubbles WO-A-9507072, these products lack vectors having affinity may also be designed to fuse, e.g. through interbubble for particular sites and thereby show comparitively poor binding promoted by interaction with the target, so as to specific retention at desired sites prior to or during drug enhance contrast in target areas. Interbubble crosslinking release. and consequent clustering may also be effected if the [0051] Therapeutic compounds used in accordance with reporter, in addition to carrying a vector leading to retention the present invention may be encapsulated in the interior of at specific sites, has unreacted linker moieties capable of the microbubbles or attached to or incorporated in the reaction with functional groups on other bubbles. stabilising membranes. Thus, the therapeutic compound [0047] Within the context of the present invention, the may be linked to a part of the membrane, for example reporter unit will usually remain attached to the vectors. through covalent or ionic bonds, or may be physically mixed However, in one type of targeting procedure, sometimes into the stabilising material, particularly if the drug-has called “pre-targeting”, the vector (often a monoclonal anti similar polarity or solubility to the membrane material, so as body) is administered alone; subsequently the reporter is to prevent it from leaking out of the product before it is administered, coupled to a moiety which is capable of intended to act in the body. The release of the drug may be specifically binding the pre-targeting vector molecule (when initiated merely by wetting contact with blood following the pre-targeting vector is an antibody, the reporter may be administration or as a consequence of other internal or coupled to an immunoglobulin-binding molecule, such as external influences, e.g. dissolution processes catalyzed by protein A or an anti-immunoglobulin antibody). The advan enzymes or the use of of ultrasound. The destruction of tage of this protocol is that time may be allowed for gas-containing microparticles using external ultrasound is a elimination of the vector molecules that do not bind their well known phenomenon in respect of ultrasound contrast targets, substantially reducing the background problems that agents, e.g. as described in WO-A-9325241; the rate of drug are connected with the presence of an excess of reporter release may be varied depending on the type of therapeutic vector conjugate. Within the context of the present inven application, using a specific amount of ultrasound energy tion, pre-targeting with one specific vector might be envis from the transducer. aged, followed by reporter units that are coupled to another [0052] The therapeutic may be covalently linked to the vector and a moiety which binds the first vector. encapsulating membrane surface using a suitable linking US 2004/0141922 A1 Jul. 22, 2004 agent, e.g. as described herein. Thus, for example, one may as alkaline phosphatase or manganese superoxide dismu initially prepare a phospholipid or lipopeptide derivative to tase; antiallergic agents such as amelexanox; inhibitors of which the drug is bonded through a biodegradable bond or tissue factor such as monoclonal antibodies and Fab frag linker, and then incorporate this derivative into the material ments thereof, synthetic peptides, nonpeptides and com used to prepare the reporter, as described above. pounds down-regulating tissue factor expression; inhibitors [0053] Representative therapeutics suitable for use in the of platelets such as GPIa, GPIb and GPIIb-IIIa, ADP recep present drug delivery compositions include any known tors, thrombin receptors, von Willebrand factor, prostaglan therapeutic drugs or active analogues thereof containing dins, aspirin, ticlopidin, clopigogrel and reopro; inhibitors of thiol groups which may be coupled to thiol-containing coagulation protein targets such as FIIa, FVa, FVIIa, microbubbles under oxidative conditions yielding disul FVIIIA, FIXa, FXa, tissue factor, heparins, hirudin, hirulog, phide groups. In combination with a vector or vectors such argatroban, DEGR-rPVIIa and annexin V: inhibitors of drug/vector-modified microbubbles may be allowed to accu fibrin formation and promoters of fibrinolysis such as t-PA, mulate in target tissue; administration of a reducing agent urokinase, Plasmin, Streptokinase, rt-Plasminogen Activator such as reduced glutathione may then liberate the drug and rStaphylokinase; antiangiogenic factors such as molecule from the targeted microbubble in the vicinity of the medroxyprogesteron, pentosan polysulphate, Suramin, taxol, target cell, increasing the local concentration of the drug and thalidomide, angiostatin, interferon-alpha, metalloprotein enhancing its therapeutic effect. Alternatively the composi ase inhibitors, platelet factor 4, somatostatin, thromobospon tion may initially be prepared without the therapeutic, which din; circulatory drugs such as propranolol; metabolic poten may then be coupled to or coated on the microbubbles tiators such as glutathione; antituberculars such as immediately prior to use; thus, for example, a therapeutic p-aminosalicylic acid, isoniazid, capreomycin sulfate, may be added to a suspension of microbubbles in aqueous cyclosexine, ethambutol, ethionamide, pyrazinamide, media and shaken in order to attach or adhere the therapeutic rifampin or streptomycin sulphate; antivirals such as acy to the microbubbles. clovir, amantadine, azidothymidine, ribavirin or vidarabine; blood vessel dilating agents such as diltiazem, nifedipine, [0054] Other drug delivery systems include vector-modi verapamil, erythritol tetranitrate, isosorbide dinitrate, nitro fied phospholipid membranes doped with lipopeptide struc glycerin or pentaerythritol tetranitrate; antibiotics such as tures comprising a poly-L-lysine or poly-D-lysine chain in dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil, combination with a targeting vector. Applied to gene cephalexin, cephradine, erythromycin, clindamycin, linco therapy/antisense technologies with particular emphasis on mycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, receptor-mediated drug delivery, the microbubble carrier is dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicil condensed with DNA or RNA via elecrostatic interaction lin, nafcillin, penicillin, polymyxin or tetracycline; antiin with the cationic polylysine. This method has the advantage flammatories such as diflunisal, ibuprofen, indomethacin, that the vector or vectors used for targeted delivery are not meclefenamate, mefenamic acid, naproxen, phenylbuta directly attached to the polylysine carrier moiety. The polyl zone, piroxicam, tolmetin, aspirin or salicylates; antiproto ysine chain is also anchored more tightly in the microbubble zoans such as chloroquine, metronidazole, quinine or meglu membrane due to the presence of the lipid chains. The use mine antimonate; antirheumatics such as penicillamine; of ultrasound to increase the effectiveness of delivery is also narcotics such as paregoric; opiates such as codeine, mor considered useful. phine or opium; cardiac glycosides such as deslaneside, [0055] Alternatively free polylysine chains are firstly digitoxin, digoxin, digitalin or digitalis; neuromuscular modified with drug or vector molecules then condensed onto blockers such as atracurium mesylate, gallamine triethio the negative surface of targeted microbubbles. dide, hexafluorenium bromide, metocurine iodide, pancuro nium bromide, succinylcholine chloride, tubocurarine chlo [0056] Representative and non-limiting examples of drugs ride or vecuronium bromide; sedatives such as amobarbital, useful in accordance with the invention include antineoplas amobarbital sodium, apropbarbital, butabarbital sodium, tic agents such as vincristine, vinblastine, vindesine, busul chloral hydrate, ethchlorvynol, ethinamate, flurazepam fan, chlorambucil, spiroplatin, cisplatin, carboplatin, meth hydrochloride, glutethimide, methotrimeprazine hydrochlo otrexate, adriamycin, mitomycin, bleomycin, cytosine ride, methyprylon, midazolam hydrochloride, paraldehyde, arabinoside, arabinosyl adenine, mercaptopurine, mitotane, pentobarbital, secobarbital sodium, talbutal, temazepam or procarbazine, dactinomycin (antinomycin D), daunorubicin, triazolam; local anaesthetics such as bupivacaine, chlorop doxorubicin hydrochloride, taxol, plicamycin, aminoglute rocaine, etidocaine, lidocaine, mepivacaine, procaine or thimide, estramustine, flutamide, leuprolide, megestrol tetracaine; general anaesthetics such as droperidol, etomi acetate, tamoxifen, testolactone, trilostane, amsacrine date, fentanyl citrate with droperidol, ketamine hydrochlo (m-AMSA), asparaginase (L-asparaginase), etoposide, ride, methohexital sodium or thiopental and pharmaceuti interferon a-2a and 2b, blood products such as hematopor cally acceptable salts (e.g. acid addition salts such as the phyrins or derivatives of the foregoing, biological response hydrochloride or hydrobromide or base salts such as sodium, modifiers such as muramylpeptides; antifungal agents such calcium or magnesium salts) or derivatives (e.g. acetates) as ketoconazole, nystatin, griseofulvin, flucytosine, micona thereof. Other examples of therapeutics include genetic zole or amphotericin B; hormones or hormone analogues material such as nucleic acids, RNA, and DNA of natural or such as growth hormone, melanocyte stimulating hormone, synthetic origin, including recombinant RNA and DNA. estradiol, beclomethasone dipropionate, betamethasone, DNA encoding certain proteins may be used in the treatment cortisone acetate, dexamethasone, flunisolide, hydrocorti of many different types of diseases. For example, tumor sone, methylprednisolone, paramethasone acetate, predniso necrosis factor or interleukin-2 genes may be provided to lone, prednisone, triamcinolone or fludrocortisone acetate; treat advanced cancers; thymidine kinase genes may be vitamins such as cyanocobalamin or retinoids; enzymes such provided to treat ovarian cancer or brain tumors; interleu US 2004/0141922 A1 Jul. 22, 2004

kin-2 genes may be provided to treat neuroblastoma, malig factors are also involved in re-vascularisation of infarcted nant melanoma or kidney cancer; and interleukin-4 genes parts of the myocardium, which occurs if a stenosis is may be provided to treat cancer. released within a short time. [0060] A number of known receptors/targets associated [0057] Lipophilic derivatives of drugs linked to the with angiogenesis are given in subsequent tables. Using the microbubble membrane through hydrophobic interactions targeting principles described in the present disclosure, may exhibit therapeutic effects as part of the microbubble or angiogenesis may be detected by the majority of the imaging after release from the microbubble, e.g. by use of ultrasound. modalities in use in medicine. Contrast-enhanced ultrasound If the drug does not possess the desired physical properties, may possess additional advantages, the contrast medium a lipophilic group may be introduced for anchoring the drug being microspheres which are restricted to the interior of to the membrane. Preferably the lipophilic group should be blood vessels. Even if the target antigens are found on many introduced in a way that does not influence the in vivo cell types, the microspheres will attach exclusively to endot potency of the molecule, or the lipophilic group may be helial cells. cleaved releasing the active drug. Lipophilic groups may be introduced by various chemical means depending on func [0061] So-called prodrugs may also be used in agents according to the invention. Thus drugs may be derivatised to tional groups available in the drug molecule. Covalent alter their physicochemical properties and to adapt them for coupling may be effected using functional groups in the drug inclusion into the reporter; such derivatised drugs may be molecule capable of reacting with appropriately functiona regarded as prodrugs and are usually inactive until cleavage lised lipophilic compounds. Examples of lipophilic moieties of the derivatising group regenerates the active form of the include branched and unbranched alkyl chains, cyclic com drug. pounds, aromatic residues and fused aromatic and non aromatic cyclic systems. In some instances the lipophilic [0062] By targeting gas-filled microbubbles containing a moiety will consist of a suitably functionalised steroid, such prodrug-activating enzyme to areas of pathology, one may as cholesterol or a related compound. Examples of func image targeting the enzyme, making it possible to visualise tional groups particularly suitable for derivatisation include when the microbubbles are targeted properly to the area of nucleophilic groups like amino, hydroxy and sulfhydryl pathology and at the same time have disappeared from groups. Suitable processes for lipophilic derivatisation of non-target areas. In this way one can determine the optimal any drug containing a sulfhydryl group, such as captopril, time for injection of prodrug into individual patients. may include direct alkylation, e.g. reaction with an alkyl [0063] Another alternative is to incorporate the prodrug, halide under basic conditions and thiol ester formation by prodrug-activating enzyme and vector in the same reaction with an activated carboxylic acid. Representative microbubbles in a system where the prodrug will only be examples of derivatisation of any drug having carboxylic activated after some external stimulus. Such a stimulus may, functions, for example atenolol or chlorambucil, include for example, be a tumour-specific protease as described amide and ester formation by coupling respectively with above, or bursting of the microbubbles by external ultra amines and alcohols possessing appropriate physical prop sound after the desired targeting has been achieved. erties. A preferred embodiment comprises attachment of [0064] Therapeutics may easily be delivered in accor cholesterol to a therapeutic compound by forming a degrad dance with the invention to diseased or necrotic areas, for able ester bond. example in the heart, general vasculature, and to the liver, [0058] A preferred application of the present invention spleen, kidneys and other regions such as the lymph system, relates to angiogenesis, which is the formation of new blood body cavities or gastrointestinal system. vessels by branching from existing vessels. The primary [0065] Products according to the present invention may be stimulus for this process may be inadequate supply of used for targeted therapeutic delivery either in vivo or in nutrients and oxygen (hypoxia) to cells in a tissue. The cells vitro. In the latter context the products may be useful in in may respond by secreting angiogenetic factors, of which vitro systems such as kits for diagnosis of different diseases there are many; one example is vascular endothelial growth or characterisation of different components in blood or tissue factor. These factors initiate the secretion of proteolytic samples. Similar techniques to those used to attach certain enzymes which break down the proteins of the basement blood components or cells to polymer particles (e.g. mono membrane, as well as inhibitors which limit the action of disperse magnetic particles) in vitro to separate them from a these potentially harmful enzymes. The combined effect of sample may be used in the present invention, using the low loss of attachment and signals from the receptors for angio density of the reporter units in agents of the present inven genetic factors is to cause the endothelial cells to move, tion to effect separation of the gas-containing material by multiply, and rearrange themselves, and finally to synthesise flotation and repeated washing. a basement membrane around the new vessels. [0066] Coupling of a reporter unit to a desired vector [0059] Tumors must initiate angiogenesis when they reach (and/or therapeutic drug) may be achieved by covalent or millimeter size in order to keep up their rate of growth. As non-covalent means, usually involving interaction with one angiogenesis is accompanied by characteristic changes in or more functional groups located on the reporter and/or the endothelial cells and their environment, this process is a vector and/or any intervening linker group/spacer element. promising target for therapeutic intervention. The transfor Examples of chemically reactive functional groups which mations accompanying angiogenesis are also very promis may be employed for this purpose include amino, hydroxyl, ing for diagnosis, a preferred example being malignant sulfhydryl, carboxyl, and carbonyl groups, as well as car disease, but the concept also shows great promise in inflam bohydrate groups, vicinal diols, thioethers, 2-aminoalcohols, mation and a variety of inflammation-related diseases. These 2-aminothiols, guanidinyl, imidazolyl and phenolic groups. US 2004/0141922 A1 Jul. 22, 2004

[0067] Covalent coupling of reporter and vector may Adv. Cancer Res. (1954) 2, 1, which react with nucleo therefore be effected using linking agents containing reac philes such as amino groups by ring opening; tive moities capable of reaction with such functional groups. Examples of reactive moieties capable of reaction with [0077] ix) squaric acid diethyl esters as described by sulfhydryl groups include O.-haloacetyl compounds of the Tietze, L. F. in Chem. Ber. (1991) 124, 1215; and type X—CH2CO- (where X=Br, Cl or I), which show [0078] x) o-haloalkyl ethers, which are more reactive particular reactivity for sulfhydryl groups but which can also alkylating agents than normal alkyl halides because of be used to modify imidazolyl, thioether, phenol and amino the activation caused by the ether oxygen atom, e.g. as groups as described by Gurd, F. R. N. in Methods Enzymol. described by Benneche, T. et al. in Eur, J. Med. Chem. (1967) 11,532. N-Maleimide derivatives are also considered (1993) 28,463. selective towards sulfhydryl groups, but may additionaly be useful in coupling to amino groups under certain conditions. [0079] Representative amino-reactive acylating agents N-maleimides may be incorporated into linking systems for include: reporter-vector conjugation as described by Kitagawa, T. et [0080] i) isocyanates and isothiocyanates, particularly al. in Chem. Pharm. Bull. (1981) 29, 1130 or used as aromatic derivatives, Which form stable urea and thio polymer crosslinkers for bubble stabilisation as described by urea derivatives respectively and have been used for Kovacic, P. et al. in J. Am. Chem. Soc. (1959) 81, 1887. protein crosslinking as described by Schick, A. F. et al. Reagents such as 2-iminothiolane, e.g. as described by in J. Biol. Chem. (1961) 236, 2477; Traut, R. et al. in Biochemistry (1973) 12, 3266, which introduce a thiol group through conversion of an amino [0081] ii) sulfonyl chlorides, which have been described group, may be considered as sulfhydryl reagents if linking by Herzig, D. J. et al. in Biopolymers (1964) 2, 349 and occurs through the formation of disulphide bridges. Thus which may be useful for the introduction of a fluores reagents which introduce reactive disulphide bonds into cent reporter group into the linker; either the reporter or the vector may be useful, since linking [0082] iii) Acid halides; may be brought about by disulphide exchange between the vector and reporter; examples of such reagents include [0083] iv) Active esters such as nitrophenylesters or Ellman’s reagent (DTNB), 4,4'-dithiodipyridine, methyl-3 N-hydroxysuccinimidyl esters; nitro-2-pyridyl disulphide and methyl-2-pyridyl disulphide [0084] v) acid anhydrides such as mixed, symmetrical (described by Kimura, T. et al. in Analyt. Biochem. (1982) or N-carboxyanhydrides; 122, 271). [0085] vi) other useful reagents for amide bond forma [0068] Examples of reactive moieties capable of reaction tion as described by Bodansky, M. et al. in ‘Principles with amino groups include alkylating and acylating agents. of Peptide Synthesis’ (1984) Springer-Verlag, Representative alkylating agents include: [0086] vii) acylazides, e.g. wherein the azide group is [0069] i) o-haloacetyl compounds, which show speci generated from a preformed hydrazide derivative using ficity towards amino groups in the absence of reactive sodium nitrite, e.g. as described by Wetz, K. et al. in thiol groups and are of the type X—CH2CO— (where Anal. Biochem. (1974) 58, 347; X=Cl, Br or I), e.g. as described by Wong, Y-H. H. in [0087] viii) azlactones attached to polymers such as Biochemistry (1979) 24, 5337; bis-acrylamide, e.g. as described by Rasmussen, J. K. [0070] ii) N-maleimide derivatives, which may react in Reactive Polymers (1991) 16, 199; and with amino groups either through a Michael type reaction or through acylation by addition to the ring [0088] ix) Imidoesters, which form stable amidines on carbonyl group as described by Smyth, D. G. et al. in reaction with amino groups, e.g. as described by J. Am. Chem. Soc. (1960) 82, 4600 and Biochem. J. Hunter, M. J. and Ludwig, M. L. in J. Am. Chem. Soc. (1964) 91, 589; (1962) 84, 3491. [0071] iii) aryl halides such as reactive nitrohaloaro [0089] Carbonyl groups such as aldehyde functions may matic compounds; be reacted with weak protein bases at a pH such that nucleophilic protein side-chain functions are protonated. [0072] iv) alkyl halides as described by McKenzie, J. A. Weak bases include 1,2-aminothiols such as those found in et al. in J. Protein Chem. (1988) 7, 581; N-terminal cysteine residues, which selectively form stable [0073] v) aldehydes and ketones capable of Schiff's 5-membered thiazolidine rings with aldehyde groups, e.g. as base formation with amino groups, the adducts formed described by Ratner, S. et al. in J. Am. Chem. Soc. (1937) 59, usually being stabilised through reduction to give a 200. Other weak bases such as phenyl hydrazones may be used, e.g. as described by Heitzman, H. et al. in Proc. Natl. stable amine; Acad. Sci. USA (1974) 71, 3537. [0074] vi) epoxide derivatives such as epichiorohydrin and bisoxiranes, which may react with amino, sulfhy [0090] Aldehydes and ketones may also be reacted with dryl or phenolic hydroxyl groups; amines to form Schiff’s bases, which may advantageously be stabilised through reductive amination. Alkoxylamino moi [0075] vii) chlorine-containing derivatives of s-triaz eties readily react with ketones and aldehydes to produce ines, which are very reactive towards nucleophiles such stable alkoxamines, e.g. as described by Webb, R. et al. in as amino, sufhydryl and hydroxy groups; Bioconjugate Chem. (1990) 1, 96. [0076] viii) aziridines based on s-triazine compounds [0091] Examples of reactive moieties capable of reaction detailed above, e.g. as described by Ross, W. C. J. in with carboxyl groups include diazo compounds such as US 2004/0141922 A1 Jul. 22, 2004

diazoacetate esters and diazoacetamides, which react with consisting mainly of phospholipid head groups and so may high specificity to generate ester groups, e.g. as described by be used to attach vectors to phospholipid microspheres; one Herriot R. M. in Adv. Protein Chem. (1947) 3, 169. Car example of such a protein is [2-glycoprotein I (Chonn, A., boxylic acid modifying reagents such as carbodiimides, Semple, S. C. and Cullis, P. R., Journal of Biological which react through O-acylurea formation followed by Chemistry (1995) 270, 25845-25849). Phosphatidylserine amide bond formation, may also usefully be employed; binding proteins have been described, e.g. by Igarashi, K. et linking may be facilitated through addition of an amine or al. in Journal of Biological Chemistry 270(49), 29075 may result in direct vector-receptor coupling. Useful water 29078; a conjugate of a vector with such a phosphati soluble carbodiimides include 1-cyclohexyl-3-(2-morpholi dylserine-binding protein may therefore be used to attach the nyl-4-ethyl)carbodiimide (CMC) and 1-ethyl-3-(3-dimethy vector to phosphatidylserine-encapsulated microbubbles. laminopropyl)carbodiimide (EDC), e.g. as described by Zot, When the amino acid sequence of a binding protein is H. G. and Puett, D. in J. Biol. Chem. (1989) 264, 15552. known, the phospholipid-binding portion may be synthe Other useful carboxylic acid modifying reagents include sised or isolated and used for conjugation with a vector, thus isoxazolium derivatives such as Woodwards reagent K, avoiding the biological activity which may be located else chloroformates such as p-nitrophenylchloroformate; carbo where in the molecule. nyldiimidazoles such as 1,1'-carbonyldiimidazole; and [0096] It is also possible to obtain molecules that bind N-carbalkoxydihydroquinolines such as N-(ethoxycarbo specifically to the surface (or in the “membrane”) of micro nyl)-2-ethoxy-1,2-dihydroquinoline. spheres by direct screening of molecular libraries for micro [0092] Other potentially useful reactive moieties include sphere-binding molecules. For example, phage libraries dis vicinal diones such as p-phenylenediglyoxal, which may be playing small peptides may be used for such selection. The used to react with guanidinyl groups, e.g. as described by selection may be made by simply mixing the microspheres Wagner et al. in Nucleic acid Res. (1978) 5, 4065; and and the phage display library and eluting the phages binding diazonium salts, which may undergo electrophilic substitu to the floating microspheres. If desired, the selection may be tion reactions, e.g. as described by Ishizaka, K. and Ishizaka done under “physiological conditions” (e.g. in blood) to T. in J. Immunol. (1960) 85, 163. Bis-diazonium compounds eliminate peptides which cross-react with blood compo are readily prepared by treatment of aryl diamines with ments. An advantage of this type of selection procedure is sodium nitrite in acidic solutions. It will be appreciated that that only binding molecules that do not destabilise the functional groups in the reporter and/or vector may if microspheres should be selected, since only binding mol desired be converted to other functional groups prior to ecules attached to intact floating microspheres will rise to reaction, e.g. to confer additional reactivity or selectivity. the top. It may also be possible to introduce some kind of Examples of methods useful for this purpose include con “stress” during the selection procedure (e.g. pressure) to version of amines to carboxylic acids using reagents such as ensure that destabilising binding moieties are not selected. dicarboxylic anhydrides; conversion of amines to thiols Furthermore the selection may be done under shear condi using reagents such as N-acetylhomocysteine thiolactone, tions, for example by first letting the phages react with the S-acetylmercaptosuccinic anhydride, 2-iminothiolane or microspheres and then letting the microspheres pass through thiol-containing succinimidyl derivatives; conversion of thi a surface coated with anti-phage antibodies under flow ols to carboxylic acids using reagents such as O-haloac conditions. In this way it may be possible to select binders etates; conversion of thiols to amines using reagents such as which may resist shear conditions present in vivo. Binding ethylenimine or 2-bromoethylamine; conversion of carboxy moieties identified in this way may be coupled (by chemical lic acids to amines using reagents such as carbodiimides conjugation or via peptide synthesis, or at the DNA-level for followed by diamines; and conversion of alcohols to thiols recombinant vectors) to a vector molecule, constituting a using reagents such as tosyl chloride followed by transes general tool for attaching any vector molecule to the micro terification with thioacetate and hydrolysis to the thiol with spheres. sodium acetate. [0097] A vector which comprises or is coupled to a [0093] Vector-reporter coupling may also be effected peptide, lipo-oligosaccharide or lipopeptide linker which using enzymes as zero-length linking agents; thus, for contains a element capable of mediating membrane insertion example, transglutaminase, peroxidase and xanthine oxidase may also be useful. One example is described by Leenhouts, may be used to produce linked products. Reverse proteolysis J. M. et al. in Febs Letters (1995) 370(3), 189-192. Non may also be used for linking through amide bond formation. bioactive molecules consisting of known membrane inser tion anchor/signal groups may also be used as vectors for [0094] Non-covalent vector-reporter coupling may, for certain applications, an example being the Hi hydrophobic example, be effected by electrostatic charge interactions e.g. segment from the Na, K-ATPase O-subunit described by between a polylysinyl-functionalised reporter and a poly Xie, Y. and Morimoto, T. in J. Biol. Chem. (1995) 270(20), glutamyl-functionalised vector, through chelation in the 11985-11991. The anchor group may also be fatty acid(s) or form of stable metal complexes or through high affinity cholesterol. binding interaction such as avidin/biotin binding. Polyl ysine, coated non-covalently to a negatively charged mem [0098] Coupling may also be effected using avidin or brane surface may also increase non-specifically the affinity streptavidin, which have four high affinity binding sites for biotin. Avidin may therefore be used to conjugate vector to of a microbubble for a cell through charge interactions. reporter if both vector and reporter are biotinylated. [0095] Alternatively, a vector may be coupled to a protein Examples are described by Bayer, E. A. and Wilchek, M. in known to bind phospholipids. In many instances, a single Methods Biochem. Anal. (1980) 26, 1. This method may also molecule of phospholipid may attach to a protein such as a be extended to include linking of reporter to reporter, a translocase, while other proteins may attach to surfaces process which may encourage bubble association and con US 2004/0141922 A1 Jul. 22, 2004 sequent potentially increased echogenicity. Alternatively, [0105] Contrast agents according to the present invention avidin or streptavidin may be attached directly to the surface are therefore useful in all imaging modalities since contrast of reporter microparticles. elements such as X-ray contrast agents, light imaging [0099] Non-covalent coupling may also utilise the bifunc probes, spin labels or radioactive units may readily be tional nature of bispecific immunoglobulins. These mol incorporated in or attached to the reporter units. ecules can specifically bind two antigens, thus linking them. [0106] Spacer elements may typically consist of aliphatic For example, either bispecific IgG or chemically engineered chains which effectively separate the reactive moieties of the bispecific F(ab')2 fragments may be used as linking agents. linker by distances of between 5 and 30 Å. They may also Heterobifunctional bispecific antibodies have also been comprise macromolecular structures such as PEGs, which reported for linking two different antigens, e.g. as described have been given much attention in biotechnical and bio by Bode, C. et al. in J. Biol. Chem. (1989) 264,944 and by medical applications (see e.g. Milton Harris, J. (ed) “Poly Staerz, U. D. et al. in Proc. Natl. Acad. Sci. USA (1986) 83, (ethylene glycol) chemistry, biotechnical and biomedical 1453. Similarly, any reporter and/or vector containing two or applications” Plenum Press, New York, 1992). PEGs are more antigenic determinants (e.g. as described by Chen, Aa soluble in most solvents, including water, and are highly et al. in Am. J. Pathol. (1988) 130, 216) may be crosslinked hydrated in aqueous environments, with two or three water by antibody molecules and lead to formation of multi-bubble molecules bound to each ethylene glycol segment; this has cross-linked assemblies of potentially increased echogenic the effect of preventing adsorption either of other polymers ity. or of proteins onto PEG-modified surfaces. PEGs are known [0100] Linking agents used in accordance with the inven to be nontoxic and not to harm active proteins or cells, whilst tion will in general bring about linking of vector to reporter covalently linked PEGs are known to be non-immunogenic or reporter to reporter with some degree of specificity, and and non-antigenic. Furthermore, PEGs may readily be modi fied and bound to other molecules with only little effect on may also be used to attach one or more therapeutically active their chemistry. Their advantageous solubility and biological agents. properties are apparent from the many possible uses of PEGs [0101] In some instances it is considered advantageous to and copolymers thereof, including block copolymers such as include a PEG component as a stabiliser in conjunction with PEG-polyurethanes and PEG-polypropylenes. a vector or vectors or directly to the reporter in the same [0107] Appropriate molecular weights for PEG spacers molecule where the PEG does not serve as a spacer. used in accordance with the invention may, for example, be [0102] So-called zero-length linking agents, which induce between 120 Daltons and 20 kDaltons. direct covalent joining of two reactive chemical groups [0108] The major mechanism for uptake of particles by the without introducing additional linking material (e.g. as in cells of the reticuloendothelial system (RES) is opsonisation amide bond formation induced using carbodiimides or enzy by plasma proteins in blood; these mark foreign particles matically) may, if desired, be used in accordance with the invention, as may agents such as biotin/avidin systems which are then taken up by the RES. The biological prop which induce non-covalent reporter-vector linking and erties of PEG spacer elements used in accordance with the agents which induce hydrophobic or electrostatic interac invention may serve to increase contrast agent circulation tions. time in a similar manner to that observed for PEGylated [0103] Most commonly, however, the linking agent will liposomes (see e.g. Klibanov, A. L. et al. in FEBS Letters comprise two or more reactive moieties, e.g. as described (1990) 268, 235-237 and Blume, G. and Ceve, G. in Bio above, connected by a spacer element. The presence of such chim. Biophys. Acta (1990) 1029, 91-97). Increased cou a spacer permits bifunctional linkers to react with specific pling efficiency to areas of interest may also be achieved functional groups within a molecule or between two differ using antibodies bound to the terminii of PEG spacers (see ent molecules, resulting in a bond between these two com e.g. Maruyama, K. et al. in Biochim. Biophys. Acta (1995) ponents and introducing extrinsic linker-derived material 1234, 74-80 and Hansen, C. B. et al. in Biochim. Biophys. into the reporter-vector conjugate. The reactive moieties in Acta (1995) 1239, 133-144). a linking agent may be the same (homobifunctional agents) or different (heterobifunctional agents or, where several [0109] In some instances it is considered advantageous to dissimilar reactive moieties are present, heteromultifunc include a PEG component as a stabiliser in conjunction with tional agents), providing a diversity of potential reagents a vector or vectors or directly to the reporter in the same that may bring about covalent bonding between any chemi molecule where the PEG does not serve as a spacer. cal species, either intramolecularly or intermolecularly. [0110] Other representative spacer elements include struc [0104] The nature of extrinsic material introduced by the tural-type polysaccharides such as polygalacturonic acid, linking agent may have a critical bearing on the targeting glydosaminoglycans, heparinoids, cellulose and marine ability and general stability of the ultimate product. Thus it polysaccharides such as alginates, chitosans and carrageen may be desirable to introduce labile linkages, e.g. containing ans; storage-type polysaccharides such as starch, glycogen, spacer arms which are biodegradable or chemically sensitive dextran and aminodextrans; polyamino acids and methyl and or which incorporate enzymatic cleavage sites. Alternatively ethyl esters thereof, as in homo- and co-polymers of lysine, the spacer may include polymeric components, e.g. to act as glutamic acid and aspartic acid; and polypeptides, oligosac surfactants and enhance bubble stability. The spacer may charides and oligonucleotides, which may or may not con also contain reactive moieties, e.g. as described above to enhance surface crosslinking, or it may contain a tracer tain enzyme cleavage sites. element such as a fluorescent probe, spin label or radioactive [0111] In general, spacer elements may contain cleavable material. groups such as vicinal glycol, azo, sulfone, ester, thioester or US 2004/0141922 A1 Jul. 22, 2004 disulphide groups. Spacers containing biodegradable meth their degradation rate (see Imasaki, K., Yoshida, M., ylene diester or diamide groups of formula Fukuzaki, H., Asano, M., Kumakura, M., Mashimo, T., Yamanaka, H. and Nagai. T. in Int. J. Pharm. (1992) 81, 31-38); [0112] [where X and Z are selected from —O—, -S-, [0122] viii) polyesters consisting of alternating units of and —NR— (where R is hydrogen or an organic group); ethylene glycol and terephthalic acid, e.g. Dacron", each Y is a carbonyl, thiocarbonyl, sulphonyl, phosphoryl or which are non-degradable but highly biocompatible; similar acid-forming group: m and n are each zero or 1; and R" and R are each hydrogen, an organic group or a group [0123] ix) block copolymers comprising biodegrad -X,Y(Z), , or together form a divalent organic group] may able segments of aliphatic hydroxyacid polymers (see e.g. Younes, H., Nataf, P. R., Cohn, D., Appel also be useful; as discussed in, for example, WO-A-9217436 baum, Y. J., Pizov, G. and Uretzky, G. in Biomater. such groups are readily biodegraded in the presence of Artif. Cells Artif. Organs (1988) 16, 705-719), for esterases, e.g. in vivo, but are stable in the absence of such instance in conjunction with polyurethanes (see enzymes. They may therefore advantageously be linked to Kobayashi, H., Hyon, S. H. and Ikada, Y. in “Water therapeutic agents to permit slow release thereof. curable and biodegradable prepolymers”—J. [0113] Poly[N-(2-hydroxyethyl)methacrylamides] &l?º Biomed. Mater Res. (1991) 25, 1481-1494); potentially useful spacer materials by virtue of their low [0124] x) polyurethanes, which are known to be well degree of interaction with cells and tissues (see e.g. Volfová, tolerated in implants, and which may be combined with I., Ríhová, B. and V. R. and Vetvicka, P. in J. Bioact. Comp. flexible “soft” segments, e.g. comprising poly(tetra Polymers (1992) 7, 175-190). Work on a similar polymer methylene glycol), poly(propylene glycol) or poly(eth consisting mainly of the closely related 2-hydroxypropyl ylene glycol) and aromatic “hard” segments, e.g. com derivative showed that it was endocytosed by the mono prising 4,4'-methylenebis(phenylene isocyanate) (see nuclear phagocyte system only to a rather low extent (see e.g. Ratner, B. D., Johnston, A. B. and Lenk, T. J. in J. Goddard, P, Williamson, I., Bron, J., Hutchkinson, L. E., Biomed. Mater. Res: Applied Biomaterials (1987) 21, Nicholls, J. and Petrak, K. in J. Bioct. Compat. Polym. 59-90; Sa Da Costa, V. et al. in J. Coll. Interface Sci. (1991) 6, 4-24). (1981) 80,445-452 and Affrossman, S. et al. in Clinical [0114] Other potentially useful polymeric spacer materials Materials (1991) 8, 25-31); include: [0125] xi) poly(1,4-dioxan-2-ones), which may be [0115] i) copolymers of methyl methacrylate with meth regarded as biodegradable esters in view of their acrylic acid; these may be erodible (see Lee, P. I. in hydrolysable ester linkages (see e.g. Song, C. X., Cui, Pharm. Res. (1993) 10, 980) and the carboxylate sub X. M. and Schindler, A. in Med. Biol. Eng. Comput. stituents may cause a higher degree of swelling than (1993) 31, S147-150), and which may include gly with neutral polymers; colide units to improve their absorbability (see Bezwada, R. S., Shalaby, S. W. and Newman, H. D. J. [0116] ii) block copolymers of polymethacrylates with in Agricultural and synthetic polymers: Biodegradabil biodegradable polyesters (see e.g. San Roman, J. and ity and utilization (1990) (ed Glass, J. E. and Swift, G.), Guillen-Garcia, P. in Biomaterials (1991) 12,236-241); 167-174—ACS symposium Series, #433, Washington [0117] iii) cyanoacrylates, i.e. polymers of esters of D.C., U.S.A.—American Chemical Society); 2-cyanoacrylic acid—these are biodegradable and have [0126] xii) polyanhydrides such as copolymers of seba been used in the form of nanoparticles for selective cic acid (octanedioic acid) with bis(4-carboxy-phenox drug delivery (see Forestier, F., Gerrier, P., Chaumard, y)propane, which have been shown in rabbit studies C., Quero, A. M., Couvreur, P. and Labarre, C. in J. (see Brem, H., Kader, A., Epstein, J. I., Tamargo, R. J., Antimicrob. Chemoter. (1992) 30, 173-179); Domb, A., Langer, R. and Leong, K. W. in Sel. Cancer [0118] iv) polyvinyl alcohols, which are water-soluble Ther. (1989) 5, 55-65) and rat studies (see Tamargo, R. and generally regarded as biocompatible (see e.g. J., Epstein, J. I., Reinhard, C. S., Chasin, M. and Brem, Langer, R. in J. Control. Release (1991) 16, 53–60); H. in J. Biomed. Mater Res. (1989) 23, 253-266) to be useful for controlled release of drugs in the brain [0119) v) copolymers of vinyl methyl ether with maleic without evident toxic effects; anhydride, which have been stated to be bioerodible (see Finne, U., Hannus, M. and Urtti, A. in Int. J. [0127] xiii) biodegradable polymers containing ortho Pharm. (1992) 78. 237-241), ester groups, which have been employed for controlled [0120] vi) polyvinylpyrrolidones, e.g. with molecular release in vivo (see Maa, Y. F. and Heller, J. in J. weight less than about 25,000, which are rapidly fil Control. Release (1990) 14, 21-28); and tered by the kidneys (see Hespe, W., Meier, A. M. and [0128] xiv) polyphosphazenes, which are inorganic Blankwater, Y. M. in Arzeim.-Forsch./Drug Res. (1977) polymers consisting of alternate phosphorus and nitro 27, 1158-1162); gen atoms (see Crommen, J. H., Vandorpe, J. and [0121] vii) polymers and copolymers of short-chain Schacht, E. H. in J. Control. Release (1993) 24, 167 aliphatic hydroxyacids such as glycolic, lactic, butyric, 180). valeric and caproic acids (see e.g. Carli, F. in Chim. Ind. [0129] The following tables list linking agents and agents (Milan) (1993) 75,494-9), including copolymers which for protein modification which may be useful in preparing incorporate aromatic hydroxyacids in order to increase targetable agents in accordance with the invention.

US 2004/0141922 A1 Jul. 22, 2004

[0134] Other potentially useful protein modifications scription factors which are very often highly overexpressed include partial or complete deglycosidation by neuramini or activated in tumour cells or in activated immune or dase, endoglycosydases or periodate, since deglycosidation endothelial cells. Combinatorial libraries may be used to often results in less uptake by liver, spleen, macrophages select oligonucleotides which bind specifically to any pos etc., whereas neo-glycosylation of proteins often results in sible target molecules and which therefore may be employed increased uptake by the liver and macrophages); preparation as vectors for targeting. of truncated forms by proteolytic cleavage, leading to reduced size and shorter half life in circulation; and cationi [0140] v) DNA-binding drugs may behave similarly to sation, e.g. as described by Kumagi et al. in J. Biol. Chem. oligonuclotides, but may exhibit biological acitvity and/or (1987) 262, 15214-15219; Triguero et al. in Proc. Natl. toxic effects if taken up by cells. Acad. Sci. USA (1989) 86,4761-4765; Pardridge et al. in J. [0141] vi) Protease substrates/inhibitors. Proteases are Pharmacol. Exp. Therap. (1989) 251, 821-826 and Par involved in many pathological conditions. Many substrates/ dridge and Boado, Febs Lett. (1991) 288, 30-32. inhibitors are non-peptidic but, at least in the case of [0135] Vectors which may be usefully employed in tar inhibitors, are often bioactive. getable agents according to the invention include the fol [0142] vii) Vector molecules may be generated from com lowing: binatorial libraries without necessarily knowing the exact [0136] i) Antibodies, which can be used as vectors for a molecular target, by functionally selecting (in vitro, ex vivo very wide range of targets, and which have advantageous or in vivo) for molecules binding to the region/structure to properties such as very high specificity, high affinity (if be imaged. desired), the possiblity of modifying affinity according to need etc. Whether or not antibodies will be bioactive will [0143] viii) Various small molecules, including bioactive depend on the specific vector/target combination. Both con compounds known to bind to biological receptors of various ventional and genetically engineered antibodies may be kinds. Such vectors or their targets may be used for generate employed, the latter permitting engineering of antibodies to non-bioactive compounds binding to the same targets. particular needs, e.g. as regards affinity and specificity. The [0144] ix) Proteins or peptides which bind to glucosami use of human antibodies may be preferred to avoid possible oglycan side chains e.g. heparan sulphate, including gluco immune reactions against the vector molecule. A further soaminoglycan-binding portions of larger molecules, as useful class of antibodies comprises so-called bi- and multi binding to glucosoaminoglycans does not result in a bio specific antibodies, i.e. antibodies having specificity for two logical response. Proteoglycans are not found on red blood or more different antigens in one antibody molecule. Such cells, which eliminates undesirable adsorption to these cells. antibodies may, for example, be useful in promoting forma tion of bubble clusters and may also be used for various [0145] Other peptide vectors and lipopeptides thereof of therapeutic purposes, e.g. for carrying toxic moieties to the particular interest for targeted ultrasound imaging are listed target. Various aspects of bispecific antibodies are described below: Atherosclerotic plaque binding peptides such as YRALVDTLK (SEQ ID NO:26), YAKFRE by McGuinness, B. T. et al. in Nat. Biotechnol. (1996) 14, TLEDTRDRMY (SEQ ID NO:27) and 1149-1154; by George, A.J. et al. in J. Immunol. (1994) 152, RALVDTEFKVKQEAGAK (SEQ ID NO:28); Thrombus 1802-1811; by Bonardi et al. in Cancer Res. (1993) 53, binding peptides such as NDGDFEEIPEEYLQ (SEQ ID 3015-3021; and by French, R. R. et al. in Cancer Res. (1991) NO:29) and GPRG (SEQ ID NO:30), Platelet binding pep 51, 2353-2361. tides such as PLYKKIIKKLLES (SEQ ID NO:31); and [0137] ii) Cell adhesion molecules, their receptors, cytok cholecystokinin, O-melanocyte-stimulating hormone, heat ines, growth factors, peptide hormones and pieces thereof. stable enterotoxin 1, vasoactive intestinal peptide, synthetic Such vectors rely on normal biological protein-protein inter alpha-M2 peptide from the third heavy chain complemen actions with target molecule receptors, and so in many cases tarity-determininig region and analogues thereof for tumour will generate a biological response on binding with the targeting. targets and thus be bioactive; this may be a relatively insignificant concern with vectors which target proteogly [0146] The following tables identify various vectors CàIlS. which may be targeted to particular types of targets and indicated areas of use for targetable diagnostic and/or thera [0138] iii) Non-peptide agonists/antagonists or non-bioac peutic agents according to the invention which contain such tive binders of receptors for cell adhesion molecules, cytok VectorS. ines, growth factors and peptide hormones. This category may include non-bioactive vectors which will be neither [0147] Protein and Peptide Vectors—Antibodies agonists nor antagonist but which may nonetheless exhibit valuable targeting ability. [0139] iv) Oligonucleotides and modified oligonucle Vector type Target Comments/areas of use Ref otides which bind DNA or RNA through Watson-Crick or antibodies CD34 vascular diseases in general, 1 other types of base-pairing. DNA is usually only present in (general) normal vessel wall (e.g extracelluar space as a consequence of cell damage, so that myocardium), activated such oligonucleotides, which will usually be non-bioactive, endothelium, immune cells antibodies ICAM-1 vascular diseases in general, 1 may be useful in, for example, targeting of necrotic regions, (general) normal vessel wall (e.g which are associated with many different pathological con myocardium), activated ditions. Oligonucleotides may also be designed to bind to endothelium, immune cells specific DNA- or RNA-binding proteins, for example tran US 2004/0141922 A1 Jul. 22, 2004 14

-continued -continued Vector type Target Comments/areas of use Ref Vector type Target Comments/areas of use Ref antibodies ICAM-2 vascular diseases in general, cells/tissue (general) normal vessel wall (e.g CD44 myocardium), activated antibodies £2-micro- general b endothelium, immune cells antibodies globulin general b antibodies ICAM-3 vascular diseases in general, MHC class 1 (general) normal vessel wall (e.g antibodies integrin tumours; angiogenesis Ç myocardium), activated antibodies ov[3 endothelium, immune cells antibodies E-selectin vascular diseases in general, (general) normal vessel wall (e.g myocardium), activated REFERENCES endothelium, immune cells antibodies P-selectin vascular diseases in general, [0148] a) Heider, K. H., M. Sproll, S. Susani, E. Patzelt, (general) normal vessel wall (e.g P. Beaumier, E. Ostermann, H. Ahorn, and G. R. Adolf. myocardium), activated 1996. “Characterization of a high-affinity monoclonal antibodies PECAM vascularendothelium, diseases Immune in general, cells antibody- specific- for CD44v6 as candidate- for immu- (general) normal vessel wall (e.g notherapy of squamous cell carcinomas”. Cancer myocardium), activated Immunology Immunotherapy 43: 245-253. endothelium, Immune cells antibodies Integrins, vascular diseases in general, 2 [0149] b) I. Roitt, J. Brostoff, and D. Male. 1985. (general)€ineja §A3,e.g. VLA-1 via myocardium),in Ojima I WeSSè| Wallas ! ? e. . Immunology, London: Gower Medical- Publishing,- - - p. 3, VLA-4, endothelium, immune cells 4.7 VLA-5,6 pº VLA [0150] c) Stromblad, S., and D. A. Cheresh. 1996. ficts, ?ºciv, “Integrins, angiogenesis and vascular cell survival”. LFA-1, Mac- Chemistry & Biology 3: 881-885. 1, CD41a, etc. [0151] Protein and Peptide Vectors–Cell Adhesion Mol antibodies GlycAM Vessel wall in lymph nodes 3 (general) (quite- specific- for lymph nodes) ecules etc. antibodies MadCara 1 Vessel wall in lymph nodes 3 (general) (quite specific for lymph nodes) antibodies fibrin Thrombi 4 (general) Vector type Target Comments/areas of use Ref antibodies Tissue Activated endothelium, tumours 5 (general) Factor L-selectin CD34 vascular diseases in 3 antibodies Myosin Necrosis, myocardial infaction 3 MadCAM1 general, normal (general) Glycam 1 vessel wall antibodies CEA Tumours 7 (e.g. myocardium), (general) (carcino- activated embryonal endothelium, antigen) Lymph nodes antibodies Mucins Tumours 8 Other selectins carbohydrate vascular diseases in 14 (general) ligands general, normal antibodies Multiple Tumours 9 (sialyl Lewis x) vessel wall (general) drug heparan sulfate (e.g. myocardium), resistance activated protein endothelium antibodies Prostate Prostate cancer RGD-peptides integrins vascular diseases in 2 (general) specific general, normal antigen vessel wall antibodies Cathepsin B Tumours (proteases of various 10 (e.g. myocardium), (general) kinds are often more or less activated specifically overexpressed in a endothelium variety of tumours - Cathepsin B PECAM PECAM, Endothelium, 15 is such a protease) and other Cells in antibodies Transferrin Tumors, 11 immune system (general) receptor vessel wall Integrins, Laminin, Endothelium, 16 MOAb 9.2.27 Tumours 12 e.g. VLA-1, VLA- collagen, Vessel wall Antigen upregulated 2, VLA-3, VLA-4, fibronectin, etc. on cell growth VLA-5, VLA-6, vcAM-1, thrombo. VAP-1 Adhesion molecule 13 ?ºlo), ficts, spondin, Band 3 Upregulated during phagocytic [31Ov, LFA-1, Vitronectin etc. protein- activity- - etc.Mac-1, CD41a, antibodies CD34 - endothelial cells Integrin Integrins, Cells in 17 (sialomucin) receptors, e.g. VLA-l, VLA- immune system 18 antibodies CD31 endothelial cells e.g. Laminin, 2, VLA-3, VLA-4, vessel wall (PECAM-1) collagen, VLA-5, VLA-6, etc. antibodies intermediate tumour cells 3. fibronectin, ?ºlo), ficts, filaments VCAM-1, [31Ov, LFA-1, necrotic thrombospondin, Mac-1, CD41a,

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Comments/ Comments/ Vector type Target areas of use Ref Vector type Target areas of use Ref plasminogen umors, M inflammation És and És, inflammation subunit of the fibro- P plasminogen activators umors, D integrin Cºv?ås, nectin receptor inflammation integrin Cºs?i, , plasminogen activator umors, U, V integrins Cle, inhibitors inflammation integrins fºr, platelet activating umors, inhibitors of A. integrin Croft, actor antagonists inflammation angiogenesis integrin Cºv?ås, platelet-derived growth umors, E actOr inflammation integrin Cls pleiotropin umors, ZA integrin Cºv?ås, inflammation fibrin receptors. proliferin umors, E Intercellular adhesion umors, P inflammation molecule-1 and -2 inflammation proliferin related umors, E Jagged gene product umors, T protein inflammation inflammation selectins umors, e.g., E-selectin D Ly-6 umors, a lymphocyte activation N inflammation SPARC umors, M inflammation protein inflammation matrix umors, D snake venoms umors, Q metalloproteinases inflammation (RGD-containing) inflammation MHC class II umors, Tissue inhibitor of umors, eg., TIMP-2 U inflammation metalloproteinases inflammation Notch gene product mors, T thrombin umors, H inflammation inflammation Osteopontin llim Ojºs Z thrombin-receptor- umors, H activating inflammation PECAM umors, alias CD31 P tetradecapeptide inflammation thymidine phosphorylase umors, D plasminogen activator umors, ZC inflammation receptor inflammation tumor growth factor umors, ZA platelet-derived growth tumors, E inflammation factor receptors inflammation Selectins: E-, P- umors, D inflammation Sialyl Lewis-X umors, blood group antigen M [0158] Receptors/Targets Associated with Angiogenesis inflammation stress proteins: umors, molecular chaperones glucose regulated, inflammation heat shock families and Comments/ others Vector type Target areas of use Ref syndecan umors, T inflammation biglycan umors, dermatan sulfate X inflammation proteoglycan hrombospondin umors, M CD34 umors, L inflammation inflammation TIE receptors umors, tyrosine kinases with Ig- E CD44 umors, F inflammation and EGF-like domains inflammation issue factor umors, Z collagen type I, IV, umors, A. inflammation VI, VIII inflammation issue inhibitor of umors, e.g., TIMP-2 U decorin umors, dermatan sulfate Y metalloproteinases inflammation inflammation proteoglycan dermatan sulfate umors, X ransforming growth umors, E proteoglycans inflammation actor receptor inflammation endothelin umors, G urokinase-type umors, D inflammation plasminogen activator inflammation endothelin umors, G receptor receptors inflammation Vascular cellular umors, D fibronectin llim Ojºs P adhesion molecule inflammation Flk-1/KDR, Flt-4 umors, VEGF receptor D inflammation (VCAM) FLT-1 (fms-like umors, VEGF-A receptor O Vascular endothelial umors, yrosine kinase) inflammation growth factor related inflammation heparan sulfate umors, p protein inflammation Vascular endothelial umors, K hepatocyte growth umors, I growth factor-A inflammation actor receptor (c-met) inflammation receptor insulin-like growth umors, R actor/mannose-6- inflammation von Willebrand factor- umors, L phosphate receptor related antigen inflammation Integrins: Tumors, laminin receptor D,

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Comments/ Comments/ Vector type Target areas of use Ref Vector type Target areas of use Ref bestatin Aminopeptidases Tumours, to chosen myocardial ([(2S,3R)-3 e.g. On diseased vessels Amino-2-hydroxy cell surfaces Structure 4-phenyl Other chemicals Any of above Any diseased 58, butanoyl]-L with structure targets - or may or normal 59, leucine determined be unknown when structure of 60 hydrochloride) during make functional interest, e.g. Pefabloc (4-(2 Serine proteases Tumours, generation selection of thrombi, tumours aminoethyl) vessel wall process vector binding or walls of benzenesulfonyl etc. to chosen myocardial fluoride diseased vessels hydrochloride) Structure Commercially Angiotensin Endothelial available converting cells inhibitors enzyme e.g. kaptopril [0166] Carbohydrate Vectors enalapri ricionopril Low specificity Coagulation Vessel wall non-peptidic factors injury, Vector type Target Comments/areas of use Ref compounds tumours, etc. Ineo- macrophages general activation/ Protease nexins proteoglycans 56 glycoproteins inflammation (extracellular oligosaccharides Asialo- liver 61 protease with terminal glycoprotein inhibitors) galactose receptor Antithrombin proteoglycans, 57 Hyaluronan aggrecan (a 62 Coagulation proteoglycan) factors “link proteins” cell-surface receptors: CD44 Mannose Blood brain barrier, 63 Brain tumours and other [0165] Vectors From Combinatorial Libraries diseases causing changes in BBB Bacterial Blood brain barrier, 64 glycopeptides Brain tumours and other Comments/ diseases causing changes in Vector type Target areas of use Ref BBB Antibodies with Any of above Any diseased 58, Structure argets - or may or normal 59, determined be unknown when structure of 60 [0167] (Glyco)Lipid Vectors during make functional interest, e.g. generation selection of thrombi, tumours process vector binding or walls of o chosen myocardial Vector type Target Comments/areas of use Ref diseased vessels Structure GM1 gangliosides cholera bacteria diagnosis/treatment of Peptides with Any of above Any diseased 58, in the cholera sequence argets - or may or normal 59, gastrointestinal determined be unknown when structure of 60 tract during make functional interest, e.g. platelet PAF receptors diagnosis of inflammation generation selection of thrombi, tumours activating process vector binding or walls of factor (PAF) o chosen myocardial antagonists diseased vessels Prostoglandin Prostoglandin diagnosis of inflammation Structure antagonists of receptors Oligonucleotides Any of above Any diseased 58, inflammation with sequence argets - or may or normal 59, Thromboxane Leukotriene diagnosis of inflammation determined be unknown when structure of 60 antagonists of receptors during make functional interest, e.g. inflammation generation selection of thrombi, tumours process vector binding or walls of o chosen myocardial diseased vessels [0168] Small Molecule Vectors Structure Modifications of Any of above Any diseased 58, oligos obtained argets - or may or normal 59, as above be unknown when structure of 60 Vector type Target Comments/areas of use Ref make functional interest, e.g. selection of thrombi, tumours Adrenalin Corresponding vector binding or walls of receptors US 2004/0141922 A1 Jul. 22, 2004 20

chloride, ambenoxan, ambroxol, ambruticin, ambucaine, -continued ambucetamide, ambuphylline, ambuside, ambutonium bro mide, amcinafal, amcinafide, amcinomide, amdinocillin, Vector type Target Comments/areas of use Ref amdinocillin pivoxil, amebucort, amedalin, ametantrone, Betablockers Adrenergic beta- Myocardium for beta-1 amezepine, amezinium metilsulfate, amfenac, amfepen receptors blockers Alpha-blockers Adrenergic Vessel wall torex, amfetaminil, amflutizole, amfonelic acid, amicar alpha-receptors balide, amicibone, amicloral, amicycline, amidantel, ami benzodiazepines dapsone, amidephrine, amiflamine, amifloverine, serotonin- Serotonin analogues receptors amifloxacin, amifostine, amikacin, amikhelline, amiloride, anti-histamines Histamine- Vessel wall aminacrine, amindocate, amineptine, aminobenzoic acid, receptors aminocaproic acid, aminoethyl nitrate, aminoglutethimide, Acetyl-choline ACh-receptors aminohippuric acid, aminometradine, aminopentamide, receptor antagonists aminophylline, aminopromazine, aminopterin, aminopyrine, verapamil Caº-channel Heart muscle aminoquinol, aminoquinuride, aminorex, aminosalicyclic blocker acid, aminothiadiazole, aminothiazole, amiodarone, amiper nifedipin Caº-channel Heart muscle one, amipheazole, amipizone, amiprilose, amiquinsin, ami blocker sometradine, amisulpride, amiterol, amithiozone, amitraz, amitriptyline, amitriptylinoxide, amixetrine, amlexanox, amlodipine, amobarbital, amodiaquine, amogastrin, amol [0169] Representative examples of drugs useful in accor anone, amonofide, amoproxan, amopyroquin, amorolfine, dance with the invention include: abamectin, abundiazole, amocanate, amosulalol, amotriphene, amoxapine, amox acaprazine, acabrose, acebrochol, aceburic acid, acebutolol, ecaine, amoxicillin, amoxydramine camsilate, amperozide, acecainide, acecarbromal, aceclidine, aceclofenac, acedap amphecloral, amphenidone, amphetamine, amphotalide, sone, acediasulfone, acedoben, acefluranol, acefurtiamine, amphotericin B, ampicillin, ampiroxicam, amprolium, acefylline clofibrol, acefylline piperazine, aceglatone, ace ampyrimine, ampyzine, amduinate, amrinone, amsacrine, glutamide, aceglutamide aluminium, acemetacin, acenocou amygdalin, amylene, amylmetacresol, amyl nitrite, anage marol, aceperone, acepromazine, aceprometazine, acequino stone acetate, anagrelide, anaxirone, anazocine, anazolene, line, acesulfame, acetaminophen, acetaminosalol, ancarolol, ancitabine, androstanediol, androstanol propi acetanilide, acetarSone, acetazolamide, acetergamine, ace onate, androstenetrione, androstenonol propionate, anethole, tiamine, acetiromate, acetohexamide, acetohydroxamic anguidine, anidoxime, anilamate, anileridine, aniline, anilo acid, acetomeroctol, acetophenazine, acetorphine, acetosul pam, anipamil, aniracetam, anirolac, anisacril, anisindione, fone, acetriozate, acetryptine, acetylcholine chloride, ace anisopirol, anisoylbromacrylic acid, anitrazafen, anpirtoline, tylcolchinol, acetylcysteine, acetyldigitoxin, acetylleucine, ansoxetine, antafenite, antazoline, antazonite, anthelmycin, anthiolimine, anthralin, anthramycin, antienite, antimony acetylsalicyclic acid, acevaltrate, acexamic acid, acifran, potassium tartrate, antimony thioglycollate, antipyrine, acipimox, acitemate, acitretin, acivicin, aclantate, aclarubi antrafenine, apalcillin, apazone, apicycline, apomorphine, cin, aclatonium napadisilate, acodazole, aconiazide, apovincamine, apraclonidine, apramycin, aprindine, aconitine, acoxatrine, acridorex, acrihellin, acrisorcin, acriv aprobarbital, aprofene, aptazapine, aptocaine, arabinosylm astine, acrocinide, acronine, actinoquinol, actodigin, acyclo ercaptopurine, aranotin, arbaprostil, arbekacin, arclofenin, vir, adafenoxate, adamexine, ademetionine, adenosine phos arfendazam, arginine, arginine glutamat, arildone, arnolol, phate, adibendan, adicillin, adimolol, adinazolam, aronixil, arotinolol, arpinocid, arpromidine, arsanilic acid, adiphenine, aditeren, aditoprim, adrafinil, adrenalone, aflo arsthinol, artemisinin, articaine, asaley, ascorbic acid, ascor qualone, afurolol, aganodine, ajmaline, aklomide, alacepril, byl palmitate, asocainol, aspartame, aspartic acid, asperlin, alafosfalin, alanine mustard, alanosine, alaproclate, alaza aspoxicillin, astemizole, atamestane, atenolol, atipamezole, nine triclofenate, albendazole, albendazole oxide, albuterol, atiprosin, atolide, atracurium besilate, atromepine, atropine, albutoin, alclofenac, alcometasome dipropionate, alcloxa, atropine oxide, auranofin, aurothoiglucose, aurothiogly alcuronium chloride, aldioxa, aldosterone, alepride, ale canide, avilamycin-A, avridine, axamozide, azabon, azabu tamine, alexidine, alfacalcidol, alfadex, alfadolone, perone, azacitodine, azaclorzine, azaconazole, azacosterol, alfaprostol, alfaxalone, alfentanil, alfuzosin, algestone azacyclonol, azaftozine, azaguanidine, azaloxan, azametho acetonide, algestone acetophenide, alibendol, aliconazole, nium bromide, azamulin, azanator, azanidazole, azaperone, alifedrine, aliflurane, alimadol, alinidine, alipamide, alitame, azapicyl, azaprocin, azaquinzole, azaribine, azarole, azaser alizapride, allantoin, alletorphine, allobarbital, alloclamide, ine, azaspirium chloride, azastene, azastrptonigrin, azatod allocupreide, allomethadione, allopurinol, allylestrenol, ine, azathioprine, azauridine, azelastine, azepexole, azepin allyl isothicyanate, allylprodine, allylthiourea, almadrate dole, azetepa, azidamfenicol, azidocillin, azimexon, sulfate, almasilate, almecillin, almestrone, alminoprofen, azintamide, azipramine, azithromycin, azlocillin, azolimine, almitrine, almoxatone, alonacic, alonimid, aloxistatin, aloz azosemide, azotomycin, aztreonam, azumolene, bacampicil afone, alpertine, alphacetylmethadol, alphameprodine, lin, baclofen, bacmecillinam, balsalazide, bamaluzole, bam alphamethadol, alphaprodine, alphavinylaziridinoethyl buterol, bamethan, bamifylline, bamipine, bamnidazole, acetate, alpidem, alpiropride, alprazolam, alprenolol, alpros baquiloprim, barbexaclone, barbital, barucainide, batilol, tadil, alrestatin, altanserin, altapizone, alteconazole, althiaz bazinaprine, becamthone, beclamide, beclobrate, beclom ide, altrenogest, altretamine, aluminium acetate, aluminium ethasone dipropionate, beclotiamine, befiperide, befunolol, clofibrate, aluminium subacetate, alverine, amadinone befuraline, bekanamycin, belarizine, beloxamide, bemar acetate, amafolone, amanozine, amantadine, amantanium inone, bemegride, bemetizide, bemitradine, benactyzine, bromide, amantocillin, ambasilide, ambazone, ambenonium benafentrine, benanserin, benapryzine, benaxibine, US 2004/0141922 A1 Jul. 22, 2004

benazepril, bencianol, bencisteine, benclonidine, bency bufogenin, buformin, bufrolin, bufuralol, bumadizone, clane, bendamustine, bendazac, bendazol, benderizine, ben bumecaine, bumepidil, bumetanide, bumetrizole, bunaftine, droflumethiazide, benethamide penicillin, benexate, benflo bunamidine, bunamiodyl, bunaprolast, bunazosin, buni rex, benfosformin, benfotiamine, benfurodil hemisuccinate, trolol, bunolol, buparvaquone, bupicomide, bupivacaine, benhepazone, benidipine, benmoxin, benolizime, benorilate, bupranolol, buprenorphine, bupropion, buquineran, benorterone, benoxafos, benoxaprofen, benoximate, benperi buquinolate, buquiterine, buramate, burodiline, buspirone, dol, benproperine, benrixate, bensalan, benserazide, bensul busulfan, butabarbital, butacaine, butacetin, butaclamol, dazic acid, bentazepam, bentemazole, bentiamine, bentipi butadiazamide, butafosfan, butalamine, butalbital, butam mine, bentiromide, benurestat, benzaldehyde, benzalkonium ben, butamirate, butamisole, butamoxane, butanediol cyclic chloride, benzaprinoxide, benzarone, benzbromarone, ben sulfite, butanilicaine, butanixin, butanserin, butantrone, Zestrol, benzethidine, benzethonium chloride, benzetimide, butaperazine, butaprost, butaverine, butedronate, buterizine, benzilonium bromide, benzindopyrine, benziodarone, ben butetamate, butethamine, buthiazide, butibufen, butidrine, Zmalecene, benznidazole, benzobarbital, benzocaine, ben butikacin, butilfenin, butinazocine, butinoline, butirosin, zoclidine, benzoctamide, benzodepa, benzododecinium butixirate, butobendine, butoconazole, butocrolol, butocta chloride, benzoic acid, benzoin, benzonatate, benzopyrro mide, butofilolol, butonate, butopamine, butopiprine, buto nium bromide, benzoquinium chloride, benzotript, benzox prozine, butopyrammonium iodide, butorphanol, butoxam iquine, benzoxonium chloride, benzoyl peroxide, ben ine, butoxylate, butriptyline, butropium bromide, butylated zoylpas, benzphetamine, benzpiperylon, benzpyrinium hydroxyanisole, butylated hydroxytoluene, butylparaben, bromide, benzquercin, benzquinamide, benzthiazide, benz butynamine, buzepide metiodide, cabastine, cabergoline, tropine, benzydamine, benzylpenicillin, benzylsulfamide, cadralazine, cafaminol, cafedrine, caffeine, calcifediol, cal beperidium iodide, bephenium naphtoate, bepiastine, bepri citrol, calcium citrate, calcium dobesilate, calcium glubion dil, beraprost, berberine sulfate, bermastine, bermoprofen, ate, calcium gluceptate, calcium gluconate, calcium glyc berythromycin, besulpamide, beslunide, beta carotene, beta erophosphate, calcium hypophosphite, calcium lactate, cetylmethadol, betahistine, betaine, betameprodine, calcium lactobionate, calcium levulinate, calcium mande betamethadol, betamethasone, betamethasone acetate, late, calcium pantothenate, calcium phosphate dibasic, cal betamethasone acibutate, betamethasone benzoate, cium phophate tribasic, calcium saccharate, calcium stear betamethasone dipropionate, betamethasone phosphate, ate, calusterone, camazepam, cambendazole, camiverine, betamethasone valerate, betamicin, betaprodine, betaxolol, camostast, camphotamide, camptothecin, camylofin, can betazole, bethanechol chloride, bethanidine, betiatide, bisol, cannabinol, cannenoic acid, cannenone, cantharidine, betoxycaine, bevantolol, bevonium metilsulfate, bezafibrate, capobenic acid, capreomycin, caproxamine, capsaicine, bezitramide, bialamicol, bibenzonium bromide, bibrocathol, captamine, captodiame, captopril, capuride, caracemide, bicifadine, biclodil, biclofibrate, biclotymol, bicozamycin, caramiphen, carazolol, carbachol, carbadox, carbaldrate, bidimazium iodine, bietamiverine, bietaserpine, bifemelane, carbamazepine, carbamide peroxide, carbantel lauryl sul bifepramide, bifluranol, bifonazole, binedaline, binfloxacin, fate, carbaril, carbarsone, carbaspirin calcium, carbazeran, binfibrate, bioallethrin, bioresmethrin, biotin, bipenamol, carbazochrome, carbazachrome salicylate, carbazachrome biperiden, biphenamine, biriperone, bisacodyl, bisantrene, sulfonate, carbazocine, carbeniciltin, carbenicillin indanyl, bis(aziridinyl) butanediol, bisbendazole, bisbentiamine, bis carbencillin phenyl, carbenoxolone, carbenzide, carbestrol, femazone, bisfentidine, bismuth betanaphthol, bismuth-trig carbetapentane, carbidopa, carbimazole, carbinoxamine, lycollamate, bismuth subgallate, bismuth subsalicylate, carbiphene, carbocloral, carbocysteine, carbofenotion, car bisorbin, bisoprolol, bisorcic, bioxatin acetate, bispyrithione bol-fuschin, carbomycin, carboplatin, carboprost, carbo magsulfex, bithionol, bithionoloxide, bitipazone, bitoterol, prost methyl, carboquone, carbromal, carbubarb, car bitoscantate, bleomycin, bluensomycin, bofumustine, bolan burazepam, carbutamide, carbuterol, carcainium chloride, diol dipropionate, bolasterone, bolazine, boldenone unde carebastine, carfentanil, carfimate, carisoprodol, carmanta cylenate, bolenol, bolmantalate, bometolol, bopindolol, bor dine, carmetizide, carmofur, carmustine, carnidazole, car naprine, bornaprolol, bornelone, botiacrine, boxidine, mitine, carocainide, caroverine, caroxazone, carperidine, brallobarbital, brazergoline, brefonalol, bremazocine, bre caperone, carphenazine, carpindolol, carpiramine, carpro quinar, bretylium tosylate, brindoxime, brivundine, brobac fen, carpronium chloride, carsalam, cartazolate, carteolol, tam, broclepride, brocresine, brocrinat, brodimoprim, bro carubicin, carumonam, carvedilol, carzenide, carzolamide, faromine, brofezil, brofoxine, brolaconazole, cathine, cathinone, cefaclor, cefadroxil, cefalonium, cefalo brolamfetamine, bromacrylide, bromadoline, bromamid, ram, cefamandole, cefamandole naftate, cefaparole, cefatriz bromazepam, bromchlorenone, bromebric acid, bromergu ine, cefazaflur, cefazedone, cefazolin, cefbuperazone, cef ride, brometenamine, bromfenac, bromhexine, bromindione, canel, cefcanel daloxate, cefedrolor, cefenmpidone, cefepime, bromisovalum, bromociclen, bromocriptine, bromodiphen cefetamet, cefetrizole, cefvitril, cefixime, cefinenoxime, hydramine, bromofenofos, bromopride, bromoxandide, bro cefinepidium chloride, cefinetazole, cefnminox, cefodizime, mperidol, bromperidol decanoate, brompheniramine, bro cefonizid, cefoperazone, ceforamide, cefotaxime, cefotetan, nopol, broparestrol, broperamole, bropirimine, cefotiam, cefoxazole, cefoxitin, cefpimizole, cefpiramide, broquinaldol, broSotamide, broSuximide, brotianide, broti cefpirome, cefpodoxime, cefpodoxime proxetil, cefaui zolam, brovanexine, brovincamine, broxaldine, broxaterol, nome, cefrotil, cefroxadine, cefsulodin, cefsumide, ceftazi broxitalamic acid, broxuridine, broxyquinoline, bruceantin, dime, cefteram, ceftezole, ceftiofur, ceftiolene, ceftioxide, brucine, bucainide, bucetin, buciclovir, bucillamine, bucin ceftizoxime, ceftriaxone, cefuracetime, cefuroxime, dolol, bucladesine, buclizine, buclosamide, bucloxic acid, cefuraxime axetil, cefurzonam, celiprolol, cephacetrile, bucolome, bucricaine, bucromarone, bucrylate, bucumolol, cephalexin, cephaloglycin, cephaloridine, cephalothin, budesonide, budipine, budotitane, budralazine, bufenadrine, cephapirin, cephradine, cetaben, cetamolol, cethexonium bufeniode, bufetolol, bufexamac, bufezolac, buflomedil, chloride, cetiedil, cetirizine, cetocycline, cetohexazine, US 2004/0141922 A1 Jul. 22, 2004 22 cetophenicol, cetotiamine, cetoxime, cetraxate, chaulmosul clomifenoxide, clominorex, clomiphene, clomipramine, clo fone, chendiol, chiniofon, chlophedianol, chloracyzine, mocycline, clomoxir, clonazepam, clonazoline, clonidine, chloral betaine, chloral hydrate, chloralose, chlorambucil, clonitazene, clonitrate, clonixeril, clonixin, clopamide, clo chloramine, chloramphenicol, chloramphenicol palmitate, penthixol, cloperastine, cloperidone, clopidogrel, clopidol, chloramphenicol succinate, chlorazanil, chlorbenzoxamine, clopimozide, clopipazan, clopirac, cloponone, cloprednol, chlorbetamide, chlorcyclizine, chlordantoin, chlordiazep cloprostenol, cloprothiazole, cloquinate, cloquinozine, clo oxide, chlordimorine, chlorhexadol, chlorhexidine, chlo racetadol, cloranolol, clorazepate, clorethate, clorexolone, rhexidine phosphanilate, chlorindanol, chlorisondamine clorgiline, cloricromen, cloridarol, clorindanic acid, clorin chloride, chlormadinone acetate, chlormerodrin, chlormeza dione, clormecaine, cloroperone, clorophene, cloroqualone, none, chlormidazole, chloronaphazine, chloroazodin, chlo clorotepine, clorprenaline, clorsulon, clortermine, closantel, robutanol, chlorocresol, chlorodihydroxyandrostenone, closiramine, clostebol, clothiapine, clothixamide, clotiaz chloroethyl mesylate, 5-chloro-3'-fluoro-2'3-dideoxyuri epam, cloticasone propionate, clotioxone, clotrimazole, clo dine, chloroguanide, chlorophenothane, chloroprednisone voxamine, cloxacepride, cloxacillin, cloxacillin benzathine, acetate, chloroprocaine, chloropyramine, chloroquine, chlo cloxazolam, cloxestradiol, cloximate, cloxotestosterone, roserpidine, chlorothen, chlorothiazide, chlorotriansene, cloxypendyl, cloxyquin, clozapine, cobamide, cocaine, chloroxine, chloroxylenol, chlorozotocin, chlorphemesin, cocarboxylase, codeine, codoxime, cofisatin, cogazocine, chlorphemesin carbamate, chlorpheniramine, chlorphenoc colchicine, colestolone, colfenamate, colforsin, colterol, tium amsonate, chlorphenoxamine, chlorphentermine, chlo conessine, conorphone, copper gluconate, cormethasone rproethazine, chlorproguanil, chlorpromazine, chlorpropam acetate, corticosterone, cortisone acetate, cortisuzol, corti ide, chlorprothixene, chlorquinaldol, chlortetracycline, vazol, cortodoxone, cotarnine chloride, cotinine, cotrip chlorthalidone, chlorthenoxazine, chlorZoaxazone, chloecal tyline, coumaphos, coumazoline, coumermycin, coumetarol, ciferol, cholic acid, choline chloride, choline glycerophos creatinolfosfate, crismatol, croconazole, cromakalim, cromi phate, chromocarb, chromonar, ciadox, ciamexon, cianer trile, cromolyn, cropropamide, crospovidone, crotamiton, goline, cianidol, cianopramine, ciapilome, cicaprost, crotetamide, crotoniazide, crufomate, cuprimyxin, cuproxo cicarperOne, ciclactate, ciclafrine, ciclazindol, cicletanine, line, cyacetacide, cyamemazine, cyanocobalamine, cyclacil ciclomenol, ciclonicate, ciclonium bromide, ciclopirox, lin, cyclandelate, cyclarbamate, cyclazocine, cyclazodone, ciclopramine, cicloprofen, cicloprolol, ciclosidomine, ciclo cyclexanone, cyclindole, cycliramine, cyclizine, cyclobar tizolam, ciclotropium bromide, cicloxilic acid, cicloxolone, bital, cyclobendazole, cyclobenzaprine, cyclobutoic acid, cicortonide, cicrotic acid, cidoxepin, cifenline, cifostodine, cyclobutyrol, cyclofenil, cycloguanil, cloheximide, cyclo ciglitazone, ciheptolane, ciladopa, cilastatine, cilazapril, leucine, cyclomenol, cyclomethicone, cyclomethycaine, cilazaprilat, cilobamine, cilofungin, cilostamide, cilostazol, cyclopentamine, cyclopenthiazide, cyclopentolate, cyclo ciltoprazine, cimaterol, cimenoxin, cimepanol, cimetidine, penazine, cyclophosphamide, cyclopregnol, cyclopyrro cimetropium bromide, cimoxatone, cinchonine, cinchophen, nium bromide, cycloserine, cyclosporine, cyclothiazide, cinecromen, cinepaxadil, cinepazet, cinepazic acid, cyclovalone, cycotiamine, cycrimine, cyheptamide, cyhep cinepazide, cinfenine, cinfenoac, cinflumide, cingestol, cini tropine, cynarine, cypenamine, cypothrin, cyprazepam, tapride, cinmetacin, cinnamaverine, cinnamedrine, cinnariz cyprenophine, cyprodenate, cyproheptadine, cyprolidol, ine, cinnarizine clofibrate, cinnofuradione, cincotramide, cyproduinate, cyproterone acetate, cyproximide, cystine, cinodine, cinolazepam, cinoquidox, cinoaxin, cinoxate, cytarabine, dacarbazine, dacemazine, dacisteine, dacinomy cinoxolone, cinooxopazide, cinperene, cinprazole, cinpro cin, dacuronium bromide, dagapamil, dalbraminol, daleda pazide, cinromide, cintazone, cintriamide, cinperone, lin, daltroban, dametralast, damotepine, danazol, danitracen, ciprafamide, ciprafazone, ciprefadol, ciprocinomide, ciprofi danosteine, danthron, dantrolene, dapiprazole, dapsone, brate, ciprofloxacin, cipropride, ciproduaZone, ciprostene, daptomycin, darenzepine, darodipine, datelliptium chloride, ciramadol, cirazoline, cisapride, cisconazole, cismadinone, dunorubicin, dazadrol, dazepinil, dazidamine, dazmegrel, cisplatin, cistinexine, citalopram, citatepine, citenamide, cit dazolicine, dazopride, dazoquinast, dacoxiben, deanol ace enazone, citicoline, citiolone, clamidoxic acid, clamox glumate, deanol acetaminobenzoate, deazauridine, debox yguin, clanfenur, clanobutin, clantifen, clarithromycin, cla amet, debrisoquin, decamethonium bromide, decimenide, vulanic acid, clazolam, clazolimine, clazuril, clebopride, decitropine, declaben, declenperone, decloxizine, decomi clefamide, clemastine, clemeprol, clemizole, clenbuterol, nol, decoquinate, deditonium bromide, deferoxamine, clempirin, cletoquine, clibucaine, clidafidine, clidanac, clidi deflazacort, defosfamide, dehydroacetic acid, dehydroemet num bromide, climazolam, climbazole, climiqualine, clin ine, dehydro-7-methyltestosterone, delanterone, delapril, damycin, clindamycin palmitate, clindamycin phosphate, delergotrile, delfantrine, delmadinone acetate, delmetacin, clinofibrate, clinolamide, cliquinol, clioxanide, clipoxamine, delmopinol, delorazepam, deloxone, delprostenate, dem cliprofen, clobazam, clobenoside, clobenzepam, cloben brexine, demecarium bromide, demeclocycline, demecol zorex, clobenztropine, clobetasol propionate, clobetasone cine, demecycline, demegestone, demelverine, demexip butyrate, clobutinol, clobuzarit, clocanfamide, cloca tiline, democonazole, demoxepam, denaverine, pramine, clociguanil, clocinizine, clocortolone acetate, clo denbufylline, denipride, denopamine, denpidazone, denzi cortolone pivalate, clocoumarol, clodacaine, clodanolene, mol, deoxyspergualin, depramine, deprodone, deprostil, clodazon, clodoxopone, clodronic acid, clofazimine, clofe deptropine, derpanicate, desacetylcolchicine tartrate, desas namic acid, clofenamide, clofenciclan, clofenetamine, pidin, desiclovir, descinolone acetonide, deserpidine, clofenoxyde, clofenvinfos, clofeverine, clofexamide, clof desipramine, deslanoside, desmethylcolchicine, desmeth eZone, clofibrate, clofibric acid, clofibride, clofilium phos ylmisonidazole, desmethylmoramide, desocriptine, phate, cloflucarban, clofoctol, cloforex, clofurac, clogestone desogestrel, desomorphine, desonide, desoximetasone, des acetate, cloguanamil, clomacran, clomegestone acetate, oxycorticosterone acetate, desoxycorticosterone pivalate, clometacin, clometherone, clomethiazole, clometocillin, desoxypyridoxine, detajmium bitartrate, detanosal, deter US 2004/0141922 A1 Jul. 22, 2004 23 enol, detomidine, detorubicin, detrothronine, devapamil, mide, disulfiram, disuprazole, ditazole, ditercalinium chlo dexamethasone, dexamethasone acefurate, dexamethasone ride, dithiazanine iodide, ditiocarb, ditionustine, acetate, dexamethasone dipropionate, dexamethasone phos ditolamide, ditophal, divabuterol, dixanthogen, dizatrifone, phate, dexamisole, dexbrompheniramine, dexchlorphe dizocilpine, dobupride, dobutamine, docarpamine, docona niramine, dexclamol, dexetimide, dexetozoline, dexfenflu zole, docusate, dolinacetam, domazoline, domiodol, domi ramine, deximafen, dexindoprofen, dexivacaine, phen bromide, domipizone, domoprednate, domoxin, dom dexlofexidine, dexmedetomidine, dexoxadrol, dexpan peridone, don, donetidine, dopamantine, dopamine, thenol, dexpropranolol, dexproxibutene, dexecoverine, dex dopexamine, dopropidil, dogualast, dorastine, doreptide, tilidine, dextroamphetamine, dextrofemine, dextromethor dosergoside, dotarizine, dotefonium bromide, dothiepin, phan, dextromoramide, dextrorphan, dextrothyroxine, doxacurium chloride, doxaminol, doxapram, doxaprost, dezaguanine, dezocine, diacerein, diacetamate, diacetolol, doxazosin, doxefazepam, doxenitoin, doxepin, doxibetasol, diacetylmorphine, diamfenetide, diaminomethylphena doxifluridine, doxofylline, doxorubicin, doxpicomine, doxy zinium chloride, diamocaine, diampromide, diamthazole, cycline, doxylamine, dramedilol, draquinolol, deazidox, dianhydrogalactitol, diapamide, diarbarone, diathymosul dribendazole, drindene, drobuline, drocinonide, droclid fone, diatrizoic acid, diaveridine, diazepam, diaziquone, inium bromide, drocode, drofenine, droloxifene, drometri diazoacetylglycine hydrazide, diazouracil, diazoxide, zole, dromostanolone, dromostanolone propionate, dronab dibekacin, dibemethine, dibenamine, dibenzepin, dibro inol, dropempine, droperidol, droprenilamine, dropropizine, mpropamidine, dibromsalan, dibrospidium chloride, drotaverine, drotebanol, droxacin, droxicainide, droxicam, dibucaine, dibuprol, dibupyrone, dibusadol, dicarbine, droxidopa, droxypropine, dulofibrate, dulozafone, duometa dicarfen, dichlorallyl lawsone, dichlorisone acetate, dichlo cin, duoperone, dupracetam, durapatite, dyclonine, rmezanone, dichlorofluormethane, dichloromethotrexate, dydrogesterone, dymanthine, dyphylline, ebastine, ebroti dichlorophen, dichlorophenarsine, dichlorotetrafluoroet dine, ebselen, ecastolol, echinomycin, echothiophate iodide, hane, dichloroxylenol, dichlorphenamide, dichlorvos, dici ecipramidil, eclanamine, eclazolast, econazole, ectylurea, ferron, dicirenone, diclazuril, diclofenac, diclofensine, edelfosine, edetic acid, edetol, edifolone, edogestrone, diclofurime, diclometide, diclonixin, dicloxacillin, dicobalt edoxudine, edrophonicum chloride, efaroxan, efetozole, edetate, dicolinium iodide, dicresulene, dicumarol, dicyclo eflornithine, efloxate, efrotomycin, elantrine, elanzepine, mine, didemnin, dideoxycytidine, didrovaltrate, dieldrin, elderfield’s pyrimidine mustard, elfazepam, ellagic acid, dienestrol, dienogest, diethadione, diethazine, diethylpro elliptinium acetate, elmustine, elnadipine, eltenac, eltopra pion, diethylstilbestrol, diethylstilbestrol diphosphate, dieth zine, elucaine, elziverine, embramine, embutramide, eme ylstilbestrol dipropionate, diethylthiambutene, diethyltolua pronium bromide, emetime, emiglitate, emilium tosylate, mide, dietifen, difebarbamate, difemerine, difemetorex, emopanil, emorfazone, emylcamate, enalapril, enalaprilat, difenamizole, difencloxazine, difenoximide, difenoxin, dife enbucrilate, encainide, enciprazine, enclomiphene, tarsone, difeterol, diflorasone diacetate, difloxacin, diflua encyprate, endomide, endralazine, endrysone, enefexine, nine, diflucortolone, diflurcortolone pivalate, diflumidone, enestebol, enfenamic acid, enflurane, eniclobrate, enilcona diflunisal, difluprednate, diftalone, digalloyl trioleate, digi zole, enilospirone, enisoprost, enocitabine, enolicam, enoxa toxin, digoxin, dihexyverine, dihydralazine, dihydroazacy cin, enoxamast, enoximone, enoxolone, eniprazole, enipro tidine, dihydroergotamine, dihydrolemperone, dihydrostrep line, emprazepine, emprofylline, empromate, emprostil, tomycin, dihydrotachysterol, dihydroxyfluoroprogestrone, enrofloxacin, entsufon sodium, enviomycin, enviradene, diisopromine, diisopropanolamine, dilazep, dilevalol, epalretat, epanolol, eperisone, ephedrine, epicainide, epicil dilmefone, diloxanide, diltiazem, dimabefylline, dimecam lin, epicriptine, epiestriol, epimestrol, epinastine, epineph ine, dimecolonium iodide, dimecrotic acid, dimefadane, rine, epinephryl borate, epipropidine, epirizole, epiroprim, dimefline, dimelazine, dimenorfan, dimenhydrinate, epirubicin, epithiazide, epitiostanol, epoprostenol, epostane, dimenoxadol, dimeheptanol, dimepranol, dimepregnen, eprazinone, eprovafen, eproxindine, eprozinol, epsiprantel, dimeprozan, dimercaprol, dimesna, dimesone, dimetacrine, eptaloprost, eptazocine, equilin, erdosteine, ergocalciferol, dimetamfetamine, dimethadione, dimethaminostyrylquino ergoloid mesylates, ergonovine, ergosterol, ergotamine, eri line, dimethazan, dimethindene, dimethiodal, dimethiso colol, erizepine, erocainide, erythrityl tetranitrate, erythro quin, dimethisterone, dimetholizine, dimethoxanate, dim mycin, erythromycin acistrate, erythromycin ethylsuccinate, ethylhydroxytestosterone, dimethylnorandrostadienone, erythromycin propionate, erythrosine, esaprazole, escu dimethylnortestosterone, dimethylstilbestrol, dimethyl, dim lamine, eseridine, esflurbiprofen, esmolol, esorubicin, esp ethylthiambutene, dimethyltubocurarinium chloride, roquin, estazolam, estradiol, estradiol benzoate, estradiol dimetipirium bromide, dimetofrine, dimetridazole, dimina cypionate, estradiol dipropionate, estradiol enanthate, estra zene, dimoxamine, dimoxaprost, dimoxyline, dimpylate, diol undecylate, estradiol valerate, estramustine, estramus dinaline, dinazafone, diniprofylline, dinitolmide, dinoprost, tine phosphate, estrapronicate, estrazinol, estriol, estrofu dinoprostone, dinsed, diosmin, dioxadilol, dioxadrol, diox rate, estrone, estrone hydrogen sulfate, estropipate, amate, dioxaphetyl butyrate, dioxethedrin, dioxifedrine, esuprone, etabenzarone, etacepride, etafedrine, etafenone, dioxybenzone, dipenine bromide, diperodon, diphemanil etamestrol, etamiline, etamiphyllin, etamocycline, etanida methylsulfate, diphenadione, diphenan, diphenhydramine, zole, etanterol, etaqualone, etasuline, etazepine, etazolate, diphendiol, diphenoxylate, diphenylpraline, diphoxazide, etebenecid, eterobarb, etersalate, ethacridine, ethacrynic dipipanone, dipipoverine, dipiverin, diprafenone, diprenor acid, ethambutol, ethamivan, ethamsylate, ethanolamine phine, diprobutine, diprofene, diprogulic acid, diproleando oleate, ethaverine, ethchlorvynol, ethenzamide, ethazide, mycin, diprodualone, diproteverine, diprotriozate, diproxa ethidium chloride, ethinamate, ethinyl estradiol, ethiofos, dol, dipyridamole, dipyrithione, dipyrocetyl, dipyrone, ethionamide, ethsterone, ethoheptazine, ethomoxane, etho dirithromycin, disobutamide, disofenin, disogluside, disopy nam, ethopropazine, ethosuximide, ethotoin, ethoxazene, ramide, disoxaril, distigmine bromide, disulergine, disulfa ethoxazorutoside, ethoxzolamide, ethyybenztropine, ethyl US 2004/0141922 A1 Jul. 22, 2004 24 biscoumacetate, ethyl carfluzepate, ethyl cartrizoate, ethyl drostanol, fluorodopane, fluorohydroxyandrosterone, fluo dibunate, ethyl dirazepate, ethylenediamine, ethylestrenol, rometholone, fluorometholone acetate, fluorosalan, 6-fluo ethylhydrocupreine, ethyl loflazepate, ethylmethylthiam roteStoSterOne propionate, fluorouracil, butene, ethylmorphine, 9-ethyl-6-mercaptopurine, ethyl 9-fluoroxotestenololactone, 9-fluoroxotestololacetone, flu nitrite, ethylnorepinephrine, ethylparaben, ethylphenace otracen, fluoxetine, fluoxymesterone, fluparoxan, flupen mide, ethylstibamine, ethynerone, ethynodiol diacetate, tixol, fluperamide, fluperlapine, fluperolone acetate, ethypicone, etibendazole, eticlopride, eticyclidine, eti fluphenazine, fluphenazine enanthate, flupimazine, flupir docaine, etidronic acid, etifelmine, etifenin, etifoxine, etil time, flupranone, fluprazine, fluprednidene, fluprednisolone, amfetamine, etilefrine, etilefrine pivalate, etintidine, etiochl fluprednisolone valerate, fluprofen, fluprofylline, fluprodua anolone, etipirium iodide, etiproston, etiracetam, etiroxate, zone, fluprostenol, fluquazone, fluradoline, flurandrenoline, etisazole, etisomicin, etisulergine, etizolam, etocarlide, etoc flurantel, flurazepam, flurbiprofen, fluretofen, flurithromy rylene, etodolac, etodroxzine, etofamide, etofenamate, cin, flurocitabine, flurofamide, flurogestone acetate, etofemprox, etofibrate, etoformin, etofuradine, etofylline, flurothyl, fluroxene, flusoxolol, fluspiperone, fluspirilene, etoglucid, etolorex, etolotifen, etoloxamine, etomidate, eto flutamide, flutazolam, flutemazepam, flutiazin, fluticasone midoline, etomoxir, etonitazene, etoperidone, etoposide, propionate, flutizenol, flutonidine, flutoprazepam, flutroline, etoprindole, etoprine, etorphine, etosalamide, etoxadrol, flutropium bromide, fluvoxamine, fluzinamide, fluzoperine, etoxeridine, etozolin, etrabamine, etretinate, etryptamine, folescutol, folic acid, fomidacillin, fominoben, fomocaine, etymemazine, eucalyptol, eucatropine, eugenol, euprocin, fonazine, fopirtoline, forfenimex, formebolone, formetorex, evandamine, Evans blue, exalamide, exametazine, formintrazole, formocortal, formoterol, fosarilate, fos exaprolol, exepanol, exifone, exiproben, falintolol, fali azepam, foscarnet, foscolic acid, fosenazide, fosfocreatine, pamil, famiraprinium chloride, famotidine, famotine, fami fosfomycin, fosfonet, fosfosal, fosinapril, fosmenic acid, profazone, fanetizole, fantridone, fazadinium bromide, faz fosmidomycin, forgirate, fostedil, fostriecin, fotemustine, aribine, febantel, febarbamate, februpol, febuverine, fotreamine, frabuprofen, frentizole, fronepidil, froxiprost, feclemine, feclobuzone, fedrilate, felbamate, felbinac, ftaxilide, ftivazide, ftorafur, ftormetazine, ftorpropazine, felipyrine, felodipine, femoxetine, femabutene, fenacetinol, fubrogonium iodide, fuchsin, fumagillin, fumoxcillin, fupra fenaclon, fenadiazole, fenaptic acid, femalamide, femalcom zole, furacrinic acid, furafylline, furalazine, furaltadone, ine, fenamifuril, penamole, fenaperone, fenbendazole, fen furaprofen, furazabol, furazolidone, furazolium chloride, bencillin, fenbufen, fenbutrazate, fencamfamine, fenci furbucillin, furcloprofen, furegrelate, furethidine, furfeno butirol, fenclexonium metilsulfate, fenclofenac, fenclonine, rex, furidarone, furmethoxadone, furobufen, furodazole, fenclorac, fenlozic acid, fendiline, fendosal, feneritrol, furofenac, furomazine, furosemide, furostilbestrol, fursalan, fenestrel, fenethazine, fenethylline, fenetradil, fenflumizole, fursultiamine, furterene, furtrethonium iodide, fusidic acid, fenfluramine, fenfluthrin, fengabine, fenharmane, fenimide, fuzlocillin, gabapentin, gabexate, gaboxadol, galantamine, feniodium chloride, femipentol, femirofibrate, fenisorex, fen gallamine triethodide, gallopamil, galosemide, galtifenin, metozole, femmetramide, fenobam, fenocinol, fenoctimine, gampexine, gamolenic acid, ganciclovir, ganglefene, gapi fenofibrate, fenoldopam, fenoprofen, fenoterol, fenoverine, comine, gapromidine, gefarnate, gemazocine, gemcadiol, fenoxazoline, fenoxedil, fenozolone, fenpentadiol, femper gemeprost, gemfibrozil, gentamicin, gentian violet, gepe ate, fenipalone, femipramide, femiprane, fempiverinium bro frine, gepirone, geroquinol, gestaclone, gestadienol, mide, femprinast, femproporex, femprostalene, fenduizone, gestodene, gestonorone caproate, gestrinone, giparmen, fenretinide, fenspiride, fentanyl, fentiazac, fenticlor, fenti gitaloxin, gitoformate, glafenine, glaziovine, gliamilide, gli conazole, fentonium bromide, fenyripol, fepentolic acid, bornuride, glibutimine, glicaramide, glicetanile, geroquinol, fepitrizol, fepradinol, feprazone, fepromide, feprosidnine, gestaclone, gestadienol, gestodene, gestonorone caproate, ferriclate calcium, ferrotremine, ferrous fumarate, ferrous gestrinone, giparmen, gitaloxin, gitoformate, glafenine, gluconate, fetoxylate, fexicaine, fexinidazole, fezatione, glaziovine, gliamilide, glibornuride, glibutimine, glicara fezolamine, fiacitabine, fibracillin, filenadol, filipin, fifexide, mide, glicetanile, gliclazide, glicondamide, glidazamide, flamenol, flavamine, flavodic acid, flavodil, flavoneactic gliflumide, glimepiride, glipentide, glipizide, gliquidone, acid, flavoxate, flazalone, flecainide, flerobuterol, fleroxacin, glisamuride, glisindamide, glisolamide, glisoxepide, glox flesinoxan, flestolol, fletazepam, floctafenine, flomoxef, azone, gloximonam, glucametacin, glucosamine., glucosul flopropione, florantyrone, flordipine, floredil, florfenicol, famide, glucosulfone, glucurolactone, glucuronamide, glu florifemine, flosequinan, flotrenizine, floverine, floxacillin, nicate, glutamic acid, glutaral, glutarimide, glutaurine, floxacrine, floxuridine, fluacizine, flualamide, fluanisone, glutethimide, glyburide, glybuthiazol, glybuzole, glyceryl fluazacort, flubanilate, flubendazole, flubepride, flucabril, monostearate, glycidyl methacrylate, glycine, glyclopyra flucetorex, flucindole, fluciprazine, flucloronide, flucona mide, glybiarsol, glycopyrrolate, glycyclamide, glyhexam zole, flucrylate, flucytosine, fludalanine, fludarabine phos ide, glymidine, glyoctamide, glypinamide, glyprothiazol, phate, fludazonium chloride, fludiazepam, fludorex, fludox glysobuzole, gold thiomalate, gold sodium thiosulfate, gra opone, fludrocortisone acetate, flufenamic acid, flufemisal, nisetron, griseofulvin, guabenxan, guacetisal, guafecainol, flufosal, flufylline, fluindarol, fluindione, flumazenil, flume guaiactamine, guaiapate, guaietolin, guaifenesin, guaimeSal, cinol, flumedroxone-17-acetate, flumequine, flumeridone, guaisteine, guaithylline, guamecycline, guanabenz, guana flumethasone, flumethasone pivalate, flumethiazide, flume cline, guanadrel, guanazodine, guanazole, guanclofine, tramide, flumexadol, flumezapine, fluminorex, flumizole, guancydine, guanethidine, guanfacine, guanisoquin, guano flumoxonide, flunamine, flunarizine, flunidazole, clor, guanoctine, guanoxabenz, guanoxan, guanoxyfen, flunisolide, flunisolide acetate, flunitrazepan, flunixin, fluno hadacidin, halazepam, halazone, halcinomide, halethazole, prost, flunoxaprofen, fluocinolone acetonide, fluocinomide, halocortolone, halofantrine, halofenate, halofuginone, flourcortin butyrate, fluocortolone, fluocortolone caproate, halometasone, halonamine, halopemide, halopenium chlo fluorescein, fluoresone, fluoroadenosine, 3-fluoroan ride, haloperidol, haloperidol decanoate, haloperidone US 2004/0141922 A1 Jul. 22, 2004 25 acetate, haloprogesterone, haloprogin, halothane, halox acid, iozomic acid, ipexidine, ipodic acid, ipragratine, ipra azolam, haloxon, halquinols, hedaquinium chloride, hep midil, ipratropium bromide, iprazochrome, ipriflavone, ronicate, heptabarbital, heptaminol, heptaverine, heptola iprindole, iprocinodine, iproclozide, iprocrolol, iprofenin, mide, hepzidine, hetacillin, hetaflur, heteronium bromide, iproheptine, iproniazid, iproidazole, iproplatin, iprotiazem, hexachlorophene, hexacyclonate, hexacyprone, hexadiline, iproxamine, iprozilamine, ipsalazide, ipsapirone, iguin hexadimethrine bromide, hexafluorenium bromide, hexam damine, irindalone, irloxacin, irolapride, irsogladine, isam ethonium bromide, hexamidine, hexapradol, hexaprofen, fazone, isamoltan, isamoxole, isaxonine, isbogrel, isepami hexapropymate, hexasonium iodide, hexacarbacholine bro cin, isoaminile, isobromindione, isobucaine, isobutamben, mide, hexedine, hexestrol, hexetidine, hexobarbital, hex isocarboxazid, isoconazole, isocromil, isoetharine, isof obendine, hexocyclium methylsulfate, hexoprenaline, hex ezolac, isoflupredone acetate, isoflurane, isoflurophate, iso opyrronium bromide, hexylcaine, hexylene glycol, leucine, isomazole, isomerol, isometamidium, isometha hexylresorcinol, histamine, histapyrrodine, homarylamine, done, isometheptene, isomylamine, isoniazid, isomixin, homatropine, homatropine methylbromide, homidium bro isoprazone, isoprednidene, isoprofen, isoprofamide iodide, mide, homochlorcyclizine, homofenazine, homoharringto isopropicillin, isopropyl myristate, isopropyl palmitate, iso nine, homopipramol, homosalate, homotestosterone propi proterenol, isosorbide, isosorbide dinitrate, isosorbide onate, homprenorphine, hopantenic acid, hoquizil, mononitrate, isospalglumic acid, isosulfan blue, isosulpride, hycanthone, hydracarbazine, hydralazine, hydrargaphen, isothipendyl, isotic, isotiquimide, isotretinoin, isoxaprolol, hydrobentizide, hydrochlorthiazide, hydrocodone, hydro isoxepac, isoxicam, isoxSuprine, isradipine, itanoxone, itazi cortamate, hydrocortisone, hydrocortisone aceponate, grel, itraconazole, itrocainide, ivermectin bib, ivoqualine, hydrocortisone acetate, hydrocortisone butyrate, hydrocor josamycin, kainic acid, kalafungin, kanamycin, kebuzone, tisone cypionate, hydrocortisone-phosphate, hydrocortisone keracyanin, ketamine, ketanserin, ketazocine, ketazolam, succinate, hydrocortisone valerate, hydroflumethiazide, kethoxal, ketipramine, ketobemidone, ketocaine, ketocainol, hydromadinone, hydromorphinol, hydromorphone, hydro ketoconazole, ketoprofen, ketorfanol, ketorolac, ketotifen, quinone, hydroximdasate, hydroximdasol, hydroxyoxocobal ketotrexate, khellin, khelloside, kitasamycin, labetalol, laci amin, hydroxy amphetamine, hydroxychloroquine, dipine, lactalfate, lactose, lactulose, lamotrigine, lamtidine, hydroxydimethandrostadienone, hydroxydione succinate, lanatoside, lapachol, lapinone, lapyrium chloride, lasalocid, hydroxymethylandrostanone, 10-hydroxymorehisterone, laudexium methyl sulfate, lauralkonium chloride, laureth, hydroxypethidine, hydroxyphenamate, hydroxyprocaine, laurixamine, laurocapram, lauroguadine, laurolinium hydroxyprogeserone, hydroxyprogesterone caproate, acetate, lauryl isoquinolinium, lefetamine, leflunomide, hydroxypyridine tartrate, hydroxystilbamidine, 7-hydrox leiopyrrole, lemidosul, lenampicillin, leniquinsin, lemper ytestololacetone, hydroxytestosterone propionate, hydrox one, leptacline, lergotrile, letimide, letosteine, leucine, leu ytetracaine, hydroxytoluic acid, hydroxyurea, hydroxyzine, cinocaine, leucocianidol, leucovorin, levacecarnine, leval hymecromone, hyoscyamine, hypericin, ibacitabine, iba lorphan, levamfetamine, levamisole, levdropropizine, floxacin, ibazocine, ibopamine, ibrotamide, ibudilast, levisoprenaline, levlofexidine, levobunolol, levocabastine, ibufenac, ibuprofen, ibuprofen piconol, ibuproxam, ibuterol, levocarnitine, levodopa, levofacetoperane, levofenflu ibuverine, icazepam, icosipiramide, icotidine, idarubicin, ramine, levofuraltadone, levoglutamide, levomenol, idaverine, idazoxan, idebenone, idenast, idoxuridine, idral levomethadone, levomethadyl acetate, levomethorphan, fidine, idrocilamide, idropranolol, ifenprodil, ifosfamide, levometionmeprazine, levomopranol, levomoramide, levo ifoxetine, ilmofosine, iloprost, imafen, imanixil, imazodan, nantradol, levonordeprin, levonorgestrel, levophenacyl mor imcarbofos, imexon, imiclopazine, imidazole salicylate, phan, levopropoxyphene, levopropylcillin, levopropyl imidazopyrazole, imidecyl iodine, imidocarb, imidoline, hexedrine, levoprotiline, levorin, levorphanol, imidurea, imiloxan, iminophendimide, imipenem, imi levothyroxime, levoxadrol, lexofenac, libecillide, libenza pramine, imipraminoxide, imirestat, imolamine, imoxiterol, pril, lidamidine, lidocaine, lidofenin, lidoflazine, lifibrate, impacarzine, impromidine, improsulfan, imuracetam, ina lilopristone, limaprost, lincomycin, lindane, linsidomine, perisone, indacrinone, indalpine, indanazoline, indanidine, liothyronine, liroldine, lisinopril, lisuride, lithium carbonate, indanorex, indapamide, indatraline, indacainide, indelox lithium citrate, litracen, lividomycin, lixazinone, lobeline, azine, indenolol, indicine-N-oxide, indigotindisulfonic acid, lobendazole, lobenzarit, lobuprofen, locicortone, lodax indobufen, indocate, indocyanine green, indolapril, indol aprine, lodacezarlodinixil, lodiperone, lodoxamide, lodoxa idan, indomethacin, indopanolol, indopine, indoprofen, mide ethyl, lofemizole, lofendazam, lofentanil, lofepramine, indoramin, indorenate, indoxole, indriline, inicarone, inoco lofexidine, loflucarban, lombazole, lomefloxacin, lometra terone, inosine, inosine dialdehyde, inositol niacinate, inp line, lomevactone, lomifylline, lomofungin, lomustine, lona roquone, intrazole, intriptyline, iobenzamic acid, iobutic palene, lonaprofen, lonazolac, lonidamine, loperamide, lop acid, iocarmic acid, iocetamic acid, iodamide, iodecimol, eramide oxide, lopirazepam, loprazolam, loprodiol, iodetryl, iodipamide, iodixanol, iodoalphionic acid, iodol, lorajmine, lorapride, loratadine, lorazepam, lorbamate, iodophthalein, iodoquinol, iodothiouracil, iodoxamic acid, lorcainide, lorcinadol, lorglumide, lormetazepam, lorta ioglicic acid, ioglucol, ioglucomide, ioglunide, ioglycamic lamine, lorzafone, losindole, losulazine, lotifazole, lotrifen, acid, iogulamide, iohexol, iodlidonic acid, iolixanic acid, lotucaine, lovastatin, loxanast, loxapine, loxiglumide, loxo iomeglamic acid, iomeprol, iomorinic acid, iopamidol, profen, loxtidine, lozilurea, lucanthone, lucartamide, luci iopanoic acid, iopentol, iophendylate, iophenoxic acid, mycin, lufuradom, lupitidine, luprostiol, luxabendazole, ioprocemic acid, iopromide, iopronic acid, iopydol, iopy lyapolate sodium, lycetamine, lydimycin, lymecycline, done, iosarcol, iosefamic acid, ioseric acid, iosimide, iosu lynestrenol, lysergide, lysine, mabuterol, maduramicin, lamide, iosumetic acid, iotasul, iotetric acid, iothalamic acid, mafenide, mafoprazine, mafosfamide, magnesium citrate, iotranic acid, iotrizoic acid, iotrolan, iotroxic acid, ioversol, magnesium gluconate, magnesium salicylate, malathion, ioxabrolic acid, ioxaglic acid, ioxitalamic acid, ioxotrizoic malethamer, malic acid, malotilate, manidipine, manganese US 2004/0141922 A1 Jul. 22, 2004 26 gluconate, mannitol, mannitol hexanitrate, mannomustine, mamide, methyl nicotinate, 2-methyl-19-nortestosterone, mannosulfan, manozodil, maprotiline, maridomycin, marip 2-methyl-11-oxoprogestrone, methyl palmoxirate, meth tiline, maroxepin, maytansine, mazaticol, mazindol, ylparaben, methylphendiate, methylprednisolone, methyl mazipredone, mebanazine, mebendazole, mebenoside, prednisolone aceponate, methylprednisolone acetate, meth mebeverine, mebezonium iodide, mebhydrolin, mebiquine, ylprednisolone hemisuccinate, methylprednisolone mebolazine, mebrofenin, mebutamate, mebutizide, phosphate, methylprednisolone suleptanate, methyl salicy mecamylamine, mecarbinate, mecetronium ethylsulfate, late, methylstreptonigrin, 4-methyltestosterone, 7-methylt mechlorethamine, meciadanol, mecinarone, meclizine, estosterone, 17-methyltestosterone, 7-methyltesosterone meclocycline, meclocycline sulfosalicylate, meclofenamic propionate, methylthionosine, 16-methylthioprogestone, acid, meclofenoxate, meclonazepam, mecloqualone, meclo methylthiouracil, methynodiol diacetate, methyprylon, ralurea, meclorisone dibutyrate, mecloxamine, mecobal methysergide, metiamide, metiapine, metiazinic acid, amin, mecrylate, mecysteine, medazepam, medazomide, metibride, meticrane, metildigoxin, metindizate, metioprim, medetomidine, medibazine, medifoxamine, medorinone, metioxate, metipirox, metipranolol, metiprenaline, medorubicin, medrogestone, medronic acid, medroxalol, metitepine, metizoline, metkephamid, metochalcone, meto medroxyprogestrone, medroxyprogestrone acetate, medry cinium iodide, metoclopramide, metocurine iodide, lamine, medrysone, mefeclorazine, mefenamic acid, mefeni metofenazate, metogest, metolazone, metomidate, metopi dil, mefenidramium metilsulfate, mefenorex, mefeserpine, mazine, metopon, metoprine, metoprolol, metoquizine, mefexamide, mefloquine, mefruside, megalomicin, mege metoserpate, metostilenol, metoxepin, metrafazoline, strol acetate, meglitimide, megucycline, meglumine, meglu metralindole, metrazifone, metrenperone, metribolone, met tol, meladrazine, melarsonyl, melarsoprol, melengestrol rifonate, metrifudil, metrizamide, metrizoic acid, metronida acetate, meletimide, melinamide, melitracen, melizame, zole, meturedepa, metyrapone, metyridine, metyrosine, meloxicam, melperone, melphalan, memantine, memotine, mevastatin, mexafylline, mexazolam, mexenone, mexilet menabitan, menadiol, menadiol diphosphate, menadiol dis ine, mexiprostil, mexoprofen, mexrenoate, mezacopride, ulfate, menadione, menadione sodium bisulfite, menate mezepine, mezilamine, mezlocillin, mianserin, mibolerone, trenone, menbutone, menfegol, menglytate, menitrazepam, micinicate, miconazole, micronomicin, midaflur, midagli menoctone, menogaril, menthol, meobentine, meparfynol, zole, midalcipran, midamaline, midazogrel, midazolam, mepazine, mepenzolate bromide, meperidine, mephenesin, midecamycin, midodrine, mifentidine, mifepristone, mifo mephenoxalone, mephentermine, mephenyton, mephobar bate, miglitol, mikamycin, milacemide, milemperone, mili bital, mepindolol, mepiprazole, mepiroxol, mepitiostane, pertine, miloxacin, milrinone, milverine, mimbane, mepivacaine, mepixanox, mepramidil, meprednisone, mep minaprine, minaxolone, mindolilol, mindoperone, minepen robamate, meproscillarin, meproxitol, meprylcaine, tate, minocromil, minocycline, minoxidil, mioflazine, mipi meptazinol, meduidox, meduinol, meguitazine, meralein, mazole, mirincamycin, miristalkonium chloride, miropro meralluride, merbarone, merbromin, mercaptamine, mer fen, mirosamicin, misonidazole, misoprostol, mitindomide, captomerin, mercaptopurine, mercuderamide, mercufenol mitobronitol, mitoclomine, mitoguazone, mitolactol, mito chloride, mercumatilin, mercurobutol, mergocriptine, mycin, mitonafide, mitopodozide, mitoguidone, mitotane, merophan, mersalyl, mesabolone, mesalamine, mesecla mitotenamine, mitoxantrone, mitozolomide, mivacurium zone, mesna, mesocarb, meso-hexestrol, mesoridazine, chloride, mixidine, misoprostol, mitindomide, mitobronitol, mesipirenone, mestanolone, mesterolone, mestranol, mesu mitoclomine, mitoguazone, mitolactol, mitomycin, mitona dipine, mesulergine, mesulfamide, mesulfen, mesuprine, fide, mitopodozide, mitoguidone, mitotane, mitotenamine, metabromsalan, metacetamol, metaclazepam, metaglycodol, mitoxantrone, mitozolomide, mivacurium chloride, mixi metahexamide, metamelfalan, metamfazone, metamfepra dine, mizoribine, mobecarb, mobenzoxamine, mocimycin, mone, metampicillin, metamixin, metapramine, metaproter mociprazine, moclobemide, moctamide, modafinil, moda enol, metaraminol, metaterol, metaxalone, metazamide, line, mofebutazone, mofloverine, mofoxime, molfarnate, metazide, metazocine, metbufen, meteneprost, metergoline, molinazone, molindone, molracetam, molsidomine, metergotamine, metescufylline, metesculetol, metethohep mometasone furoate, monalazone disodium, monensin, tazine, metformin, methacholine chloride, methacycline, monobenzone, monoethanolamine, monometacrine, mono methadone, methadyl acetate, methallenestril, methallibure, phosphothiamine, monothioglycerol, monoxerutin, montine methalthiazide, methamphetamine, methandriol, meth lin, moperone, mopidamol, mopidralazine, moprolol, androstenolone, methaniazide, methantheline bromide, moduizone, morantel, morazone, morclofone, morforex, methaphenilene, methapyrilene, methaqualone, metharbital, moricizine, morinamide, morniflumate, morocromen, methastyridone, methazolamide, methdilazine, meth moroxydine, morpheridine, morphine, morsuximide, mot enamine, methenolone acetate, methenolone enanthate, apizone, motrazepam, motretinide, moveltipril, moxadolen, metheptazine, methestrol, methetoin, methicillin, methima moxalactam, moxaprindine, moxastine, moxaverine, mox zole, methiodal sodium, methioguanine, methiomeprazine, azocine, moxestrol, moxicoumone, moxipraquine, moxisy methionine, methisazone, methitural, methixene, methocar lyte, moxnidazole, moxonidine, mupirocin, murabutide, bamol, methohexital, methopholine, methoserpidine, meth murocainide, muzolimine, mycophenolic acid, my fadol, otrexate, methotrimeprazine, methoxamine, methoxsalen, myralact, myrophine, myrtecaine, nabazenil, nabilone, nabi methoxyflurane, methoxyphedrine, methoxyphenamine, tan, naboctate, nabumetone, nadide, nadolol, nadoxolol, methoxypromazine, methscopolamine bromide, methsuxim naepaine, nafamostat, nafazatrom, nafcaproic acid, nafcillin, ide, methylclothiazide, N-methyladrealone hel, methyl alco nafenodone, nafenopin, nafetolol, nafimidone, nafiverine, hol, methylatropine nitrate, methylbenactyzium bromide, naflocort, nafonine, nafoxadol, nafoxidine, nafronyl, nafta methylbenzethonium, methylchromone, methyldesorphine, lofos, naftazone, naftifine, naftopidil, naftoxate, naftypra methyldihydromorphine, methyldopa, methyldopate, meth mide, nalbuphine, nalidixic acid, nalmefene, nalmexone, ylene blue, methylphedrine, methylergonovine, methylfor malorphine, naltrexone, naminterol, namoxyrate, nanapro US 2004/0141922 A1 Jul. 22, 2004 27 cin, nandrolone cyclotate; nandrolone decanoate, nan oxaceprol, oxacillin, oxadimedine, oxaflozane, oxaflu drolone phenpropionate, nanofin, mantradol, napactadine, mazine, oxagrelate, Oxalinast, oxaliplatin, oxamarin, oxam napamezole, naphazoline, naphthonone, naprodoxime, etacin, oxamisole, oxamniquine, oxanamide, oxandrolone, naproxen, naproxol, naranol, narasin, natamycin, nax oxantel, oxapadol, oxapium iodide, oxapropanium iodide, agolide, naxaprostene, nealbarbital, nebidrazine, nebivolol, oxaprotiline, oxaprozin, oxarbazole, oxatomide, oxazafone, nebracetam, nedocromil, nefazodone, neflumozide, nefo oxazepam, oxazidione, oxazolam, oxazorone, oxcarba pam, melezaprine, neoarsphenamine, neocinchophen, neo zepine, oxdralazine, oxeladin, oxendolone, oxepinac, oxet mycin, neostigmine bromide, nequinate, neraminol, ner acillin, oxethazaine, oxetorone, oxfendazole, oxfenicine, bacadol, nesapidil, mesosteine, netilmicin, netobimin, oxibendazole, oxibetaine, oxiconazole, oxidopamine, neutramycin, nexeridine, niacin, niacinamide, nialamide, oxidronic acid, oxifentorex, oxifungin, oxilorphan, oxi niaprazine, nibroxane, micafenine, nicainoprol, nicametate, monam, oxindanac, oxiniacic acid, oxiperomide, oxirac nicarbazin, nicarpidine, nicergoline, niceritrol, niceverine, etam, oxiramide, oxisopred, oxisuran, oxitefonium bromide, niclofolan, niclosamide, nicoboxil, nicoclonate, nicocodine, oxitriptan, oxitriptyline, oxitropium bromide, oxmetidine, nicocortonide, nicodicodine, nicofibrate, nicofuranose, nico oxodipine, oxogestone phenpropionate, oxolamine, oxolinic furate, nicogrelate, nicomol, nicomorphine, nicopholine, acid, oxomemazine, oxonazine, oxophenarsine, oxoprostol, nicorandil, nicothiazone, nicotinyl alcohol, nicoxamat, nic oxpheneridine, oxpremoate potassium, oxprenolol, oxtriph tiazem, nictindole, nodroxyzone, nifedipine, nifemalol, ylline, oxybenzone, oxybutynin, oxychlorosene, oxycin nifemazone, niflumic acid, nifluridide, nifuradene, nifurald chophen, oxyclozanide, oxycodone, oxydipentonium chlo eZone, nifuralide, nifuratel, nifuratrone, nifurdazil, nifuret ride, oxyfedrine, oxymesterone, oxymetazoline, hazone, nifurfoline, nifurimide, nifurizone, nifurmazole, oxymetholone, oxymorphone, oxypendyl, oxypertine, nifurmerone, nifuroquine, nifuroxazide, nifuroxime, nifurpi oxyphenbutazone, oxyphenonium bromide, oxypurinol, pone, nifurpirinol, nifurprazine, nifurduinazole, nifursemi oxypyrronium bromide, oxyquinoline, oxyridazine, oxyso zone, nifursol, nifurthiazole, nifurtimox, nifurtoinol, nifur nium iodide, oxytetracycline, oxytiocin, ozagrel, ozolinone, vidine, nifurzide, niguldipine, nihydrazone, nikethamide, pacrinolol, pactamycin, padimate, pafenolol, palatrigine, mileprost, nilprazole, niludipine, nilutamide, nilvadipine, paldimycin, palmidrol, palmoxiric acid, pamabrom, pam nimazone, nimeSulide, nimetazepam, nimidane, nimodipine, aquine, pamatolol, pamidronic acid, pancuronium bromide, nimorazole, nimustine, niometacin, niperotidine, nipradilol, panidazole, panomifene, patenicate, panthenol, pantothenic niprofazone, niridazole, nisbuterol, nisobamate, nisoldipine, acid, panuramine, papaverine, papaveroline, parachlorophe misoxetine, nisterime acetate, nitarsone, nitazoxanide, nithia mol, paraflutizide, paraldehyde, paramethadione, parametha mide, nitracrine, nitrafudam, nitralamine., nitramisole, some acetate, paranyline, parapenzolate bromide, para nitraquazone, nitrazepam, nitrefazole, nitrendipine, nitricho propamol, pararosaniline, pararosaniline embonate, line, nitrochlofene, nitrocycline, nitrodan, nitrofurantoin, paraxazone, parbendazole, parconazole, pareptide, pare nitrofurazone, nitroglycerin, nitromersol, nitromide, thoxycaine, pargeverine, pargolol, pargyline, paridocaine, nitromifene, nitroscanate, nitrosulfathiazole, nitroximil, parodilol, paromomycin, paroxetine, paroxypropione, nitroxoline, nivazol, nivimeldone, mixylic acid, nizatidine, parsalmide, partricin, parvaquone, pasiniazid, paulomycin, nizofenone, noberastine, nocloprost, nocodazole, nofecain paxamate, pazelliptine, pazoxide, pcnu, pecilocin, pecocy ide, nogalamycin, nolinium bromide, nomegestrol, nomeli cline, pefloxacin, pelanserin, pelretin, pelrinone, pemedolac, dine, nomifensine, nonabine, nonaperone, nonapyrimine, pemerid, pemoline, pempidine, penamecillin, penbutolol, monoxynol-4, nonoxynol-9, noracymethadol, norbolethone, pendecamaine, penfluridol, penflutizide, pengitoxin, peni norbudrine, norclostebol, norcodeine, nordazepam, norde cillamine, penicillin procaine, penicillin, penimepicycline, frin, nordinone, norepinephrine, norethandrolone, norethin penimocycline, penirolol, penmesterol, penoctonium bro drone, norethindrone acetate, norethynodrel, noreximide, mide, penprostene, pentabamate, pentacynium chloride, norfenefrine, norfloxacin, norfloxacin succinil, norflurane, pentaerythritol tetranitrate, pentafluranol, pentagastrin, pent norgesterone, norgestimate, norgestomet, norgestrel, norg agestrone, pentalamide, pentamethonium bromide, pentam estrienone, norletimol, norlevorphanol, normethadone, ethylmelamine, pentamidine, pentamoxane, pentamustine, normethandrone, normorphine, norpipanone, nortestoster pentapiperide, pentapiperium methylsulfate, pentaquine, one propionate, nortetrazepam, nortriptyline, norvinister pentazocine, pentetate calcium trisodium, pentetic acid, one, nosantine, noscapine, nosiheptide, novobiocin, noxip penthienate bromide, penthrichloral, pentiapine maleate, tiline, noxytiolin, nuclomedone, nuclotixine, mufenoxole, pentifylline, pentigetide, pentisomicin, pentisomide, pentizi nuvenzepine, nylestriol, nylidrin, nystatin, obidoxime, ocilt done, pentobarbital, pentolinium tartrate, pentomone, pen ide, ocrylate, octabenzone, octacaine, octafonium chloride, topril, pentorex, pentosan polysulfate sodium, pentostatin, octamoxin, octamylamine, octanoic acid, octapinol, octast pentoxifylline, pentrinitrol, pentylenetrazole, peplomycin, ine, octaverine, octazamide, octenidine, octenidine saccha pepstatin, peraclopone, peradoxime, perafensine, peralo rin, octicizer, octimibate, octorylene, octodrine, octopamine, pride, peraquinsin, perastine, peratizole, perbufylline, per octotiamine, octoxynol-9, octriptyline, octrizole, ofioxacin, fluamine, perflunafene, pergolide, perhexilene, periciazine, oformine, oftasceine, olaflur, olaquindox, oleanomycin, ole perimetazine, perindopril, perindoprilat, perisoxal, perlap timol, oleyl alcohol, olivomycin a, olmidine, olpimedone, ine, permethrin, perphenazine, persilic acid, petrichloral, olsalazine, oltipraz, olvanil, omeprazole, omidoline, omo pexantel, phanquone, phenacaine, phenacemide, phenacetin, conazole, omonasteine, onapristone, ondansetron, ontianil, phenacttropinium chloride, phenadoxone, phenaglycodol, opiniazide, opipramol, orazamide, orbutopril, orconazole, phenamazoline, phenampromide, phenarsone sulfoxylate, orestrate, ormetoprim, ornidazole, ornipressin, ornithine, phenazocine, phenazopyridine, phencarbamide, phencyclid ornoprostil, orotic acid, orotirelin, orpanoxin, orphenadrine, ine, phendimetrazine, phenelzine, pheneridine, phenesterin, ortetamine, osalmid, osmadizone, otilonium bromide, otim penethicillin, phenformin, phenglutarimide, phenicarbazide, erate sodium, ouabain, oxabolone cipionate, oxabrexine, phenindamine, phenindione, pheniprazine, pheniramine, US 2004/0141922 A1 Jul. 22, 2004 28 phenisonone, phenmetrazine, phenobarbital, phenobutiodil, prideperone, pridinol, prifelone, pri?inium bromide, prifuro phenolphtalein, phenolsulfonphthalein, phenomorphan, line, prilocalne, primaperone, primaquine, primidolol, phenoperidine, phenothiazine, phenothrin, phenoxyben primidone, primycin, prinomide, pristinamycin, prizidilol, zamine, phenoxypropazine, phenprobamate, phenprocou proadifen, probarbital, probenecid, probicromil, probucol, mon, phenpromethamine, phensuzimide, phentermine, procainamide, procaine, procarbazine, procaterol, prochlor phentolamine, phenylalanine, phenyl aminosalicylate, phe perazine, procinolol, procinomide, proclonol, procodazole, nylbutazone, phenylrphrine, phenylethyl alcohol, phenylm procyclidine, procymate, prodeconium bromide, prodili ercuric acetate, phenylmercuric borate, phenylmercuric dine, prodipine, prodolic acid, profadol, profexalone, profla chloride, phenylmercuric nitrate, phenylmethylbarbituric vine, proflazepam, progabide, progesterone, proglumetacin, acid, phenylpropanolamine, phenylthilone, phenyltoloxam proglumide, proheptazine, proligestone, proline, prolintane, ine, phenyramidol, phenytoin, phetharbital, pholcodine, prolonium iodide, promazine, promegestone, promestriene, pholedrine, phosphoramide mustard, phoxim, phthalofyne, promethazine, promolate, promoxolane, pronetalol, propac phthalysulfacetamide, phthalylsulfamethizole, phthalylsul etamol, propafenone, propamidine, propanidid, pro fathiazole, physostigmine, phytic acid, phytonadiol diphos panocaine, propantheline bromide, proparacaine, propatyl phate, phytonadione, pibecarb, pibenzimol, pibecarb, piben nitrate, propazolamide, propendiazole, propentofylline, pro zimol, piberaline, picafibrate, picartamide, picenadol, penzolate, properidine, propetamide, propetandrol, propicil picilorex, piclonidine, piclopastine, picloxydine, picoben lin, propikacin, propinetidine, propiolactone, propiomazine, Zide, picodralazine, picolamine, piconol, picoperine, pico propipocaine, propiram, propisergide, propiverine, prazole, picotamide, picotrin diolamine, picumast, pidolic propizepine, propofol, propoxate, propoxycaine, pro acid, pifarnine, pifenate, pifexole, piflutixole, pifoxime, poxyphene, propranolol, propyl docetrizoate, propylene gly piketoprofen, pildralazine, pilocarpine, pimoclone, pimefyl col, propylene glycol monostearate, propyl gallate, propyl line, pimelautde, pimetacin, pimethixene, pimetime, pime hexedrine, propyliodone, propylparaben, propylthiouracil, tremide, piminodine, pimobendan, pimondiazole, pimozide, propyperone, propyphenazone, propyromazine bromide, pinacidil, pinadoline, pinafide, pinaverium bromide, proguazone, produinolate, prorenoate potassium, proroxan, pinazepam, pincainide, pindolol, pinolcaine, pinoxepin, proscillaridin, prospidium chloride, prostalene, prosulpride, pioglitazone, pipacycline, pipamazine, pipaperone, prosultiamine, proterguride, protheobromine, prothipendyl, pipazethate, pipebuzone, pipecuronium bromide, pipemidic prothixene, protiofate, protionamide, protirelin, protizinic acid, pipenzolate bromide, pipequaline, piperacetazine, pip acid, protokylol, protoveratine, protriptyline, proxazole, eracillin, piperamide, piperazine, piperazinedione, piperi proxibarbal, proxibutene, proxicromil, proxifezone, proxor dolate, piperilate, piperocaine, piperoxan, piperylone, pipo phan, proxyphylline, prozapine, pseudoephedrine, psilocy broman, pipoctanone, pipofezine, piposulfan, pipotiazine bine, pumiteba, puromycin, pyrabrom, pyran copolymer, palmiate, pipoxizine, pipoxolan, pipradimadol, pipradol, pyrantel, pyrathiazine, pyrazinamide, pyrazofurin, pyricar pipramadol, pipratecol, piprinhydrinate, piprocurarium bate, pyridarone, pyridofylline, pyridostigmine bromide, iodide, piprofurol, piprozolin, piquindone, piquizil, pirac pyridoxine, pyrilamine, pyrimethamine, pyrimitate, pyrino etam, pirandamine, pirarubicin, piraxelate, pirazmonam, line, pyrithione zinc, pyrithyldione, pyritidium bromide, pirazolac, pirbenicillin, pirbuterol, pirdonium bromide, pyritinol, pyronine, pyrophenindane, pyrovalerone, pyrox pirenoxine, piremperone, pirenzepine, pirepolol, piretanide, amine, pyrrobutamine, pyrrocaine, pyrroliphene, pyrrolni pirfenidone, piribedil, piridicillin, piridocaine, piridoxilate, trin, pyrvinium chloride, pytamine, quadazocine, quadrosi piridronic acid, pirifibrate, pirindazole, pirinixic acid, pirin lan, quatacaine, quazepam, quazinone, quaZodine, ixil, piriprost, piriqualone, pirisudanol, piritramide, piritr quaZolast, quifenadine, quillifoline, quinacainol, quinacillin, exim, pirlimycin, pirlindole, pirmagrel, pirmenol, pirnabine, quinacrine, quinaldine blue, quinapril, quinaprilat, piroctone, pirogliride, piroheptine, pirolate, pirolazamide, quinazosin, quinbolone, quincarbate, quindecamine, quin piromidic acid, piroxantrone hel, piroxicam, piroxicam cin donium bromide, quindoxin, quinestradol, quinestrol, namate, piroxicillin, piroximone, pirozadil, pirprofen, quinethazone, quinetolate, quinezamide, quinfamide, pirquinozol, pirralkonium bromide, pirtenidine, pitenodil, quingestanol acetate, quingestrone, quindine, quinine, pitofenone, pituxate, pivampicillin, pivenfrine, pivopril, piv quinocide, quinpirole, quinterenol, quintiofos, quinuclium oxazepam, pizotyline, plafibride, plaunotol, pleuromulin, bromide, quinupramine, quipazine, quisultazine, racefen plicamycin, podilfen, podophylloxoxin, poldine methylsul ine, racemethionine, racemethorphan, racemetirosine, raclo fate, polidocanol, ploymyxin, polythiazide, ponalrestat, pon pride, ractopamine, rafoxanide, ralitoline, raloxifene, ram fibrate, porfiromycin, poskine, potassium guaiacolsulfonate, ciclane, ramefenazone, ramipril, ramiprilat, ramixotidine, potassium nitrazepate, potassium sodium tartrate, potassium ramnodignin, ranimustine, ranimycin, ranitidine, ranolazine, sorbate, potassium thiocyanate, practolol, prajmalium, prali rathyronine, razinodil, razobazam, razoxane, reboxetine, doxime chloride, pramipexole, pramiracetam, pramiverine, recainam, reclazepam, relomycin, remoxipride, renanolone, pramoxime, prampine, pranolium chloride, pranoprofen, rentiapril, repirinast, repromicin, reproterol, recimetol, res pranosal, prasterone, pravastatin, praxadine, prazepam, cinnamine, reserpine, resorantel, resorcinol, resorcinol prazepine, praziquantel, prazitone, prazocillin, prazosin, monoacetate, retelliptine, retinol, revenast, ribavirin, ribo preclamol, prednazate, prednazoline, prednicarbate, predni flavin, riboflavin 5'-phosphate, riboprine, ribostamycin, mustine, prednisolamate, prednisolone, prednisolone ridazolol, ridiflone, rifabutin, rifamide, rifampin, rifamycin, acetate, prednisolone hemisuccinate, prednisolone phos rifapentine, rifaximin, rilapine, rilmazafone, rilmenidine, phate, prednisolone steaglate, prednisolone tebutate, pred rilopirox, rilozarone, rimantadine, rimazolium metilsulfate, nisone, prednival, prednylidene, preferlamate, preg rim.cazole, rimexolone, rimiterol, rimoprogin, riodipine, rio menolone, pregnenolone succinate, premazepam, prostil, ripazepam, risocaine, risperidone, ristianol, ristoce prenalterol, premisteine, prenoverine, prenoxdiazine, preny tin, ritanserin, ritionetan, ritodrine, ritropirronium bromide, lamine, pretamazium iodide, pretiadil, pribecaine, pridefine, ritrosulfan, robenidine, rocastine, rociverine, rodocaine, US 2004/0141922 A1 Jul. 22, 2004 29 rodorubicin, rofelodine, roflurante, rokitamycin, roletamide, sulfamethizole, sulfamethoxazole, sulfamethoxypyridazine, rolgamidine, rolicyclidine, rolicyprine, rolipram, rolitetra sulfamethoxypyridazine acetyl, sulfametomidine, sulfame cycline, rolodine, rolziracetam, romifenone, romifidine, trole, sulfamonomethoxine, sulfamoxole, sulfanil amide, romactolol, ronidazole, ronifibrate, ronipamil, ronnel, ropi sulfanitran, sulfaperin, sulfaphenazole, sulfaproxyline, sul toin, ropivacaine, ropizine, roquinimex, rosaprostol, rosa fapyridine, sulfaquinoxaline, sulfarsphenamine, sulfasala ramicin, rosaramicin butyrate, rosaramicin propionate, zine, sulfasomizole, sulfasuccinamide, sulfasymazine, sul rosoxacin, rosterolone, rotamicillin, rotoxamine, rotraxate, fathiazole, sulfathiourea, sulfatolamide, sulfatroxazole, roxarsone, roxatidine acetate, roxibolone, roxindole, sulfatrozole, sulfazamet, sulfinalol, sulfinpyrazone, sulfiram, roxithromycin, roxolonium metilsulfate, roxoperone, sulfisomidine, sulfisoxazole, sulfisoxazole, sulfobromoph rufloxacin, rutamycin, rutin, ruvazone, sabeluzole, saccha thalein, sulfonethylmethane, sulfonmethane, sulfonterol, rin, salacetamide, salafibrate, salantel, Salazodine, Salazos sulforidazine, sulfoxone sodium, Sulicrinat, sulindac, Suli sulfadimedine, salazosulfamide, salazosulfathiazole, sale satin, sulisobenzone, sulmarin, Sulmazole, Sulmepride, thamide, Salfluverine, salicin, salicyl alcohol, salicylamide, sulinidazole, Sulocarbilate, Suloctidil, Sulosemide, salicylanilide, salicylic acid, salinazid, salinomycin, salme sulotroban, Suloxifen, Sulpiride, sulprosal, sulprostone, sul fanol, salmeterol, salmisteine, salprotoside, salsalate, salver tamicillin, sulthiame, sultopride, sultosilic acid, sultropo ine, sancycline, Sangivamycin, Saperconazole, sarcolysin, nium, Sulverapride, Sumacetamol, Sumatriptan, Sumetizide, sarmazenil, sarmoxicillin, sarpicillin, saterinone, satranida sunagrel, suncillin, supidimide, suproclone, suprofen, zole, Savoxepin, Scarlet red, scopafungin, Scopolamine, suramin, Suricainide, Suriclone, suxemerid, suxethonium seclazone, secnidazole, secobarbital, secoverine, securinine, chloride, suxibuzone, symclosene, symetime, synephrine, sedecamycin, Seganserin, seglitide, selegiline, selenium sul syrisingopine, taclamine, tacrine, taglutimide, talampicillin, fide, selprazine, sematilide, semustine, sepazonium chloride, talastine, talbutal, taleranol, talinolol, talipexole, talisomy seperidol, sequifenadine, serfibrate, Sergolexole, serine, ser cin, talmetacin, talmetoprim, talniflumate, talopram, talos metacin, serotonin, Sertaconazole, sertraline, setastine, set alate, taloximine, talsupram, taltrimide, tameridone, tameti azindol, setiptiline, setoperone, sevitropium mesilate, sevof cillin, tametraline, tamitinol, tamoxipen, tampramine, lurane, sevopramide, siagoside, sibutramine, siccanin, tandamine, taprostene, tartaric acid, tasuldine, taurocholic silandrone, silibinin, silicristin, silidianin, silver sulfadiaz acid, taurolidine, tauromustine, tauroselcholic acid, taurul ine, simetride, simfibrate, simtrazene, simvastatin, sinefun tam, taxol, tazadolene, tazanolast, tazaburate, tazeprofen, gin, sintropium bromide, sisomicin, sitalidone, sitofibrate, tazifylline, taziprinone, tazolol, tebatizole, tebuquine, sitogluside, sodium benzoate, sodium dibunate, sodium teclothiazide, teclozan, tedisamil, tefazoline, tefenperate, ethasulfate, sodium formaldehyde sulfoxylate, sodium gen tefludazine, teflurane, teflutixol, tegafur, telenzepine, tema tisate, sodium gualenate, sodium nitrite, sodium nitroprus floxacin, temarotene, temazepam, temefos, temelastine, side, sodium oxybate, sodium phenylacetate, sodium pico temocillin, temodox, temozolomide, temurtide, tenamfe fosfate, sodium picosulfate, sodium propionate, sodium tamine, tenilapine, teniloxazine, tenilsetam, teniposide, stibocaptate, sodium stibogluconate, sodium tetradecyl sul tenocyclidine, tenonitrozole, tenoxicam, tenylidone, teop fate, sodium thiosulfate, Sofalcone, solasulfone, Solpecainol, ranitol, teoprolol, tepirindole, tepoxalin, terazosin, terbin solypertine, somantadine, sopitazine, sopromidine, Sodui afine, terbucromil, terbufibrol, terbuficin, terbuprol, terbuta nolol, sorbic acid, sorbinicate, sorbinil, sorbitan monolau line, terciprazine, terconazole, terfenadine, terfluranol, rate, sorbitan monooleate, sorbitan monopalmitate, sorbitan terguride, terizidone, termidazole, terodiline, terofenamate, monostearate, sorbitan trioleate, sorbitan tristearate, sorbi teroxalene, teroxirone, terpin hydrate, tertatolol, tesicam, tol, Sorndipine, Sotalol, soterenol, spaglumic acid, sparfosic tesimide, testolactone, testosterone, testosterone cypionate, acid, sparsomycin, Sparteine, spectinomycin, spiclamine, testosterone enanthate, testosterone ketolaurate, testosterone spiclomazine, spiperone, spiradoline, spiramide, spiramy phenylacetate, testosterone propionate, tetrabarbital, tetra cin, spirapril, spiraprilat, spirendolol, spirgetine, spirilene, benazine, tetracaine, tetrachloroethylene, tetracycline, tetra spirofylline, spirogermanium, spiromustine, spironolactone, donium bromide, tetraethylammonium chloride, tetrahydro spiroplatin, spirorenone, spirotriazine, spiroxasone, spirox zoline, tetramethrin, tetramisole, tetrandrine, tetrantoin, atrine, spiroxepin, spizofurone, stallimycin, stanolone, stan tetrazepam, tetriprofen, tetronasin 5930, tetroquinone, zolol, stearic acid, stearyl alcohol, stearylsulfamide, Steffi tetroxoprim, tetrydamine, texacromil, thalicarpine, thalido mycin, stenbolone acetate, stepronin, stercuronium iodide, mide, thebacon, thebaine, thenalidine, thenium closylate, stevaladil, stibamine glucoside, stibophen, stilbamidine, stil thenyldiamine, theobromine, theodrenaline, theofibrate, bazium iodide, stilonium iodide, stirimazole, stiripentol, theophylline, thiabendazole, thiacetarsamide, thialbarbital, stirocainide, stirifos, streptomycin, streptonicozid, streptoni thiambutosine, thiamine, thiamiprine, thiamphenicol., thia grin, streptovarycin, streptozocin, strinoline, Strychnine, mcylal, thiazesim, thiazinamium chloride, thiazolsulfone, styramate, subathizome, subendazole, succimer, succinyl thiethyperazine, thihexinol methylbromide, thimerfonate, choline chloride, succinylsulfathiazole, succisulfone, thimerosal, thiocarbanidin, thiocarzolamide, thiocolchio Suclofenide, sucralfate, sucrose octaacetate, Sudexanox, side, thiofuradene, thioguanine, thioguanine alpha-deoxyri sudoxicam, sufentanil, sufosfamide, sufotidine, sulazepam, boside, thioguanine beta-deoxyriboside, thioguanosine, sulbactam, sulbactam pivoxil, sulbenicillin, sulbenox, sul thiohexamide, thioinosine, thiopental, thiopropazate, thio bentine, sulbutiamine, sulclamide, sulconazole, sulfabenz, properazine, thioridazine, thiosalan, thiotepa, thiotetrabar sulfabenzamide, sulfacarbamide, sulfacecole, sulfaceta bital, thiothixene, thiouracil, thiphenamil, thiphencillin, mide, sulfachlorpyridazine, sulfachrysoidine, sulfaclomide, thiram, thonzonium bromide, thonzylamine, thozalinone, sulfaclorazole, sulfaclozine, sulfacytine, sulfadiazine, sul threonine, thymidine, thymol, thymol iodide, thymopentin, fadicramide, sulfadimethoxine, sulfadoxine, sulfaethidole, thyromedan, thyropropic acid, tiacrilast, tiadenol, tiafibrate, sulfaguandide, sulfaguanole, sulfalene, sulfaloxic acid, tiamenidine, tiametonium iodide, tiamulin, tianafac, tianep sulfamazone, sulfamerazine, sulfameter, sulfamethazine, time, tiapamil, tiapirinol, tiapride, tiaprofenic acid, tiaprost, US 2004/0141922 A1 Jul. 22, 2004 30 tiaramide, tiazofurin, tiazuril, tibalosin, tibenalast sodium, prim, trimetozine, trimetrexate, trimexiline, trimipramine, tibenzate, tibezonium iodide, tibolone, tibric acid, tibrofan, trimoprostil, trimoxamine, trioxifene, trioxsalem, tripamide, tic-mustard, ticabesone propionate, ticarbodine, ticarcillin, triparanol, tripelennamine, tripotassium dicitratobismuthate, ticarcillin cresyl, ticlatone, ticlopidine, ticrynafen, tidiacic, triprolidine, tritiozine, tritoqualine, trityl cysteine, trixolane, tiemoium iodide, tienocarbine, tienopramine, tienoxolol, trizoxime, trocimine, troclosene potassium, trofosfamide, tifemoxone, tiflamizole, tiflorex, tifluadom, tiflucarbine, troleandomycin, troInitrate, tromantadine, tromethamine, tiformin, tifurac, tigemonam, tigestol, tigloidine, tilbro tropabazate, tropanserin, tropapride, tropatepine, tropen Ziline bromide, tropicamide, tropigline, tropiprine, tropod quinol, tiletamine, tilidine, tiliquinol, tilisolol, tilmicosin, ifene, trospectomycin, trospium chloride, troxerutin, troxip tilomisole, tilorone, tilozepine, tilsuprost, timefurone, tim ide, troxolamide, troxonium tosilate, troxypyrrolium egadine, timelotem, timepidium bromide, timiperone, tosilate, troxypyrrolium tosilate, truxicurium iodide, truxi timobesone acetate, timofibrate, timolol, timonacic, timo picurium iodide, tryparsamide, tryptophan, tryptophane prazole, tinabinol, tinazoline, tinidazole, tinisulpride, mustard, tuaminoheptane, tubercidine, tubocurarine chlo tinofedrine, tinoridine, tiocarlide, tioclomarol, tioconazole, ride, tubulozole, tuclazepam, tulobutrol, tuvatidine, tybam tioctilate, tiodazosin, tiodonium chloride, tionmergine, ate, tylocrebin, tylosin, tyramine, tyropanic acid, tyrosine, tiomesterone, tioperidone, tiopinac, tiopronin, tiopropamine, ubenimex, ubidecarenone, ubisindine, ufenamate, ufipra tiospirone, tiotidine, tioicacin, tioxamast, tioxaprofen, tioxi zole, uldazepam, ulobetasol, undecoylium chloride, unde dazole, tioxolone, tipentosin, tipepidine, tipetropium bro cyclenic acid, uracil mustard, urapidil, urea, uredepa, ure mide, tipindole, tipredane, tiprenolol, tiprinast, tipropidil, dofos, urefibrate, urethane, uridine, ursodeoxycholic acid, tiprostanide, tiprotimod, tiquinamide, tiquizium bromide, ursucholic acid, vadocaine, Valconazole, valdetamide, val tiratricol, tiropramide, tisocromide, tisopurine, tisoquone, dipromide, valine, valnoctamide, valofane, valperinol, val tivandizole, tixadil, tixanox, tixocortol pivalate, tizabrin, proate pivoxil, valproic acid, valpromide, valtrate, vanco tianidine, tizolemide, tizoprolic acid, tobramycin, tobuterol, mycin hel, vaneprim, vanillin, vanitolide, vanyldisulfamide, tocainide, tocamphyl, tocofenoxate, tocofibrate, tocopher vapiprost, vecuronium bromide, velnacrine maleate, ven Solan, todralazine, tofenacin, tofetridine, tofisoline, tofiso lafaxine, veradoline, veralipride, verapamil, verazide, ver pam, tolamolol, tolazamide, tolazoline, tolboxane, tolbuta ilopam, verofylline, vesnarinone, vetrabutine, vidarabine, mide, tolciclate, toldimfos, tolfamide, tolfenamic acid, vidarabine phophate, vigabatrin, viloxazine, viminol, vin tolgabide, tolimidone, tolindate, toliodium chloride, tolip barbital, vinblastine, vinburnine, vincamine, vincanol, vin rolol, tolmesoxide, tolmetin, tolnaftate, tolnapersine, cantril, vincofos, vinconate, vincristine, vindrburnol, vin tolnidamine, toloconium metilsulfate, tolonidine, tolonium desine, vindepidine, vinformide, vinglycinate, vinorelbine, chloride, toloxatone, toloxychlorinol, tolpadol, tolpentam vinpocetine, vinpoline, vinrosidine, vintiamol, vintriptol, ide, tolperisone, toliprazole, tolpromine, tolpropamine, vinylbital, vinylether, vinzolidine, viomycin, viprostol, tolpyrramide, tolduinzole, tolrestat, toltraZuril, tolufazepam, vidualine, viguidil, virginiamycin factors, viroxime, visna tolycaine, tomelukast, tomoglumide, tomoxetine, tomox dine, visnafylline, vitamin e, volazocine, warfarin, xamot iprole, tonazocine, topiramate, toprilidine, tonazocine, topi erol, xanoxic acid, xanthinol niacinate, xanthiol, xantifi ramate, toprilidine, topterone, toguizine, torasemide, toe brate, xantocillin, xenalipin, xenazoic acid, xenbucin, bafylline, toremifene, tosifen, tosufloxacin, tosulur, xenipentone, xenthiorate, xenygloxal, xenyhexenic acid, toyocamycin, toyomycin, traboxepine, tracazolate, tral xenytropium bromide, xibenolol, xibornol, xilobam, ximo onide, tramadol, tramazoline, trandolapril, tranexamic acid, profen, xinidamine, xinomiline, xipamide, xipranolol, xor tranilast, transcainide, trantelinium bromide, tranylcyprom phanol, xylamidine, xylazine, xylocoumarol, xylometazo ine, trapencaine, trapidil, traxanox, trazilitime, trazium esi line, xyloxemine, yohimbic acid, Zabicipril, zacopride, late, trazodone, trazolopride, trebenzomine, trecadrine, tre Zafuleptine, zaltidine, zapizolam, Zaprinast, Zardaverine, loxinate, trenbolone acetate, trengestone, trenizine, Zenazocine mesylate, zepastine, Zeranol, Zetidoline, Zidapa trosulfan, trepibutone, trepipam, trepirium iodide, treptil mide, zidometacin.., zidovudine, zilantel, Zimeldine, zimi amine, trequensin, trestolone acetate, trethinium tosilate, doben, zinc acetate, zinc phenolsulfonate, zinc unde trethocanoic acid, tretinoin, tretoquinol, triacetin, triafungin, cylenate, zindotrine, zindoxifene, Zinoconazole, Zinterol, triamcinolone, triamcinolone acetonide, triamcinolone Zinviroxime, zipeprol, zocainone, zofenopril, Zoficonazole, acetonide-phosphate, triamcinolone benetonide, triamcino zolamine, Zolazepam, Zolenzepine, zolertine, zolimidine, lone diacetate, triamcinolone furetonide, triamcinolone zoliprofen, Zoloperone, zolpidem, Zomebazam, Zomepirac, hexacetonide, triampyzine, triamterene, triazinate, triazi zometapine, zonisamide, zopiclone, Zorubicin, Zotepine, quone, triazolam, tribendilol, tribenoside, tribromoethanol, zoxazolamine, Zuclomiphene, Zuclophenthixol, Zylo tribromsalan, tribuzone, triacetamide, trichlormethiazide, furamine. trichlormethine, trichloroacetic acid, trichloroethylene, tric ribine phosphate, triclabendazole, triclacetamol, triclazate, [0170] The following non-limitative examples serve to triclobisonicum chloride, triclocarban, triclodazol, tri illustrate the invention. Confirmation of the microparticulate clofenol, piperazine, triclofos, triclofylline, triclonide, tri nature of products is performed using microscopy as closan, tricyclamol chloride, tridihexethyl chloride, trien described in WO-A-9607434. Ultrasonic transmission mea time, triethylenemelamine, triethylenephosphoramide, surements may be made using a broadband transducer to trifenagrel, trifezolac, triflocin, triflubazam, triflumidate, tri indicate microbubble suspensions giving an increased sound fluomeprazine, trifluoperazine, trifluperidol, triflupro beam attenuation compared to a standard. Flow cytometric mazine, trifluridine, triflusal, trigevolol, trihexyphenidyl, analysis of products can be used to confirm attachment of triletide, trilostane, trimazosin, trimebutine, trimecaine, tri macromolecules thereto. The ability of targeted medoxime bromide, trimeperidine, trimeprazine, trimetazi microbubbles to bind specifically to cells expressing a target dine, trimethadione, trimethamide, trimethaphan camsylate, may be studied in vitro by microscopy and/or using a flow trimethidinium methosulfate, trimethobenzamide, trimetho chamber containing immobilised cells, for example employ US 2004/0141922 A1 Jul. 22, 2004 ing a population of cells expressing the target structure and [0177] A solution of N-succinimidyl-3-maleimidopropi a further population of cells not expressing the target. onate (5.6 mg, 0.018 mmol) in tetrahydrofuran (0.2 ml) is Radioactive, fluorescent or enzyme-labelled streptavidin/ added to H2N-PEGs,00-DSPE (65 mg, 0.012 mmol) dis avidin may be used to analyse biotin attachment. solved in tetrahydrofuran (1 ml) and 0.1 M sodium phos phate buffer pH 7.5 (2 ml). The reaction mixture is heated to EXAMPLE 1. 30° C. and the reaction is followed to completion by TLC, whereafter the solvent is evaporated. Adhesion of poly-L-lysine-coated Phosphatidylserine-Encapsulated Microbubbles to [0178] d) Synthesis of RGDC-Mal-PEGs loo-DSPE (SEQ Endothelial Cells ID NO:1) [0179] Mal-PEGaloo-DSPE (0.010 mmol) in 0.1 M [0171] Poly-L-lysine (8 mg) having a molecular weight of sodium phosphate buffer having a pH of 7.5 is added to the 115 kDa was dissolved in water (400 ul). Freshly redispersed peptide RGDC (SEQ ID NO:1) (0.010 mmol). The reaction microbubbles of phosphatidylserine-encapsulated perfluo mixture is heated to 37° C. if necessary and the reaction is robutane (40 ul) were incubated in either water (400 ul) or followed by TLC to completion, whereafter the solvent is the poly-L-lysine solution for 15 minutes at room tempera removed. ture. Zeta potential measurements confirmed that the poly L-lysine-coated microbubbles were positively charged while [0180] e) Preparation of Gas-Filled Microbubbles Encap the uncoated bubbles were negatively charged. A cell adhe sulated by Phosphatidylserine and RGDC-Mal-PEGsaoo sion study using human endothelial cells grown in culture DSPE (SEQ ID NO:1) dishes was performed with the above-described [0181] To a mixture (5 mg) of phosphatidylserine (90-99.9 microbubbles, the uncoated microbubbles being used as a mol %) and Mal-PEGsaoo-DSPE (10-0.1 mol %) is added control. Microscopy of the endothelial cells after incubation 5% propylene glycol-glycerol in water (1 ml). The disper showed a much increased number of poly-L-lysine-coated sion is heated to not more than 80° C. for 5 minutes and then microbubbles adhering to endothelial cells in comparison to cooled to ambient temperature. The dispersion (0.8 ml) is the uncoated microbubbles. then transferred to a vial (1 ml) and the head space is flushed with perfluorobutane. The vial is shaken in a cap-mixer for EXAMPLE 2 45 seconds, whereafter the sample is put on a roller table. After centrifugation the infranatant is exchanged with 0.1 M Gas-Filled Microbubbles Comprising sodium phosphate buffer having a pH of 7.5. The peptide Phosphatidylserine and RGDC-Mal-PEGsaoo-DSPE RGDC (SEQ ID NO:1), dissolved in 0.1 M sodium phos (SEQ ID NO:1) phate buffer having a pH of 7.5, is added to the washed microbubbles, which are placed on the roller table. The [0172] a) Synthesis of Boc-NH-PEGaoo-DSPE (t-butyl washing procedure is then repeated. Carbamate poly(ethylene glycol)distearoylphosphati dylethanolamine) [0182] f) Alternative Preparation of Gas-Filled Microbubbles Encapsulated by Phosphatidylserine and [0173] DSPE (distearoylphosphatidylethanolamine) (31 RGDC-Mal-PEG, co-DSPE (SEQ ID NO:1) mg, Sygena Inc.) was added to a solution of Boc-NH PEGsaoo-SC (t-butyl carbamate poly(ethylene glycol)-suc [0183] To phosphatidylserine (5 mg) is added 5% propy cinimidyl carbonate) (150 mg) in chloroform (2 ml), fol lene glycol-glycerol in water (1 ml). The dispersion is heated lowed by triethylamine (33 ul). The mixture formed a clear to not more than 80° C. for 5 minutes and then cooled to solution after stirring at 41° C. for 10 minutes. The solvent ambient temperature. The dispersion (0.8 ml) is transferred was rotary evaporated and the residue taken up in acetoni to a vial (1 ml) and the head space is flushed with perfluo trile (5 ml). The thus-obtained dispersion was cooled to 4° robutane. The vial is shaken in a cap-mixer for 45 seconds, C. and centrifuged, whereafter the solution was separated whereafter the sample is put on a roller table. After cen from the undissolved material and evaporated to dryness. trifugation the infranatant is exchanged with 0.1 M sodium The structure of the resulting product was confirmed by phosphate buffer having a pH of 7.5. RGDC-Mal-PEGs loo NMR. DSPE (SEQ ID NO:1) dissolved in 0.1 M sodium phosphate buffer having a pH of 7.5 is added to the washed [0174] b) Synthesis of H2N-PEGsaoo-DSPE (amino-poly microbubbles, which are then placed on the roller table. The (ethylene glycol)-distearoylphosphatidylethanolamine) washing procedure is repeated following incorporation of the RGDC-Mal-PEGsaoo-DSPE (SEQ ID NO:1) into the [0175] Boc-NH-PEGs oo-DSPE (167 mg) was stirred in 4 microbubble membranes. M hydrochloric acid in dioxane (5 ml) for 2.5 hours at ambient temperature. The solvent was removed by rotary EXAMPLE 3 evaporation and the residue was taken up in chloroform (1.5 ml) and washed with water (2×1.5 ml). The organic phase Gas-Filled Microbubbles Encapsulated with was removed by rotary evaporation. TLC (chloroform/ Phosphatidylserine, Phosphatidylcholine and methanol/water 13:5:0.8) gave the title product with Rf=0.6; biotin-amidocaproate-PEGsaoo-Ala-cholesterol the structure of the product, which was ninhydrin positive, was confirmed by NMR. [0184] a) Synthesis of Z-Ala-cholesterol (3-O-(carboben zyloxy-L-alanyl)cholesterol) [0176] c) Synthesis of Mal-PEGs loo-DSPE (3-maleimi dopropionate poly(ethylene glycol)distearoylphosphati [0185] Cholesterol (4 mmol), Z-alanine (5 mmol) and dylethanolamine) dimethylaminopyridine (4 mmol) were dissolved in dimeth US 2004/0141922 A1 Jul. 22, 2004 32 ylformamide/tetrahydrofuran (20 ml+5 ml) and dicyclo [0197] To a mixture (5 mg) of phosphatidylserine and hexylcarbodiimide was added. The reaction mixture was phosphatidylcholine is added 5% propylene glycol-glycerol stirred at ambient temperature overnight. Dicyclohexylurea in water (1 ml). The dispersion is heated to not more than was filtered off and the solvent was rotary evaporated. The 80° C. for 5 minutes and then cooled to ambient temperature. residue was taken up in chloroform, undissolved dicyclo The dispersion (0.8 ml) is then transferred to a vial (1 ml) hexylurea was filtered off and the solvent was removed by and the head space is flushed with perfluorobutane. The vial rotary evaporation. The residue was placed on a column of is shaken in a cap-mixer for 45 seconds, whereafter the silica gel, and Z-Ala-cholesterol was eluted with toluene/ sample is put on a roller table. After centrifugation the petroleum ether (20:2) followed by toluene/diethyl ether infranatant is exchanged with water. Biotinamidocaproate (20:2). The fractions containing the title compound were PEGsaoo-Ala-cholesterol dissolved in water is added to the combined and the solvent was removed by rotary evapora washed microbubbles, which are placed on a roller table for tion. The structure of the product was confirmed by NMR. several hours. The washing procedure is repeated following [0186] b) Synthesis of Ala-cholesterol (3-O-(L-alanyl) incorporation of the biotinamidocaproate-PEGsaoo-Ala-cho cholesterol) lesterol into the microbubble membranes. [0187] Z-Ala-cholesterol (0.48 mmol) is placed in tetrahy EXAMPLE 4 drofuran (20 ml) and glacial acetic acid (3 ml) and hydro genated in the presence of 5% palladium on charcoal for 2 Gas-Filled Microbubbles Comprising Phosphati hours. The reaction mixture is filtered and concentrated in dylserine, Phosphatidylcholine, biotinamidoca Vacul{O. proate-PEGsaoo-Ala-cholesterol and Drug-Choles [0188] c) Synthesis of Boc-NH-PEGsaoo-Ala-cholesterol terol [0189] Ala-cholesterol is added to a solution of Boc-NH [0198] a) Synthesis of Drug-Cholesterol PEGsaoo-SC (t-butyl carbamate poly(ethylene glycol)-suc [0199] Cholesterol (4 mmol), a drug having an acid group cinimidyl carbonate) in chloroform, followed by triethy and dimethylaminopyridine (4 mmol) are dissolved in dim lamine. The suspension is stirred at 41° C. for 10 minutes. ethylformamide/tetrahydrofuran (20 ml+5 ml) and dicyclo The crude product is purified by chromatography. hexylcarbodiimide is added. The reaction mixture is stirred at ambient temperature overnight. Dicyclohexylurea is fil [0190] d) Synthesis of H2N-PEGsaoo-Ala-cholesterol tered off and the solvent is rotary evaporated. The title [0191] Boc-NH-PEGs loo-Ala-cholesterol is stirred in 4 M compound is purified by chromatography. hydrochloric acid in dioxane for 2.5 hours at ambient [02001 b) Preparation of Gas-Filled Microbubbles Encap temperature. The solvent is removed by rotary evaporation sulated with Phosphatidylserine, Phosphatidylcholine, bioti and the residue is taken up in chloroform and washed with namidocaproate-PEGsaoo-Ala-cholesterol and Drug-Choles water. The organic phase is rotary evaporated to dryness. terol The crude product may be purified by chromatography. [0201] To a mixture (5 mg) of phosphatidylserine and [0192] e) Synthesis of biotinamidocaproate-PEGs loo-Ala phosphatidylcholine (in total 90-99.9 mol %) and biotina cholesterol midocaproate-PEGsaoo-Ala-cholesterol (prepared as in Example 3) and drug-cholesterol (in total 10-0.1 mol %) is [0193] A solution of biotinamidocaproate N-hydroxysuc added 5% propylene glycol-glycerol in water (1 ml). The cinimide ester in tetrahydrofuran is added to H2N-PEGsaoo dispersion is heated to not more than 80° C. for 5 minutes Ala-cholesterol dissolved in tetrahydrofuran and 0.1 M and then cooled to ambient temperature. The dispersion (0.8 sodium phosphate buffer having a pH of 7.5 (2 ml). The ml) is transferred to a vial (1 ml) and the head space is reaction mixture is heated to 30° C. and the reaction is flushed with perfluorobutane. The vial is shaken in a cap followed to completion by TLC, whereafter the solvent is mixer for 45 seconds whereafter the sample is put on a roller evaporated. table. After centrifugation the infranatant is exchanged with [0194 f) Preparation of Gas-Filled Microbubbles Encap water and the washing is repeated. sulated with Phosphatidylserine, Phosphatidylcholine and biotinamidocaproate-PEGsaoo-Ala-cholesterol EXAMPLE 5 [0195] To a mixture (5 mg) of phosphatidylserine and Gas-Filled Microbubbles Encapsulated with phosphatidylcholine (in total 90-99.9 mol %) and biotina Phosphatidylserine and midocaproate-PEGsaoo-Ala-cholesterol (10-0.1 mol %) is thiolated-Anti-CD34-Mal-PEGs loo-DSPE added 5% propylene glycol-glycerol in water (1 ml). The dispersion is heated to not more than. 80° C. for 5 minutes [0202) a) Preparation of Thiolated anti-CD34 Antibodies and then cooled to ambient temperature. The dispersion (0.8 [0203] Thiolation of anti-CD34 antibodies may be ml) is then transferred to a vial (1 ml) and the head space is effected as described by Hansen, C. B. et al. (1995) Biochim. flushed with perfluorobutane. The vial is shaken in a cap Biophys. Acta 1239, 133-144. mixer for 45 seconds, whereafter the sample is put on a roller [0204] b) Preparation of Gas-Filled Microbubbles Encap table. After centrifugation the infranatant is exchanged with sulated with Phosphatidylserine and thiolated-Anti-CD34 water and the washing is repeated. Mal-PEGs loo-DSPE [0196] g) Alternative Preparation of Gas-Filled [0205] To a mixture (5 mg) of phosphatidylserine (90-99.9 Microbubbles Encapsulated with Phosphatidylserine, Phos mol %) and Mal-PEGsaoo-DSPE (10-0.1 mol %, prepared as phatidylcholine and biotinamidocaproate-PEGsaoo-Ala-cho in Example 2) is added 5% propylene glycol-glycerol in lesterol water (1 ml). The dispersion is heated to not more than 80° US 2004/0141922 A1 Jul. 22, 2004 33

C. for 5 minutes and then cooled to ambient temperature. method to that described by Nayar, R. and Schroit, A. J. in The dispersion (0.8 ml) is transferred to a vial (1 ml) and the Biochemistry (1985) 24, 5967-71. head space is flushed with perfluorobutane. The vial is shaken in a cap-mixer for 45 seconds, whereafter the sample [0210] b) Preparation of Gas-Filled Microbubbles Encap is put on a roller table. After centrifugation the infranatant is sulated with Phosphatidylserine and Succ-PEGsaoo-DSPE exchanged with an appropriate buffer and coupling of the thiolated antibody to the microbubbles is performed, e.g. as [0211] To a mixture (5 mg) of phosphatidylserine (90-99.9 described by Goundalkar, A., Ghose, T. and Mezei, M. in J. mol %) and Succ-PEGsaoo-DSPE (10-0.1 mol %) is added Pharm. Pharmacol. (1984) 36.465-66 or Hansen, C. B. et al. 5% propylene glycol-glycerol in water (1 ml). The disper (1995) Biochim. Biophys. Acta 1239 133-144. The sion is heated to not more than 80° C. for 5 minutes and then microbubbles are then placed on a roller table for several coooled to ambient temperature. The dispersion (0.8 ml) is hours and are washed. Flow cytometric analysis of the transferred to a vial (1 ml) and the head space is flushed with resulting microbubbles (employing a fluorescently labeled perfluorobutane. The vial is shaken in a cap-mixer for 45 secondary antibody) is used to confirm attachment of the seconds, whereafter the sample is put on a roller table. After anti-CD34 antibody to the bubbles. The ability of the centrifugation the infranatant is exchanged with water and bubbles to bind specifically to CD34-expressing cells is the washing is repeated. Alternatively the microbubbles may studied by microscopy employing one population of cells be prepared as described in Example 2(f). expressing CD34 and one population that do not express CD34. [0212] c) Coupling of Streptavidin to Gas-Filled Microbubbles Encapsulated with Phosphatidylserine and EXAMPLE 6 Succ-PEGsaoo-DSPE Biotin Attached to Gas-Filled Microbubbles [0213] Streptavidin is covalently bound to Succ-PEGs loo DSPE in the microbubble membranes by standard coupling [0206] Biotin may be attached to microbubbles in many methods using a water-soluble carbodiimide. The sample is different ways, e.g. in a similar way to that described by placed on a roller table during the reaction. After centrifu Corley, P. and Loughrey, H. C. in (1994) Biochim. Biophys. gation the infranatant is exchanged with water and the Acta 1195, 149-156. The resulting bubbles are analysed by washing is repeated. The functionality of the attached flow cytometry, e.g. by employing fluorescent streptavidin streptavidin is analysed by binding, e.g. to fluorescently to detect attachment of biotin to the bubbles. Alternatively labeled biotin, biotinylated antibodies (detected with a fluo radioactive or enzyme-labelled streptavidin/avidin is used to rescently labeled secondary antibody) or biotinylated and analyse biotin attachment. fluorescence- or radioactively-labeled oligonucleotides. EXAMPLE 7 Analysis is performed by fluorescence microscopy or scin tillation counting. Gas-Filled Microbubbles Encapsulated with [0214] d) Preparation of Gas-Filled Microbubbles Encap Distearoylphosphatidylserine and Biotin-DPPE sulated with Phosphatidylserine and Biotin Non-Covalently [0207] To distearoylphosphatidylserine (DSPS) (22.6 mg) Bound to Streptavidin-Succ-PEGs loo-DSPE was added 4% propylene glycol-glycerol in water (4 ml). The dispersion was heated to not more than 80° C. for five [0215] Microbubbles from Example 8(c) are incubated in minutes and then cooled to ambient temperature. An aque a solution containing biotinylated vectors, e.g. biotinylated ous dispersion of biotin-DPPE (1.5 mg) in 4% propylene antibodies. The vector-coated microbubbles are washed as glycol-glycerol (1 ml) was added and the sample was put on described above. a roller table for 1–2 hours. The suspension was filled into vials and the head spaces were flushed with perfluorobutane. EXAMPLE 9 The vials were shaken for 45 seconds, whereafter they were put on a roller table. After centrifugation for 7 minutes the infranatant was exchanged with water and the washing was Gas-Filled Microbubbles Encapsulated with repeated twice. Normal phase HPLC with an Evaporative Phosphatidylserine and Biotinylated Light Scattering Detector confirmed that the membranes of Oligonucleotide Non-Covalently Bound to the microbubbles contained 4 mol % biotin-DPPE. The Streptavidin-Succ-PEG-DSPE mean particle diameter of the microbubbles was 4 um measured by Coulter Counter. Ultrasound transmission mea [0216] a) Synthesis of Succ-PEGsaoo-DSPE surements using a 3.5 MHz broadband transducer showed [0217] NH2-PEG,00-DSPE (prepared as in Example 2) is that a particle dispersion of ~2 mg/ml gave a sound beam carboxylated using succinic anhydride, e.g. by a similar attenuation higher than 5 dB/cm. method to that described by Nayar, R. and Schroit, A. J. in EXAMPLE 8 Biochemistry (1985) 24, 5967-71. [0218] b) Preparation of Gas-Filled Microbubbles Encap Gas-Filled Microbubbles Encapsulated with Phosphatidylserine and Biotinylated Antibody sulated with Phosphatidylserine and Succ-PEGsaoo-DSPE Non-Covalently Bound to [0219] To a mixture (5 mg) of phosphatidylserine (90-99.9 streptavidin-Succ-PEG-DSPE mol %) and Succ-PEGs loo-DSPE (10-0.1 mol %) is added 5% propylene glycol-glycerol in water (1 ml). The disper [0208] a) Synthesis of Succ-PEGs loo-DSPE sion is heated to not more than 86° C. for 5 minutes and then [0209] NH2-PEGs loo-DSPE (prepared as in Example 2) is cooled to ambient temperature. The dispersion (0.8 ml) is carboxylated using succinic anhydride, e.g. by a similar transferred to a vial (1 ml) and the head space is flushed with US 2004/0141922 A1 Jul. 22, 2004 34 perfluorobutane. The vial is shaken in a cap-mixer for 45 centrifugation the infranatant is exchanged with water and seconds, whereafter the sample is put on a roller table. After the washing is repeated. Alternatively the microbubbles are centrifugation the infranatant is exchanged with water and prepared as described in Example 2(e) or 2(f). Analysis of the washing is repeated. Alternatively the microbubbles may folate attachment may for example be done by microscopic be prepared as described in Example 2(f). study of the binding of the folate-containing microbubbles to [0220, c) Coupling of Streptavidin to Gas-Filled cells expressing different levels of folate receptors. Microbubbles Encapsulated with Phosphatidylserine and EXAMPLE 11 Succ-PEGs loo-DSPE [0221] Streptavidin is covalently bound to Succ-PEGsao,0 Gas-Filled Microbubbles Encapsulated with Phos DSPE in the microbubble membraness by standard coupling phatidylserine and thiolated-anti-CD34-Mal methods using a water-soluble carbodiimide. The sample is PEGsaoo-DSPE, thiolated-anti-ICAM-1-Mal placed on a roller table during the reaction. After centrifu PEGsaoo-DSPE and thiolated-anti-E-Selectin-Mal gation the infranatant is exchanged with water and the PEGsaoo-DSPE washing is repeated. The functionality of the attached streptavidin is analyzed by binding, e.g. to fluorescently [0228] a) Preparation of thiolated-anti-CD34 Antibodies labeled biotin, biotinylated antibodies (detected with a fluo [0229] Thiolation of anti-CD34 antibodies may be rescently labeled secondary antibody) or biotinylated and effected as described by Hansen, C. B. et al. in (1995) fluorescence- or radioactively-labeled oligonucleotides. Biochim. Biophys. Acta 1239, 133-144. Analysis is performed by fluorescence microscopy or scin [0230] b) Preparation of thiolated-anti-ICAM-1 Antibod tillation counting. ies [0222] d) Preparation of Gas-Filled Microbubbles Encap sulated with Phosphatidylserine and a Biotinylated Oligo [0231] Thiolation of anti-ICAM-1 antibodies may be nucleotide Non-Covalently Bound to streptavidin-Succ effected as described by Hansen, C. B. et al. in (1995) PEGsaoo-DSPE Biochim. Biophys. Acta 1239, 133-144. [0223] Microbubbles from Example 9(c) are incubated in [0232] c) Preparation of thiolated-anti-E-selectin Antibod a solution containing a biotinylated oligonucleotide. The ies oligonucleotide-coated bubbles are washed as described [0233] Thiolation of anti-E-selectin antibodies may be above. Binding of the oligonucleotide to the bubbles is effected as described by Hansen, C. B. et al. in (1995) detected e.g. by using fluorescent-labeled oligonucleotides Biochim. Biophys. Acta 1239, 133-144. for attachment to the bubbles, or by hybridising the attached oligonucleotide to a labeled (fluorescence or radioactivity) [0234] d) Preparation of Gas-Filled Microbubbles Encap complementary oligonucleotide. The functionality of the sulated with Phosphatidylserine and thiolated-anti-CD34 oligonucleotide-carrying microbubbles is analysed, e.g. by Mal-PEGs loo-DSPE, thiolated-anti-ICAM-1-Mal-PEGs loo hybridising the bubbles with immobilized DNA-containing DSPE, thiolated-anti-E-selectin-Mal-PEGsaoo-DSPE sequences complementary to the attached oligonucleotide. [0235] To a mixture (5 mg) of phosphatidylserine (90-99.9 As examples, an oligonucleotide complementary to riboso mol %) and Mal-PEGsaoo-DSPE (10-0.1 mol %, prepared as mal DNA (of which there are many copies per haploid in Example 2) is added 5% propylene glycol-glycerol in genome) and an oligonucleotide complementary to an onco water (1 ml). The dispersion is heated to not more than 80° gene (e.g. ras of which there is one copy per haploid C. for 5 minutes and is then cooled to ambient temperature. genome) may be used. The dispersion (0.8 ml) is transferred to a vial (1 ml) and the head space is flushed with perfluorobutane. The vial is EXAMPLE 10 shaken in a cap-mixer for 45 seconds, whereafter the sample is put on a roller table. After centrifugation the infranatant is Gas-Filled Microbubbles Encapsulated with exchanged with an appropriate buffer, and coupling of the Phosphatidylserine and folate-PEG-Succ-DSPE antibodies from Example 11(a), 11(b) and 11(c) to the microbubbles is performed, e.g. as described by Goundalkar, [0224] a) Preparation of folate-PEG-Succ-DSPE A., Ghose, T. and Mezei, M. in J. Pharm. Pharmacol. (1984) [0225] Folate-PEG-Succ-DSPE is synthesised as 36, 465-466 or by Hansen, C. B. et al. in (1995) Biochim. described by Lee, R. J. and Low, P. S. in (1995) Biochimica. Biophys. Acta 1239, 133-144. The microbubbles are placed Biophysica. Acta 1233, 134-144. on a roller table for several hours and are then washed. [0226] b) Preparation of Gas-Filled Microbubbles Encap EXAMPLE 12 sulated with Phosphatidylserine and folate-PEG-Succ DSPE The Peptide [0227] To a mixture (5 mg) of phosphatidylserine (90-99.9 FNFRLKAGOKIRFGAAAWEPPRARI (SEQ ID mol %) and folate-PEG-DSPE (10-0.1 mol %) is added 5% NO:2) Attached to Gas-Filled Microbubbles propylene glycol-glycerol in water (1 ml). The dispersion is Encapsulated with Phosphatidylserine heated to not more than 80° C. for 5 minutes and is then [0236] The peptide FNFRLKAGQKIRFGAAAWEP cooled to ambient temperature. The dispersion (0.8 ml) is PRARI (SEQ ID NO:2), comprising phosphatidylserine transferred to a vial (1 ml) and the head space is flushed with binding and heparin-binding sections, is synthesised. The perfluorobutane. The vial is shaken in a cap-mixer for 45 peptide is added to preformed phosphatidylserine-encapsu seconds, whereafter the sample is put on a roller table. After lated perfluorobutane microbubbles and thoroughly mixed. US 2004/0141922 A1 Jul. 22, 2004 35

EXAMPLE 1.3 flushed with perfluorobutane. The vial was shaken on a cap-mixer at 4450 oscillations/minute for 45 seconds and Fibronectin Covalently Bound to Gas-Filled put on a roller table. The sample was washed by centrifuging Microbubbles Encapsulated with Phosphatidylserine at 2000 rpm for 5 minutes. The infranatant was removed by and Phosphatidylethanolamine a syringe and distilled water was added to the same volume. The headspace was again flushed with perfluorobutane and [0237] a) Microbubbles Preparation the sample was kept on a roller table until a homogeneous [0238] DSPS (25 mg) and DSPE (5.0 mg) were weighed appearance was obtained. The washing procedure was into a clean vial and 5 ml of a solution of 1.4% propylene repeated again. glycol/2.4% glycerol was added. The mixture was warmed to 80° C. for 5 minutes. The sample was cooled to room EXAMPLE 1.5 temperature and the head space was flushed with perfluo robutane gas. The vials were shaken in a cap mixer for 45 Gas-Filled Microbubbles Encapsulated with seconds and the microbubbles were twice washed with Phosphatidylserine and WEPPRARI-PE (SEQ ID distilled water then resuspended in 0.1 M sodium borate NO:3) buffer, pH 9. [0249] Phosphatidylethanolamine (PE) is reacted with an [0239] b) Modification of Fibronectin equimolar amount of the crosslinker N-hydroxysuccinim idyl-2,3-dibromopropionate in a 1:1 mixture of dioxane and [0240] Fibronectin (1.0 mg) in 5 ml 0.01 M Hepes buffer, 0.02 M HEPES buffer, pH 8.0. Following incubation for 2 pH 8, was added to 0.1 mmol of the crosslinker SDBP. The hours on ice, an equimolar amount of the heparin-binding mixture was incubated on ice for 2 hours. peptide WEPPRARI (SEQ ID NO:3) is added, the pH is [0241] c) Microbubble Modification. brought to 9 by the addition of 0.2 M disodium tetraborate, and the incubation is continued for 2 hours at room tem [0242] To the protein solution from (b) was added the perature. The reaction product is purified by chromatogra microbubble suspension from (a) and incubation was phy. Monolayer-encapsulated microbubbles containing per allowed to proceed for 2 hours at room temperature on a fluorobutane are made from a mixture of 80-95% roller table. Unreacted material was removed by allowing phosphatidylserine (PS) and 5-20% of peptide-substituted the microbubbles to float and then replacing the buffer with PE. 0.1 M sodium borate buffer, pH 9. This process was repeated three times. EXAMPLE 16 [0243] d) In Vitro Analysis. Gas-Filled Microbubbles Encapsulated with [0244] The microbubbles were tested in the in vitro assay Phosphatidylserine and Inactivated Human detailed in Example 21. A gradual accumulation of thrombin-Succ-PEGs loo-DSPE microbubbles binding to the cells was observed. [0250) a) Inactivation of Human Thrombin EXAMPLE 1.4 [0251] Human thrombin was inactivated by incubation with a 20% molar excess of D-Phe-L-Pro-L-Arg-chlorom Gas-Filled Microbubbles Encapsulated with ethyl ketone in 0.05 M HEPES buffer, pH 8.0, at 37° C. for Phosphatidylserine, and 30 minutes. 33-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol [0252] b) Preparation of Gas-Filled Microbubbles Encap [0245] a) Synthesis of 33-[N-(N',N'-dimethylaminoet sulated with Phosphatidylserine and Succ-PEGsaoo-DSPE hane)-carbamoyl]cholesterol (DC-chol) (Farhood, H., Gao, [0253] To a mixture (5 mg) of phosphatidylserine (90-99.9 X. Barsoum, J. and Huang, L., Anal. Biochem. 225, 89-93 mol %) and Succ-PEGsaoo-DSPE (10-0.1 mol %, prepared (1995) as in Example 9(a)) was added 5% propylene glycol [0246] To a stirred solution of 2-dimethylaminoethy glycerol in water (1 ml). The dispersion was heated to not more than 80° C. for 5 minutes and was then cooled to lamine (19.40 mg, 24:1, 0.22 mmol) and triethylamine (310 ambient temperature. The dispersion (0.8 ml) was trans Aul, 2.23 mmol) in dichloromethane (3 ml) at room tempera ferred to a vial (1 ml) and the head space was flushed with ture was slowly added a solution of cholesteryl chlorofor perfluorobutane. The vial was shaken in a cap-mixer for 45 mate (100 mg, 0.22 mmol) in 1,4-dioxane. When the reac seconds, whereafter the sample was put on a roller table. tion was completed, the mixture was evaporated to dryness After centrifugation the infranatant was exchanged with and the residue was purified by flash chromatography water and the washing was repeated. Alternatively the (CHCls/MeOH, 4:1). A white solid was obtained, yield 105 microbubbles may be prepared as described in Example 2(f). mg (95%). The structure was verified by NMR and MALDI. [0254] c) Preparation of Gas-Filled Microbubbles Encap [0247] b) Preparation of Microbubble Dispersion sulated with Phosphatidylserine and Inactivated Human [0248] Monolayer-encapsulated microbubbles containing thrombin-Succ-PEGsaoo-DSPE perfluorobutane are made from a mixture of 90% phosphati [0255] Inactivated human thrombin was covalently bound dylserine and 10% (DC-chol) by weighing DSPS (4.5 mg) to Succ-PEGsaoo-DSPE in the microbubbles from Example and (DC-chol) (0.5 mg) into a 2 ml vial. 0.8 ml propylene 16(b) by standard coupling methods using a water-soluble glycol/glycerol (4%) in water was added. The solution was carbodiimide. The sample was placed on a roller table heated at 80° C. for 5 minutes and shaken. The solution was during the reaction. After centrifugation the infranatant was then cooled to ambient temperature and the headspace was exchanged with water and the washing was repeated. US 2004/0141922 A1 Jul. 22, 2004 36

EXAMPLE 1.7 [0263] To a mixture (5 mg) of phosphatidylserine (90-99.9 mol %), Mal-PEGsaoo-DSPE (10-0.1 mol %, prepared as in Gas-Filled Microbubbles Having Methotrexate and Example 2) and the anticancer prodrug 3',5'-O-dipamitoyl Prodrug-Activating Enzyme Attached 5-fluoro-2'-deoxyuridine (Mori, A. et al. (1995) Cancer [0256] a) Methotrexate Attached via a Peptide Linker to Chemother. Pharmacol. 35,447-456) is added 5% propylene Gas-Filled Micrububbles glycol-glycerol in water (1 ml). The dispersion is heated to [0257] Methods for attaching aminoacids to the anticancer not more than 80° C. for 5 minutes and is then cooled to drug methotrexate (MTX) are well described in the literature ambient temperature. The dispersion (0.8 ml) is transferred (see e.g. Huennekens, F. M. (1994), TIBTECH 12, 234-239 to a vial (1 ml) and the head space is flushed with perfluo and references therein). Instead of a single amino acid a robutane. The vial is shaken in a cap-mixer for 45 seconds, peptide may be attached to MTX using the same technology. whereafter the sample is put on a roller table. After cen Such a peptide may constitute a linker for the attachment of trifugation the infranatant is exchanged with an approperiate MTX to the surface of microbubbles. One class of such buffer, and coupling of the antibody to the microbubble is linkers comprises peptides of the general structure (MTX) performed, e.g. as described by Goundalkar, A., Ghose, T. F-K/R-X-R-Z-C where X is any amino acid and Z is a and Mezei, M. in J. Pharm. Pharmacol. (1984) 36.465-466 hydrophobic amino acid. Aspecific example of such a linker or by Hansen, C. B. et al. in (1995) Biochim. Biophys. Acta is (MTX)-F-K-L-R-L-C (SEQ ID NO:4). The SH-group in 1239 133-144. The microbubbles are placed on a roller table the Cys-residue is employed for attachment of the MTX for several hours and are then washed. peptide to the microbubbles (e.g. composed of phosphati dylserine and Mal-PEG-DSPE) using standard technology, EXAMPLE 1.9 e.g. as in Example 2. A linker of this kind is expected to be cleaved by the enzyme cathepsin B which often is selec Gas-Filled Microbubbles Encapsulated with tively overexpressed outside and on the surface of tumour Phosphatidylserine, cells (Panchal, R. G. et al. (1996), Nat. Biotechnol. 14, thiolated-anti-CEA-Mal-PEGsaoo-DSPE and the 852-856). Thus, the potential prodrug (MTX)-F-K/R-X-R Anticancer Prodrug would be liberated selectively in tumours. This prodrug can N-trifluoroacetyl-adriamycin-14-valerate further be activated to the active drug MTX by the action of carboxypeptidases, either present endogeneously in the [0264] a) Preparation of Thiolated anti-CEA Antibodies tumour or targeted to the tumour e.g. by tumour-associated antibodies (see below). [0265] Thiolation of anti-CEA antibodies may be effected [0258] b) Prodrug-Activating Enzyme Covalently as described by Hansen, C. B. et al. in (1995) Biochim. Attached to the Surface of Gas-Filled Microbubbles Biophys. Acta 1239 133-144. [0259] An example of a prodrug-activating enzyme is [0266] b) Preparation of Gas-Filled Microbubbles Encap carboxypeptidase A (CPA), which may be conjugated to the sulated with Phosphatidylserine, thiolated-anti-CEA-Mal surface of microbubbles encapsulated by, for example, a PEGsaoo-DSPE and the Anticancer Prodrug N-trifluoro mixture of phosphatidylserine and phosphatidylethanola acetyl-adriamycin-14-valerate mine, e.g. by using a 3400 Da poly(ethylene glycol) chain [0267] To a mixture (5 mg) of phosphatidylserine (90-99.9 bearing an N-hydroxysuccinimide group at both ends (Per molt), Mal-PEGsaoo-DSPE (10-0.1 molt, prepared as in ron, M. J. and Page, M., Br. J. Cancer 73, 281-287); the Example 2) and the anticancer prodrug N-trifluoroacetyl microbubbles may be prepared by standard methods. Microbubbles containing CPA may be targeted to areas of adriamycin-14-valerate (Mori, A. et al. (1993) Pharm. Res. pathology by incorporating a suitable targeting vector in the 10, 507–514), is added 5% propylene glycol-glycerol in CPA-containing bubbles. Alternatively CPA may be attached water (1 ml). The dispersion is heated to not more than 80° directly to a vector (e.g. an antibody), for example by the C. for 5 minutes and is then cooled to ambient temperature. method as described above. In this latter case the CPA The dispersion (0.8 ml) is transferred to a vial (1 ml) and the vector conjugate will be attached to the surface of the head space is flushed with perfluorobutane. The vial is microbubbles as described in Hansen, C. B. et al. (1995) shaken in a cap-mixer for 45 seconds, whereafter the sample Biochim. Biophys. Acta 1239 133-144. Examples of the is put on a roller table. After centrifugation the infranatant is many possible prodrug-enzyme pairs are described in e.g. exchanged with an appropriate buffer, and coupling of the Huennekens, F. M. (1994) TIBTECH 12, 234-239. antibody to the microbubble is performed, e.g. as described by Goundalkar, A., Ghose, T. and Mezei, M. in J. Pharm. EXAMPLE 1.8 Pharmacol. (1984) 36 465-66 or by Hansen, C. B. et al. in Gas-Filled Microbubbles Encapsulated with Phos (1995) Biochim. Biophys. Acta 1239 133-144. The phatidylserine, thiolated-anti-CEA-Mal-PEGsaoo microbubbles are placed on a roller table for several hours DSPE and the Anticancer Prodrug 3',5'-O-dipami and are then washed. toyl-5-fluoro-2'-deoxyuridine EXAMPLE 20 [0260] a) Preparation of Thiolated anti-CEA Antibodies [0261] Thiolation of anti-CEA antibodies may be effected Method of Use as described by Hansen, C. B. et al. in (1995) Biochim. [0268] An agent comprising phosphatidylserine-encapsu Biophys. Acta 1239, 133-144. lated microbubbles having inactivated human thrombin [0262] b) Preparation of Gas-Filled Microbubbles Encap Succ-PEGsaoo-DSPE incorporated into the encapsulating sulated with Phosphatidylserine, thiolated-anti-CEA-Mal membrane is lyophilised from 0.01 M phosphate buffer, pH PEGsaoo-DSPE and the Anticancer Prodrug 3',5'-O-dipami 7.4. The product is redispersed in sterile water and injected toyl-5-fluoro-2'-deoxyuridine intravenously into a patient with suspected venous throm US 2004/0141922 A1 Jul. 22, 2004 37 bosis in a leg vein. The leg is examined by standard Ex2so, Emsso-product retention time=19.44 minutes). Fur ultrasound techniques. The thrombus is located by increased ther product characterisation was carried out using MALDI contrast as compared with surrounding tissue. mass spectrometry: expected M+H at 2198, found at 2199. [0272] b) Preparation of Gas-Filled Microbubbles of EXAMPLE 21 DSPS ‘Doped’ with a Multiple-Specific Lipopeptide Con Preparation and Biological Evaluation of sisting of a Heparin Sulphate Binding Peptide (KRKR) Gas-Containing Microbubbles of DSPS ‘Doped’ (SEQ ID NO:5) and Fibronectin Peptide (WOPPRARI) with a Lipopeptide Comprising a Heparin Sulphate (SEQ ID NO:6) Binding Peptide (KRKR) (SEQ ID NO:5) and a [0273] DSPS (4.5 mg) and lipopeptide from (a) (0.5 mg) Fibronectin Peptide (WOPPRARI) (SEQ ID NO:6) were weighed into each of two vials and 0.8 ml of a solution of 1.4% propylene glycol/2.4% glycerol was added to each [0269] This example is directed at the preparation of vial. The mixtures were warmed to 80° C. for 5 minutes targeted microbubbles comprising multiple peptidic vectors (vials shaken during warming). The samples were cooled to arranged in a linear sequence. room temperature and the head spaces flushed with perfluo [0270] a) Synthesis of a Lipopeptide Consisting of a robutane gas. The vials were shaken in a cap mixer for 45 Heparin Sulphate Binding Peptide (KRKR) (SEQ ID NO:5) seconds and rolled overnight. The resulting microbubbles and Fibronectin Peptide (WOPPRARI) (SEQ ID NO:6) were washed several times with deionised water and analy

(SEQ ID NO: 7) O ~~~~~~~~ NH º NH2 º NH2 NH NH OS 2. NH2

O O O O H H H N ~~~~~~~~ N N N N N N H H H H O O O O ~~ NH S

H2N H2N

O O § OH N N N H H O O O J. J. HN NH2 HN NH2

[0271] The lipopeptide was synthesised on an ABI 433A sed by Coulter counter [size: 1-3 micron (87%), 3-5 micron automatic peptide synthesiser starting with Fmoc-Ile-Wang (11.5%)] and acoustic attenuation (frequency at maximum resin on a 0.1 mmol scale using 1 mmol amino acid attenuation: 3.5 MHz). The microbubbles were stable at 120 cartridges. All amino acids and palmitic acid were preacti vated using HBTU before coupling. The simultaneous mm Hg. MALDI mass spectral analysis was used to confirm removal of peptide from the resin and side-chain protecting incorporation of lipopeptide into DSPS microbubbles as groups was carried out in TFA containing 5% phenol, 5% follows; ca. 0.05-0.1 ml of microbubble suspension was EDT, 5% anisole and 5% H2O for 2 hours, giving a crude transferred to a clean vial and 0.05-0.1 ml methanol was product yield of 150 mg. Purification by preparative HPLC added. The suspension was sonicated for 30 seconds and the of a 40 mg aliquot of crude material was carried out using solution was analysed by MALDI MS. Positive mode gave a gradient of 70 to 100% B over 40 minutes (A=0.1% M+H at 2200 (expected for lipopeptide, 2198). TFA/water and B=MeOH) at a flow rate of 9 ml/min. After lyophilisation, 16 mg of pure material were obtained (ana [0274] c) In Vitro Study of Gas-Filled Microbubbles of lytical HPLC, gradient 70-100% B where B=MeOH, DSPS ‘Doped’ with a Multiple-Specific Lipopeptide Con A=0.01% TFA/water; detection—UV 260 and fluorescence, sisting of a Heparin Sulphate-Binding Peptide (KRKR) US 2004/0141922 A1 Jul. 22, 2004 38

(SEQ ID NO:5) and Fibronectin Peptide (WOPPRARI) [0279] Case 2) [Comparative] (SEQ ID NO:6): Binding to Endothelial Cells Under Flow [0280] A net volume of 2 ml microbubbles prepared in an Conditions identical manner to (b) above with the exception that no lipopeptide was included in the preparation was injected, [0275] The human endothelial cell line ECV 304, derived using the same imaging procedure as above. The myocardial from a normal umbilical cord (ATCC CRL-1998) was echo enhancement was far less intense and of shorter cultured in 260 mL Nunc culture flasks (chutney 153732) in duration than that observed in Case 1. At the completion of RPMI 1640 medium to which L-glutamine (200 mM), the left ventricular attenuation phase, there was also almost penicillin/streptomycin (10,000 U/ml and 10,000 ug/ml) and complete loss of myocardial contrast effects, and the myo 10% fetal bovine serum were added. The cells were subcul cardial echo increases in the posterior part of the left tured with a split ratio of 1:5 to 1:7 when reaching conflu ventricle noted in Case 1 were not observed. ence. Cover-glasses, 22 mm in diameter, were sterilised and placed on the bottom of 12 well culture plates, whereafter EXAMPLE 22 cells in 0.5 ml complete medium with serum were added Preparation of Gas-Filled Microbubbles Encapsuled above the plates. When the cells reached confluence the with DSPS Comprising Thiolated coverslips were placed in a custom-made flow chamber anti-CD34-MAL-PEG2000-PE consisting of a groove carved into a glass plate upon which [0281] a) Preparation of Gas-Filled Microbubbles Encap the cover slip with cells was placed, with the cells facing the suled with DSPS and PE-PEG-ooo-Mal groove, so as to form a flow channel. Microbubbles prepared as in (b) were passed from a reservoir held at 37° C. through [0282] DSPS (4.5 mg, 3.9 mmol) and PE-PEGeooo-Mal the flow chamber and back to the reservoir using a peristaltic from Example 50 (0.5 mg) were weighed into a clean vial pump. The flow rate was adjusted to simulate physiologi and 1 ml of a solution of 1.4% propylene glycol/2.4% cally relevant shear rates. The flow chamber was placed glycerol was added. The mixture was warmed to 80° C. for under a microscope and the interaction between the 5 minutes then filtered through a 4.5 micron filter. The microbubbles and cells was viewed directly. A camera sample was cooled to room temperature and the head space mounted on the microscope was connected to a colour video was flushed with perfluorbutane gas. The vials were shaken printer and a monitor. A gradual accumulation of in a cap mixer for 45 seconds and the resulting microbubbles microbubbles on the cells took place at a rate dependent on were washed three times with distilled water. the flow rate. On further increasing the flow rate, cells [0283] b) Thiolation of anti-CD34 Antibodies started to become detached from the coverslip, but the [0284] To 0.3 mg of anti-CD34 antibody dissolved in 0.5 microbubbles remained bound to the cells. Control bubbles ml phosphate buffered saline (PBS), pH7, was added 0.3 mg not carrying the vector did not adhere to the endothelial cells Traut’s reagent and the solution was stirred at room tem and disappeared from the chamber under minimal flow perature for 1 hour. Excess reagent was separated from the conditions. modified protein on a NAP-5 column. [0276] d) In Vivo Experiment in Dog [0285] c) Conjugation of Thiolated anti-CD34Antibody to Gas-Filled Microbubbles Encapsuled with DSPS and Com [0277] Case 1) prising DSPE-PEG 2000-MAL [0278] A 22 kg mongrel dog was anaesthetised with [0286] 0.5 ml of the thiolated antibody praparation from pentobarbital and mechanically ventilated. The chest was (b) was added to an aliquot of microbubbles from (a) and the opened by a midline sternotomy, the anterior pericardium conjugation reaction was allowed to proceed for 30 minutes was removed, and a 30 mm gelled silicone rubber spacer on a roller table. Following centifugation at 2000 rpm for 5 was inserted between the heart and a P5-3 transducer of an minutes the infranatant was removed. The microbubbles ATL HDI-3000 ultrasound scanner. The scanner was set for were washed a further three times with water. intermittent short axis imaging once in each end-systole by [0287] d) Detection of the Antibody Encapsulated in the delayed EGC triggering. A net volume of 2 ml of Microbubbles Using a FITC-Conjugated Secondary Anti microbubbles from (b) was injected as a rapid intravenous body bolus; 3 seconds later, the imaged right ventricle was seen to contain contrast material, and another 3 seconds later the left [0288] To the microbubble suspension from (c) was added ventricle was also filled and a transient attenuation shadow 0.025 mL FITC-conjugated goat-anti-mouse antibody. The which obscured the view of the posterior parts of the left mixture was incubated in the dark at room temperature for ventricle was observed. Substantial increases in brightness 30 minutes on a roller table and was then centrifuged at 2000 were seen in the myocardium and, when the attenuation rpm for 5 minutes. The infranatant was then removed and the shadow subsided, in the portions of the heart distal to the left microbubbles were washed a further three times with water. ventricle. After passage of the inital bolus, the ultrasound Flow cytometric analysis of the microbubble suspension scanner was set to continuous, high frame rate, high output showed that 98% of the population was fluorescent. power imaging, a procedure known to cause destruction of EXAMPLE 23 ultrasound contrast agent microbubbles in the imaged tissue regions. After a few seconds, the scanner was adjusted back Preparation of Gas-Filled Microbubbles Encapsuled to its initial setting. The myocardium was then darker, and with DSPS Comprising Thiolated closer to the baseline value. Moving the imaged slice to a anti-CD62-MAL-PEG2000-PE new position resulted in re-appearance of contrast effects; [0289] An identical procedure to that described in moving the slice back to the initial position again resulted in Example 22 was used to prepare microbubbles comprising a tissue brightness close to baseline. anti-CD62 antibodies. US 2004/0141922 A1 Jul. 22, 2004 39

EXAMPLE 24 [0300] The peptide was synthesised on an ABI 433A automatic peptide synthesiser starting with Fmoc-Ile-Wang Preparation of Gas-Filled Microbubbles Encapsuled resin on a 0.1 mmol scale using 1 mmol amino acid with DSPS Comprising Thiolated cartridges. All amino acids were preactivated using HBTU anti-ICAM-1-MAL-PEG2000-PE before coupling. The simultaneous removal of peptide from [0290] An identical procedure to that described in the resin and side-chain protecting groups was carried out in Example 22 was used to prepare microbubbles comprising TFA containing 5% phenol, 5% EDT and 5% H2O for 2 anti-ICAM-1 antibodies. hours, giving a crude product yield of 302 mg. Purification EXAMPLE 25 by preparative HPLC of a 25 mg aliquot of crude material was carried out using a gradient of 20 to 40% B over 40 Preparation of Gas-Filled Microbubbles Encapsu minutes (A=0.1% TFA/water and B=0.1% TFA/acetonitrile) lated with DSPS and Thiolated anti-CD62-Mal at a flow rate of 9 ml/min. After lyophilisation 10 mg of pure PEG2000-PE and thiolated-anti-ICAM-1-Mal-PEG material was obtained (analytical HPLC, gradient 20 to 50% 2000-PE B where B=0.1% TFA/acetonitrile, A=0.01% TFA/water: [0291] This example is directed to the preparation of detection—UV 214 and 260 nm—product retention time=12.4 microbubbles comprising multiple antibody vectors for tar minutes). Further product characterization was carried out geted ultrasound imaging. using MALDI mass spectrometry: expected M+H at 2856, [0292] a) Preparation of Gas-Filled Microbubbles Encap found at 2866. sulated with DSPS and PE-PEG2000-Mal [0301] b) Preparation of Gas-Filled Microbubbles Com [0293] DSPS (4.5 mg) and PE-PEGeooo-Mal from prising DSPS Coated Non-Covalently with Polylysine and Example 2 (a) (0.5 mg) were weighed into a clean vial and 1 ml of a solution of 1.4% propylene glycol/2.4% glycerol the PS-Binding/Fibronectin Fragment Fusion Peptide was added. The mixture was warmed to 80° C. for 5 minutes FNFRLKAGOKIRFGGGGWOPPRAI (SEQ ID NO:8) and then filtered through a 4.5 micron filter. The sample was [0302] DSPS (5 mg) was weighed into a clean vial along cooled to room temperature and the head space was flushed with perfluorobutane gas. The vials were shaken in a cap with poly-L-lysine (0.2 mg) and peptide from (a) above (0.2 mixer for 45 seconds and the microbubbles were washed mg). To the vial was added 1.0 ml of a solution of 1.4% three times with distilled water. propylene glycol/2.4% glycerol. The mixture was warmed to 80° C. for 5 minutes. The sample was cooled to room [0294] b) Thiolation of anti-CD62 and anti-ICAM-1 Anti temperature and the head space was flushed with perfluo bodies robutane gas. The vials were shaken in a cap mixer for 45 [0295] To 0.3 mg each of anti-CD62 and anti-ICAM-1 seconds and the resulting microbubbles were centrifuged at antibodies dissolved in PBS buffer (pH 7, 0.5 ml) was added 1000 rpm for 3 minutes. Following extensive washing with Traut’s reagent and the solutions were stirred at room water, PBS and water, the final solution was examined for temperature for 1 hour. Excess reagent was separated from polylysine and peptide content using MALDI MS. No the modified protein on a NAP-5 column. polypeptide material was observed in the final wash solu [0296] c) Conjugation of Thiolated anti-CD62 and anti tion. Acetonitrile (0.5 ml) was then added and the ICAM-1 Antibodies to Gas-Filled Microbubbles Encapsu microbubbles were destroyed by sonication. Analysis of the lated with DSPS and DSPE-PEGeooo-Mal resulting solution for polylysine and PS-binding/fibronectin [0297] 0.5 ml of the mixed thiolated antibody preparation fusion peptide was then carried out using MALDI MS. The from (b) was added to an aliquot of microbubbles from (a) results were as follows: and the conjugation reaction was allowed to proceed for 30 minutes on a roller table. Following centrifugation at 2000 rpm for 5 minutes, the infranatant was removed. The microbubbles were washed a further three times with water. MALDI expected MALDI found [0298] The PEG spacer length may be varied to include longer (e.g. PEGsaoo and PEGsooo) or shorter (e.g. PEGooo Poly-L-lysine 786,914, 790, 919, or PEG 800) chains. Addition of a third antibody such as 1042, 1170 1048, 1177 thiolated-anti-CD34 is also possible. DSPS-binding peptide 2856 2866 EXAMPLE 26 Targeted Gas-Filled Microbubbles Comprising DSPS Coated Non-Covalently with Polylysine and [0303] The spacer element contained within the PS-bind a Fusion Peptide Comprising a PS-Binding ing/fibronectin fusion peptide (-GGG-) may also be replaced Component and a Fibronectin Peptide Sequence with other spacers such as PEG2000 or poly alanine (-AAA-). FNFRLKAGOKIRFGGGGWOPPRAI (SEQ ID A form of pre-targeting may also be employed, whereby the NO:8) DSPS-binding/fibronectin fragment fusion peptide is firstly [0299) a) Synthesis of PS-Binding/Fibronectin Fragment allowed to associate with cells via fibronectin peptide bind Fusion Peptide FNFRLKAGOKIRFGGGGWOPPRAI ing, followed by administration of PS microbubbles which (SEQ ID NO:8) then bind to the PS-binding peptide. US 2004/0141922 A1 Jul. 22, 2004 40

EXAMPLE 27 ml of a solution of 1.4% propylene glycol/2.4% glycerol was Gas-Filled Microbubbles Encapsulated with Phos added. The mixture was warmed to 80° C. for 5 minutes phatidylserine and biotin-PEG-alanyl-cholesterol (vials shaken during warming). The sample was cooled to and Functionalised with streptavidin/biotinyl-endot room temperature and the head space was flushed with helin-1 Peptide (biotin-D-Trp-Leu-Asp-Ile-Ile-Tr perfluorobutane gas. The vial was shaken in a cap-mixer for p.OH) (SEQ ID NO:9) and biotinyl-fibrin-anti 45 seconds and the vial was rolled overnight. The polymerant Peptide (biotin-GPRPPERHOS.NH2) microbubble suspension was washed several times with (SEQ ID NO:10) deionised water and analysed by Coulter counter and acous [0304] This example is directed at the preparation of tic attenuation. targeted ultrasound microbubbles whereby streptavidin is [0313] e) Conjugation with Fluorescein-Labelled Strepta used as a linker between biotinylated reporter(s) and vec vidin and Biotinylated Peptides From (b) and (c) tor(s). [0314] To the microbubble preparation from (d) was added [0305] a) Synthesis of biotin-PEGsaoo-b-Alanine Choles fluorescein-conjugated streptavidin (0.2 mg) dissolved in terol PBS (1 ml). The bubbles were placed on a roller table for 3 [0306] To a solution of cholesteryl-b-alanine hydrochlo hours at room temperature. Following extensive washing ride (as described in Example 59) (15 mg, 0.03 mmol) in 3 with water and analysis by fluorescence microscopy, the ml chloroform/wet methanol (2.6:1) was added triethy microbubbles were incubated in 1 ml of PBS containing lamine (42 ml, 0.30 mmol). The mixture was stired for 10 biotinyl-endothelin-1 peptide (0.5 mg) and biotinyl-fibrin minutes at room temperature and a solution of biotin anti-polymerant peptide (0.5 mg) from (b) and (c) respec PEGsaoo-NHS (100 mg, 0.03 mmol) in 1,4-dioxane (1 ml) tively for 2 hours. Extensive washing of the microbubbles was added dropwise. After stirring at room temperature for was performed to remove unconjugated peptide. 3 hours, the mixture was evaporated to dryness and the residue purified by flash chromatography to give white EXAMPLE 28 crystals, yield 102 mg (89%). The structure was verified by Gas-Filled Microbubbles Encapsulated with Phos MALDI-MS and NMR. phatidylserine and biotin-DPPE Used to Prepare a Streptavidin “Sandwich’ with a Mixture of biotinyl [0307] b) Synthesis of Biotinylated Endothelin-1 Peptide endothelin-1 Peptide (biotin-D-Trp-Leu-Asp-Ile-Ile (biotin-D-Trp-Leu-Asp-Ile-Ile-Trp.OH) (SEQ ID NO:9) Trp.OH) (SEQ ID NO:9) and biotinyl-fibrin-anti [0308] The peptide was synthesised on a ABI 433A auto polymerant Peptide (biotin-GPRPPERHOS.NH2) matic peptide synthesiser starting with Fmoc-Trp(Boc) (SEQ ID NO:10) Wang resin on a 0.1 mmol scale using 1 mmol amino acid [0315] a) Preparation of Biotin-Containing Microbubbles cartridges. All amino acids were preactivated using HBTU before coupling. The simultaneous removal of peptide from [0316] To a mixture of phosphatidylserine (5 mg) and the resin and side-chain protecting groups was carried out in biotin-DPPE (0.6 mg) in a clean vial was added 5% propy TFA containing 5% anisole and 5% H2O for 2 hours giving lene glycol-glycerol in water (1 ml). The dispersion was a crude product yield of 75 mg. Purification by preparative heated to 80° C. for 5 minutes and then cooled to ambient HPLC of a 20 mg aliquot of crude material was carried out temperature. The head space was then flushed with perfluo using a gradient of 30 to 80% B over 40 minutes (A=0.1% robutane and the vial was shaken in a cap-mixer for 45 TFA/water and B=0.1% TFA/acetonitrile) and a flow rate of seconds. After centrifugation the infranatant was removed 9 ml/min. After lyophilisation of the pure fractions 2 mg of and the microbubbles were washed extensively with water. pure material was obtained (analytical HPLC, gradient [0317] b) Conjugation of Gas-Filled Microbubbles Encap 30-80% B where B=0.1% TFA/acetonitrile, A=0.01% TFA/ sulated with Phosphatidylserine and Biotin-DPPE with water; detection—UV 214 nm—product retention time=12.6 Streptavidin and a Mixture of biotinyl-endothelin-1 (biotin minutes). Further product characterization was carried out D-Trp-Leu-Asp-Ile-Ile-Trp.OH) (SEQ ID NO:9) and bioti using MALDI mass spectrometry: expected M+H at 1077, nyl-fibrin-anti-polymerant Peptide (biotin-GPRPPERHO found at 1077. SNH) (SEQ ID NO:10) [0309] c) Synthesis of Biotinyl-Fibrin-Anti-Polymerant [0318] The procedure detailed in Example 27 was fol Peptide (biotin-GPRPPERHOS.NH2)(SEQ ID NO:10) lowed. [0310] This peptide was synthesised and purified using EXAMPLE 29 similar protocols to those described in (b) above. The pure product was characterised by HPLC and MALDI MS. PFB Gas-Containing Microbubbles of DSPS Functionalised with Heparin Sulphate Binding [0311] d) Preparation of Multiple-Specific Gas-Filled Peptide/Fibronectin Peptide/RGD Peptide and Microbubbles Encapsulated with Phosphatidylserine and Fluorescein. biotin-PEGsaoo-b-Alanine Cholesterol [0319] a) Synthesis of a Lipopeptide Containing the RGD [0312] DSPS (4.5 mg) and biotin-PEGsaoo-b-alanine cho Sequence and a Fluorescein Reporter Group: Dipalmitoyl lesterol from (a) (0.5 mg) were weighed into a vial and 0.8 Lys-Lys-Lys-Lysfacetyl-Arg-Gly-Asp US 2004/0141922 A1 Jul. 22, 2004 41

#

•–(=

=

)–:

ZEE US 2004/0141922 A1 Jul. 22, 2004 42

[0320] The lipopeptide was synthesised as described in seconds and then rolled overnight. The microbubbles so Example 21(a) using commercially available amino acids obtained were washed several times with deionised water and polymers. The lipopeptide was cleaved from the resin in and analysed by MALDI mass spectrometry as described in TFA containing 5% water, 5% phenol and 5% EDT for 2 Example 21(b). The microbubbles were investigated by hours. Following evaporation in vacuo the crude product microscopy and were seen to have a range of sizes between was precipitated and triturated with diethyl ether. Purifica 1 and 5 microns. Furthermore the microbubbles were fluo tion by preparative HPLC of a 40 mg aliquot of crude rescent. material was carried out using a gradient of 60 to 100% B over 40 minutes (A=0.1% TFA/water and B=0.1% TFA/ EXAMPLE 30 acetonitrile) at a flow rate of 9 ml/min. After lyophilisation 10 mg of pure material (analytical HPLC, gradient 60-100% Gas-Filled Microbubbles Comprising DSPS B where B=0.1% TFA/acetonitrile, A=0.01 TFA/water: Covalently Modified with CD71 FITC-Labelled detection—V 260-product retention time=20-22 minutes). anti-transferrin Receptor Antibody and ‘Doped’ Further product characterisation was carried out using with a Lipopeptide with Affinity for Endothelial MALDI mass spectrometry: expected M+H at 1922, found Cells at 1920. [0325] This example is directed at the preparation of [0321] b) Synthesis of a Lipopeptide Containing a Heparin multiple vector targeted ultrasound agents. Sulphate-Binding Sequence and a Fibronectin Peptide [0326] a) Synthesis of an Endothelial Cell Binding Lipopeptide: 2-n-hexadecylstearyl-Lys-Leu-Ala-Leu-Lys [0322] Synthesis and purification were carried out as Leu-Ala-Leu-Lys-Ala-Leu-Lys-Ala-Ala-Leu-Lys-Leu-Ala described in Example 21 (a). NH, (SEQ ID NO:12) [0323] c) Preparation of Multiple-Specific Gas-Filled [0327] The lipopeptide shown below was synthesised on a Microbubbles of DSPS Functionalised with a Heparin Sul ABI 433A automatic peptide synthesiser starting with a Rink phate-Binding Peptide, a Fibronectin Peptide, acetyl-RGD amide resin on a 0.1 mmol scale using 1 mmol amino acid Peptide and Fluorescein cartridges.

# # # # SJ's: ) &J's: ) SJ's: N SJ's* N O = H O = H & 5 H & H O

Hss-H Hss-H

h O h O h O h O h O

: N 2 N : N : N : N * E | | E | E | E | H H O H * > H * > H * > H *

[0324] DSPS (4 mg, 3.9 mmol), lipopeptide from (a) (0.5 [0328] All amino acids and 2-m-hexadecylstearic acid mg, 0.2 mmol) and lipopeptide from (b) (0.5 mg) were were preactivated using HBTU before coupling. The simul weighed into each of two vials and 0.8 ml of a solution of taneous removal of peptide from the resin and side-chain 1.46 propylene glycol/2.4% glycerol was added to each vial. protecting groups was carried out in TFA containing 5% EDT and 5% H2O for 2 hours, giving a crude product yield The mixtures were warmed to 80° C. for 5 minutes (vials of 150 mg. Purification by preparative HPLC of a 40 mg shaken during warming). The samples were cooled to room aliquot of crude material was carried out using a gradient of temperature and the head spaces were flushed with perfluo 90 to 100% B over 50 minutes (A=0.1% TFA/water and robutane gas. The vials were shaken in a cap mixer for 45 B=MeOH) at a flow rate of 9 ml/min. After lyophilisation, US 2004/0141922 A1 Jul. 22, 2004 43

10 mg of pure material was obtained (analytical HPLC, analysed for the presence of antibody by flow cytometry and gradient 90-100% B where B=MeOH, A=0.01% TFA/water: microscopy. A fluorescent population of >92% was observed detection—UV 214 mm—product retention time=23 minutes). (see FIG. 1). Further product characterisation was carried out using MALDI mass spectrometry: expected M+H at 2369, found [0335] Incorporation of lipopeptide into the microbubbles at 2373. was confirmed by MALDI mass spectrometry as described [0329] b) Preparation of Gas-Filled Microbubbles Com in Example 21 (b). prising DSPS ‘Doped’ with a Endothelial Cell-Binding Lipopeptide and PE-PEG2000-Mal EXAMPLE 31 [0330] DSPS (4.5 mg) and lipopeptide from (a) (0.5 mg) Gas-Filled Microbubbles Comprising DSPS, a along with PE-PEGeooo-Mal from Example 50 (0.5 mg) Lipopeptide for Endothelial Cell Targeting and a were weighed into a clean vial and 1 ml of a solution of 1.4% Captopril-Containing Molecule propylene glycol/2.4% glycerol was added. The mixture was warmed to 80° C. for 5 minutes and then filtered through a [0336] This example is directed to the preparation of 4.5 micron filter. The sample was cooled to room tempera ultrasound agents for combined targeting and therapeutic ture and the head space was flushed with perfluorobutane applications. gas. The vial was shaken in a cap mixer for 45 seconds and the resulting microbubbles were washed three times with [0337] a) Synthesis of a Lipopeptide Functionalised with distilled water. Captopril

(SEQ ID NO: 13)

~~~~~~~~ NH2

O O O ~~~~~~~~ sº "J'sN H # Nº. - O -

O OH NH2 HN sº~'s O E

[0331] c) Thiolation of FITC-Labelled Anti-Transferrin [0338] The structure shown above was synthesised using Receptor Antibody a manual nitrogen bubbler apparatus starting with Fmoc [0332] FITC-labelled CD71 anti-transferrin receptor Ab protected Rink Amide MBHA resin on a 0.125 mmol scale. (100 mg/ml in PBS, 0.7 ml) was reacted with Traut’s reagent Coupling was carried out using standard TBTU/HOBt/DIEA (0.9 mg) at room temperature for 1 hour. Excess reagent was protocols. Bromoacetic acid was coupled through the side separated from modified protein on a NAP-5 column. chain of Lys as a symmetrical anhydride using DIC preac [0333] d) Conjugation of Thiolated FITC-Labelled Anti tivation. Captopril dissolved in DMF was introduced on the Transferrin Receptor Antibody to Gas-Filled Microbubbles solid-phase using DBU as base. Simultaneous removal of Comprising DSPS ‘Doped’ with an Endothelial Cell-Bind the peptide from the resin and deprotection of side-chain ing Lipopeptide and DSPE-PEG2000-Mal protecting groups was carried out in TFA containing 5% EDT, 5% water and 5% ethyl methyl sulphide for 2 hours. [0334] A 0.5 ml aliquot of the protein fraction (2 ml in An aliquot of 10 mg of the crude material was purified by total) from (c) above was added to the microbubbles from (b) and the conjugation reaction was allowed to proceed for preparative liquid chromatography using a gradient of 70 to 10 minutes on a roller table. Following centrifugation at 100% B over 60 minutes (A=0.1% TFA/water and B=0.1% 1000 rpm for 3 minutes the protein solution was removed TFA/acetonitrile) at a flow rate of 10 ml/min. After lyophi and the conjugation repeated twice more with 1 ml and 0.5 lisation a yield of 2 mg of pure material was obtained ml aliquots of protein solution respectively. The bubbles (analytical HPLC, gradient 70-100% B over 20 minutes, were then washed four times in distilled water and a sample A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow rate US 2004/0141922 A1 Jul. 22, 2004 44

1 ml/min., detection UV 214 nm, retention time 26 minutes). [0341] c) Preparation of Gas-Filled Microbubbles Com Further characterisation was carried out using MALDI mass prising DSPS, a Lipopeptide for Endothelial Cell Targeting spectrometry, giving M+H at 1265 as expected. and a Captopril-Containing Molecule for Drug Delivery [0339] b) Synthesis of a Lipopeptide with Affinity for Endothelial Cells: Dipalmitoyl-Lys-Lys-Lys-Aca-Ile-Arg [0342] DSPS (4.5 mg), product from (a) (0.5 mg) and Arg-Val-Ala-Arg-Pro-Pro-Leu-NH. (SEQ ID NO:14) product from (b) (0.5 mg) were weighed into a vial and 1.0 ~~~~~~~~O

"J's, SJTS_2^_^ à H

NH2

O O O O NH2 H H H N N N i N - N - N - N H # H # H # O O F. O 2's O = N *NH *NH

[0340] The lipopeptide was synthesised on a ABI 433A ml of a solution of 1.4% propylene glycol/2.4% glycerol was automatic peptide synthesiser starting with Rink amide resin added. The mixture was warmed to 80° C. for 5 minutes on a 0.1 mmol scale using 1 mmol amino acid cartridges. All (vial shaken during warming). The sample was cooled to amino acids and palmitic acid were preactivated using room temperature and the head space was flushed with HBTU before coupling. The simultaneous removal of pep perfluorobutane gas. The vial was firstly shaken in a cap tide from the resin and side-chain protecting groups was mixer for 45 seconds then rolled for 1 hour, whereafter the carried out in TFA containing 5% phenol, 5% EDT and 5' contents were extensively washed with deionised water. No H2O for 2 hours, giving a crude product yield of 160 mg. detectable level of starting material was found in the final Purification by preparative HPLC of a 35 mg aliquot of wash solution as evidenced by MALDI MS. MALDI mass crude material was carried out using a gradient of 70 to spectral analysis was used to confirm incorporation of the 100% B over 40 minutes (A=0.1% TFA/water and products from (a) and (b) into the microbubbles as described B=MeOH) at a flow rate of 9 ml/min. After lyophilisation, in Example 21(b). 20 mg of pure material was obtained (analytical HPLC, gradient 70-100% B where B=MeOH, A=0.01% TFA/water: detection—UV214 and 260 nm—product retention time=16 [0343] d) In Vitro Study of Gas-Filled Microbubbles Com minutes). Further product characterisation was carried out prising DSPS, a Lipopepitole for Endothelial Cell Targeting using MALDI mass spectrometry: expected M+H at 2050, and a Captopril-Containing Molecule for Therapeutic Appli found at 2055. cations US 2004/0141922 A1 Jul. 22, 2004 45

[0344] The in vitro assay decribed in Example 21(c) was glycerol was added. The mixture was sonicated for 3-5 used to examine cell binding under flow conditions. A minutes, warmed to 80° C. for 5 minutes and then filtered gradual accumulation of microbubbles on the cells took through a 4.5 micron filter. The mixture was cooled to room place, depending on the flow rate. On further increasing the temperature and the head space was flushed with perfluo flow rate cells started to become detached from the cover robutane gas. The vial was shaken in a cap-mixer for 45 slip, but the microbubbles remained bound to the cells. seconds and the resulting microbubbles were centrifuged at Control microbubbles not carrying the vector did not adhere 1000 rpm for 3 minutes. The microbubbles were washed to the endothelial cells and disappeared from the chamber with water until no polymixin B sulphate or lipopeptide under minimal flow conditions. could be detected in the infranatant by MALDI-MS. Micros copy showed that the size distribution of the bubble popu EXAMPLE 32 lation was in the desired range of 1-8 micron. To the washed Preparation of Gas-Filled Microbubbles Comprising bubbles (ca. 0.2 ml) was added methanol (0.5 ml), and the DSPS Loaded with a Lipopeptide Comprising a mixture was placed in a sonicator bath for 2 minutes. The Helical Peptide with Affinity for Cell Membranes resulting clear solution, on analysis by MALDI-MS, was and the Peptide Antibiotic Polymixin B Sulphate found to contain both lipopeptide and polymixin B sulphate (expected 1203, found 1207). [0345] This example is directed to the preparation of targeted microbubbles comprising multiple peptidic vectors EXAMPLE 33 having a combined targeting and therapeutic application. Preparation of Gas-Filled Microbubbles Comprising [0346] a) Synthesis of a Lipopeptide Comprising a Helical DSPS ‘Doped with a Lipopeptide Comprising a Peptide with Affinity for Cell Membranes: hexadecylstearyl IL-1 Receptor-Binding Sequence and Modified with Lys-Leu-Ala-Leu-Lys-Leu-Ala-Leu-Lys-Ala-Leu-Lys-Ala a Branched Structure Containing the Drug Ala-Leu-Lys-Leu-Ala-NH. (SEQ ID NO.12) Methotrexate [0350] This example is directed to the preparation of [0347] This is prepared as described in Example 30(a). targeted microbubbles comprising multiple vectors for tar [0348] b) Preparation of Multiple-Specific Gas-Filled geted/therapeutic applications. Microbubbles [0351] a) Synthesis of a Lipopeptide Comprising an Inter [0349] DSPS (5.0 mg), lipopeptide from (a)(0.3 mg) and leukin-1 Receptor-Binding Peptide: Dipalmitoyl-Lys-Gly polymixin B sulphate (0.5 mg) were weighed into a clean Asp-Trp-Asp-Gln-Phe-Gly-Leu-Trp-Arg-Gly-Ala-Ala.OH vial and 1.0 ml of a solution of 1.4% propylene glycol/2.4% (SEQ ID NO:15)

~~~~~~~~O o” H 2.

O H O

H O US 2004/0141922 A1 Jul. 22, 2004 46

-continued H - Sr. N—H N—H ~\,O sº,O sº,O º,O sºO # | | s | | # | - H O H O = H

[0352] The lipopeptide was synthesised on a ABI 433A characterisation was carried out using MALDI mass spec automatic peptide synthesiser starting with Fmoc-Ala-Wang trometry: expected M+H at 2083, found at 2088. resin on a 0.1 mmol scale using 1 mmol amino acid [0354] b) Synthesis of a Branched Methotrexate Core cartridges. Structure Containing a Thiol Moiety

H2N N N Sr. | S | COOH N 22 N S N O

NH2 N N N "J's N H H F. H >~~ O F O O H2N N N N COOH COOH O Sriºrs H N O HN : COOH QC N H H NH2 N N O - N s : H O SH s O COOH

[0353] All amino acids and palmitic acid were preacti [0355] The methotrexate structure was synthesised on an vated using HBTU before coupling. The simultaneous ABI 433A automatic peptide synthesiser starting with Fmoc removal of lipopeptide from the resin and side-chain pro Cys(Trt) Tentagel resin on a 0.1 mmol scale. The simulta tecting groups was carried out in TFA containing 5% H2O, neous removal of product from the resin and deprotection of protecting groups was carried out in TFA containing 5% 5% anisole, 5% phenol and 5% EDT for 2 hours, giving a EDT and 5% H2O for 2 hours, giving a crude product yield crude product yield of 150 mg. Purification by preparative of 160 mg. Purification by preparative HPLC of a 30 mg HPLC of a 30 mg aliquot of crude material was carried out aliquot of crude material was carried out using a gradient of using a gradient of 90 to 100% B over 40 minutes (A=0.1% 10 to 30% B over 40 minutes (A=0.1% TFA/water and TFA/water and B=MeOH) at a flow rate of 9 mlnin. After B=0.1% TFA/acetonitrile) and a flow rate of 9 ml/min. After lyophilisation, 4 mg of pure material was obtained (analyti lyophilisation of the pure fractions, 9 mg of pure material cal HPLC, gradient 90-100% B over 20 minutes where was obtained (analytical HPLC, gradient 5-50% B where B=MeOH, A=0.01% TFA/water detection—UV 214 B=0.1% TFA/acetonitrile, A=0.01% TFA/water detection— nm—product retention time=23 minutes). Further product UV 214 nm—product retention time=9.5 minutes). Further US 2004/0141922 A1 Jul. 22, 2004 47 product characterisation was carried out using MALDI mass microbubble membranes with vector-modified lipid struc spectrometry: expected M+H at 1523, found at 1523. tures as described in Example 21. [0356] c) Preparation of Multiple-Specific Gas-Filled [0362] a) Preparation of DSPS-Encapsulated Gas-Filled Microbubbles Microbubbles [0357] DSPS (4.5 mg), thiol-containing lipopeptide from [0363] DSPS (4.5 mg) was weighed into a clean vial. 1.0 Example 64(a) (0.5 mg) and lipopeptide from (a) (0.2 mg) ml of a solution of 1.4% propylene glycol/2.4% glycerol was were weighed into a clean vial and 1.0 ml of a solution of added and the mixture was sonicated for 2 minutes and then 1.4% propylene glycol/2.4% glycerol was added. The mix warmed to 80° C. for 5 minutes. Immediately following ture was sonicated for 3-5 minsutes, warmed to 80° C. for 5 warming the solution was filtered through a 4 micron filter. minutes and then filtered through a 4.5 micron filter. The The sample was cooled to room temperature and the head mixture was cooled to room temperature and the head space space was flushed with perfluorobutane gas. The vial was was flushed with perfluorobutane gas. The vial was shaken shaken in a cap mixer for 45 seconds. The resulting in a cap mixer for 45 seconds and the resulting microbubbles microbubbles were then washed once with deionised water were centrifuged at 1000 rpm for 3 minutes, whereafter the and the infranatant was discarded. The microbubbles were infranatant was discarded. then resuspended in 0.5 ml water. [0358] d) Conjugation of Methotrexate Branched Struc [0364] b) Preparation of poly-L-lysine/DNA Complex and ture to Thiolated Microbubbles Loading of DSPS-Encapsulated Microbubbles [0359] The methotrexate structure from (b) above (0.5 [0365] To 1 mg of poly-L-lysine (70-150 kD) in a clean mg) was dissolved in PBS, pH 8.0. The solution was then vial was added 0.1 ml of a fluorescein-labeled digest of added to the thiol-containing microbubbles from (c) and plasmid pBR322 dissolved in TE buffer (10 mM tris-HCl, disulphide bond formation was allowed to proceed for 16 pH 8). The solution was made up to a total of 0.6 ml by hours. Following extensive washing with PBS and water the addition of water and the pH was adjusted to 8. Complexing bubbles were analysed by microscopy and MALDI MS. was allowed to proceed for 1 hour, after which 0.05 mL of the polylysine-DNA solution was added to the microbubble [0360] The disulphide bond linking the methotrexate suspension from (a) above. After 1 hour microscopy was structure to the microbubbles may be reduced in vivo to used to show that the bubbles were fluorescent, confirming liberate the free drug molecule, so that such microbubbles in combination with a tumour specific vector comprise a drug the presence of DNA. delivery system. A physiologically acceptable reducing EXAMPLE 35 agent such as glutathione may be used to bring about drug release. Preparation of Gas-Filled Microbubbles Containing a Branched Core Peptide Comprising a EXAMPLE 34 Dabsylated-Atherosclerotic Plaque-Binding Sequence and RGDS Preparation of Gas-Filled Microbubbles Coated [0366] This example is directed to the preparation of with poly-L-lysine Complexed to microbubbles having a thiol group on the surface for modi Fluorescein-Labeled DNA Fragments From Plasmid fication with thiol-containing vectors for targeting/drug pBR322 delivery and drug release. [0361] This example is directed to the preparation of [0367] a) Synthesis of the Branched Peptide Dabsyl-Tyr microbubbles for gene therapy/anti-sense applications. Spe Arg-Ala-Leu-Val-Asp-Thr-leu-Lys-Lys(NH3-Arg-Gly-Asp cific targeting may be achieved by further doping of Ser)-Gly-Cys OH (SEQ ID NO:16)

OH

i i H

COOH

NH

H2N NH US 2004/0141922 A1 Jul. 22, 2004 48

-continued NH2

OH O O O

à à Xs SH HO O H NH N N : N H = H O =`coon O

NH

[0368] The peptide was synthesised on an ABI 433A water souble reducing agent tris-(2-carboxyethyl)-phos automatic peptide synthesiser starting with Fmoc-Cys(Trt) phine. Following reduction, the infranatant was found to Tentagel resin on a 0.1 mmol scale using 1 mmol amino acid contain free dabsyl-peptide as evidenced by HPLC and cartridges. All amino acids were preactivated using HBTU MALDI MS. before coupling. The simultaneous removal of peptide from the resin and side-chain protecting groups was carried out in [0373] Other physiologically acceptable reducing agents TFA containing 5% phenol, 5% EDT and 5% H2O for 2 such as reduced glutathione may also be useful for initiating hours, giving a crude product yield of 160 mg. Purification release. by preparative HPLC of a 30 mg aliquot of crude material was carried out using a gradient of 10 to 60% B over 40 EXAMPLE 36 minutes (where A=0.1% TFA/water and B=acetonitrile) at a flow rate of 9 ml/min. After lyophilisation, 2.5 mg of pure Preparation of Gas-Filled Microbubbles Encapsu material was obtained (analytical HPLC, gradient 10-50% B lated with DSPS and Biotin-PEGsaoo-acyl-phos over 20 minutes where B=0.1% TFA/acetonitrile and phatidylethanolamine and Functionalised with A=0.01% TFA/water; detection—UV 214 and 435 mm—prod Streptavidin, Oligonucleotide biotin-GAAAGG uct retention time=21 minutes). Further product characteri TAGTGGGGTCGTGTGCCGG (SEQ ID NO:17) sation was carried out using MALDI mass spectrometry: and Biotinylated Fibrin-Anti-Polymerant Peptide expected M+H at 2070, found at 2073. (Biotin-GPRPPERHOS, NH2)(SEQ ID NO:10) [0369] b) Preparation of Thiol-Containing Gas-Filled º, a) Synthesis of Biotin-PEGao-Acyl-Phosphatidyl Microbubbles thanolamine [0375] A mixture of dipalmitoyl phosphatidyl ethanola [0370] These were prepared as described in Example mine, (21.00 mg, 0.03 mmol), biotin-PEG-CO2-NHS, (100 64(a). mg, 0.03 mmol) and triethylamine (42 ul, 0.30 mmol) in a [0371] c) Oxidative Coupling of Thiolated Microbubbles solution of chloroform/methanol (3:1) was stirred at room with Multiple-Specific Peptide via Disulphide Bond Forma temperature for 2 hours. After evaporation of the solvents tion under reduced pressure, the residue was flash chromato graphed (methylene chloride/methanol/water, 40.8:1). The [0372] The infranatant from the microbubbles from (b) product was obtained as a yellow gum (112 mg, 94%), and above was discarded and replaced with a solution of dabsyl structure was verified by NMR and MALDI-MS. peptide from (a) (1 mg) in 0.7 ml dilute ammonia solution (pH 8). To this was added 0.2 ml of a stock solution [0376] b) Binding of Fluorescein-Conjugated Streptavidin containing 6 mg of potassium ferricyanate dissolved in 2 ml to Gas-Filled Microbubbles of water. The vial was placed on a roller table and thiol [0377] Gas-filled microbubbles were prepared by mixing oxidation allowed to proceed for 2 hours. The bubbles were DSPS and biotin-PEGsaoo-acyl-phosphatidylethanolamine then washed extensively with water until the infranatant was as described in previous examples. The microbubble sus free of the dabsyl-peptide as evidenced by HPLC and pension was divided into 0.2 ml aliquots and fluorescein MALDI MS. Detection of microbubble-bound peptide was conjugated streptavidin was added as shown in the table carried out by reduction of the disulphide bond using the below. The samples were incubated on a roller table for 15 US 2004/0141922 A1 Jul. 22, 2004 49 or 30 minutes at ambient temperature before removal of [0380] The particles from aliquot no. 6 above were cen excess protein by washing in PBS. The samples were trifuged and the supernatant was replaced with 1 ml PBS analysed by flow cytometry and Coulter Counter. The results buffer, pH 7.5, containing 0.2 mg of biotin-GAAAGG are summarized in the table below. TAGTGGGGTCGTGTGCCGG (SEQ ID NO:17) and 0.2 [0378] Results: mg of biotin-GPRPPERHQS (SEQ ID NO:10) (prepared as in Example 27(b) and (c)). After incubation for 24 hours the particles were washed extensively with PBS and water.

Added Particle [0381] Other biotinylated vectors or therapeutic agents Streptavidin Incubation % median may be conjugated to streptavidin- or avidin-coated Aliquot (mg/200:1 time (amb. Fluorescent diameter in O. sample temp.) particles (microns) microbubbles using this procedure.

1 O 2.0 - 2 O - 12 (foam) EXAMPLE 37 3 0.2 (3 x 10° mmol) 30 min 7.8 3.9 4 2 (3 x 10-9 mmol) 30 min 26.2 4.2 5 10 (1.5 x 107 mmol) 15 min 30.5 ima Preparation of Gas-Filled Microbubbles 6 20 (3 x 10-7 mmol) 30 min 97.9 5.2 Encapsulated with DSPS and Functionalised with a 7 40 (6 x 10-7 mmol) 15 min 96.7 5.1 Thrombi-Targeting Lipopeptide and the 8 20 (3 x 10-7 mmol) 15 min 0.6 3.7 Thrombolytic Enzyme Tissue Plasminogen DSPS control Activator [0382] This example is directed at the preparation of thrombus targeted ultrasound contrast agents comprising a [0379] c) Conjugation of Streptavin-Coated Microbubbles therapeutic thromolytic agent. with the Oligonucleotide biotin-GAAAGG TAGTGGGGTCGTGTGCCGG (SEQ ID NO:17) and [0383] a) Synthesis of a Lipopeptide with Affinity for Biotinylated Fibrin-Anti-Polymerant Peptide Biotin-GPRP Thrombi (Dipalmitoyl-Lys-Asn-Asp-Gly-Asp-Phe-Glu PERHOS (SEQ ID NO:10) Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln.NH2) (SEQ ID NO:18) ~~~~~~~~O

COOH COOH O O H H N N N : N N O FYoosu, O

O O O H H N N : N : N i NH2 ? H E H E

COOH

OH US 2004/0141922 A1 Jul. 22, 2004 50

[0384] The lipopeptide was synthesised on an ABI 433 A resulting microbubbles were washed twice with deionised automatic peptide synthesiser starting with Rink amide resin water. The infranatant was discarded and replaced with a 1 on a 0.1 mmol scale using 1 mmol amino acid cartridges. All ml aliquot of the protein solution from (b) above. The amino acids and palmitic acid were preactivated using conjugation reaction was allowed to proceed for 1 hour. The HBTU before coupling. The simultaneous removal of pep microbubbles were centrifuged and the infranatant was tide from the resin and side-chain protecting groups was exchanged with a further 1 ml of protein solution. The carried out in TFA containing 5% phenol, 5% EDT, 5% incubation step was repeated until all protein solution was anisole and 5% H2O for 2 hours, giving a crude product used up. The microbubbles were then washed extensively yield of 80 mg. Purification by preparative HPLC of a 20 mg with water and analysed by Coulter counter. The aliquot of the crude material was carried out. After lyophi microbubbles were tested in the flow chamber assay lisation, 6 mg of pure material was obtained. The product described in Example 21(c). Microbubbles modified with was characterised by MALDI mass spectrometry and ana protein were found to bind in higher numbers than those lytical HPLC. comprising either lipopeptide/DSPS or DSPS alone. [0385] b) Modification of Tissue Plasminogen Activator [0389] The targeting/therapeutic/ultrasound activities of with Sulpho-SMPB these microbubbles be evaluated in models of both in vitro [0386] A solution of 0.1 ml of ammonim carbonate buffer and in vivo thrombogenisis. containing 0.1 mg of t-PA was made up to 0.2 ml by the EXAMPLE 38 addition of water. To this solution was added 0.4 mg of Sulpho-SMPB (dissolved in 0.05 ml DMSO. The protein solution was left standing at room temperature for 45 Preparation of Gas-Filled Microbubbles Comprising minutes, whereafter purification was carried out on a Super DSPS Loaded with a Lipopeptide Comprising a dex 200 column. The product was eluted in PBS and the Helical Peptide with Affinity for Cell Membranes modified protein fraction was collected. [0390] This example is directed to the preparation of [0387] c) Preparation of Gas-Filled Microbubbles Encap targeted microbubbles comprising a peptidic vector for sulated with DSPS/Thrombi-Binding Lipopeptide and targeting of cell membrane structures. Thiol-Containing Lipoeptide and Conjugation of Modified [0391] a) Synthesis of a Lipopeptide Comprising a Helical Tissue Plasminogen Activator Peptide with Affinity for Cell Membranes (SEQ ID NO:12)

# # # # SJ's: ) &J's: ) SJ's: N SJ's* N O = H O = H & 5 H & H O

Hss-H Hss-H

h O h O h O h O h O

: N 2 N : N : N : N * E | | E | E | E | H H O H ° SH v SSH v SSH

[0388] DSPS (5.0 mg) was weighed into a clean vial along [0392] The lipopeptide was synthesised on an ABI 433A with 0.5 mg of the lipopeptide from (a) and 0.5 mg of the automatic peptide synthesiser starting with Rink amide resin thiol-containing lipopeptide from Example 64(a). To this on a 0.2 mmol scale using 1 mmol amino acid cartridges. All was added 1.0 ml of a solution of 1.4% propylene glycol/ amino acids and 2-m-hexadecylstearic acid were preacti 2.4% glycerol and the mixture was sonicated for 2 minutes vated using HBTU before coupling. The simultaneous and then warmed to 80° C. for 5 minutes. Immediately removal of lipopeptide from the resin and side-chain pro following warming, the solution was filtered through a 4 tecting groups was carried out in TFA containing 5% H2O micron filter. The sample was cooled to room temperature for 2 hours, giving a crude product yield of 520 mg. and the head space flushed with perfluorobutane gas. The Purification by preparative HPLC of a 30 mg aliqout of vial was shaken in a cap mixer for 45 seconds and the crude material was carried out using a gradient of 90 to US 2004/0141922 A1 Jul. 22, 2004 51

100% B over 40 minutes (A=0.1% TFA/water and EDT, 5% anisole and 5% H2O for 2 hours, giving a crude B=MeOH) at a flow rate of 9 ml/min. After lyophilisation, product yield of 150 mg. Purification by preparative HPLC 10 mg of pure material was obtained (analytical HPLC, of a 40 mg aliqout of crude material was carred out using a gradient 90-100% B over 20 minutes where B=MeOH, gradient of 70 to 100% B over 40 minutes (A=0.1% TFA/ A=0.01% TFA/water; detection—UV 214 mm—product reten water and B MeOH) at a flow rate of 9 ml/min. After tion time=23 minutes). Further product characterisation was lyophilisation. 14 mg of pure material (analytical HPLC, carried out using MALDI mass spectrometry: expected gradient 70-100% B where B=MeOH, A=0.01% TFA/Wa M+H at 2369, found at 2375. ter; detection—UV 260 and fluorescence, Ex280, Em350– product retention time=22 minutes). Further product char [0393] b) Preparation of Gas-Filled Microbubbles acterisation was carried out using MALDI mass [0394] DSPS (4.5 mg) and lipopeptide from (a) (0.5 mg) spectrometry: expected M+H at 1478, found at 1471. were weighed into a clean vial and 1.0 ml of a solution of [0399] b) Preparation of Gas-Filled Microbubbles Com 1.4% propylene glycol/2.4% glycerol was added. The mix prising DSPS ‘Doped’ with the Biotinylated Lipopeptide ture was sonicated for 3-5 minutes, warmed to 80° C. for 5 Sequence From (a) minutes and then filtered through a 4.5 mm filter. The mixture was cooled to room temperature and the head space [0400] DSPS (4.5 mg) and lipopeptide from (a) (0.5 mg, was flushed with perfluorobutane gas. The vial was shaken 0.2 mmol) were weighed into each of two vials, and 0.8 ml in a cap mixer for 45 seconds and the resulting microbubbles of a solution of 1.4% propylene glycol/2.4% glycerol was were centrifuged at 1000 rpm for 3 minutes. The added to each vial. The mixtures were warmed to 80° C. for microbubbles were then washed with water until no lipopep 5 minutes (vials shaken during warming). The samples were tide could be detected by MALDI-MS. Coulter counter, cooled to room temperature and the head spaces were acoustic attenuation and pressure stability studies were flushed with perfluorobutane gas. The vials were shaken in performed. To an aliquot of the washed bubbles (ca. 0.2 ml) a cap mixer for 45 seconds and then rolled overnight. The was added methanol (0.5 ml), and the mixture was placed in resulting microbubbles were washed several times with a sonicator bath for 2 minutes. The resulting clear solution, deionised water and analysed by Coulter counter and acous on analysis by MALDI-MS, was found to contain the tic attenuation. MALDI mass spectral analysis was used to lipopeptide. confirm incorporation of lipopeptide into DSPS microbubbles as follows: ca. 50-100 ml of microbubbles [0395] c) In Vitro and In Vivo Tests were transferred to a clean vial and 50-100 ml water were [0396] The microbubbles had similar characteristics in added. The mixture was sonicated for 30 seconds and vitro and in vivo as was found for the microbubbles made in spotted onto a clean target disc (1 ml+0.5 ml ACH matrix). Example 21. Positive mode gave M+H at 1474, expected for lipopeptide at 1478. EXAMPLE 39 EXAMPLE 40 Gas-Filled Microbubbles Encapsulated with Phosphatidylserine and a Biotinylated Lipopeptide Preparation of Multiple-Specific Gas-Filled Microbubbles Comprising DSPS Loaded with a [0397] a) Synthesis of Lipopeptide dipalmitoyl-lysinyl Lipopeptide Comprising a Non-Bioactive tryptophanyl-lysinyl-lysinyl-lysinyl(biotinyl)-glycine (SEQ Interleukin-1 Receptor-Binding Peptide ID NO:19) [0401] This example is directed to the preparation of [0398] The lipopeptide was synthesised on an ABI 433A targeted microbubbles comprising a non-bioactive peptidic automatic peptide synthesiser starting with Fmoc-Gly-Wang vector for targeting at the IL-1 recptor which does not induce resin on a 0.1 mmol scale using 1 mmol amino acid cartridges. All amino acids and palmitic acid were preacti signal tranduction or prevent IL-1 binding. vated using HBTU before coupling. The simultaneous [0402] a) Synthesis of a Lipopeptide Comprising a Non removal of peptide from the resin and side-chain protecting Bioactive Interleukin-1 Receptor-Binding Peptide (SEQ ID groups was carried out in TFA containing 5% phenol, 5% NO:15)

O ~~~~~~~~ _-H H o” H os / 2. O O O O

N Js N Js• N | | s H O H O US 2004/0141922 A1 Jul. 22, 2004 52

-continued H–N Sr. N—H N—H

O O O O O N Js Js Js Js ~,B H O |H |! {| H|| O iH O =| H? 2 >N—i?

H–N O

H

[0403] The lipopeptide was synthesised on an ABI 433A EXAMPLE 41 automatic peptide synthesiser starting with Fmoc-Ala-Wang Preparation of Perfluoropropane-Filled resin on a 0.1 mmol scale using 0.1 mmol amino acid Microbubbles Comprising DSPC, DSPS and cartridges. All amino acids and palmitic acid were preacti Endothelial Cell-Binding Lipopeptide for Targeted vated using HBTU before coupling. The simultaneous Ultrasound Imaging removal of lipopeptide from the resin and side-chain pro [0406] To 0.8 ml of a solution containing DSPC:DSPS tecting groups was carried out in TFA containing 5% H2O, (3:1) (5 mg/ml) in propylene glycol/glycerol (4% in water) 5% anisole, 5% phenol and 5% EDT for 2 hours, giving a was added 0.5 mg of the lipopeptide from Example 31(b). crude product yield of 150 mg. Purification by preparative The mixture was heated to 80° C. for 5 minutes and shaken. HPLC of a 30 mg aliquot of crude material was carried out The solution was then cooled to ambient temperature and the using a gradient of 90 to 100% B over 40 minutes (A=0.1% headspace was flushed with perfluoropropane. The vial was TFA/water and B=MeOH) at a flow rate of 9 ml/min. After shaken on a cap-mixer for 45 seconds and placed on a roller lyophilisation, 4 mg of pure material was obtained (analyti table for 5 minutes. The sample was centrifuged at 2000 rpm cal HPLC, gradient 90-100 B over 20 minutes where for 5 minutes and the infranatant was removed and replaced B=MeOH, A=0.01% TFA/water detection—UV 214 with distilled water. The headspace was again flushed with nm—product retention time=23 minutes). Further product perfluoropropane and the sample was kept on a roller table characterisation was carried out using MALDI mass spec until a homogeneous appearance was obtained. The washing trometry: expected M+H at 2083, found at 2088. procedure was repeated. The resulting ultrasound contrast agent was characterised by Coulter counter analysis, acous [0404] b) Preparation of Gas-Filled Microbubbles tic attenuation measurements and resistance to external [0405] DSPS (4.5 mg) and lipopeptide from (a) (0.5 mg) pressure. The microbubbles were tested in the in vitro assay were weighed into a clean vial and 1.0 ml of a solution of as detailed in Example 21. A gradual accumulation of 1.4% propylene glycol/2.4% glycerol was added. The mix microbubbles binding to the cells was observed. ture was sonicated for 3-5 mins, warmed to 80° C. for 5 EXAMPLE 42 minutes and then filtered through a 4.5 micron filter. The mixture was cooled to room temperature and the head space Preparation of Sulphur Hexafluoride-Containing was flushed with perfluorobutane gas. The vials were shaken Microbubbles Comprising DSPC, DSPS and in a cap mixer for 45 seconds and the resulting microbubbles Endothelial Cell-Binding Lipopeptide for Targeted were centrifuged at 1000 rpm for 3 minutes. The Ultrasound Imaging microbubbles were then washed with water until no lipopep [0407] To 0.8 ml of a solution containing DSPC:DSPS tide could be detected by MALDI-MS. To the washed (3:1) (5 mg/ml) in propylene glycol/glycerol (4% in water) microbubbles (ca. 0.2 ml) was added methanol (0.5 ml), and was added 0.5 mg of the lipopeptide from Example 31(b). the mixture was placed in a sonicator bath for 2 minutes. The The mixture was heated to 80° C. for 5 minutes and shaken. resulting clear solution, on analysis by MALDI-MS, was The solution was then cooled to ambient temperature and the found to contain lipopeptide (expected 2083, found 2088). headspace was flushed with sulphur hexafluoride gas. The US 2004/0141922 A1 Jul. 22, 2004 53 vial was shaken on a cap-mixer for 45 seconds and placed EXAMPLE 45 on a roller table for 5 minutes. The sample was centrifuged at 2000 rpm for 5 minutes and the infranatant was removed Preparation of Sulphur Hexafluoride-Containing Microbubbles Comprising DSPG and Endothelial and replaced with distilled water. The headspace was again Cell-Binding Lipopeptide for Targeted Ultrasound flushed with sulphur hexafluoride and the sample was kept Imaging on a roller table until a homogenous appearance was obtained. The washing procedure was repeated. The result [0410] To 0.8 ml of a solution containing DSPG (5 mg/ml) ing ultrasound contrast agent was confirmed by Coulter in propylene glycol/glycerol (4% in water) was added 0.5 counter, acoustic attenuation measurements and resistance mg of the lipopeptide from Example 31(b). The mixture was to external pressure. heated to 80° C. for 5 minutes and shaken. The solution was then cooled to ambient temperature and the headspace was EXAMPLE 43 flushed with sulphur hexafluoride gas. The vial was shaken on a cap-mixer for 45 seconds and placed on a roller table Preparation of Gas-Filled Microbubbles Comprising for 5 minutes. The sample was centrifuged at 2000 rpm for DSPG and Endothelial Cell-Binding Lipopeptide 5 minutes and the infranatant was removed and replaced for Targeted Ultrasound Imaging with distilled water. The headspace was again flushed with [0408] To 0.8 ml of a solution containing DSPG (5 mg/ml) sulphur hexafluoride and the sample was kept on a roller in propylene glycol/glycerol (4% in water) was added 0.5 table until a homogeneous appearance was obtained. The mg of the lipopeptide from Example 31(b). The mixture was washing procedure was repeated. The resulting ultrasound heated to 80° C. for 5 minutes and shaken. The solution was contrast agent was characterised by Coulter counter analy then cooled to ambient temperature and the headspace was sis, acoustic attenuation measurements and resistance to flushed with perfluorobutane. The vial was shaken on a external pressure. cap-mixer for 45 seconds and placed on a roller table for 5 minutes. The sample was centrifuged at 2000 rpm for 5 EXAMPLE 46 minutes and the infranatant was removed and replaced with distilled water. The headspace was again flushed with per Targeted Gas-Filled Microbubbles Comprising fluorobutane and the sample was kept on a roller table until DSPS Coated Non-Covalently with Polylysine a homogenous appearance was obtained. The washing pro [0411] DSPS (5 mg) was weighed into a clean vial along cedure was repeated. The resulting ultrasound contrast agent with poly-L-lysine (0.2 mg). To the vial was added 1.0 ml of was characterised by Coulter counter analysis, acoustic a solution of 1.4% propylene glycol/2.4% glycerol. The attenuation measurements and resistance to external pres mixture was warmed to 80° C. for 5 minutes. The sample sure. The microbubbles were tested in the in vitro assay as was cooled to room temperature and the head space flushed detailed in Example 21; a gradual accumulation of with perfluorobutane gas. The vial was shaken in a cap mixer microbubbles binding to the cells was observed. for 45 seconds and the resulting microbubbles were centri fuged at 1000 rpm for 3 minutes. Following extensive EXAMPLE 44 washing with water, PBS and water, the final solution was examined for polylysine content using MALDI MS. No Preparation of Perfluoropropane-Filled polypeptide material was observed in the final wash solu Microbubbles Comprising DSPG and Endothelial Cell Binding Lipopeptide for Targeted Ultrasound tion. Acetonitrile (0.5 ml) was then added and the Imaging microbubbles were sonicated until all bubbles had burst. Analysis of the resulting solution for polylysine was again [0409] To 0.8 ml of a solution containing DSPG (5 mg/ml) carried out using MALDI MS. The results were as follows: in propylene glycol/glycerol (4% in water) was added 0.5 mg of the lipopeptide from Example 31(b). The mixture was heated to 80° C. for 5 minutes and then shaken. The solution was then cooled to ambient temperature and the headspace MALDI expected MALDI found was flushed with perfluoropropane. The vial was shaken on Poly-L-lysine 786,914, 1042, 1170 790, 919, 1048, 1177 a cap-mixer for 45 seconds and placed on a roller table for 5 minutes. The sample was centrifuged at 2000 rpm for 5 minutes and the infranatant was removed and replaced with EXAMPLE 47 distilled water. The headspace was again flushed with per fluorobutane and the sample was kept on a roller table until Preparation of Functionalised Gas-Filled a homogeneous appearance was obtained. The washing Microbubbles for Targeted Ultrasound Imaging procedure was repeated. The resulting ultrasound contrast (SEQ ID NO:20) agent was characterised by Coulter counter analysis, acous [0412] This example is directed to the preparation of tic attenuation measurements and resistance to external microbubbles having a reactive group on the surface for pressure. The microbubbles were tested in the in vitro assay non-specific targeting, principally utilising disulphide as detailed in Example 21; a gradual accumulation of exchange reactions to effect binding to a multiplicity of microbubbles binding to the cells was observed. cellular targets. US 2004/0141922 A1 Jul. 22, 2004 54

[0413] a) Synthesis of a Thiol-Functionalised Lipid Mol ecule (SEQ ID NO:20)

O

~~~~~~~~H NH2

S H O O H H

N "J's- N "sº-sºº's N OH º,O - O O

NH2

[0414] The lipid structure shown above was synthesised [0416] DSPS (4.5 mg) and the lipid structure from (a) on an ABI 433A automatic peptide synthesiser starting with above (0.5 mg, 0.4 mmol) were weighed into a clean vial and Fmoc-Cys(Trt)-Wang resin on a 0.25 mmol scale using 1 0.8 ml of a solution containing 1.4% propylene glycol/2.4% mmol amino acid cartridges. All amino acids and palmitic glycerol in water was added. The mixture was warmed to acid were preactivated using HBTU coupling chemistry. The 80° C. for 5 minutes (vial shaken during warming) and simultaneous removal of peptide from the resin and depro filtered while still hot through a 40 mm filter. The sample tection of side-chain protecting groups was carried out in was cooled to room temperature and the head space was TFA containing 5% EDT and 5% H2O for 2 hours, giving a flushed with perfluorobutane gas. The vial was shaken in a crude product yield of 250 mg. Purification by preparative cap mixer for 45 seconds and then placed on roller table HPLC of a 40 mg aliquot of crude material was carried out overnight. The resulting microbubbles were washed several using a gradient of 90 to 100% B over 50 minutes (A=0.1% times with deionised water and analysed for thiol group TFA/water and B=MeOH) at a flow rate of 9 ml/min. After incorporation using Ellmans Reagent. lyophilisation, 24 mg of pure material was obtained (ana lytical HPLC, gradient 70-100% B where B=0.1% TFA/ EXAMPLE 48 acetonitrile, A=0.01% TFA/water: detection—UV 214 nm—product retention time=23 minutes). Further product Preparation of Gas-Filled Microbubbles Comprising characterisation was carried out using MALDI mass spec DSPS Doped with a Thrombus-Binding trometry: expected M+H at 1096, found at 1099. Lipopeptide [0415] b) Preparation of Gas-Filled Microbubbles Com [0417] a) Synthesis of a Lipopeptide with Affinity for prising DSPS ‘Doped’ with a Thiol-Containing Lipid Struc Thrombi (Dipalmitoyl-Lys-Asn-Asp-Gly-Asp-Phe-Glu ture Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln.NH2) (SEQ ID NO:18) ~~~~~~~~O

COOH COOH O O O H H

~~~~~~~~ N "J's: N "J's N H - H H O O O CONH2 US 2004/0141922 A1 Jul. 22, 2004 55

-continued COOH

COOH O O

E O = O = COOH N CONH2

OH

[0418] The lipopeptide was synthesised on an ABI 433 A confirm the presence of lipopeptide as described in previous automatic peptide synthesiser starting with Rink amide resin examples. on a 0.1 mmol scale using 1 mmol amino acid cartridges. All EXAMPLE 49 amino acids and palmitic acid were preactivated using HBTU before coupling. The simultaneous removal of pep Preparation of Transferrin-Coated Gas-Filled tide from the resin and side-chain protecting groups was Microbubbles for Targeted Ultrasound Imaging carried out in TFA containing 5% phenol, 5% EDT, 5% [0421] a) Synthesis of a Thiol-Functionalised Lipid Mol anisole and 5% H2O for 2 hours, giving a crude product ecule (SEQ ID NO:20)

O

~~~~~~~~ NH2

S O O O H H

~~~~~~~~ "J's- N sº N OH à # H H O - O O

yield of 80 mg. Purification by preparative HPLC of a 20 mg [0422] The lipid structure shown above was synthesised aliquot of the crude material was carried out. After lyophi on an ABI 433A automatic peptide synthesiser starting with lisation, 6 mg of pure material were obtained. The product Fmoc-Cys(Trt)-Wang resin on a 0.25 mmol scale using 1 was characterised by MALDI mass spectrometry and ana mmol amino acid cartridges. All amino acids and palmitic lytical HPLC. acid were preactivated using HBTU before coupling. The [0419] b) Preparation of Thromi-Targeting Ultrasound simultaneous removal of peptide from the resin and depro Microbubbles tection of side-chain protecting groups was carried out in TFA containing 5% EDT and 5% H2O for 2 hours, giving a [0420] DSPS (4.5 mg) and lipopeptide from (a) (1.0 mg) crude product yield of 250 mg. Purification by preparative were weighed into a vial and 0.8 ml of a solution of 1.4% HPLC of a 40 mg aliquot of crude material was carried out propylene glycol/2.4% glycerol was added. The mixture was using a gradient of 90 to 100% B over 50 minutes (A=0.1% warmed to 80° C. for 5 minutes and then filtered through a TFA/water and B=MeOH) at a flow rate of 9 ml/min. After 4 micron filter. After cooling to room temperature the head lyophilisation, 24 mg of pure material was obtained (ana space was flushed with perfluorobutane gas. The vial was lytical HPLC, gradient 70-100% B where B=0.1% TFA/ shaken in a cap mixer for 45 seconds and the resulting acetonitrile, A=0.01% TFA/water: detection—UV 214 microbubbles were washed extensively with deionised nm—product retention time=23 minutes). Further product water. The microbubbles were characterised by microscopy characterisation was carried out using MALDI mass spec and Coulter counter analysis. MALDI-MS was used to trometry: expected M+H at 1096, found at 1099. US 2004/0141922 A1 Jul. 22, 2004 56

[0423] b) Preparation of Gas-Filled Microbubbles Com [0433] c) Synthesis of Mal-PEGeooo-DSPE (3-maleimi prising DSPS ‘Doped’ with a Thiol-Containing Lipid Struc dopropionate poly(ethylene glycol)distearoylphosphati ture dylethanolamine) [0424] DSPS (4.5 mg) and lipid structure from (a) above (0.5 mg, 0.4 mmol) were weighed into a clean vial and 0.8 [0434] A solution of N-succinimidyl-3-maleimidopropi ml of a solution of 1.4% propylene glycol/2.4% glycerol was onate (5.6 mg, 0.018 mmol) in tetrahydrofuran (0.2 ml) was added. The mixture was warmed to 80° C. for 5 minutes added to H2N-PEG-20oo-DSPE (65 mg, 0.012 mmol) dis (vial shaken during warming) and filtered while still hot solved in tetrahydrofuran (1 ml) and 0.1 M sodium phos through a 40 mm filter. The sample was cooled to room phate buffer pH 7.5 (2 ml). The mixture was warmed to 30° temperature and the head space was flushed with perfluo C. and the reaction was followed to completion by TLC, robutane gas. The vial was shaken in a cap mixer for 45 whereafter the solvent was removed in vacuo. The title seconds and then placed on roller table overnight. The material was purified on a flash silica column using 80:20 resulting microbubbles were washed several times with deionised water and analysed for thiol group incorporation chloroform:methanol as eluent. The structure of the pure using Ellmans Reagent. product was confirmed by NMR and mass spectrometry. [0425] c) Modification of Transferrin with Fluorescein [0435] d) Preparation of Gas-Filled Microbubbles of NHS and Sulpho-SMPB DSPS ‘Doped’ with PE-PEG2000-Mal [0426] To 4 mg of transferrin (Holo, human) in PBS (1 ml) [0436] DSPS (4.5 mg) and PE-PEGeooo-Mal from (c) was added 0.5 ml DMSO solution containing 1 mg Sulpho above (0.5 mg) were weighed into a clean vial and 1 ml of SMPB and 0.5 mg fluorescein-NHS. The mixture was stirred a solution of 1.4% propylene glycol/2.4% glycerol was for 45 minutes at room temperature and then passed through added. The mixture was warmed to 80° C. for 5 minutes and a Sephadex 200 column using PBS as eluent. The protein then filtered through a 4.5 mm filter. The sample was cooled fraction was collected and stored at 4° C. prior to use. to room temperature and the head space was flushed with [0427] d) Microbubble Conjugation with Transferrin perfluorobutane gas. The vial was shaken in a cap-mixer for [0428] To the thiol-containing microbubbles from (b) was 45 seconds and the resulting microbubbles were washed added 1 ml of the modified transferrin protein solution from three times with distilled water. (c). After adjusting the pH of the solution to 9 the conjuga tion reaction was allowed to proceed for 2 hours at room [0437] e) Preparation of Fluorescein-Labelled Trypsin temperature. Following extensive washing with deionised water the microbubbles were analysed by Coulter counter [0438] To 5 mg of trypsin in PBS (1 ml) was added 0.2 ml (97% between 1 and 5 mm) and fluorescence microscopy DMSO solution containing 1 mg of fluorescein-NHS. The (highly fluorescent microbubbles). mixture was stirred for 45 minutes at room temperature. A Sephadex 200 column was then charged with the modified EXAMPLE 50 protein mixture and product was eluted at a flow rate of 1 Gas-Filled Microbubbles Comprising DSPS ml/min using PBS. The protein fraction (5 ml) was collected Incorporating PE-PEG2000-Mal Conjugated to and stored at 4° C. Thiolated Trypsin Fluorescein [0439] f) Preparation of Thiolated, Fluorescein-Labelled [0429] a) Synthesis of Boc-NH-PEGeooo-DSPE (t-butyl Trypsin carbamate poly(ethylene glycol)distearoylphosphati dylethanolamine) [0440] To the protein fraction from (e) was added 1 mg of Traut’s reagent and the mixture stirred at room temperature [0430] DSPE (31 mg) was added to a solution of Boc NH-PEG2000-SC (150 mg) in chloroform (2 ml), followed for a further 1 hour. 4 ml of the Traut’s-modified product was by triethylamine (33 ul). The mixture was stirred at 41° C. then charged on a Sephadex 200 column and the product was for 10 minutes until the starting material had dissolved. The eluted with PBS. The protein fraction containing maximum solvent was rotary evaporated and the residue was taken up fluorescent intensity was collected in a total volume of 6 ml. in acetonitrile (5 ml). The resulting dispersion was cooled to [0441] g) Conjugation of Microbubbles with Thiolated. 4° C. and centrifuged, whereafter the solution was filtered and evaporated to dryness. The structure of the resulting Fluorescein-Labelled Trypsin product was confirmed by NMR. [0442] Microbubbles from (d) were incubated on a roller [0431] b) Synthesis of H2N-PEGeooo-DSPE (amino-poly table in 1 ml of protein solution from (f) above. The (ethylene glycol)-distearoylphosphatidylethanolamine) conjugation was allowed to proceed at pH 7.3–7.8 for 10 minutes before centrifugation and removal of the infrana [0432] Boc-NH-PEGeooo-DSPE (167 mg) was stirred in 4 tant. The process was repeated a further three times, after M hydrochloric acid in dioxane (5 ml) for 2.5 hours at which the bubbles were washed four times with water to ambient temperature. The solvent was removed by rotary remove unconjugated protein. evaporation and the residue was taken up in chloroform (1.5 ml) and washed with water (2×1.5 ml). The organic phase [0443] D. Bubbles contained active enzyme as evidenced was evaporated in vacuo. TILC analysis (chloroform/metha by the cleavage of an Arg-pMA derivative in PBS. mol/water 13:5:0.8) gave a single ninhydrin positive spot with Rf=0.6; confirmation of the structure was obtained by [0444] E. Analysis of the bubbles by Coulter and mea NMR. surement of echogenicity was carried out. US 2004/0141922 A1 Jul. 22, 2004 57

[0445] Bubbles were pressure stable (see FIG. 2) [0448] b) Preparation of Gas-Filled Microbubbles Com prising DSPS and a Compound Containing Captopril [0449] A solution of 1.4% propylene glycol/2.4% glycerol FEK-022–015 (1.0 ml) was added to a mixture of DSPS (4.5 mg) and Total 0.83 product from (a) (0.5 mg) in a vial. The mixture was concentration Diameter 1–3 mm 40 sonicated for 5 minutes and then warmed to 80° C. for 5 Diameter 3–5 mm 28 minutes (vial was shaken during warming). The vial was Diameter 5–7 mm 13 Freq of max Atten. 3.3 then cooled and the head space was flushed with perfluo Atten at 2. Mhz 4.9 robutane gas. The vial was shaken in a cap mixer for 45 Atten at 3.5 Mhz 7.8 seconds and the resulting microbubbles were extensively Atten at 5.0 MHz 7.2 washed with deionised water. MALDI mass spectrometry showed no detectable level of compound from (a) in the final solution. EXAMPLE 51 wash Incorporation of captopril-containing lipopeptide into the microbubbles was confirmed by Gas-Filled Microbubbles Comprising DSPS and a MALDI-MS as follows; ca. 50 ul of microbubbles were Captopril-Containing Molecule for Diagnostic and transferred to a clean vial containing ca. 100 ul of 90% Therapeutic Applications methanol. The mixture was sonicated for 30 seconds and [0446] a) Synthesis of a Lipopeptide Functionalised with analysed by MALDI mass spectrometry, giving a M+H peak Captopril (SEQ ID NO:13) corresponding to lipopeptide from (a).

O

~~~~~~~~ NH2

O O O N ~~~~~~~~ à sº# "J's Nº. - O TS l l Q O\von NH2 HN ~'s s sºO E Ö

[0447] The structure shown above was synthesised by the EXAMPLE 52 manual “bubbler’ method starting with Fmoc-protected Rink Amide MBHA resin on a 0.125 mmol scale. Coupling was Gas-Filled Microbubbles Comprising DSPS and a carried out using standard TBTU/HOBt/DIEA protocol. Vector with Affinity for Adrenergic Receptors for Bromoacetic acid was coupled through the side-chain of Lys Diagnostic and Therapeutic Applications as a symmetrical anhydride using DIC preactivation. Cap [0450] a) Synthesis of a Protected Atenolol Derivative topril dissolved in DMF was introduced on the solid phase Suitable for Solid Phase Coupling using DBU as base. Simultaneous removal of the peptide from the resin and deprotection of side-chain protecting [0451] i) Synthesis of Methyl 4-[(2,3-epoxy)propoxy] groups was carried out in TFA containing 5% EDT, 5% phenylacetate water and 5% ethyl methyl sulphide for 2 hours. An aliquot [0452] A mixture of methyl 4-hydroxyphenylacetate (4.98 of 10 mg of the crude material was purified by preparative g, 0.030 mol), epichlorohydrin (23.5 ml, 0.30 mol) and liquid chromatography using a gradient of 70 to 100% B pyridine (121 ul, 1.5 mmol) was stirred at 85°C. for 2 hours. over 60 minutes (A=0.1% TFA/water and B=0.1% TFA/ The reaction mixture was cooled and excess epichlorohydrin acetonitrile) at a flow rate of 10 ml/min. After lyophilisation, was distilled off (rotavapor). The residue was taken up in a yield of 2 mg of pure material was obtained (analytical ethyl acetate, washed with brine and dried (Na2SO4). The HPLC, gradient 70-100% B over 20 minutes, A=0.1% solution was filtered and concentrated. The dark residue was TFA/water and B=0.1% TFA/acetonitrile, flow rate 1 chromatographed (silica, hexane/ethyl acetate 7:3) to give ml/min, detection UV 214 nm, retention time 26 minutes). 2.25 g (34%) of a colourless oil. "H (300 MHz) and **C Further characterisation was carried out using MALDI mass NMR (75 MHz) spectra were in accordance with the struc spectrometry, giving M+H at 1265 as expected. ture. US 2004/0141922 A1 Jul. 22, 2004 58

[0453] ii) Synthesis of Methyl 4-[2-hydroxy-3-[(1-meth lised. Yield 415 mg (56%), white solid. The structure was ylethyl)-aminolpropoxy]phenylacetate confirmed by *H and **C NMR analysis. [0454] A mixture of methyl 4-[(2,3-epoxy)propoxy]phe [0459] b) Synthesis of a Lipopeptide Functionalised with nylacetate (2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 Atenolol (SEQ ID NO:21)

~~~~~~~~O

O O ~~~~~~~~ § i i # NH

NH2

mol) and water (1.35 ml, 74.7 mmol) was stirred at room [0460] The structure shown above was synthesised by the temperature overnight. The reaction mixture was concen manual bubbler method starting with Fmoc-protected Rink trated (rotavapor) and the oily residue was dissolved in Amide MBHA resin on a 0.125 mmol scale, using the chloroform and dried (Na2SO4). Filtration and concentration compound from (a). Coupling was carried out using stan gave quantitative yield of a yellow oil that was used in the dard TBTU/HOBt/DIEA protocols. Simultaneous removal next step without further purification. The structure was of the peptide from the resin and deprotection of side-chain verified by *H and **C NMR analysis. protecting groups was carried out in TFA containing 5% [0455] iii) Synthesis of 4-[2-hydroxy-3-[(1-methylethyl) EDT and 5% water for 2 hours. Crude material was pre aminolpropoxy]phenylacetic acid hydrochloride cipitated from ether and purified by preparative liquid chro matography using a gradient of 70 to 100% B over 60 [0456] A solution of methyl 4-[2-hydroxy-3-[(1-methyl minutes (A=0.1% TFA/water and B=0.1% TFA/acetonitrile) ethyl)-aminolpropoxy]phenylacetate (563 mg, 2.00 mmol) at a flow rate of 10 ml/min. After lyophilisation, a yield of in 6M hydrochloric acid (15 ml) was heated at 100° C. for 38 mg of pure material was obtained (analytical HPLC, 4 hours. The reaction mixture was concentrated (rotavapor) gradient 70–100% B over 20 minutes, A=0.1% TFA/water and the residue was taken up in water and lyophilised. "H and B=0.1% TFA/acetonitrile, flow rate 1 ml/minute, detec and **C NMR spectra were in accordance with the strucure tion UV 214 nm, retention time 25 minutes). Further char and MALDI mass spectrometry gave a M+H at 268 as acterisation was carried out using MALDI mass spectrom expected. etry (ACH matrix), giving M+H at 1258, expected 1257. [0457] iv) Synthesis of N-Boc-4-[(2-hydroxy-3-[(1-meth [0461] c) Preparation of Gas-Filled Microbubbles Com ylethyl)-aminolpropoxy]phenylacetic acid prising DSPS and a Lipopeptide Containing Atenolol [0458] A solution of the 4-[2-hydroxy-3-[(1-methylethyl) [0462] Asolution of 1.4% propylene glycol/2.4% glycerol aminolpropoxy]phenylacetic acid hydrochloride (2.0 mmol) (1.0 ml) was added to a mixture of DSPS (4.5 mg) and in water (2 ml) was added to a solution of sodium bicar product from (b) (0.5 mg) in a vial. The mixture was bonate (0.60 g, 7.2 mmol) in water/dioxane (2:1, 15 ml). A sonicated for 5 minutes, heated at 80° C. for 5 minutes (vial solution of di-tert-butyl dicarbonate (0.48 g, 2.2 mmol) in was shaken during warming) and then cooled. The head dioxane (5 ml) was added. Progress of the reaction was space was flushed with perfluorobutane gas and the vial was monitored by TLC analysis (silica, CHCls/MeOH/AcOH shaken in a cap mixer for 45 seconds, whereafter the 85:10:5), and portions of di-tert-butyl dicarbonate were contents were extensively washed with deionised water. added until conversion was complete. The reaction mixture MALDI mass spectrometry showed no detectable level of was poured onto water saturated with potassium hydrogen compound from (b) in the final wash solution. Incorporation sulphate and organic material was extracted into ethyl of atenolol-containing lipopeptide into the microbubbles acetate. The organic phase was washed with water and brine, was confirmed by MALDI-MS as follows: ca. 50 ul of dried (Na2SO4) and filtered to give 0.6 g of crude material. microbubbles were transferred to a clean vial containing ca. The product was purified by chromatography (silica, CHCls/ 100 ul of 90% methanol. The mixture was sonicated for 30 MeOH/AcOH 85:10:5). The solution was concentrated and seconds and analysed by MALDI-MS (ACH-matrix), giving the residue was taken up in glacial acetic acid and lyophi a M+H peak at 1259 corresponding to lipopeptide (b). US 2004/0141922 A1 Jul. 22, 2004 59

[0463] d) In Vitro Analysis lyophilisation, 16 mg of pure material was obtained (ana lytical HPLC, gradient 70-100% B where B=MeOH, [0464] The microbubbles were tested in the in vitro assay A=0.01% TFA/water: detection—UV 260 and fluorescence, as detailed in Example 21. A gradual accumulation of Ex280, Em350—product retention time=19.44 minutes). Fur microbubbles binding to the cells was observed. ther product characterisation was carried out using MALDI EXAMPLE 53 mass spectrometry: expected M+H at 2198, found at 2199. [0467] b) Synthesis of a Protected Atenolol Derivative Gas-Filled Microbubbles Comprising DSPS and a Lipopeptide Consisting of a Heparin Suitable for Solid Phase Coupling Sulphate-Binding Peptide (KRKR) (SEQ ID NO:5) [0468] i) Synthesis of Methyl 4-[(2,3-epoxy)propoxy]phe and a Fibronectin Peptide (WOPPRARI) (SEQ ID nylacetate NO:6) for Targeting and a Lipopeptide Containing [0469] A mixture of methyl 4-hydroxyphenylacetate (4.98 Atenolol for Therapeutic Application g, 0.030 mol), epichlorohydrin (23.5 ml, 0.30 mol) and [0465] a) Synthesis of a Lipopeptide Consisting of a pyridine (121 ul, 1.5 mmol) was stirred at 85°C. for 2 hours. Heparin Sulphate-Binding Peptide (KRKR) (SEQ ID NO:5) The reaction mixture was cooled, and excess epichlorohy and a Fibronectin Peptide (WOPPRARI) (SEQ ID NO:6) drin was distilled off (rotavapor). The residue was taken up

N H = H = H O E O E O

NH2 NH2 H2N O

O O O H H § N O N N NH2 2 Sº SS =- NH ;- NH É H ”, = O = O - O %

~~ NH & \ } *NH *NH

[0466] The lipopeptide was synthesised on an ABI 433A in ethyl acetate, washed with brine and dried (Na2SO4). The automatic peptide synthesiser starting with Fmoc-Ile-Wang solution was filtered and concentrated. The dark residue was resin on a 0.1 mmol scale using 1 mmol amino acid chromatographed (silica, hexane/ethyl acetate 7:3) to give cartridges. All amino acids and palmitic acid were preacti 2.25 g (34%) of a colourless oil. "H (300 MHz) and **C vated using HBTU before coupling. The simultaneous NMR (75 MHz) spectra were in accordance with the struc removal of peptide from the resin and side-chain protecting ture. groups was carried out in TFA containing 5% phenol, 5% [0470] ii) Synthesis of Methyl 4-[2-hydroxy-3-[(1-meth EDT, 5% anisole and 5% H2O for 2 hours, giving a crude ylethyl)-aminolpropoxy]phenylacetate product yield of 150 mg. Purification by preparative HPLC [0471] A mixture of methyl 4-[(2,3-epoxy)propoxy]phe of a 40 mg aliquot of crude material was carried out using nylacetate (2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 a gradient of 70 to 100% B over 40 minutes (A=0.1% mol) and water (1.35 ml, 74.7 mmol) was stirred at room TFA/water and B=MeOH) at a flow rate of 9 ml/min. After temperature overnight. The reaction mixture was concen US 2004/0141922 A1 Jul. 22, 2004 60 trated (rotavapor) and the oily residue was dissolved in priate amine acids, palmitic acid and the compound from (a). chloroform and dried (Na2SO4). Filtration and concentration Coupling was carried out using standard TBTU/HOBt/DIEA gave quantitative yield of a yellow oil that was used in the protocols. Simultaneous removal of the peptide from the next step without further purification. The structure was resin and deprotection of side-chain protecting groups was verified by *H and **C NMR analysis. carried out in TFA containing 5% EDT and 5% water for 2 [0472] iii) Synthesis of 4-[2-hydroxy-3-[(1-methylethyl) hours. Crude material was precipitated from ether and aminolpropoxy]phenylacetic acid hydrochloride purified by preparative liquid chromatography using a gra [0473] A solution of methyl 4-[2-hydroxy-3-[(1-methyl dient of 70 to 100% B over 60 minutes (A=0.1% TFA/water ethyl)-aminolpropoxy]phenylacetate (563 mg, 2.00 mmol) and B=0.1% TFA/acetonitrile) at a flow rate of 10 ml/min. in 6M hydrochloric acid (15 ml) was heated at 100° C. for After lyophilisation, a yield of 38 mg of pure material was 4 hours. The reaction mixture was concentrated (rotavapor) and the residue was taken up in water and lyophilised. 1H obtained (analytical HPLC, gradient 70-100% B over 20 and **C NMR spectra were in accordance with the strucure minutes, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, and MALDI mass spectrometry gave a M+H at 268 as flow rate 1 ml/minute, detection UV 214 nm, retention time expected. 25 minutes). Further characterisation was carried out using [0474] iv) Synthesis of N-Boc-4-[2-hydroxy-3-[(1-meth MALDI mass spectrometry (ACH matrix), giving M+H at ylethyl)-aminolpropoxy]phenylacetic acid 1258, expected 1257. [0475] A solution of the 4-[2-hydroxy-3-[(1-methylethyl) [0478] d) Preparation of Gas-Filled Microbubbles Com aminolpropoxy]phenylacetic acid hydrochloride (2.0 mmol) prising DSPS and a Lipopeptide Consisting of a Heparin in water (2 ml) was added to a solution of sodium bicar Sulphate-Binding Peptide (KRKR) (SEQ ID NO:5), a bonate (0.60 g, 7.2 mmol) in water/dioxane (2:1, 15 ml). A solution of di-tert-butyl dicarbonate (0.48 g, 2.2 mmol) in Fibronectin Peptide (WOPPRARI) (SEQ ID NO:6) and a dioxane (5 ml) was added. Progress of the reaction was Lipopeptide Containing Atenolol monitored by TLC analysis (silica, CHCls/MeOH/AcOH [0479] A solution of 1.4% propylene glycol/2.4% glycerol 85:10:5), and portions of di-tert-butyl dicarbonate were added until conversion was complete. The reaction mixture (1.0 ml) was added to a mixture of DSPS (5.0 mg), product was poured onto water saturated with potassium hydrogen from (a) (0.5 mg) and product from (c) (0.5 mg) in a vial. sulphate and organic material was extracted into ethyl The mixture was sonicated for 5 minutes and then heated at acetate. The organic phase was washed with water and brine, 80° C. for 5 minutes (vial was shaken during warming). The dried (Na2SO4) and filtered to give 0.6 g of crude material. solution was filtered and cooled. The head space was flushed The product was purified by chromatography (silica, CHCls/ with perfluorobutane gas and the vial was shaken in a cap MeOH/AcOH 85:10:5). The solution was concentrated and mixer for 45 seconds, whereafter the contents were exten the residue was taken up in glacial acetic acid and lyophi sively washed with deionised water. Incorporation of lised. Yield 415 mg (56%), white solid. The structure was atenolol-containing lipopeptide into the microbubbles was confirmed by *H and **C NMR analysis. confirmed by MALDI-MS as follows; ca. 50 ul of [0476] c) Synthesis of a Lipopeptide Functionalised with microbubbles were transferred to a clean vial containing ca. Atenolol (SEQ ID NO:21) 100 ul of 90% methanol. The mixture was sonicated for 30

~~~~~~~~O

O O O

~~~~~~~~ N - N H H - H 2 O

OH

[0477] The structure shown above was synthesised by the seconds and analysed by MALDI-MS (ACH matrix), giving manual bubbler method starting with Fmoc-protected Rink two M+H peaks at 2202 and 1259, corresponding to lipopep Amide MBHA resin on a 0.125 mmol scale, using appro tide. (a) and to lipopeptide (c) respectively. US 2004/0141922 A1 Jul. 22, 2004

[0480] e) In Vitro Analysis hydrogen sulphate and organic material was extracted into ethyl acetate. The organic phase was washed with water and [0481] The microbubbles were tested in the in vitro assay brine, dried (Na2SO) and filtered to give 0.6 g of crude as detailed in example 21. A gradual accumulation of material. The product was purified by chromatography microbubbles binding to the cells was observed. (silica, CHCla/MeOH/AcOH 85:10:5). The solution was concentrated and the residue was taken up in glacial acetic EXAMPLE 54 acid and lyophilised. Yield 415 mg (56%), white solid. The structure was confirmed by "H and **C NMR analysis. Gas-Filled Microbubbles Comprising DSPS and a Lipophilic Derivative of Atenolol with Affinity for [0491] v) Synthesis of N'-Boc, N-hexadecyl-4-[2-hy Adrenergic Receptors for Diagnostic and droxy-3-[(1-methylethyl)aminolpropoxy]phenylacetamide Therapeutic Applications [0492] A solution of N-Boc-4-[2-hydroxy-3-[(1-methyl [0482] a) Synthesis of N-hexadecyl-4-[2-hydroxy-3-[(1 ethyl)-aminolpropoxy]phenylacetic acid (92 mg, 0.25 methyl-ethyl)aminolpropoxy]phenylacetamide mmol) and hexadecylamine (60 mg, 0.25 mmol) in DMF (5 ml) was cooled to 0° C. HOBt (39 mg, 0.25 mmol) and [0483] i) Synthesis of Methyl 4-[(2,3-epoxy)propoxy] N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydro phenylacetate chloride (water soluble carbodiimide) (48 mg, 0.25 mmol) [0484] A mixture of methyl 4-hydroxyphenylacetate (4.98 were added. The reaction mixture was stirred at 0° C. for 1 g, 0.030 mol), epichlorohydrin (23.5 ml, 0.30 mol) and hour and then at room temperature overnight. The reaction pyridine (121 ul, 1.5 mmol) was stirred at 85°C. for 2 hours. mixture was poured onto water (25 ml) containing sodium The reaction mixture was cooled and excess epichlorohydrin carbonate (2.5 g) and sodium chloride (4.0 g). Precipitated was distilled off (rotavapor). The residue was taken up in material was filtered off, washed with water and taken up in ethyl acetate, washed with brine and dried (Na2SO4). The chloroform. The chloroform phase was washed with 5% solution was filtered and concentrated. The dark residue was sodium carbonate and water and dried (Na2SO4). The solu chromatographed (silica, hexane/ethyl acetate 7:3) to give tion was filtered and concentrated to give 150 mg of yellow 2.25 g (34%) of a colourless oil. "H (300 MHz) and **C white crude material. The product was purified by column NMR (75 MHz) spectra were in accordance with the struc chromatography (silica, chloroform/methanol 95:5) to give 118 mg (80%) of white material. The structure was verified ture. by "H (500 MHz) and **C (125 MHz) NMR. The product [0485] ii) Synthesis of Methyl 4-[2-hydroxy-3-[(1-me was further characterised by MALDI mass spectrometry, thyl-ethyl)aminolpropoxy]phenylacetate giving a M+Na peak at 614 as expected. [0486] A mixture of methyl 4-[(2,3-epoxy)propoxy]phe [0493] vi) Synthesis of N-hexadecyl-4-[2-hydroxy-3-[(1 nylacetate (2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 methyl-ethyl)aminolpropoxy]phenylacetamide mol) and water (1.35 ml, 74.7 mmol) was stirred at room temperature overnight. The reaction mixture was concen [0494] To a solution of N'-Boc-N-hexadecyl-4-[2-hy trated (rotavapor) and the oily residue was dissolved in droxy-3-[(1-methyl-ethyl)amino]propoxy]phenylacetamide chloroform and dried (Na2SO4). Filtration and concentration (10 mg) in dichloromethane (9 ml) was added trifluoroacetic gave quantitative yield of a yellow oil that was used in the acid (1 ml). The reaction mixture was stirred for 2 hours at next step without further purification. The structure was room temperature. TLC (silica, chloroform/methanol 95:5) verified by *H and **C NMR analysis. showed complete conversion of starting material. Solvents were evaporated off and the residue was taken up in water/ [0487] iii) Synthesis of 4-[2-hydroxy-3-[(1-methyl-ethy acetonitrile and lyophilised to give a quantitative yield of l)aminolpropoxy]phenylacetic Acid Hydrochloride white solid material. The structure was verified by "H (500 [0488] A solution of methyl 4-[2-hydroxy-3-[(1-methyl MHz) and **C (125 MHz) NMR analysis and further char ethyl)aminolpropoxy]phenylacetate (563 mg, 2.00 mmol) in acterised by MALDI mass spectrometry, giving M+H at 492 6M hydrochloric acid (15 ml) was heated at 100° C. for 4 and M+Na at 514 as expected. hours. The reaction mixture was concentrated (rotavapor) and the residue was taken up in water and lyophilised. "H [0495] b) Preparation of Gas-Filled Microbubbles Com and **C NMR spectra were in accordance with the strucure prising DSPS and N-hexadecyl-4-[2-hydroxy-3-[(1-methyl and MALDI mass spectrometry gave a M+H at 268 as ethyl)-aminolpropoxy]phenylacetamide for Diagnostic and expected. Therapeutic Applications [0489] iv) Synthesis of N-Boc-4-[2-hydroxy-3-[(1-me [0496] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml) was added to a mixture of DSPS (4.5 mg) and thyl-ethyl)aminolpropoxy]phenylacetic acid N-hexadecyl-4-[2-hydroxy-3-[(1-methylethyl)-amino]pro [0490] A solution of the 4-[2-hydroxy-3-[(1-methyl-ethy poxy]phenylacetamide (0.5 mg) in a vial. The mixture was l)aminolpropoxy]phenylacetic acid hydrochloride (2.0 sonicated for 5 minutes and then heated at 80° C. for 5 mmol) in water (2 ml) was added to a solution of sodium minutes (vial was shaken during warming) The solution was bicarbonate (0.60 g, 7.2 mmol) in water/dioxane (2:1, 15 filtered and cooled. The head space was flushed with per ml). A solution of di-tert-butyl dicarbonate (0.48 g, 2.2 fluorobutane gas and the vial was shaken in a cap mixer for mmol) in dioxane (5 ml) was added. Progress of the reaction 45 seconds, whereafter the contents were extensively was monitored by TLC analysis (silica, CHCls/MeOH/ washed with deionised water. Incorporation of compound AcOH 85:10:5), and portions of di-tert-butyl dicarbonate from (a) into the microbubbles was confirmed by MALDI were added until conversion was complete. The reaction MS as follows; ca. 50 ul of microbubbles were transferred mixture was poured onto water saturated with potassium to a clean vial containing ca. 100 ul of 90% methanol. The US 2004/0141922 A1 Jul. 22, 2004 62 mixture was sonicated for 30 seconds and analysed by Amide MBHA resin on a 0.125 mmol scale, using appro MALDI-MS, giving a M+H peak at 492 corresponding to priate amino acids, palmitic acid and folic acid. Coupling N-hexadecyl-4-[2-hydroxy-3-[(1-methylethyl)amino]pro was carried out using standard TBTU/HOBt/DIEA proto poxy]phenylacetamide. cols. Simultaneous removal of the peptide from the resin and EXAMPLE 55 deprotection of side-chain protecting groups was carried out in TFA containing 5% EDT and 5% water for 2 hours. Crude Gas-Filled Microbubbles Encapsulated with DSPS material was precipitated from ether and analysed by and a Compound Containing Folic Acid for MALDI mass spectrometry, giving a M+H peak correspond Diagnostic Applications ing to the structure at 1435, expected 1430. The material was [0497] a) Synthesis of a Lipopeptide Containing Folic further characterised by analytical HPLC, gradient 70-100% Acid B over 20 minutes, A=0.1% TFA/water and B=0.1% TFA/

(SEQ ID NO: 22)

~~~~~~~~ NH NH2

§ § à à N H

NH2 HN

N NH2

and/or O S O

HO N O H O N N S N i | 2 Js| N N NH2 H

[0498] The structure shown above was synthesised by the acetonitrile, flow rate 1.0 ml/minute, giving a product peak manual bubbler method starting with Fmoc-protected Rink with retention time 27 minutes detected at UV 368 mm. US 2004/0141922 A1 Jul. 22, 2004

[0499] b) Preparation of Gas-Filled Microbubbles Com microbubbles were transferred to a clean vial containing ca. prising DSPS and a Lipopeptide Containing Folic Acid 100 ul of 90% methanol. The mixture was sonicated for 30 [0500] A solution of 1.4% propylene glycol/2.4% glycerol seconds and analysed by MALDI-MS, giving a M+H peak (1.0 ml) was added to a mixture of DSPS (4.5 mg) and at 668 corresponding to structure from (a). product from (a) (0.5 mg) in a vial. Dilute ammonia (to pH 8) and DMSO (40 ul) were added and the mixture was EXAMPLE 57 sonicated for 5 minutes and then heated at 80° C. for 5 minutes (vial was shaken during warming). The solution was Gas-Filled Microbubbles Comprising DSPS and a filtered and cooled. The head space was flushed with per Lipopeptide Containing Atenolol and a Cholesterol fluorobutane gas and the vial was shaken in a cap mixer for Derivative of Chlorambucil for Diagnostic and 45 seconds, whereafter the contents were extensively Therapeutic Applications washed with deionised water. Incorporation of structure from (a) into the bubbles was confirmed by MALDI-MS as [0507] a) Synthesis of a Protected Atenolol Derivative follows; ca. 50 ul of microbubbles were transferred to a Suitable for Solid Phase Coupling clean vial containing ca. 100 ul of 90% methanol. The [0508] i) Synthesis of Methyl 4-[(2,3-epoxy)propoxy] mixture was sonicated for 30 seconds and analysed by phenylacetate MALDI-MS (ACH matrix), giving a M+H peak at 1238 corresponding to structure from (a). [0509] A mixture of methyl 4-hydroxyphenylacetate (4.98 g, 0.030 mol), epichlorohydrin (23.5 ml, 0.30 mol) and [0501] c) In Vitro Analysis pyridine (121 ul, 1.5 mmol) was stirred at 85°C. for 2 hours. [0502] The microbubbles were tested in the in vitro assay The reaction mixture was cooled and excess epichlorohydrin as detailed in Example 21. A gradual accumulation of was distilled off (rotavapor). The residue was taken up in microbubbles binding to the cells was observed. ethyl acetate, washed with brine and dried (Na2SO4). The solution was filtered and concentrated. The dark residue was EXAMPLE 56 chromatographed (silica, hexane/ethyl acetate 7:3) to give Gas-Filled Microbubbles Comprising DSPS and a 2.25 g (34%) of a colourless oil. "H (300 MHz) and **C Cholesteryl Ester of Chlorambucil for Diagnostic NMR (75 MHz) spectra were in accordance with the struc and Therapeutic Applications ture. [0503] a) Synthesis of Cholesterol 4-[4-(bis(2-chloroethy [0510] ii) Synthesis of Methyl 4-[2-hydroxy-3-[(1-me l)aminolphenyl]butanoate thyl-ethyl)aminolpropoxy]phenylacetate [0504] DIC (170 ul, 1.10 mmol) was added to a solution [0511] A mixture of methyl 4-[(2,3-epoxy)propoxy]phe of chlorambucil (669 mg, 2.20 mmol) in dry dichlo nylacetate (2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 romethane (15 ml). The mixture was stirred at room tem perature for 0.5 hour and added to a solution of cholesterol mol) and water (1.35 ml, 74.7 mmol) was stirred at room (387 mg, 1.00 mmol) and DMAP (122 mg, 1.00 mmol) in temperature overnight. The reaction mixture was concen dichloromethane (10 ml). The reaction mixture was stirred trated (rotavapor) and the oily residue was dissolved in overnight and then poured onto 5% sodium bicarbonate. The chloroform and dried (Na2SO4). Filtration and concentration phases were separated and the organic phase was washed gave quantitative yield of a yellow oil that was used in the with brine and dried (MgSO4). The solution was filtered and next step without further purification. The structure was concentrated and the product was purified by column chro verified by *H and **C NMR analysis. matography (silica, chloroform) to give 560 mg (83%) of colouless oil. The product was characterised by MALDI [0512] iii) Synthesis of 4-[2-hydroxy-3-[(1-methyl-ethy mass spectrometry, giving M+H at 674 as expected. Further l)aminolpropoxy]phenylacetic acid hydrochloride characterisation was carried out using "H (500 MHz) and [0513] A solution of methyl 4-[2-hydroxy-3-[(1-methyl *C (125 MHz) NMR analysis, giving spectra in accordance ethyl)aminolpropoxy]phenylacetate (563 mg, 2.00 mmol) in with the structure. 6M hydrochloric acid (15 ml) was heated at 100° C. for 4 [0505] b) Preparation of Gas-Filled Microbubbles Com hours. The reaction mixture was concentrated (rotavapor) prising DSPS and a Cholesterol Ester of Chlorambucil for and the residue was taken up in water and lyophilised. "H Diagnostic and/or Therapeutic Applications and **CNMR spectra were in accordance with the structure [0506] A solution of 1.4% propylene glycol/2.4% glycerol and MALDI mass spectrometry gave a M+H at 268 as (1.0 ml) was added to a mixture of DSPS (4.5 mg) and expected. product from (a) (0.5 mg) in a vial. The mixture was sonicated for 5 minutes and then heated at 80° C. for 5 [0514] iv) Synthesis of N-Boc-4-[2-hydroxy-3-[(1-me minutes (vial was shaken during warming) and cooled. The thyl-ethyl)aminolpropoxy]phenylacetic acid head space was flushed with perfluorobutane gas and the vial was shaken in a cap mixer for 45 seconds, whereafter the [0515] A solution of the 4-[2-hydroxy-3-[(1-methyl-ethy contents were extensively washed with deionised water. l)aminolpropoxy]phenylacetic acid hydrochloride (2.0 MALDI mass spectrometry showed no detectable level of mmol) in water (2 ml) was added to a solution of sodium compound from (a) in the final wash solution. Incorporation bicarbonate (0.60 g, 7.2 mmol) in water/dioxane (2:1, 15 of chlorambucil cholesteryl ester into the bubbles was ml). A solution of di-tert-butyl dicarbonate (0.48 g, 2.2 confirmed by MALDI-MS as follows; ca. 50 ul of mmol) in dioxane (5 ml) was added. Progress of the reaction US 2004/0141922 A1 Jul. 22, 2004 64 was monitored by TLC analysis (silica, CHCls/MeOH/ romethane (15 ml). The mixture was stirred at room tem AcOH 85:10:5), and portions of di-tert-butyl dicarbonate perature for 0.5 hour and added to a solution of cholesterol were added until conversion was complete. The reaction (387 mg, 1.00 mmol) and DMAP (122 mg, 1.00 mmol) in mixture was poured onto water saturated with potassium dichloromethane (10 ml). The reaction mixture was stirred hydrogen sulphate and organic material was extracted into overnight and then poured onto 5% sodium bicarbonate. The ethyl acetate. The organic phase was washed with water and phases were separated and the organic phase was washed brine, dried (Na2SO) and filtered to give 0.6 g of crude with brine and dried (MgSO4). The solution was filtered and material. The product was purified by chromatography concentrated and the product was purified by column chro (silica, CHCla/MeOH/AcOH 85:10:5). The solution was matography (silica, chloroform) to give 560 mg (83%) of concentrated and the residue was taken up in glacial acetic colouless oil. The product was characterised by MALDI acid and lyophilised. Yield 415 mg (56%), white solid. The mass spectrometry, giving M+H at 674 as expected. Further structure was confirmed by "H and **C NMR analysis. characterisation was carried out using "H (500 MHz) and [0516] b) Synthesis of a Lipopeptide Functionalised with *C (125 MHz) NMR analysis, giving spectra in accordance Atenolol (SEQ ID NO:21) with the structure.

~~~~~~~~O

O O O

~~~~~~~~ N - N H H - H 2 O O

H2

[0517] The Structure shown above was synthesised by the [0520] d) Preparation of Gas-Filled Microbubbles Com manual bubbler method starting with Fmoc-protected Rink prising DSPS and a Lipopeptide Containing Atenolol and a Amide MBHA resin on a 0.125 mmol scale, using appro Cholesteryl Ester of Chloambucil priate amino acids, palmitic acid and the compound from (a). Coupling was carried out using standard TBTU/HOBt/ [0521] A solution of 1.4% propylene glycol/2.4% glycerol DIEA protocols. Simultaneous removal of the peptide from (1.0 ml) was added to a mixture of DSPS (5.0 mg), product the resin and deprotection of side-chain protecting groups from (b) (0.5 mg) and product from (c) (0.5 mg) in a vial. was carried out in TFA containing 5% EDT and 5% water for The mixture was sonicated for 5 minutes and then warmed 2 hours. Crude material was precipitated from ether and to 80° C. for 5 minutes (vial was shaken during warming). purified by preparative liquid chromatography using a gra The solution was filtered and cooled. The head space was dient of 70 to 100% B over 60 minutes (A=0.1% TFA/water flushed with perfluorobutane gas and the vial was shaken in and B=0.1% TFA/acetonitrile) at a flow rate of 10 ml/min. a cap mixer for 45 seconds, whereafter the contents were After lyophilisation, a yield of 38 mg of pure material was extensively washed with deionised water. Incorporation of obtained (analytical HPLC, gradient 70-100% B over 20 compounds (b) and (c) into the microbubbles was confirmed minutes, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, by MALDI-MS as follows: ca. 50 ul of microbubbles were flow rate 1 ml/minute, detection UV 214 nm, retention time transferred to a clean vial containing ca. 100 ul of 90% 25 minutes). Further characterisation was carried out using methanol. The mixture was sonicated for 30 seconds and MALDI mass spectrometry (ACH matrix), giving M+H at analysed by MALDI-MS (ACH-matrix), giving a M+H peak 1258, expected 1257. corresponding to lipopeptide (b) and cholesteryl ester (c). [0518] c) Synthesis of Cholesterol 4-[4-(bis(2-chloroethy [0522] e) In Vitro Analysis l)aminolphenyl]butanoate [0523] The microbubbles were tested in the in vitro assay [0519) DIC (170 ul, 1.10 mmol) was added to a solution as detailed in Example 21. A gradual accumulation of of chlorambucil (669 mg, 2.20 mmol) in dry dichlo microbubbles binding to the cells was observed. US 2004/0141922 A1 Jul. 22, 2004 65

EXAMPLE 58 [0529] iii) Synthesis of 4-[2-hydroxy-3-[(1-methyl-ethy l)aminolpropoxy]phenylacetic Acid Hydrochloride Gas-Fiiled Microbubbles Comprising DSPS and a Lipopeptide Containing Atenolol for Cell Targeting [0530] A solution of methyl 4-[2-hydroxy-3-[(1-methyl and a Lipophilic Thiol Ester of Captopril for ethyl)aminolpropoxy]phenylacetate (563 mg, 2.00 mmol) in Therapeutic Use 6M hydrochloric acid (15 ml) was heated at 100° C. for 4 hours. The reaction mixture was concentrated (rotavapor) [0524] a) Synthesis of a Protected Atenolol Derivative and the residue was taken up in water and lyophilised. "H Suitable for Solid Phase Coupling and **C NMR spectra were in accordance with the strucure [0525] i) Synthesis of Methyl 4-[(2,3-epoxy)propoxy] and MALDI mass spectrometry gave a M+H at 268 as phenylacetate expected. [0531] iv) Synthesis of N-Boc-4-[2-hydroxy-3-[(1-me [0526] A mixture of methyl 4-hydroxyphenylacetate (4.98 thyl-ethyl)aminolpropoxy]phenylacetic acid g, 0.030 mol), epichlorohydrin (23.5 ml, 0.30 mol) and pyridine (121 ul, 1.5 mmol) was stirred at 85°C. for 2 hours. [0532] A solution of the 4-[2-hydroxy-3-[(1-methyl-ethy The reaction mixture was cooled and excess epichlorohydrin l)aminolpropoxy]phenylacetic acid hydrochloride (2.0 was distilled off (rotavapor). The residue was taken up in mmol) in water (2 ml) was added to a solution of sodium ethyl acetate, washed with brine and dried (Na2SO4). The bicarbonate (0.60 g, 7.2 mmol) in water/dioxane (2:1, 15 solution was filtered and concentrated. The dark residue was chromatographed (silica, hexane/ethyl acetate 7:3) to give ml). A solution of di-tert-butyl dicarbonate (0.48 g, 2.2 2.25 g (34%) of a colourless oil. "H (300 MHz) and **C mmol) in dioxane (5 ml) was added. Progress of the reaction NMR (75 MHz) spectra were in accordance with the struc was monitored by TLC analysis (silica, CHCls/MeOH/ ture. AcOH 85:10:5), and portions of di-tert-butyl dicarbonate were added until conversion was complete. The reaction [0527] ii) Synthesis of Methyl 4-[2-hydroxy-3-[(1-me mixture was poured onto water saturated with potassium thyl-ethyl)aminolpropoxy]phenylacetate hydrogen sulphate and organic material was extracted into [0528] A mixture of methyl 4-[(2,3-epoxy)propoxy]phe ethyl acetate. The organic phase was washed with water and nylacetate (2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 brine, dried (Na2SO) and filtered to give 0.6 g of crude mol) and water (1.35 ml, 74.7 mmol) was stirred at room material. The product was purified by chromatography temperature overnight. The reaction mixture was concen (silica, CHCla/MeOH/AcOH 85:10:5). The solution was trated (rotavapor) and the oily residue was dissolved in concentrated and the residue was taken up in glacial acetic chloroform and dried (Na2SO4). Filtration and concentration acid and lyophilised. Yield 415 mg (56%), white solid. The gave quantitative yield of a yellow oil that was used in the structure was confirmed by *H and **C NMR analysis. next step without further purification. The structure was [0533] b) Synthesis of a Lipopeptide Functionalised with verified by *H and **C NMR analysis. Atenolol (SEQ ID NO:21)

~~~~~~~~

à US 2004/0141922 A1 Jul. 22, 2004 66

[0534] The structure shown above was synthesised by the sonicated for 30 seconds and analysed by MALDI-MS manual bubbler method starting with Fmoc-protected Rink (ACH-matrix), giving peaks according to structures from (b) Amide MBHA resin on a 0.125 mmol scale, using appro and (c) respectively. priate amino acids, palmitic acid and the compound from [0539] e) In Vitro Analysis (a). Coupling was carried out using standard TBTU/HOBt/ DIEA protocols. Simultaneous removal of the peptide from [0540] The microbubbles were tested in the in vitro assay the resin and deprotection of side-chain protecting groups as detailed in Example 21. A gradual accumulation of was carried out in TFA containing 5% EDT and 5% water for microbubbles binding to the cells was observed. 2 hours. Crude material was precipitated from ether and purified by preparative liquid chromatography using a gra EXAMPLE 59 dient of 70 to 100% B over 60 minutes (A=0.1% TFA/water Gas-Filled Microbubbles Comprising and B=0.1% TFA/acetonitrile) at a flow rate of 10 ml/min. Phosphatidylserine and After lyophilisation, a yield of 38 mg of pure material was Biotinamide-PEG-fl-Ala-cholesterol and a obtained (analytical HPLC, gradient 70-100% B over 20 Cholesteryl Ester of Chlorambucil for Diagnostic minutes, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, and Therapeutic Applications flow rate 1 ml/minute, detection UV 214 nm, retention time [0541] a) Synthesis of Cholesteryl N-Boc-f-alaninate 25 minutes). Further characterisation was carried out using MALDI mass spectrometry (ACH matrix), giving M+H at [0542] DIC (510 ul) was added to a solution of Boc-5 1258, expected 1257. Ala-OH (1.25 g, 6.60 mmol) in dichloromethane (15 ml) under an inert atmosphere. The reaction mixture was stirred [0535.] c) Synthesis of Cholanic Acid Thiol Ester of Cap for 30 minutes and then transferred to a flask containing a topril solution of cholesterol (1.16 g, 3.00 mmol) and DMAP (367 [0536] A mixture of 5-3-cholanic acid (361 mg, 1.00 mg, 3.00 mmol) in dichloromethane (15 ml). The reaction mmol) and DIC (77 ul, 0.50 mmol) in dichloromethane (5 mixture was stirred for 2 hours and then poured onto an ml) was stirred for 10 minutes and then added to a solution aqeous solution of potassium hydrogen sulphate. After phase of captopril (130 mg, 0.600 mmol) and DBU (180 ul, 1.20 separation the aqueous phase was extracted with chloro mmol) in dichloromethane (10 ml). The reaction mixture form. The combined organic phases were washed with was stirred overnight and then poured onto dilute hydro aqueous potassium hydrogen sulphate and water and dried chloric acid. Chloroform (30 ml) was added. The phases (MgSO4). After filtration and evaporation the crude product were separated and the organic phase was washed with water was chromatographed (silica, chloroform/methanol 99:1) to and brine and dried (MgSO4). After filtration and concen give 1.63 g (97%) of white solid. The structure was con tration, the crude material was chromatographed (silica, firmed by *H NMR (500 MHz). chloroform/methanol/acetic acid 95:4:1). The product was [0543] b) Synthesis of Cholesteryl B-alaninate Hydrochlo lyophilised from a acetonitrile/water/ethanol mixture. Yield ride 137 mg (49%) of off-white solid. The structure was verified [0544] A solution of compound from (a) (279 mg, 0.500 by "H (500 MHz) and **C (125 MHz) NMR spectroscopy. mmol) in 1M hydrochloric acid in 1,4-dioxane (5 ml) was Further characterisation was carried out using MALDI mass stirred at room temperature for 4 hours. The reaction mixture spectrometry, giving a M+Na peak in positive mode at m/z was concentrated to give a quantitative yield of cholesteryl 584. fl-alaninate hydrochloride. The structure was confirmed by [0537] d) Preparation of Gas-Filled Microbubbles Com *H NMR (500 MHz) analysis and by MALDI mass spec prising DSPS and a Lipopeptide Containing Atenolol for trometry, giving a M+Na peak at 482, expected 481. Cell Targeting and a Lipophilic Thiol Ester of Captopril for Therapeutic Use [0545] c) Biotin-PEGs loo-B-Ala-Cholesterol [0546] To a solution of cholesteryl B-alaninate hydrochlo [0538] A solution of 1.4% propylene glycol/2.4% glycerol ride (15 mg, 0.03 mmol) in chloroform/wet methanol (2.6:1, (1.0 ml) was added to a mixture of DSPS (5.0 mg) and 3 ml) was added triethylamine (42 ul, 0.30 mmol). The products from (b) (0.5 mg) and (c) (0.5 mg) in a vial. The mixture was stirred for 10 minutes at room temperature and mixture was sonicated for 5 minutes and then heated at 80° a solution of biotin-PEGsaoo-NHS (100 mg, 0.03 mmol) in C. for 5 minutes (vial was shaken during warming) and 1,4-dioxane (1 ml) was added dropwise. After stirring at cooled. Head space was flushed with perfluorobutane gas room temperature for 3 hours the mixture was evaporated to and the vial was shaken in a cap mixer for 45 seconds dryness and the residue was purified by flash chromatogra followed by extensive washing with deionised water. phy to give white crystals, yield 102 mg (89%). The struc MALDI mass spectrometry showed no detectable level of ture was verified by MALDI-MS and by NMR analysis. compound from (b) or (c) in the final wash solution. Incor [0547] d) Synthesis of Cholesterol 4-[4-(bis(2-chloroethy poration of compounds from (b) and (c) into the l)aminolphenyl]butanoate microbubbles was confirmed by MALDI-MS as follows; ca. 50 ul of microbubbles were transferred to a clean vial [0548] DIC (170 ul, 1.10 mmol) was added to a solution containing ca. 100 ul of 90% methanol. The mixture was of chlorambucil (669 mg, 2.20 mmol) in dry dichlo US 2004/0141922 A1 Jul. 22, 2004 67 romethane (15 ml). The mixture was stirred at room tem Amide MBHA resin on a 0.125 mmol scale, using appro perature for 0.5 hour and added to a solution of cholesterol priate amino acids and palmitic acid. Coupling was carried (387 mg, 1.00 mmol) and DMAP (122 mg, 1.00 mmol) in out using standard TBTU/HOBt/DIEA protocols. Simulta dichloromethane (10 ml). The reaction mixture was stirred neous removal of the peptide from the resin and deprotection overnight and then poured onto 5% sodium bicarbonate. The of side-chain protecting groups was carried out in TFA phases were separated and the organic phase was washed with brine and dried (MgSO4). The solution was filtered and containing 5% EDT and 5% water for 2 hours. Crude concentrated and the product was purified by column chro material was precipitated from ether and purified by pre matography (silica, chloroform) to give 560 mg (83%) yield parative liquid chromatography using a gradient of 70 to of colouless oil. The product was characterised by MALDI 100% B over 60 minutes (A=0.1% TFA/water and B=0.1% mass spectrometry, giving M+H at 674 as expected. Further TFA/acetonitrile) at a flow rate of 10 ml/min. After lyophi characterisation was carried out using "H (500 MHz) and lisation, a yield of 12 mg of pure material was obtained *C (125 MHz) NMR analysis, giving spectra in accordance (analytical HPLC, gradient 70-100% B over 20 minutes, with the structure. A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow rate [0549] e) Preparation of Gas-Filled Microbubbles 1 ml/minute, detection UV 214 nm, retention time 25 [0550] A solution of 1.4% propylene glycol/2.4% glycerol minutes). Further characterisation was carried out using (1.0 ml) was added to a mixture of DSPS (5 mg) and MALDI mass spectrometry (ACH matrix), giving M+H at products from (c) (0.5 mg) and (d) (0.5 mg) in a vial. The 1315, expected 1314. mixture was sonicated for 5 minutes and then heated at 80° C. for 5 minutes (vial was shaken during warming) and [0553] b) Preparation of Gas-Filled Microbubbles Com cooled. The head space was flushed with perfluorobutane prising DSPS and a Lipopeptide Containing a Derivative of gas and the vial was shaken in a cap mixer for 45 seconds, Bestatin for Diagnostic and Therapeutic Applications whereafter the contents were extensively washed with deio mised water. MALDI mass spectrometry showed no detect [0554] A solution of 1.4% propylene glycol/2.4% glycerol able level of compound from (c) or (d) in the final wash (1.0 ml) was added to a mixture of DSPS (4.5 mg) and solution. Incorporation of compounds from (c) and (d) into product from (a) (0.5 mg) in a vial. The mixture was the microbubbles was confirmed by MALDI-MS as follows: sonicated for 5 minutes and then heated at 80° C. for 5 ca. 50 ul of microbubbles were transferred to a clean vial minutes (vial was shaken during warming) and cooled. The containing ca. 100 ul of 90% methanol. The mixture was sonicated for 30 seconds and analysed by MALDI-MS head space was flushed with perfluorobutane gas and the vial (ACH-matrix), giving M+H peaks corresponding to com was shaken in a cap mixer for 45 seconds and the contents pounds from (c) and (d). were extensively washed with deionised water. MALDI mass spectrometry showed no detectable level of compound EXAMPLE 60 from (b) in the final wash solution. Incorporation of Gas-Filled Microbubbles Comprising DSPS and a atenolol-containing lipopeptide into the microbubbles was Lipopeptide Containing a Derivative of Bestatin for confirmed by MALDI-MS as follows; ca. 50 ul of Diagnostic and Therapeutic Applications microbubbles were transferred to a clean vial containing ca. [0551] a) Synthesis of a Lipopeptide Containing a Deriva 100 ul of 90% methanol. The mixture was sonicated for 30 tive of Bestatin (SEQ ID NO:23) seconds and analysed by MALDI-MS (ACH-matrix), giving [0552] The structure shown above was synthesised by the a M+H peak at 1320, expected at 1314, corresponding to manual bubbler method starting with Fmoc-protected Rink lipopeptide from (a).

~~~~~~~~O

O # H US 2004/0141922 A1 Jul. 22, 2004 68

[0555] c) In Vitro Analysis product from (a) (0.5 mg) in a vial. The mixture was [0556] The microbubbles were tested in the in vitro assay sonicated for 5 minutes and then heated at 80° C. for 5 as detailed in Example 21. A gradual accumulation of minutes (vial was shaken during warming) and cooled. The microbubbles binding to the cells was observed. head space was flushed with perfluorobutane gas and the vial was shaken in a cap mixer for 45 seconds, whereafter the EXAMPLE 61 contents were extensively washed with deionised water. MALDI mass spectrometry showed no detectable level of Gas-Filled Microbubbles Comprising DSPS and a compound from (a) in the final wash solution. Incorporation Lipopeptide Containing Chlorambucil for of chlorambucil-containing lipopeptide into the bubbles was Diagnostic and Therapeutic Applications confirmed by MALDI-MS as follows; ca. 50 ul of [0557] a) Synthesis of a Lipopeptide Containing Chloram microbubbles were transferred to a clean vial containing ca. bucil (SEQ ID NO:24) 100 ul of 90% methanol. The mixture was sonicated for 30

~~~~~~~~O

O O O H H N N ~~~~~~~~ N N H H O O

H2

Cl

[0558] The structure shown above was synthesised by the seconds and analysed by MALDI-MS (ACH-matrix), giving manual bubbler method starting with Fmoc-protected Rink a M+H peak at 1300, expected at 0.1294 and a M+Na peak Amide MBHA resin on a 0.125 mmol scale, using appro at 1324, expected 1317. priate amino acids and palmitic acid. Coupling was carried out using standard TBTU/HOBt/DIEA protocol. Chloram [0561] c) In Vitro Analysis bucil was coupled through the side-chain of Lys as a [0562] The microbubbles were tested in the in vitro assay symmetrical anhydride using DIC preactivation. Simulta as detailed in Example 21. A gradual accumulation of neous removal of the peptide from the resin and deprotection of side-chain protecting groups was carried out in TFA microbubbles binding to the cells was observed. containing 5% EDT, 5% water and 5% ethyl methyl sulphide for 2 hours. An aliqout of 10 mg of the crude material was EXAMPLE 62 purified by preparative liquid chromatography using a gra dient of 70 to 100% B over 60 minutes (A=0.1% TFA/water Gas-Filled Microbubbles Comprising DSPS, a and B=0.1% TFA/acetonitrile) at a flow rate of 10 ml/min. Lipopeptide Containing Atenolol and a Lipophilic After lyophilisation, a yield of 30 mg of pure material was Derivative of Captopril for Diagnostic and obtained (analytical HPLC, gradient 70-100% B over 20 Therapeutic Applications minutes, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow rate 1 ml/minute, detection UV 214 nm retention time [0563] a) Synthesis of a Protected Atenolol Derivative 26.5 minutes). Further characterisation was carried out using Suitable for Solid Phase Coupling MALDI mass spectrometry, giving M+H at 1295, expected [0564] i) Synthesis of Methyl 4-[(2,3-epoxy)propoxy] 1294. phenylacetate [0559] b) Preparation of Gas-Filled Microbubbles Com prising DSPS and a Lipopeptide Containing Chlorambucil [0565] A mixture of methyl 4-hydroxyphenylacetate (4.98 for Diagnostic and Therapeutic Applications g, 0.030 mol), epichlorohydrin (23.5 ml, 0.30 mol) and pyridine (121 ul, 1.5 mmol) was stirred at 85°C. for 2 hours. [0560] A solution of 1.4% propylene glycol/2.4% glycerol The reaction mixture was cooled, and excess epichlorohy (1.0 ml) was added to a mixture of DSPS (4.5 mg) and drin was distilled off (rotavapor). The residue was taken up US 2004/0141922 A1 Jul. 22, 2004 69 in ethyl acetate, washed with brine and dried (Na2SO4). The hydrogen sulphate and organic material was extracted into solution was filtered and concentrated. The dark residue was ethyl acetate. The organic phase was washed with water and chromatographed (silica, hexane/ethyl acetate 7:3) to give brine, dried (Na2SO4) and filtered to give 0.6 g of crude 2.25 g (34%) of a colourless oil. ‘H (300 MHz) and **C material. The product was purified by chromatography NMR (75 MHz) spectra were in accordance with the struc ture. (silica, CHCla/MeOH/AcOH 85:10:5). The solution was concentrated and the residue was taken up in glacial acetic [0566] ii) Synthesis of Methyl 4-[2-hydroxy-3-[(1-me acid and lyophilised. Yield 415 mg (56%), white solid. The thyl-ethyl)aminolpropoxy]phenylacetate structure was confirmed by "H and **C NMR analysis. [0567] A mixture of methyl 4-[(2,3-epoxy)propoxy]phe nylacetate (2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 [0572] b) Synthesis of a Lipopeptide Functionalised with mol) and water (1.35 ml, 74.7 mmol) was stirred at room Atenolol (SEQ ID NO:21)

~~~~~~~~O

O O O H N ~~~~~~~~ N H O O

H2

temperature overnight. The reaction mixture was concen [0573] The structure shown above was synthesised by the trated (rotavapor) and the oily residue was dissolved in manual bubbler method starting with Fmoc-protected Rink chloroform and dried (Na2SO4). Filtration and concentration Amide MBHA resin on a 0.125 mmol scale, using appro gave quantitative yield of a yellow oil that was used in the priate amino acids, palmitic acid and the compound from next step without further purification. The structure was (a). Coupling was carried out using standard TBTU/HOBt/ verified by *H and **C NMR analysis. DIEA protocols. Simultaneous removal of the peptide from the resin and deprotection of side-chain protecting groups [0568] iii) Synthesis of 4-[2-hydroxy-3-[(1-methyl-ethy was carried out in TFA containing 5% EDT and 5% water for l)aminolpropoxy]phenylacetic acid hydrochloride 2 hours. Crude material was precipitated from ether and [0569] A solution of methyl 4-[2-hydroxy-3-[(1-methyl purified by preparative liquid chromatography using a gra ethyl)aminolpropoxy]phenylacetate (563 mg, 2.00 mmol) in dient of 70 to 100% B over 60 minutes (A=0.1% TFA/water 6M hydrochloric acid (15 ml) was heated at 100° C. for 4 and B=0.1 TFA/acetonitrile) at a flow rate of 10 ml/min. hours. The reaction mixture was concentrated (rotavapor) After lyophilisation, a yield of 38 mg of pure material was and the residue was taken up in water and lyophilised. "H obtained (analytical HPLC, gradient 70-100% B over 20 and **C NMR spectra were in accordance with the strucure minutes, A=0.1 TFA/water and B=0.1 TFA/acetonitrile, flow and MALDI mass spectrometry gave a M+H at 268 as rate 1 ml/minute, detection UV 214 nm, retention time 25 expected. minutes). Further characterisation was carried out using MALDI mass spectrometry (ACH matrix), giving M+H at [0570) iv) Synthesis of N-Boc-4-[2-hydroxy-3-[(1-me 1258, expected 1257. thyl-ethyl)aminolpropoxy]phenylacetic acid [0574] c) Synthesis of N-[(S)-3-hexadecylthio-2-methyl [0571] A solution of the 4-[2-hydroxy-3-[(1-methyl-ethy propionyl]proline l)aminolpropoxy]phenylacetic acid hydrochloride (2.0 [0575] DIEA (188 ul, 1.10 mmol) was added to a solution mmol) in water (2 ml) was added to a solution of sodium of 1-iodohexadecane (176 mg, 0.500 mmol), captopril (120 bicarbonate (0.60 g, 7.2 mmol) in water/dioxane (2:1, 15 mg, 0.550 mmol) and DBU (165 ul, 1.10 mmol) in tetrahy ml). A solution of di-tert-butyl dicarbonate (0.48 g, 2.2 drofuran (5 ml). The mixture was heated at 70° C. for 2 mmol) in dioxane (5 ml) was added. Progress of the reaction hours and then concentrated. The residue was poured onto was monitored by TLC analysis (silica, CHCls/MeOH/ water saturated with potassium hydrogen sulphate and AcOH 85:10:5), and portions of di-tert-butyl dicarbonate organic material was extracted into chloroform. The organic were added until conversion was complete. The reaction phase was washed with water and dried (MgSO4). The mixture was poured onto water saturated with potassium product was purified by chromatography (silica, CHCls/ US 2004/0141922 A1 Jul. 22, 2004 70

MeOH/AcOH 85:10:5) and lyophilised to give 105 mg [0584] c) Synthesis of 4-[2-hydroxy-3-[(1-methyl-ethy (48%) of white solid material. The structure was verified by l)aminolpropoxy]phenylacetic acid hydrochloride *H (500 Mhz) and **C (125 Mhz) NMR analysis and further [0585] A solution of methyl 4-[2-hydroxy-3-[(1-methyl characterised by MALDI mass spectrometry, giving M-H in ethyl)aminolpropoxy]phenylacetate (563 mg, 2.00 mmol) in negative mode at m/z 440 as expected. 6M hydrochloric acid (15 ml) was heated at 100° C. for 4 [0576] d) Preparation of Gas-Filled Microbubbles Com hours. The reaction mixture was concentrated (rotavapor) prising DSPS, a Lipopeptide Containing Atenolol and a and the residue was taken up in water and lyophilised. "H Lipophilic Derivative of Captopril for Diagnostic and Thera and **C NMR spectra were in accordance with the strucure peutic Applications and MALDI mass spectrometry gave a M+H at 268 as [0577] A solution of 1.4% propylene glycol/2.4% glycerol expected. (1.0 ml) was added to a mixture of DSPS (4.5 mg) and [0586] d) Synthesis of N-Boc-4-[2-hydroxy-3-[(1-methyl products from (b) (0.5 mg) and (c) in a vial. The mixture was ethyl)aminolpropoxy]phenylacetic acid sonicated for 5 minutes and then heated at 80° C. for 5 [0587] A solution of the 4-[2-hydroxy-3-[(1-methyl-ethy minutes (vial was shaken during warming) and cooled. The l)aminolpropoxy]phenylacetic acid hydrochloride (2.0 head space was flushed with perfluorobutane gas and the vial mmol) in water (2 ml) was added to a solution of sodium was shaken in a cap mixer for 45 seconds, whereafter the bicarbonate (0.60 g, 7.2 mmol) in water/dioxane (2:1, 15 contents were extensively washed with deionised water. ml). A solution of di-tert-butyl dicarbonate (0.48 g, 2.2 MALDI mass spectrometry showed no detectable level of mmol) in dioxane (5 ml) was added. Progress of the reaction compound from (b) or (c) in the final wash solution. Incor was monitored by TLC analysis (silica, CHCls/MeOH/ poration of compounds (b) and (c) into the microbubbles AcOH 85:10:5), and portions of di-tert-butyl dicarbonate was confirmed by MALDI-MS as follows; ca. 50 ul of were added until conversion was complete. The reaction microbubbles were transferred to a clean vial containing ca. mixture was poured onto water saturated with potassium 100 ul of 90% methanol. The mixture was sonicated for 30 hydrogen sulphate and organic material was extracted into seconds and analysed by MALDI-MS (ACH-matrix), giving ethyl acetate. The organic phase was washed with water and M+H peaks corresponding to structures (b) and (c) respec brine, dried (Na2SO) and filtered to give 0.6 g of crude tively. material. The product was purified by chromatography [0578] e) In Vitro Analysis (silica, CHCla/MeOH/AcOH 85:10:5). The solution was [0579] The microbubbles were tested in the in vitro assay concentrated and the residue was taken up in glacial acetic as detailed in Example 21. A gradual accumulation of acid and lyophilised. Yield 415 mg (56%), white solid. The microbubbles binding to the cells was observed. structure was confirmed by "H and **C NMR analysis. [0588] e) Synthesis of Cholesterol N-Boc-f-alaninate EXAMPLE 63 [0589] DIC (510 ul) was added to a solution of Boc-f Ala-OH (1.25 g, 6.60 mmol) in dichloromethane (15 ml) Gas-Filled Microbubbles Comprising DSPS and a under an inert atmosphere. The reaction mixture was stirred Cholesterol Derivative of Atenolol for Diagnostic for 30 minutes and then transferred to a flask containing a and Therapeutic Applications solution of cholesterol (1.16 g, 3.00 mmol) and DMAP (367 [0580] a) Synthesis of Methyl 4-[(2,3-epoxy)propoxy] mg, 3.00 mmol) in dichloromethane (15 ml). The reaction mixture was stirred for 2 hours and then poured onto an phenylacetate aqeous solution of potassium hydrogen sulphate. After phase [0581] A mixture of methyl 4-hydroxyphenylacetate (4.98 separation the aqueous phase was extracted with chloro g, 0.030 mol), epichlorohydrin (23.5 ml, 0.30 mol) and form. The combined organic phases were washed with pyridine (121 ul, 1.5 mmol) was stirred at 85°C. for 2 hours. aqueous potassium hydrogen sulphate and water and dried The reaction mixture was cooled, and excess epichlorohy (MgSO4). After filtration and evaporation the crude product drin was distilled off (rotavapor). The residue was taken up was chromatographed (silica, chloroform/methanol 99:1) to in ethyl acetate, washed with brine and dried (Na2SO4). The give 1.63 g (97%) of white solid. The structure was con solution was filtered and concentrated. The dark residue was firmed by *H NMR (500 MHz). chromatographed (silica, hexane/ethyl acetate 7:3) to give 2.25 g (34%) of a colourless oil. "H (300 MHz) and **C [0590] f) Synthesis of Cholesteryl B-alaninate Hydrochlo NMR (75 MHz) spectra were in accordance with the struc ride ture. [0591] A solution of compound from (a) (279 mg, 0.500 mmol) in 1M hydrochloric acid in 1,4-dioxane (5 ml) was [0582] b) Synthesis of Methyl 4-[2-hydroxy-3-[(1-methyl stirred at room temperature for 4 hours. The reaction mixture ethyl)aminolpropoxy]phenylacetate was concentrated to give a quantitative yield of cholesteryl [0583] A mixture of methyl 4-[(2,3-epoxy)propoxy]phe fl-alaninate hydrochloride. The structure was confirmed by nylacetate (2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 1H NMR (500 MHz) analysis and by MALDI mass spec mol) and water (1.35 ml, 74.7 mmol) was stirred at room trometry, giving a M+Na peak at 482, expected 481. temperature overnight. The reaction mixture was concen [0592] g) Synthesis of Cholesteryl N-Boc-4-[2-hydroxy trated (rotavapor) and the oily residue was dissolved in 3-[(1-methylethyl)aminolpropoxy]phenylacetyl-[3-alani chloroform and dried (Na2SO4). Filtration and concentration gave quantitative yield of a yellow oil that was used in the nate. next step without further purification. The structure was [0593] To a solution of N-Boc-4-[2-hydroxy-3-[(1-me verified by *H and **C NMR analysis. thyl-ethyl)aminolpropoxy]phenylacetic acid (55 mg, 0.15 US 2004/0141922 A1 Jul. 22, 2004 mmol) and cholesteryl fl-alaninate hydrochloride (74 mg, of compound from (h) into the microbubbles was confirmed 0.15 mmol) in DMF (5 ml) was added DIEA (26 ml, 0.15 by MALDI mass spectrometry. mmol). HOBt (23 mg, 0.15 mmol) and water-soluble car bodiimide (WSC) (29 mg, 0.15 mmol) were added. The [0598] j) In Vitro Analysis reaction mixture was stirred at room temperature overnight [0599] The microbubbles were tested in the in vitro assay and then poured onto water (25 ml) containing sodium as detailed in Example 21. A gradual accumulation of carbonate (2.5 g) and sodium chloride (4.0 g). Precipitated bubbles binding to the cells was observed. material was extracted into chloroform. The organic phase was washed with water and dried (MgSO4). After filtration EXAMPLE 64 and concentration, crude material (132 mg) was purified by Preparation of Multiple-Sepecific column chromatography (silica, chloroform/methanol/acetic Transferrin/Avidin-Coated Gas-Filled Microbubbles acid, 95:4:1). Pooled fractions were concentrated, taken up for Targeted Ultrasound Imaging in glacial acetic acid and lyophilised. Yield 83 mg (69%), [0600] This example is directed to the preparation of yellow-white solid. Structure was confirmed by "H NMR microbubbles containing vectors for targeted ultrasound/ analysis. therapy. [0594 h) Synthesis of Cholesteryl 4-[2-hydroxy-3-[(1 [0601] a) Synthesis of a Thiol-Functionalised Lipid Mol methyl-ethyl) aminolpropoxy]phenylacetyl-[3-alaninate trif ecule: Dipalmitoyl-Lys-Lys-Lys-Aca-Cys.OH (SEQ ID luoroacetate NO:20)

O

~~~~~~~~H NH2

SH O O H H N N sº- N 's-s-s-s N OH H - H H O O O

[0595] To a solution of N-Boc-4-[2-hydroxy-3-[(1-me [0602] The lipid structure shown above was synthesised thyl-ethyl) aminolpropoxy]phenylacetyl-[3-alaninate (40 on an ABI 433A automatic peptide synthesiser starting with mg, 0.05 mmol) in dry dichloromethane (4 ml) was added Fmoc-Cys(Trt)-Wang resin on a 0.25 mmol scale using 1 trifluoroacetic acid (2 ml). The reaction mixture was stirred mmol amino acid cartridges. All amino acids and palmitic for 2 hours and then concentrated. The product was lyophi acid were preactivated using HBTU coupling chemistry. The lised from a acetonitrile/water mixture to give a quantitative simultaneous removal of peptide from the resin and depro yield of white-yellow material. The product was character tection of side-chain protecting groups was carried out in ised by MALDI mass spectrometry giving M+H at 708 as TFA containing 5% EDT and 5% H2O for 2 hours, giving a expected. crude product yield of 250 mg. Purification by preparative HPLC of a 40 mg aliquot of crude material was carried out [0596] i) Preparation of Gas-Filled Microbubbles Com using a gradient of 90 to 100% B over 50 minutes (A=0.1% prising DSPS and a Cholesterol Derivative of Atenolol for TFA/water and B=MeOH) at a flow rate of 9 ml/min. After Diagnostic and Therapeutic Applications lyophilisation, 24 mg of pure material was obtained (ana lytical HPLC, gradient 70-100% B where B=0.1% TFA/ [0597] A solution of 1.4% propylene glycol/2.4% glycerol acetonitrile, A=0.01% TFA/water: detection—UV 214 (1.0 ml) was added to a mixture of DSPS (4.5 mg) and nm—product retention time=23 minutes). Further product product from (h) (0.5 mg) in a vial. The mixture was characterisation was carried out using MALDI mass spec sonicated for 5 minutes and then heated at 80° C. for 5 trometry: expected M+H at 1096, found at 1099. minutes (vial was shaken during warming) and cooled. The [0603] b) Preparation of Gas-Containing Microbubbles head space was flushed with perfluorobutane gas and the vial Comprising DSPS ‘Doped’ with a Thiol-Containing Lipid was shaken in a cap mixer for 45 seconds, whereafter the contents were extensively washed with deionised water. Structure MALDI mass spectrometry showed no detectable level of [0604] DSPS (4.5 mg) and the lipid structure from (a) compound from (b) in the final wash solution. Incorporation above (0.5 mg) were weighed into a clean vial and 0.8 ml of US 2004/0141922 A1 Jul. 22, 2004 72 a solution containing 1.4% propylene glycol/2.4% glycerol Fluorescent Protein vector from CLONTECH) and 50 ul of in water was added. The mixture was warmed to 80° C. for microbubble suspension from (a) in RPMI medium at a final 5 minutes (vial shaken during warming) and filtered while volume of 250 ul was prepared. The mixture was left still hot through a 40 micron filter. The sample was cooled standing for 15 minutes at room temperature then 1 ml of to room temperature and the head space was flushed with complete RPMI medium was added. The medium was perfluorobutane gas. The vial was shaken in a cap mixer for removed from the cell culture dish and the DNA-mi 45 seconds and then placed on aroller table overnight. The crobubble mixture was added to the cells. The cells were resulting microbubbles were washed several times with incubated in a cell culture incubator (37° C). deionised water and analysed for thiol group incorporation using Ellmans Reagent. [0614] c) Ultrasonic Treatment [0615] After 15 minutes incubation, selected wells were [0605] c) Modification of Transferrin and Avidin with exposed to continious wave ultrasound of 1 MHz, 0.5 Fluorescein-NHS and Sulpho-SMPB W/cm3, for 30 seconds. [0606] To a mixture of 2 mg of transferrin (Holo, human) and 2 mg of avidin in PBS (1 ml) was added 0.5 ml of a [0616] d) Incubation and Examination DMSO solution containing 1 mg Sulpho-SMPB and 0.5 mg [0617] The cells were further incubated in the cell culture fluorescein-NHS. The mixture was stirred for 45 minutes at incubator (37° C) for approximately 4.5 hours. The medium room temperature then passed through a Sephadex 200 containing DNA-microbubbles was then removed by aspi column using PBS as eluent. The protein fraction was ration, and 2 ml complete RPMI medium was added. The collected and stored at 4° C. prior to use. cells were incubated for 40-70 hours before examination. Most of the medium was then removed and the cells were [0607] d) Microbubble Conjugation with Modified Trans examined by fluorescence microscopy. The results were ferrin/Aidin compared to the results from control experiments where [0608] To the thiol-containing microbubbles from (b) was DNA or DNA-polylysine were added to the cells. added 1 ml of the modified transferrin/avidin protein solu tion from (c). After adjusting the pH of the solution to 9, the EXAMPLE 66 conjugation reaction was allowed to proceed for 2 hours at room temperature. Following extensive washing with deio Flotation of Endothelial Cells by Microbubbles mised water the microbubbles were analysed by Coulter with Vectors That Specifically Bind to the counter (81% between 1 and 7 micron) and fluorescence Endothelial Cells microscopy (highly fluorescent microbubbles were [0618] This experiment was carried out to show that the observed). present invention can be used for separation of cells to which the microbubbles are targeted. The human endothelial cell EXAMPLE 65 line ECV 304, derived from a normal umbilical cord (ATCC CRL-1998) was cultured in Nunc culture flasks (chutney Gene Transfer by Gas-Filled Microbubbles 153732) in RPMI 1640 medium to which L-glutamine (200 mM), penicillin/streptomycin (10,000 U/ml and 10,00 [0609] This example is directed at the preparation of Aug/ml) and 10 fetal calf serum were added. The cells were targeted microbubbles for gene transfer. subcultured following trypsination with a split ratio of 1:5 to [0610] a) Preparation of Gas-Filled Microbubbles Com 1:7 when reaching confluence. 2 million cells from trypsi prising DSPS and Lipopeptide Coated with poly-L-lysine nated confluent cultures were added to each set of five [0611] DSPS (4.5 mg) and lipopeptide from Example 41 centrifuge tubes. Then control microbubbles or (0.5 mg) were weighed in two 2 ml vials. To each vial, 0.8 microbubbles binding to endothelial cells, made as ml propylene glycol/glycerol (4%) in water was added. Each described in Example 21 and in Example 38, were added at solution was heated at 80°C. for 5 minutes, shaken and then 2, 4, 6, 8 or 10 million bubbles per tube. The cells at the cooled to ambient temperature, whereafter the headspaces bottom of the tubes after centrifugation at 400 g for 5 were flushed with perfluorobutane. The vials were shaken on minutes were counted with a Coulter counter. It was found a cap-mixer at 4450 oscillations/minute for 45 seconds and the 4 or more microbubbles binding to a cell brought the put on a roller table for 5 minutes. The content of the vials cells to the top of the fluid in the centrifugation tube. All were mixed and the resulting sample was washed by cen cells were floated by the microbbbles from Example 38 trifugation at 2000 rpm for 5 minutes. The infranatant was whereas about 50% were floated with the microbubbles from removed and the same volume of distilled water was added. Example 21. The washing procedure was repeated once. Poly-L-lysine EXAMPLE 67 HBr (20.6 mg) was dissolved in 2 ml water, then an aliquot (0.4 ml) was made up to 2 ml with water. To 1.2 ml of the Gas-Filled Microbubbles of diluted poly-L-lysine solution was added 0.12 ml of the Distearoyl-Phosphatidylserine Comprising a DSPS-lipopeptide microbubble suspension. Following incu Lipopeptide Containing a Vector with Affinity for bation, excess polylysine was removed by extensive wash Endothelin Receptors for Targeted Ultrasound ing with water. Imaging [0612] b) Transfection of Cells [0619] a) Synthesis of 4'-[(3,4-dimethyl-5-isoxazolyl)-sul famoylsuccinanilic acid [0613] Endothelial cells (ECV 304) were cultured in 6 well plates to a uniform subconfluent layer. A transfection [0620] To a solution of sulfisoxazole (267mg, 1.00 mmol) mixture consisting of 5 ug DNA (an Enhanced Green in DMF (10 ml) was added succinic anhydride (1.00 g, 10.0 US 2004/0141922 A1 Jul. 22, 2004 73 mmol) and 4-dimethylaminopyridine (122 mg, 1.00 mmol). extensive washing with deionised water. MALDI mass The reaction mixture was stirred at 80° C. for 2 hours and spectrometry showed no detectable level of compound from then concentrated. The residue was taken up in 5% aqueous (b) in the final wash solution. Incorporation of isoxazole containing lipopeptide into the microbubbles was confirmed sodium bicarbonate solution and extracted with ethyl by MALDI-MS as follows: ca. 50 ul of microbubbles were acetate. The aqueous solution was acidified with dilute transferred to a clean vial containing ca. 100 ul of 90% hydrochloric acid and organic material was extracted into methanol. The mixture was sonicated for 30 seconds and ethyl acetate. The organic phase was 1.0 washed with dilute analysed by MALDI-MS (ACH-matrix), giving a m-H peak hydrochloric acid, water and brine, treated with active at m/z 1359 corresponding to lipopeptide (b). charcoal and dried (MgSO4). The solution was filtered and concentrated to give 280 mg (76%) of white solid. The structure was verified by *H (300 MHz) and **C (75 MHz) 1. A targetable diagnostic and/or therapeutically active NMR spectroscopy. Further characterisation was carried out agent comprising a suspension in an aqueous carrier liquid using MALDI mass spectrometry (ACH matrix), giving a of a reporter comprising gas-filled microbubbles stabilised M+Na peak at m/z 390 and a M+K peak at m/z 406 as by monolayers of film-forming surfactant, said agent further expected. comprising at least one vector. 2. An agent as claimed in claim 1 wherein the gas [0621] b) Synthesis of a Lipopeptide Functionalised with comprises air, nitrogen, oxygen, carbon dioxide, hydrogen, Sulfisoxazole (SEQ ID NO:25) an inert gas, a sulphur fluoride, selenium hexafluoride, a low

O ~~~~~~~~ NH NH2

O O O

~~~~~~~". NH NH NH NH NH2 O O

NH2

O

[0622] The structure shown above was synthesised on a molecular weight hydrocarbon, a ketone, an ester, a halo manual nitrogen bubbler apparatus starting with Fmoc genated low molecular weight hydrocarbon or a mixture of protected Rink Amide BMHA resin on a 0.125 mmol scale, any of the foregoing. using appropriate amino acids, palmitic acid and the com 3. An agent as claimed in claim 2 wherein the gas pound from (a). Coupling was carried out using standard comprises a perfluorinated ketone, perfluorinated ether or TBTU/HOBt/DIEA protocols. Simultaneous removal of the perfluorocarbon. peptide from the resin and deprotection of side-chain pro tecting groups was carried out in TFA containing 5% EDT 4. An agent as claimed in claim 2 wherein the gas and 5% water for 2 hours. Crude material was precipitated comprises sulphur hexafluoride or a perfluoropropane, per from ether. The product was analysed by analytical HPLC, fluorobutane or perfluoropentane. gradient 70-100% B over 20 minutes, A=0.1% TFA/water 5. An agent as claimed in any of the preceding claims and B=0.1% TFA/acetonitrile, flow rate 1 ml/minute, detec wherein the film-forming surfactant material comprises a tion UV 214 nm, retention time 27 minutes). Further char non-polymeric and non-polymerisable wall-forming surfac acterisation was carried out using MALDI mass spectrom tant material, a polymer surfactant material or a phospho etry, giving a M+H at m/z 1359, expected 1356. lipid. 6. An agent as claimed in claim 5 wherein at least 75% of [0623] c) Preparation of Gas-Filled Microbubbles Com the film-forming surfactant material comprises phospholipid prising the Compound From (b) molecules individually bearing net overall charge. [0624] A solution of 1.4% propylene glycol/2.4% glycerol 7. An agent as claimed in claim 6 wherein at least 75% of (1.0 ml) was added to a mixture of DSPS (4.5 mg) and the film-forming surfactant material comprises one or more product from (b) (0.5 mg) in a vial. The mixture was phospholipids selected from phosphatidylserines, phosphati sonicated for 5 minutes and then heated at 80° C. for 5 dylglycerols, phosphatidylinositols, phosphatidic acids and minutes (vial was shaken during warming) and cooled. The cardiolipins. head space was flushed with perfluorobutane gas and the vial 8. An agent as claimed in claim 7 wherein at least 80% of was shaken in a cap mixer for 45 seconds followed by said phospholipids comprise phosphatidylserines. US 2004/0141922 A1 Jul. 22, 2004 74

9. An agent as claimed in any of the preceding claims ii) a second administrable composition comprising an wherein the film-forming surfactant material comprises a agent as claimed in any of the preceding claims, said lipopeptide. agent comprising a vector having affinity for said 10. An agent as claimed in any of the preceding claims pre-targeting vector. wherein the vector is selected from antibodies; cell adhesion 23. A combined formulation as claimed in claim 22 molecules; cell adhesion molecule receptors; cytokines; wherein said pre-targeting vector is a monoclonal antibody. growth factors; peptide hormones and pieces thereof; non 24. A combined formulation comprising: peptide agonists/antagonists and non-bioactive binders of i) a first administrable composition comprising an agent receptors for cell adhesion molecules, cytokines, growth as claimed in any of claims 1 to 21; and factors and peptide hormones; oligonucleotides and modi ii) a second administrable composition comprising a fied oligonucleotides; DNA-binding drugs; protease sub substance capable of displacing or releasing said agent strates/inhibitors; molecules generated from combinatorial from its target. libraries; and small bioactive molecules. 25. A combined formulation comprising: 11. An agent as claimed in any of the preceding claims wherein the vector or vectors have affinity for targets at a i) a first administrable composition comprising an agent level such that the agent interacts with but does not fixedly as claimed in claim 20; and bind to said targets. ii) a second administrable composition comprising a 12. An agent as claimed in claim 11 wherein the vector or reducing agent capable of reductively cleaving the vectors are selected from ligands for cell adhesion proteins disulphide groups coupling or linking the therapeutic and cell adhesion proteins which have corresponding ligands compound and reporter in the agent of said first admin on endothelial cell surfaces. istrable composition. 13. An agent as claimed in any of the preceding claims 26. A process for the preparation of a targetable diagnostic wherein the vector or vectors are sited such that they are not and/or therapeutically active agent as defined in claim 1 readily exposed to the target. which comprises either coupling or linking at least one 14. An agent as claimed in any of the preceding claims vector to a reporter comprising gas-filled microbubbles wherein the vector is covalently coupled or linked to the stabilised by monolayers of film-forming surfactant or gen reporter. erating gas-filled reporter microbubbles using film-forming surfactant having at least one vector attached thereto. 15. An agent as claimed in any of claims 1 to 13 wherein 27. A process as claimed in claim 26 wherein a therapeutic the vector is coupled or linked to the reporter through compound is also combined with the reporter. electrostatic charge interactions. 28. A process as claimed in claim 27 wherein a therapeutic 16. An agent as claimed in any of claims 1 to 13 wherein compound containing thiol groups is linked to thiol group the vector is coupled or linked to the reporter by means of containing surfactant monolayers stabilising the gas-filled avidin-biotin and/or streptavidin-biotin interactions. microbubbles of the reporter by reaction under oxidative 17. An agent as claimed in any of the preceding claims conditions so as to generate disulphide groups. which further contains moieties which are radioactive or are 29. Use of an agent as claimed in any of claims 1 to 21 effective as X-ray contrast agents, light imaging probes or as a targetable ultrasound contrast agent. spin labels. 30. A method of generating enhanced images of a human 18. An agent as claimed in any one of the preceding or non-human animal body which comprises administering claims further comprising a therapeutic compound. to said body an agent as claimed in any of claims 1 to 21 and 19. An agent as claimed in claim 18 wherein said thera generating an ultrasound, magnetic resonance, X-ray, radio peutic compound is an antineoplastic agent, blood product, graphic or light image of at least a part of said body. biological response modifier, antifungal agent, hormone or 31. A method as claimed in claim 30 which comprises the hormone analogue, vitamin, enzyme, antiallergic agent, tis steps: sue factor inhibitor, platelet inhibitor, coagulation protein target inhibitor, fibrin formation inhibitor, fibrinolysis pro i) administering to said body a pre-targeting vector having moter, antiangiogenic, circulatory drug, metabolic potentia affinity for a selected target; and thereafter tor, antitubercular, antiviral, vasodilator, antibiotic, antiin ii) administering an agent as claimed in any of claims 1 to flammatory, antiprotozoan, antirheumatic, narcotic, opiate, 21, said agent comprising a vector having affinity for cardiac glycoside, neuromuscular blocker, sedative, local said pre-targeting vector. anaesthetic, general anaesthetic or genetic material. 32. A method as claimed in claim 31 wherein said 20. An agent as claimed in claim 18 or claim 19 wherein pre-targeting vector is a monoclonal antibody. said therapeutic compound is covalently coupled or linked to 33. A method as claimed in claim 30 which comprises the the reporter through disulphide groups. steps: 21. An agent as claimed in claim 18 or claim 19 wherein i) administering to said body an agent as claimed in any a lipophilic or lipophilically-derivatised therapeutic com of claims 1 to 21; and thereafter pound is linked to the surfactant monolayers stabilising the gas-filled microbubbles of the reporter through hydrophobic ii) administering a substance capable of displacing or interactions. releasing said agent from its target. 34. A method as claimed in any of claims 30 to 33 wherein 22. A combined formulation comprising: said agent further comprises a therapeutic compound. i) a first administrable composition comprising a pre 35. A method as claimed in claim 34 wherein said targeting vector having affinity for a selected target; and therapeutic compound is covalently coupled or linked to the US 2004/0141922 A1 Jul. 22, 2004 75 reporter through disulphide groups, and a composition com- through said chamber, and binding of said agent to said cells prising a reducing agent capable of reductively cleaving said is examined. disulphide groups is subsequently administered. 37. A method as claimed in claim 36 wherein the flow rate 36. A method for in vitro investigation of targeting by an of carrier liquid is controlled to simulate shear rates encoun agent as defined in any of claims 1 to 21 wherein cells tered in vivo. expressing a target are fixedly positioned in a flow chamber, a suspension of said agent in a carrier liquid is passed #: :#: ::: ::: :#: