US 2002O102215A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2002/0102215 A1 Klaveness et al. (43) Pub. Date: Aug. 1, 2002

(54) DIAGNOSTIC/THERAPEUTICAGENTS (60) Provisional application No. 60/049.264, filed on Jun. 6, 1997. Provisional application No. 60/049,265, filed (75) Inventors: Jo Klaveness, Oslo (NO); Pal on Jun. 6, 1997. Provisional application No. 60/049, Rongved, Oslo (NO); Anders Hogset, 268, filed on Jun. 7, 1997. Oslo (NO); Helge Tolleshaug, Oslo (NO); Anne Naevestad, Oslo (NO); (30) Foreign Application Priority Data Halldis Hellebust, Oslo (NO); Lars Hoff, Oslo (NO); Alan Cuthbertson, Oct. 28, 1996 (GB)...... 9622.366.4 Oslo (NO); Dagfinn Lovhaug, Oslo Oct. 28, 1996 (GB). ... 96223672 (NO); Magne Solbakken, Oslo (NO) Oct. 28, 1996 (GB). 9622368.0 Jan. 15, 1997 (GB). ... 97OO699.3 Correspondence Address: Apr. 24, 1997 (GB). ... 9708265.5 BACON & THOMAS, PLLC Jun. 6, 1997 (GB). ... 9711842.6 4th Floor Jun. 6, 1997 (GB)...... 97.11846.7 625 Slaters Lane Alexandria, VA 22314-1176 (US) Publication Classification (73) Assignee: NYCOMED IMAGING AS (51) Int. Cl." ...... A61K 49/00; A61K 48/00 (52) U.S. Cl...... 424/9.52; 514/44 (21) Appl. No.: 09/765,614 (22) Filed: Jan. 22, 2001 (57) ABSTRACT Related U.S. Application Data Targetable diagnostic and/or therapeutically active agents, (63) Continuation of application No. 08/960,054, filed on e.g. ultrasound contrast agents, having reporters comprising Oct. 29, 1997, now patented, which is a continuation gas-filled microbubbles stabilized by monolayers of film in-part of application No. 08/958,993, filed on Oct. forming Surfactants, the reporter being coupled or linked to 28, 1997, now patented. at least one vector.

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DIAGNOSTIC/THERAPEUTICAGENTS 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 No. 60/049,264 and cally to a chosen target, or it may bind only Selectively, 60/049,265 both filed Jun. 6, 1997; and No. 60/049,268 filed having affinty also for a limited number of other molecules/ Jun. 7, 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 is 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, Oct. 3-5, 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 Nov. 13-16, 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 2002/0102215 A1 Aug. 1, 2002

A-94.01140, WO-A-9408.627, WO-A-9428874, U.S. Pat. thrombi and atherosclerotic lesions or for visualising neo No. 5,088,499, U.S. Pat. No. 5,348,016 and U.S. Pat. No. vascularised and inflamed tissue areas. The present inven 5,469,854. tion is particularly Suited to imaging changes which occur in 0.014. The present invention is based on the finding that normal blood vessels situated in areas of tissue necrosis. gas-filled microbubbles stabilised by monolayers of film 0019. In a further embodiment of the present invention, forming Surfactant material are particularly useful reporters one or more vectors may be attached to or included within in targeted diagnostic and/or therapeutic agents. Thus, for the reporter in a manner Such that the vectors are not readily example, the flexibility and deformability of such thin exposed to the target or target receptors. Increased tissue monolayer membranes Substantially enhances the echoge Specificity may therefore be achieved by applying an addi nicity of Such reporters relative to liposome Systems con tional process to expose the vectors, for example by expos taining lipid bilayerS or multiples of Such bilayers. This may ing the agent after administration to external ultrasound So permit the use of Very low doses of the reporter material to as to modify the diffusibility of the moieties containing the achieve high ultrasound contrast efficacy, with consequent VectOrS. safety benefits. 0.015 Thus according to one aspect of the present inven BRIEF DESCRIPTION OF THE FIGURES tion there is provided a targetable diagnostic and/or thera 0020 FIG. 1: Flow cytometric comparison of negative peutically active agent, e.g. an ultrasound contrast agent, control microbubbles of DSPS (left curve) with bubbles comprising a Suspension in an aqueous carrier liquid, e.g. an conjugated with CD71 FITC-labelled anti-transferrin anti injectable carrier liquid, of a reporter comprising gas-filled body (filled curve, right) showing that 92% of the population microbubbles stabilised by monolayers of film-forming Sur fluoresce. factant material, Said agent further comprising at least one VectOr. 0021 FIG. 2: Flow cytometry data-comparison with 0016. The term “monolayer” is used herein to denote that negative control bubbles (left curve). 98% of the bubbles the amphiphilic Surfactant moieties form monolayer films or were calculated to be fluorescent. membranes Similar to So-called Langmuir-Blodgett films at 0022. Any biocompatible gas may be present in the the gas-liquid interfaces, with the lipophilic parts of the reporter, the term "gas' as used herein including any Sub amphiphiles aligning towards the gas phase and the hydro stances (including mixtures) Substantially or completely in philic parts interacting with the water phase. gaseous (including vapour) form at the normal human body temperature of 37 C. The gas may thus, for example, 0017. As indicated in WO-A-9729783, it is believed that comprise air; nitrogen; oxygen; carbon dioxide; hydrogen; electroStatic repulsion between charged phospholipid mem an inert gas Such as helium, argon, Xenon or krypton; a branes encourages the formation of Stable and Stabilising Sulphur fluoride Such as Sulphur hexafluoride, disulphur monolayers at microbubble-carrier liquid interfaces. The decafluoride or trifluoromethylsulphur pentafluoride, Sele flexibility and deformability of such thin membranes are nium hexafluoride; an optionally halogenated Silane Such as believed to enhance the echogenicity of products according methylsilane or dimethylsilane; a low molecular weight to the invention disclosed therein relative to gas-filled lipo hydrocarbon (e.g. containing up to 7 carbon atoms), for Somes comprising one or more lipid bilayers. The amount of example an alkane Such as methane, ethane, a propane, a phospholipid used to Stabilise Such microbubble-containing butane or a pentane, a cycloalkane Such as cyclopropane, aqueous Suspensions may be as low as that necessary for cyclobutane or cyclopentane, an alkene Such as ethylene, formation of Single monolayers of Surfactant around each propene, propadiene or a butene, or an alkyne Such as gas microbubble, the resulting film-like Structure Stabilising acetylene or propyne; an ether Such as dimethyl ether; a the microbubbles against collapse or coalescence. ketone; an ester; a halogenated low molecular weight hydro Microbubbles with a liposome-like surfactant bilayer are carbon (e.g. containing up to 7 carbon atoms); or a mixture believed not to be obtained when such low phospholipid of any of the foregoing. Advantageously at least Some of the concentrations are used. halogen atoms in halogenated gases are fluorine atoms; thus 0.018. One advantageous embodiment of the invention is biocompatible halogenated hydrocarbon gases may, for based on the additional finding that limited adhesion to example, be selected from bromochlorodifluoromethane, targets is a highly useful property of diagnostic and/or chlorodifluoromethane, dichlorodifluoromethane, bromotri therapeutically active agents, which property may be fluoromethane, chlorotrifluoromethane, chloropentafluoro achieved using vectors giving temporary retention rather ethane, dichlorotetrafluoroethane, chlorotrifluoroethylene, than fixed adhesion to a target. Thus Such agents, rather than fluoroethylene, ethylfluoride, 1,1-difluoroethane and per being fixedly retained at Specific Sites, may for example fluorocarbons, e.g. perfluoroalkanes Such as perfluo effectively exhibit a form of retarded flow along the vascular romethane, perfluoroethane, perfluoropropanes, perfluo endothelium by virtue of their transient interactions with robutanes (e.g. perfluoro-n-butane, optionally in admixture endothelial cells. Such agents may thus become concen with other isomerS Such as perfluoro-iso-butane), perfluoro trated on the walls of blood vessels, in the case of ultrasound pentanes, perfluorohexanes and perfluoroheptanes, perfluo contrast agents providing enhanced echogenicity thereof roalkenes Such as perfluoropropene, perfluorobutenes (e.g. relative to the bulk of the bloodstream, which is devoid of perfluorobut-2-ene) and perfluorobutadiene; perfluoro anatomical features. They therefore may permit enhanced alkynes Such as perfluorobut-2-yne, and perfluorocycloal imaging of the capillary System, including the microvascu kanes Such as perfluorocyclobutane, perfluoromethylcy lature, and So may facilitate distinction between normal and clobutane, perfluorodimethylcyclobutanes, inadequately perfused tissue, e.g. in the heart, and may also perfluorotrimethylcyclobutanes, perfluorocyclopentane, per be useful in Visualising Structures Such as Kupffer cells, fluoromethylcyclopentane, perfluorodimethylcyclopen US 2002/0102215 A1 Aug. 1, 2002 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 palmitoyland 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-9211873, WO-A-9217212, WO-A-9222247, WO-A- charged phospholipids containing two fatty acyl groups. 9428780, WO-A-95.03835 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 0.025 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 2002/0102215 A1 Aug. 1, 2002 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 lug/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/acqueous 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. 0.036 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 2002/0102215 A1 Aug. 1, 2002 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 004.5 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 2002/0102215 A1 Aug. 1, 2002 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 downregulating tissue factor expression; inhibitors 0.053 Representative therapeutics suitable for use in the of platelets such as GPIa, GPIb and GPIb-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-rFVIIa 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, amplicillin, bacampicillin, carbenicillin, receptor-mediated drug delivery, the microbubble carrier is dicloxacillin, cyclacillin, picloxacillin, he tacillin, 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, 0.055 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, Vindesline, 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, arabinosyladenine, 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 2002/0102215 A1 Aug. 1, 2002

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 mav 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, 0.058 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 2002/0102215 A1 Aug. 1, 2002

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 C.-haloacetyl compounds of the Tietze, L. F. in Chem. Ber. (1991) 124, 1215; and type X-CHCO- (where X= Br, C1 or I), which show 0078 x) C-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 additionally 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: 0.086 vii) acylazides, e.g. wherein the azide group is 0069 i) C-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-CHCO-(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 epichlorohydrin 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 2002/0102215 A1 Aug. 1, 2002

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 f2-glycoprotein I (Chonn, A., boxylic acid modifying reagents Such as cabodiimides, 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 nents. 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-liminothiolane 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 C.-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 0.093 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 C.-Subunit described by Xie, between a polylysinyl-functionalised reporter and a poly 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. 0.095 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 2002/0102215 A1 Aug. 1, 2002

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 A. 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, tion will in general bring about linking of vector to reporter whilst covalently linked PEGs are known to be non-immu or reporter to reporter with Some degree of Specificity, and nogenic and non-antigenic. Furthermore, PEGS may readily may also be used to attach one or more therapeutically active be modified and bound to other molecules with only little agents. effect on their chemistry. Their advantageous Solubility and biological properties are apparent from the many possible 0101. In some instances it is considered advantageous to uses of PEGs and copolymers thereof, including block include a PEG component as a Stabiliser in conjunction with copolymers such as PEG-polyurethanes and PEG-polypro a vector or vectors or directly to the reporter in the same pylenes. molecule where the PEG does not serve as a Spacer. 0102 So-called Zero-length linking agents, which induce 0107 Appropriate molecular weights for PEG spacers direct covalent joining of two reactive chemical groups used in accordance with the invention may, for example, be without introducing additional linking material (e.g. as in between 120 Daltons and 20 kdaltons. amide bond formation induced using carbodiimides or enzy 0108. The major mechanism for uptake of particles by the matically) may, if desired, be used in accordance with the cells of the reticuloendothelial system (RES) is opsonisation invention, as may agents Such as biotin/avidin Systems by plasma proteins in blood; these mark foreign particles which induce non-covalent reporter-vector linking and which are then taken up by the RES. The biological prop agents which induce hydrophobic or electrostatic interac erties of PEG spacer elements used in accordance with the tions. invention may serve to increase contrast agent circulation 0103 Most commonly, however, the linking agent will time in a similar manner to that observed for PEGylated comprise two or more reactive moieties, e.g. as described liposomes (see e.g. Klibanov, A. L. et al. in FEBS Letters above, connected by a Spacer element. The presence of Such (1990) 268, 235-237 and Blume, G. and Cevc, G. in Bio a Spacer permits bifunctional linkers to react with Specific chim. Biophys. Acta (1990) 1029, 91-97). Increased cou functional groups within a molecule or between two differ pling efficiency to areas of interest may also be achieved ent molecules, resulting in a bond between these two com using antibodies bound to the termini of PEG spacers (see ponents and introducing extrinsic linker-derived material e.g. Maruyama, K. et al. in Biochim. Biophys. Acta (1995) into the reporter-vector conjugate. The reactive moieties in 1234, 74-80 and Hansen, C. B. et al. in Biochim. BiophyS. a linking agent may be the same (homobifunctional agents) Acta (1995) 1239, 133-144). 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 glycosaminoglycans, 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 tain enzyme cleavage Sites. enhance Surface crosslinking, or it may contain a tracer 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 2002/0102215 A1 Aug. 1, 2002

disulphide groups. Spacers containing biodegradable meth Fukuzaki, H., Asano, M., Kumakura, M., Mashimo, T., ylene diester or diamide groups of formula Yamanaka, H. and Nagai.T. in Int. J. Pharm. (1992) 81, 31-38); 0112 where X and Z are selected from -O-, -S-, 0.122 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 biodegradable -X.Y.(Z)- or together form a divalent organic group Segments of aliphatic hydroxyacid polymers (see e.g. may also be useful; as discussed in, for example, WO-A- Younes, H., Nataf, P. R., Cohn, D., Appelbaum, Y. J., 9217436 Such groups are readily biodegraded in the pres Pizov, G. and Uretzky, G. in Biomater. Artif Cells Artif ence of esterases, e.g. in Vivo, but are stable in the absence Organs (1988) 16, 705-719), for instance in conjunc of Such enzymes. They may therefore advantageously be tion with polyurethanes (see Kobayashi, H., Hyon, S. linked to therapeutic agents to permit slow release thereof. H. and Ikada, Y. in “Water-curable and biodegradable prepolymers' J. Biomed. Mater. Res. (1991) 25, 0113 PolyN-(2-hydroxyethyl)methacrylamides C 1481-1494); potentially useful spacer materials by Virtue of their low degree of interaction with cells and tissues (see e.g. Volfova, 0124 x) polyurethanes, which are known to be well I., Rihova, B. and V. R. and Vetvicka, P. in J. Bioact. Comp. tolerated in implants, and which may be combined with Polymers (1992) 7, 175-190). Work on a similar polymer flexible “Soft” segments, e.g. comprising poly(tetra consisting mainly of the closely related 2-hydroxypropyl methylene glycol), poly(propylene glycol) or poly(eth derivative showed that it was endocytosed by the mono ylene glycol) and aromatic "hard” segments, e.g. com nuclear phagocyte System only to a rather low extent (see prising 4,4'-methylenebis(phenylene isocyanate) (see Goddard, P., Williamson, I., Bron, J., Hutchkinson, L. E., e.g. Ratner, B. D., Johnston, A. B. and Lenk, T.J. in J. Nicholls, J. and Petrak, K. in J. Bioct. Compat. Polym. Biomed. Mater. Res: Applied Biomaterials (1987) 21, (1991) 6, 4-24). 59-90; Sa Da Costa, V. et al. in J. Coll. Interface Sci. 0114. Other potentially useful polymeric spacer materials (1981) 80, 445-452 and Affrossman, S. et al. in Clinical include: Materials (1991) 8, 25-31); 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, collide units to improve their absorbability (see 0116 ii) block copolymers of polymethacrylates with Bezwada, R. S., Shalaby, S. W. and Newman, H. D. J. biodegradable polyesters (see e.g. San Roman, J. and in Agricultural and Synthetic polymers. Biodegradabil Guillen-Garcia, P. in Biomaterials (1991) 12,236-241); ity and utilization (1990) (ed Glass, J. E. and Swift, G.), 0117 iii) cyanoacrylates, i.e. polymers of esters of 167-174-ACS symposium Series, #433, Washington 2-cyanoacrylic acid-these are biodegradable and have D.C., U.S.A.-American Chemical Society); been used in the form of nanoparticles for Selective 0126 xii) polyanhydrides such as copolymers of seba drug delivery (see Forestier, F., Gerrier, P., Chaumard, cic acid (octanedioic acid) with bis(4-carboxy-phenox C., Quero, A. M., Couvreur, P. and Labarre, C. in J. y)propane, which have been shown in rabbit studies Antimicrob. Chemoter. (1992) 30, 173-179); (see Brem, H., Kader, A., Epstein, J. I., Tamargo, R. J., 0118 iv) polyvinyl alcohols, which are water-soluble Domb, A., Langer, R. and Leong, K.W. in Sel. Cancer and generally regarded as biocompatible (see e.g. Ther. (1989) 5, 55-65) and rat studies (see Tamargo, R. 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 0119 v) copolymers of vinyl methyl ether with maleic useful for controlled release of drugs in the brain anhydride, which have been stated to be bioerodible without evident toxic effects; (see Finne, U., Hannus, M. and Urtti, A. in Int. J. Pharm. (1992) 78. 237-241); 0127 xiii) biodegradable polymers containing ortho ester groups, which have been employed for controlled 0120 vi) polyvinylpyrrollidones, 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 Blankwater, Y. M. in Arzeim-Forsch./Drug Res. (1977) 0128 xiv) polyphosphaZenes, which are inorganic 27, 1158-1162); polymers consisting of alternate phosphorus and nitro 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. (Milan) (1993) 75,494-9), including copolymers which 0129. The following tables list linking agents and agents incorporate aromatic hydroxyacids in order to increase for protein modification which may be useful in preparing their degradation rate (see Imasaki, K., Yoshida, M., targetable agents in accordance with the invention. US 2002/0102215 A1 Aug. 1, 2002 12

-continued Heterobifunctional linking agents DMA -NH. DMP -NH. Linking agent Reactivity 1 Reactivity 2 Comments DMS -NH. DPDPB -SH disulphide linker ABH carbohydrate photoreactive DSG -NH. ANB-NOS -NH. photoreactive DSP -NH. disulphide linker APDP(1) -SH photoreactive iodinable DSS -NH. disulphide DST -NH linker Sulfo-DST -NH. water-soluble APG -NH. photoreactive reacts DTBP -NH. disulphide linker selectively DTSSP -NH. disulphide linker with Arg at pH EGS -NH. 7-8 Sulfo-EGS -NH. water-soluble ASIB (1) -SH photoreactive iodinable SPBP -NH. ASBA (1) -COOH photoreactive iodinable Biotinylation agents EDC -NH. -COOH Zero-length linker biotin-BMCC -SH GMBS -NH. -SH biotin-DPPE* preparation of Sulfo-GMBS -NH -SH water-soluble biotinylated liposomes HSAB -NH. photoreactive biotin-LC-DPPE* preparation of Sulfo-HSAB -NH. photoreactive water-soluble biotinylated liposomes MBS -NH. -SH biotin-HPDP -SH disulphide linker Sulfo-MBS -NH -SH water-soluble biotin-hydrazide carbohydrate MCH carbohydrate -SH biotin-LC-hydrazide carbohydrate MPBH carbohydrate -SH iodoacetyl-LC-biotin -NH. NHS-ASA (1) -NH. photoreactive iodinable NHS-iminobiotin -NH. reduced affinity for Sulfo-NHS- -NH photoreactive water-soluble, avidin ASA (1) iodinable NHS-SS-biotin -NH. disulphide linker Sulfo-NHS-LC- -NH. photoreactive water-soluble, photoactivatable biotin nucleic ASA (1) iodinable acids PDPH carbohydrate -SH disulphide Sulfo-NHS-biotin -NH water-soluble linker Sulfo-NHS-LC-biotin -NH. PNP-DTP -NH. photoreactive Notes: SADP -NH. photoreactive disulphide DPPE = dipalmitoylphosphatidylethanolamine; linker LC = long chain Sulfo-SADP -NH. photoreactive water-soluble Agents for protein modification disulphide linker Ellman's reagent -SH quantifies/detects/protects SAED -NH. photoreactive disulphide DTT -S.S.- reduction linker 2-mercaptoethanol -S.S.- reduction SAND -NH. photoreactive water-soluble 2-mercaptylamine -S.S.- reduction disulphide Traut's reagent -NH introduces -SH linker SATA -NH. introduces protected -SH SANPAH -NH. photoreactive AMCA-NHS -NH. fluorescent labelling Sulfo-SANPAH -NH. photoreactive water-soluble AMCA-hydrazide carbohydrate fluorescent labelling SASD (1) -NH photoreactive water-soluble AMCA-HPDP -S.S.- fluorescent labelling iodinable SBF-chloride -S.S.- fluorescent detection of -SH disulphide N-ethylmaleimide -S.S.- blocks -SH linker NHS-acetate -NH. blocks and acetylates -NH. SAB -NH -SH citraconic anhydride -NH reversibly blocks and sulfo-SIAB -NH. -SH water-soluble introduces negative charges SMCC -NH. -SH DTPA -NH. introduces chelator Sulfo-SMCC -NH. -SH water-soluble BNPS-skatole tryptophan cleaves tryptophan residue SMPB -NH -SH Bolton-Hunter -NH introduces iodinable group Sulfo-SMPB -NH. -SH water-soluble SMPT -NH. -SH Sulfo-LC-SMPT -NH -SH water-soluble SPDP NH -SH 0.130. Other potentially useful protein modifications Sulfo-SPDP -NH. -SH water-soluble include partial or complete deglycosidation by neuramini Sulfo-LC-SPDP -NH. -SH water-soluble dase, endoglycosydases or periodate, Since deglycosidation sulfo-SAMCA (2) -NH. photoreactive often results in leSS uptake by liver, Spleen, macrophages Sulfo-SAPB -NH photoreactive water-soluble etc., whereas neo-glycosylation of proteins often results in Notes: increased uptake by the liver and macrophages); preparation (1) = iodinable; of truncated forms by proteolytic cleavage, leading to Linking(2) = fluorescent agent Reactivity Comments reduced size and shorter half life in circulation; and cationi sation, e.g. as described by Kumagi et al. in J. Biol. Chem. Homobifunctional linking agents (1987) 262, 15214-15219; Triguero et al. in Proc. Natl. Acad. Sci. USA (1989) 86, 4761-4765; Pardridge et al. in J. Eth N. Pharmacol. Exp. Therap. (1989) 251, 821-826 and Par BASED (1) photoreactive iodinable disuiphide linker dridge and Boado, Febs Lett. (1991) 288, 30-32. Escoes NE water-soluble 0131 Vectors which may be usefully employed in tar DFDNB -NH. getable agents according to the invention include the fol lowing: US 2002/0102215 A1 Aug. 1, 2002

0132) i) Antibodies, which can be used as vectors for 0138 vii) Vector molecules may be generated from a very wide range of targets, and which have advanta combinatorial libraries without necessarily knowing geous properties Such as very high Specificity, high the exact molecular target, by functionally selecting (in affinity (if desired), the possiblity of modifying affinity vitro, ex vivo or in vivo) for molecules binding to the according to need etc. Whether or not antibodies will be region/structure to be imaged. bioactive will depend on the Specific vector/target combination. Both conventional and genetically engi 0139 viii) Various small molecules, including bioac neered antibodies may be employed, the latter permit tive compounds known to bind to biological receptors ting engineering of antibodies to particular needs, e.g. of various kinds. Such vectors or their targets may be as regards affinity and Specificity. The use of human used for generate non-bioactive compounds binding to antibodies may be preferred to avoid possible immune the same targets. reactions against the vector molecule. A further useful 0140) ix) Proteins or peptides which bind to glucosa class of antibodies comprises So-called bi- and multi mioglycan Side chains e.g. heparan Sulphate, including Specific antibodies, i.e. antibodies having Specificity for glucosoaminoglycan-binding portions of larger mol two or more different antigens in one antibody mol ecules, as binding to glucosoaminoglycans does not ecule. Such antibodies may, for example, be useful in result in a biological response. Proteoglycans are not promoting formation of bubble clusters and may also found on red blood cells, which eliminates undesirable be used for various therapeutic purposes, e.g. for car adsorption to these cells. rying toxic moieties to the target. Various aspects of bispecific antibodies are described by McGuinness, B. 0.141. Other peptide vectors and lipopeptides thereof of T. et al. in Nat. Biotechnol. (1996) 14, 1149-1154; by particular interest for targeted ultrasound imaging are listed George, A. J. et al. in J. Immunol. (1994) 152, 1802 below: Atherosclerotic plaque binding peptides Such as 1811; by Bonardi et al. in Cancer Res. (1993) 53, YRALVDTLK (SEQ ID NO:26), YAKFRE 3015-3021; and by French, R. R. et al. in Cancer Res. TLEDTRDRMY (SEQ ID NO:27) and (1991) 51,2353-2361. RALVDTEFKVKQEAGAK (SEQ ID NO:28); Thrombus binding peptides such as NDGDFEEIPEEYLQ (SEQ ID 0133) ii) Cell adhesion molecules, their receptors, NO:29) and GPRG (SEQ ID NO:30), Platelet binding pep cytokines, growth factors, peptide hormones and pieces tides such as PLYKKIIKKLLES (SEQ ID NO:31); and thereof. Such vectors rely on normal biological protein cholecystokinin, C.-melanocyte-stimulating hormone, heat protein interactions with target molecule receptors, and Stable enterotoxin 1, vasoactive intestinal peptide, Synthetic So in many cases will generate a biological response on alpha-M2 peptide from the third heavy chain complemen binding with the targets and thus be bioactive; this may tarity-determininig region and analogues thereof for tumour be a relatively insignificant concern with vectors which target proteoglycans. targeting. 0142. The following tables identify various vectors 0134) iii) Non-peptide agonists/antagonists or non-bio which may be targeted to particular types of targets and active binders of receptors for cell adhesion molecules, indicated areas of use for targetable diagnostic and/or thera cytokines, growth factors and peptide hormones. This peutic agents according to the invention which contain Such category may include non-bioactive vectors which will be neither agonists nor antagonist but which may VectOrS. nonetheless exhibit valuable targeting ability. 0135) iv) Oligonucleotides and modified oligonucle otides which bind DNA or RNA through Watson-Crick Protein and peptide vectors - antibodies or other types of base-pairing. DNA is usually only Vector type Target Comments fareas of use Ref present in extracellular space as a consequence of cell antibodies CD34 vascular diseases in general, 1. damage, So that Such oligonucleotides, which will (general) normal vessel wall (e.g usually be non-bioactive, may be useful in, for myocardium), activated example, targeting of necrotic regions, which are asso endothelium, immune cells ciated with many different pathological conditions. ICAM-1 ICAM-2 oligonucleotides may also be designed to bind to spe ICAM-3 cific DNA- or RNA-binding proteins, for example E-selectin transcription factors which are very often highly over P-selectin expressed or activated in tumour cells or in activated PECAM immune or endothelial cells. Combinatorial libraries Integrins, e.g. VLA-1, may be used to Select oligonucleotides which bind VLA-2, VLA Specifically to any possible target molecules and which 3, VLA-4, therefore may be employed as Vectors for targeting. VLA-5, VLA 6, 1C.7, 0136 V) DNA-binding drugs may behave similarly to B1Cls, BCiv. oligonuclotides, but may exhibit biological acitvity LFA-1, Mac 1, CD41a, and/or toxic effects if taken up by cells. etc. GlyCAM Vessel wall in lymph nodes 3 0137 vi) Protease substrates/inhibitors. Proteases are (quite specific for involved in many pathological conditions. Many Sub lymph nodes) Strates/inhibitors are non-peptidic but, at least in the MadCam 1 case of inhibitors, are often bioactive. US 2002/0102215 A1 Aug. 1, 2002 14

-continued -continued Protein and peptide vectors - antibodies Protein and peptide vectors - antibodies Vector type Target Comments fareas of use Ref Vector type Target Comments fareas of use Ref fibrin Thrombi 4 antibodies intermediate Tissue Activated endothelium, 5 filaments Factor tumours necrotic Myosin Necrosis, myocardial infaction 6 cells/tissue CEA Tumours 7 CD44 tumour cells (carcino embryonal antibodies antigen) 3-micro general Mucins Tumours 8 antibodies globulin MHC class 1 Multiple Tumours 9 general drug antibodies integrin tumours; angiogenesis resistance antibodies CVB3 protein Prostate Prostate cancer specific antigen REFERENCES Cathepsin B Tumours (proteases of various 10 kinds are often more or less specifically overexpressed in a 0143 a) Heider, K. H., M. Sproll, S. Susani, E. Patzelt, variety of tumours - P. Beaumier, E. Ostermann, H. Ahorn, and G. R. Adolf. Cathepsin B is such a protease) 1996. “Characterization of a high-affinity monoclonal Transferrin Tumors, 11 antibody specific for CD44v6 as candidate for immu receptor vessel wall notherapy of Squamous cell carcinomas'. Cancer MOAb 9.2.27 Tumours 12 Antigen upregulated Immunology Immunotherapy 43: 245-253. on cell growth VAP-1 Adhesion molecule 13 014.4 b) I. Roitt, J. Brostoff, and D. Male. 1985. Band 3 Upregulated during Immunology, London: Gower Medical Publishing, p. protein phagocytic activity 4.7 antibodies CD34 (sialomu endothelial cells cin antibodies CD31 (PECAM endothelial cells 0145) c) Stromblad, S., and D. A. Cheresh. 1996. 1) “Integrins, angiogenesis and Vascular cell Survival'. Chemistry & Biology 3: 881-885.

Vector Type Target Comments fareas of use Ref Protein and peptide vectors - cell adhesion molecules etc. L-selectin CD34 vascular diseases in MadCAM1 general, normal vessel wall GlyCam 1 (e.g. myocardium), activated endothelium, Lymph nodes Other selectins carbohydrate vascular diseases in 14 ligands general, normal vessel wall (sialyl Lewis X) (e.g. myocardium), activated heparan sulfate endothelium RGD-peptides integrins PECAN PECAN, Endothelium, and other Cells in immune system Integrins, Laminin, Endothelium, 16 e.g. VLA-1, VLA collagen, Vessel wall 2. VLA-3, VLA-4, fibronectin, etc. VLA-5, VLA-6, VCAM-1, thrombo B1C-7, B1 Cls, spondin, BC, LFA-1, vitronectin etc. Mac-1, CD41a, etc. Integrin Integrins, Cells in immune system 17 receptors, e.g. VLA-1, VLA vessel wall 18 e.g. Laminin, 2. VLA-3, VLA-4, etc. collagen, VLA-5, VLA-6, fibronectin, f51C-7, f1Cls. VCAM-1, fCl, LFA-1, thrombospondin, Mac-1, CD41a, vitronectin etc. etc. US 2002/0102215 A1 Aug. 1, 2002 15

-continued Vector Type Target Comments fareas of use Ref Nerve cell proteoglycans 19 adhesion N-CAM molecule (N-CAM) (homophilic) integrin Civ3 CD31 (PECAM-1) endothelial cells RGD-peptides integrins angiogenesis Vectors comprising cytokines/growth factors/peptide hormones and fragments thereof Epidermal growth EGF-receptor or Tumours factor related receptors Nerve growth NGF-receptor Tumours 21 factor Somatostatin ST-receptor Tumours 22 Endothelin Endothelin Vessel wall receptor Interleukin-1 IL-1-receptor Inflammation, activated 23 cells of different kinds Interleukin-2 IL-2-receptor 24 Chemokines (ca. Chemokine Inflammation 25 20 different receptors, cytokines partly proteoglycans sharing receptors) Tumour necrosis TNF-receptors Inflammation factor Parathyroid PTH-receptors Bone diseases hormone Kidney diseases Bone BMP-receptors Bone Diseases Morphogenetic Protein Calcitonin CT-receptors Bone diseases Colony Corresponding Endothelium 26 stimulating specific factors (G-CSF, receptors, GM-CSF, M-CSF, proteoglycans IL-3) Insulin like IGF-I receptor Tumours, growth factor I other growing tissues Atrial ANF-receptors Kidney, Natriuretic vessel wall Factor Vasopressin Vasopressin Kidney, receptor vessel wall VEGF VEGF-receptor Endothelium, regions of angiogenesis Fibroblast FGF-receptors, Endothelium 27 growth factors Proteoglycans Angiogenesis Schwann cell proteoglycans 28 growth factor specific receptors Miscellaneous protein and peptide vectors Streptavidin Kidney Kidney diseases 29 Bacterial Fibronectin Vessel wall 3O fibronectin binding proteins Fc-part of Fc-receptors Monocytes 31 antibodies macrophages liver Transferrin transferrin Tumours 11 receptor vessel walls Streptokinase? thrombi thrombi tissue plasminogen activator Plasminogen, Fibrin Thrombi, 32 plasmin tumours Mast cell proteoglycans 33 proteinases Elastase proteoglycans 34 Lipoprotein proteoglycans 35 lipase US 2002/0102215 A1 Aug. 1, 2002 16

-continued Vector Type Target Comments fareas of use Ref Coagulation proteoglycans 36 enzymes Extracellular proteoglycans 37 Superoxide dismutase Heparin cofactor proteoglycans 38 II Retinal survival proteoglycans 39 factor specific receptors Heparin-binding proteoglycans 40 brain mitogen specific receptors Apollipoprotein, proteoglycans 41 C.S. specific apolipoprotein B receptors (e.g., LDL receptor) Apollipoprotein E LDL receptor 42 proteoglycans Adhesion proteoglycans 43 promoting proteins, e.g. Purpurin Viral coat proteoglycans 44 proteins, e.g. HIV. Herpes Microbial fibronectin, 45 adhesins, e.g. collagen, “Antigen 85' fibrinogen, complex of vitronectin, mycobacteria heparan sulfate B-amyloid proteoglycans B-amyloid accumulates in 46 precursor Alzheimer's disease Tenascin, heparan sulfate, 47 e.g. tenascin C integrins Vectors comprising non-peptide agonists/antagonists or non-bioactive binders of receptors for cytokines/growth factors/peptide hormones/cell adhesion molecules Several agonists/antagonists 48 are known for such factors 49 acting through G-protein coupled receptors Endothelin Endothelin Vessel wall antagonist receptor Desmopressin Vasopressin Kidney (vasopressin receptor Vessel wall analogue) Demoxytocin Oxytocin Reproductive organs, (Oxytocin Receptor Mammary glands, analogue) Brain Angiotensin II Angiotensin II Vessel wall receptor receptors brain antagonists adrenal gland CV-11974, TCV-116 non-peptide RGD integrins Cells in immune system 50 analogues vessel wall etc. Vectors comprising anti-angiogenic factors Angiostatin EC of tumors plasminogen fragment cartilage-derived EC of tumors inhibitor B-Cyclodextrin tumors, tetradecasulfate inflammation fumagillin and analogs tumors, E inflammation Interferon-C. EC of tumors Interferon-Y EC of tumors interleukin-12 EC of tumors linomide tumors, inflammation medroxyprogesterone EC of tumors US 2002/0102215 A1 Aug. 1, 2002 17

-continued Vector Type Targe Comments fareas of use Ref metalloproteinase EC of tumors K inhibitors pentosan polysulfate EC of tumors K platelet factor 4 EC of tumors M Somatostatin EC of tumors K Suramin EC of tumors K Taxol EC of tumors K thalidomide EC of tumors K Thrombospondin EC of tumors K Vectors comprising angiogenic factors acidic fibroblast growth EC of tumors K actor adenosine EC of tumors K Angiogenin EC of tumors K Angiotensin II EC of tumors K basement membrane tumors e.g., tenascin, M components collagen IV basic fibroblast growth EC of tumors K actor Bradykinin EC of tumors K Calcitonin gene-related EC of tumors K peptide epidermal growth factor EC of tumors K Fibrin tumors K Fibrinogen tumors K Heparin EC of tumors K histamine EC of tumors K hyaluronic acid or fragments EC of tumors K hereof Interleukin-1C. EC of tumors K aminin, laminin fragments EC of tumors K nicotinamide EC of tumors K platelet activating factor EC of tumors K Platelet-derived endothelial EC of tumors K growth factor prostaglandins E1, E2 EC of tumors K spermine EC of tumors K spermine EC of tumors K Substance P EC of tumors K ransforming growth factor-C. EC of tumors K ransforming growth factor-f EC of tumors K Tumor necrosis factor-C. EC of tumors K vascular endothelial growth EC of tumors K actor/vascular permeability actor vitronectin A. Vector molecules other than recognized angiogenetic factors with known affinity for receptors associated with angiogenesis angiopoietin umors, B inflammation C2-antiplasmin umors, inflammation combinatorial libraries, umors, for instance: compounds from inflammation compounds that bind to basement membrane after degradation endoglin umors, D inflammation endosialin umors, D inflammation endostatin collagen umors, M fragment inflammation Factor VII related umors, D antigen inflammation fibrinopeptides umors, ZC inflammation fibroblast growth factor, umors, E basic inflammation hepatocyte growth factor umors, I inflammation US 2002/0102215 A1 Aug. 1, 2002 18

-continued Vector Type Target Comments fareas of use Ref insulin-like growth tumors, R factor inflammation interleukins tumors, e.g.: IL-8 I inflammation leukemia inhibitory tumors, A. factor inflammation metalloproteinase tumors, e.g., batimastat E inhibitors inflammation Monoclonal antibodies tumors, for instance: to inflammation angiogenetic factors or their receptors, or to components of the fibrinolytic system peptides, for instance umors, B, Q cyclic RGDFV inflammation placental growth factor umors, J inflammation placental umors, E proliferin-related inflammation protein plasminogen umors, M inflammation plasminogen activators umors, D inflammation plasminogen activator umors, U, V inhibitors inflammation platelet activating umors, inhibitors of A. actor antagonists inflammation angiogenesis platelet-derived growth umors, E actor inflammation pleiotropin umors, ZA inflammation proliferin umors, E inflammation proliferin related umors, E protein inflammation selectins umors, e.g., E-selectin D inflammation SPARC umors, M inflammation snake venoms umors, O (RGD-containing) inflammation Tissue inhibitor of umors, eg, TIMP-2 U metalloproteinases inflammation thrombin umors, H inflammation thrombin-receptor-activat- umors, H ing tetradecapeptide inflammation thymidine phosphorylase umors, D inflammation tumor growth factor umors, ZA inflammation Receptors/targets associated with angiogenesis biglycan umors, dermatan sulfate X inflammation proteoglycan CD34 umors, L inflammation CD44 umors, F inflammation collagen type I, IV, umors, A. VI, VIII inflammation decorin umors, dermatan sulfate Y inflammation proteoglycan dermatan sulfate umors, X proteoglycans inflammation endothelin umors, G inflammation endothelin umors, G receptors inflammation fibronectin OS P FIk-1/KDR, Flt-4 umors, VEGF receptor D inflammation US 2002/0102215 A1 Aug. 1, 2002 19

-continued Vector Type Target Comments fareas of use Ref FLT1 (fins-like tumors, VEGF-A receptor O tyrosine kinase) inflammation heparan sulfate tumors, P inflammation hepatocyte growth tumors, I factor receptor (c-met) inflammation insulin-like growth tumors, R factorfmannose-G- inflammation phosphate receptor integrins: Tumors, D, fs and fs, inflammation P integrin Cly?is, integrin Croft, laminin receptor integrins C6, integrins B1, integrin C.2?1, integrin Cly?is, integrin C.5 subunit of the fibronectin receptor integrin Cly?is, fibrin receptors. Intercellular adhesion umors, P molecule-1 and -2 inflammation Jagged gene product umors, T inflammation Ly-6 umors, a lymphocyte activation N inflammation protein matrix umors, D metalloproteinases inflammation MHC class II umors, inflammation Notch gene product umors, T inflammation Osteopontin OS Z. PECAM umors, alias CD31 P inflammation plasminogen activator umors, ZC receptor inflammation platelet-derived growth umors, E factor receptors inflammation Selectins: E-, P- umors, D inflammation Sialyl Lewis-X umors, blood group antigen M inflammation stress proteins: umors, molecular chaperones glucose regulated, inflammation heat shock families and others syndecan umors, T inflammation hrombospondin umors, M inflammation TIE receptors umors, tyrosine kinases with Ig- E inflammation and EGF-Iike domains issue factor umors, Z. inflammation issue inhibitor of umors, e.g., TIMP-2 U metalloproteinases inflammation ransforming growth umors, E actor receptor inflammation urokinase-type umors, D plasminogen activator inflammation receptor Vascular cellular umors, D adhesion molecule inflammation (VCAM) Vascular endothelial umors, growth factor related inflammation protein Vascular endothelial umors, K growth factor-A inflammation receptor von Willebrand factor- umors, L related antigen inflammation US 2002/0102215 A1 Aug. 1, 2002 20

-continued Vector Type Target Comments fareas of use Ref Oligonucleotide vectors Oligonucleotides DNA made Tumours 51 complementary to available by Myocardial infarction repeated necrosis All other diseases that Sequences, e.g. involves necrosis genes for ribosomal RNA, Alu-sequences Oligonucleotides DNA made Tumours 51 complementary to available by disease-specific necrosis in a mutations (e.g. region of the mutated relevant disease oncogenes). oligonucleotides DNA of infective Viral or bacterial 51 complementary to agent infections DNA of infecting agent. Triple or As in above As in above examples 51 quadruple-helix examples forming oligonucleotides Oligonucleotides DNA-binding Tumours with recognition protein, e.g. Activated endothelium sequence for transcription Activated immune cells DNA-or RNA factors (often binding proteins overexpressed/ activated in tumours Or activated endotheliumf immune cells Modified oligonucleotide vectors Phosphorothioate As for As for unmodified oligos 51 oligos unmodified oligos 2'-O-methyl 51 substituted oligos circular oligos 51 oligos 51 containing hairpin Structure to decrease degradation oligos with 51 terminal phosphorothioate 2'-fluoro oligos 51 2'-amino oligos 51 DNA-binding Increased binding affinity 52 drugs conjugated as compared to pure oligos to oligos (for examples, see below) Peptide Nucleic Increased binding affinity 53 Acids (PNAs, and stability compared to oligonucleotidss standard oligos. with a peptide backbone) Nucleoside and nucleotide vectors

Adenosine or Adenosine Vessel wall 54 analogues receptors Heart ADP, UDP, UTP Various Many tissues, e.g. brain, 55 and others nucleotide spinal cord, kidney, spleen receptors Receptors comprising DNA-binding drugs acridine DNA made Tumours, derivatives available by Myocardial infarction and US 2002/0102215 A1 Aug. 1, 2002 21

-continued Vector Type Target Comments fareas of use Ref distamycin necrosis all other diseases involving netropsin necrosis or other processes actinomycin D liberating DNA from cells echinomycin bleomycin etc. Receptors comprising protease substrates Peptidic or non Cathepsin B Tumours, a variety of 1O peptidic which may more or less Substrates specifically overexpress proteases of various kinds, e.g. Cathepsin B Receptors comprising protease inhibitors Peptidic or non Cathepsin B Tumours, a variety of 1O peptidic which may more or less inhibitors specifically overexpress e.g. N-acetyl proteases of various kinds, Leu-Leu e.g. Cathepsin B norleucinal bestatin Aminopeptidases Tumours, (2S,3R)-3- e.g. on cell surfaces Amino-2-hydroxy 4-phenyl butanoyl-L- eucine hydrochloride) Pefabloc (4-(2- Serine proteases Tumours, aminoethyl)- vessel wall benzenesulfonyl etc. fluoride hydrochloride) Commercially Angiotensin Endothelial cells available converting inhibitors enzyme e.g. kaptopril enalapri ricionopril Low specificity Coagulation Vessel wall injury, non-peptidic actors tumours, compounds etc. Protease nexins proteoglycans 56 (extracellular protease inhibitors) Antithrombin proteoglycans, 57 Coagulation actors Vectors from combinatorial libraries Antibodies with Any of above Any diseased or normal 58, Structure argets - or may structure of interest, e.g. 59, determined be unknown when thrombi, tumours or walls 60 during make functional of myocardial vessels generation selection of process vector binding O chosen diseased Structure Peptides with sequence 59, determined 60 during generation process Oligonucleotides with sequence 59, determined 60 during generation process Modifications of oligos obtained 59, as above 60 US 2002/0102215 A1 Aug. 1, 2002 22

-continued Vector Type Target Comments fareas of use Ref Other chemicals 58, with structure 59, determined 60 during generation process Carbohydrate vectors CO- macrophages general activation? glycoproteins inflammation oligosaccharides Asialo- liver 61 with terminal glycoprotein galactose receptor Hyaluronan aggrecan (a 62 proteoglycan) “link proteins cell - Surface receptors: CD44 Mannose Blood brain barrier, 63 Brain tumours and other diseases causing changes in BBB Bacterial 64 glycopeptides (Glyco) Lipid vectors GM1 gangliosides cholera bacteria diagnosis/treatment of in the cholera gastrointestinal tract platelet PAF receptors diagnosis of inflammation activating factor (PAF) antagonists Prostoglandin Prostoglandin diagnosis of inflammation antagonists of receptors inflammation Thromboxane Leukotriene diagnosis of inflammation antagonists of receptors inflammation Small molecule vectors Adrenalin Corresponding receptors Betablockers Adrenergic beta- Myocardium for beta-1 receptors blockers Alpha-blockers Adrenergic Vessel wall alpha-receptors benzodiazepines serotonin- Serotonin analogues receptors anti-histamines Histamine- Vessel wall receptors Acetyl-choline ACh-receptors receptor antagonists Verapamil Ca'-channel Heart muscle blocker nifedipin Cat-channel Heart muscle blocker

0146 Representative examples of drugs useful in accor acetanilide, acetarSone, acetazolamide, acetergamine, ace dance with the invention include: abamectin, abundiazole, tiamine, acetiromate, acetohexamide, acetohydroxamic acaprazine, acabrose, acebrochol, aceburic acid, acebutolol, acid, acetomeroctol, acetophenazine, acetorphine, acetoSul acecainide, acecarbromal, aceclidine, aceclofenac, acedap fone, acetriozate, acetryptine, acetylcholine chloride, ace Sone, acediaSulfone, acedoben, acefluranol, acefurtiamine, tylcolchinol, acetylcysteine, acetyldigitoxin, acetylleucine, acefylline clofibrol, acefylline piperazine, aceglatone, ace acetylsalicyclic acid, acevaltrate, aceXamic acid, acifran, glutamide, aceglutamide aluminium, acemetacin, acenocou acipimox, acitemate, acitretin, acivicin, aclantate, aclarubi marol, aceperone, acepromazine, aceprometazine, acequino cin, aclatonium napadisilate, acodazole, aconiazide, line, aceSulfame, acetaminophen, acetaminoSalol, aconitine, acoxatrine, acridorex, acrihellin, acrisorcin, acriv US 2002/0102215 A1 Aug. 1, 2002

astine, acrocinide, acronine, actinoquinol, actodigin, acyclo ansoxetine, antafenite, antazoline, antazonite, anthelmycin, Vir, adafenoxate, adamexine, ademetionine, adenosine phoS anthiolimine, anthralin, anthramycin, antienite, antimony phate, adibendan, adicillin, adimolol, adinazolam, potassium tartrate, antimony thioglycollate, antipyrine, adiphenine, aditeren, aditoprim, adrafinil, adrenalone, aflo antrafenine, apalcillin, apaZone, apicycline, apomorphine, qualone, afurolol, aganodine, ajmaline, aklomide, alacepril, apoVincamine, apraclonidine, apramycin, aprindine, alafosfalin, alanine mustard, alanosine, alaproclate, alaza aprobarbital, aprofene, aptazapine, aptocaine, arabinosylm nine triclofenate, albendazole, albendazole oxide, albuterol, ercaptopurine, aranotin, arbaproStil, arbekacin, arclofenin, albutoin, alclofenac, alcometasone dipropionate, alcloxa, arfendaZam, arginine, arginine glutamat, arildone, arnolol, alcuronium chloride, aldioxa, aldosterone, alepride, ale aronixil, arotinolol, arpinocid, arpromidine, arsanilic acid, tamine, alexidine, alfacalcidol, alfadeX, alfadolone, arSthinol, artemisinin, articaine, asaley, ascorbic acid, ascor alfaprostol, alfaxalone, alfentanil, alfuzosin, algestone byl palmitate, asocainol, aspartame, aspartic acid, asperlin, acetonide, algestone acetophenide, alibendol, aliconazole, aspoxicillin, astemizole, atameStane, atenolol, atipamezole, alifedrine, alliflurane, alimadol, alinidine, alipamide, alitame, atiproSin, atolide, atracurium besilate, atromepine, atropine, alizapride, allantoin, alletorphine, allobarbital, alloclamide, atropine oxide, auranof in, aurothoiglucose, aurothiogly allocupreide, allomethadione, allopurinol, allylestrenol, canide, avilamycin-A, avridine, aXamozide, azabon, azabu allyl isothicyanate, allylprodine, allylthiourea, almadrate perone, azacitodine, azaclorzine, azaconazole, azacosterol, Sulfate, almasilate, almecillin, almestrone, alminoprofen, azacyclonol, azaftoZine, azaguanidine, azaloxan, azametho almitrine, almoxatone, alonacic, alonimid, aloxiStatin, aloZ nium bromide, azamulin, azanator, azanidazole, azaperone, afone, alpertine, alphacetylmethadol, alphameprodine, aZapicyl, azaprocin, azaduinzole, azaribine, azarole, aZaser alphamethadol, alphaprodine, alphavinylaziridinoethyl ine, azaspirium chloride, azastene, azastrptonigrin, azatod acetate, alpidem, alpiropride, alprazolam, alprenolol, alproS ine, azathioprine, azauridine, azelastine, azepeXole, azepin tadil, alreStatin, altanserin, altapizone, alteconazole, althiaz dole, azetepa, azidamfenicol, azidocillin, azimeXon, ide, altrenogest, altretamine, aluminium acetate, aluminium aZintamide, azipramine, azithromycin, azlocillin, azolimine, clofibrate, aluminium Subacetate, alverine, amadinone aZOSemide, azotomycin, aztreonam, azumolene, bacampicil acetate, amafolone, amanozine, amantadine, amantanium lin, baclofen, bacmecillinam, balSalazide, bamaluzole, bam bromide, amantocillin, ambasilide, ambaZone, ambenonium buterol, bamethan, bamifylline, bamipine, bamnidazole, chloride, ambenoxan, ambroXol, ambruticin, ambucaine, baguiloprim, barbexaclone, barbital, barucainide, batilol, ambucetamide, ambuphylline, ambuSide, ambutonium bro baZinaprine, becanthone, beclamide, beclobrate, beclom mide, amcinafal, amcinafide, amcinonide, amdinocillin, ethasone dipropionate, beclotiamine, befiperide, befunolol, amdinocillin pivoxil, amebucort, amedalin, ametantrone, befuraline, bekanamycin, belarizine, beloxamide, bemar ameZepine, ameZinium metilsulfate, amfenac, amfepen inone, bemegride, bemetizide, bemitradine, benactyzine, torex, amfetaminil, amflutizole, amfonelic acid, amicar benafentrine, benanSerin, benapry Zine, benaxibine, balide, amicibone, amicloral, amicycline, amidantel, ami benazepril, bencianol, bencisteine, benclonidine, bency dapSone, amidephrine, amiflamine, amifloverine, clane, bendamustine, bendaZac, bendaZol, benderizine, ben amifloxacin, amifostine, amikacin, amikhelline, amiloride, droflumethiazide, benethamide penicillin, benexate, benflo aminacrine, amindocate, amineptine, aminobenzoic acid, reX, benfoSformin, benfotiamine, benfurodil hemisuccinate, aminocaproic acid, aminoethyl nitrate, aminoglutethimide, benhepaZone, benidipine, benmoxin, benolizime, benorilate, aminohippuric acid, aminometradine, aminopentamide, benorterone, benoxafos, benoxaprofen, benoXinate, benperi aminophylline, aminopromazine, aminopterin, aminopyrine, dol, beinproperine, benrixate, benSalan, benSerazide, benSul aminoquinol, aminoquinuride, aminorex, aminosalicyclic dazic acid, bentazepam, bentemazole, bentiamine, bentipi acid, aminothiadiazole, aminothiazole, amiodarone, amiper mine, bentiromide, benurestat, benzaldehyde, benzalkonium one, amipheaZole, amipizOne, amiprilose, amiquinsin, ami chloride, benzaprinoxide, benzarone, benzbromarone, ben Sometradine, amisulpride, amiterol, amithioZone, amitraz, Zestrol, benzethidine, benzethonium chloride, benzetimide, amitriptyline, amitriptylinoxide, amiXetrine, amlexanox, benzilonium bromide, benzindopyrine, benziodarone, ben amlodipine, amobarbital, amodiacquine, amogastrin, amol Zmalecene, benznidazole, benzobarbital, benzocaine, ben anone, amonofide, amoproxan, amopyroquin, amorolfine, Zoclidine, benzoctamide, benzodepa, benzododecinium amocanate, amoSulalol, amotriphene, amoxapine, amox chloride, benzoic acid, benzoin, benzonatate, benzopyrro ecaine, amoxicillin, amoxydramine camsilate, amperozide, nium bromide, benzoquinium chloride, benzotript, benzox amphecloral, amphenidone, amphetamine, amphotalide, iquine, benzoxonium chloride, benzoyl peroxide, ben amphotericin B, amplicillin, ampiroXicam, amprolium, Zoylpas, benzphetamine, benzpiperylon, benzpyrinium ampyrimine, ampyZine, amduinate, amrinone, amsacrine, bromide, benzauercin, benzcuinamide, benzthiazide, benz amygdalin, amylene, amylmetacresol, amyl nitrite, anage tropine, benzydamine, benzylpenicillin, benzylsulfamide, Stone acetate, anagrelide, anaXirone, anazocine, anazolene, beperidium iodide, bephenium naphtoate, bepiastine, bepri ancarolol, ancitabine, androStanediol, androstanol propi dil, beraprost, berberine Sulfate, bermastine, bermoprofen, onate, androstenetrione, androstenonol propionate, anethole, berythromycin, beSulpamide, beslunide, beta caroteine, beta anguidine, anidoxime, anilamate, anilleridine, aniline, anilo cetylmethadol, betahistine, betaine, betameprodine, pam, anipamil, aniracetam, anirolac, anisacril, anisindione, betamethadol, betamethasone, betamethasone acetate, aniSopirol, anisoylbromacrylic acid, anitraZafen, anpirtoline, betamethasone acibutate, betamethasone benzoate, US 2002/0102215 A1 Aug. 1, 2002 24 betamethasone dipropionate, betamethasone phosphate, citrol, calcium citrate, calcium dobesilate, calcium glubion betamethasone Valerate, betamicin, betaprodine, betaxolol, ate, calcium gluceptate, calcium gluconate, calcium glyc betazole, bethanechol chloride, bethanidine, betiatide, erophosphate, calcium hypophosphite, calcium lactate, betoxycaine, bevantolol, bevonium metilsulfate, beZafibrate, calcium lactobionate, calcium levulinate, calcium mande bezitramide, bialamicol, bibenzonium bromide, bibrocathol, late, calcium pantothenate, calcium phosphate dibasic, cal bicifadine, biclodil, biclofibrate, biclotymol, bicozamycin, cium phophate tribasic, calcium Saccharate, calcium Stear bidimazium iodine, bietamiverine, bietaserpine, bifemelane, ate, calusterone, camazepam, cambendazole, camiverine, bifepramide, bifluranol, bifonazole, binedaline, binfloxacin, camoStast, camphotamide, camptothecin, camylofin, can binfibrate, bioallethrin, bioresmethrin, biotin, bipenamol, bisol, cannabinol, canrenoic acid, canrenone, cantharidine, biperiden, biphenamine, biriperone, bisacodyl, bisantrene, capobenic acid, capreomycin, caproxamine, capsaicine, bis(aziridinyl)butanediol, bisbendazole, bisbentiamine, bis captamine, captodiame, captopril, capuride, caracemide, fenaZone, bisfentidine, bismuth betanaphthol, bismuth-trig caramiphen, carazolol, carbachol, carbadox, carbaldrate, lycollamate, bismuth Subgallate, bismuth SubSalicylate, carbamazepine, carbamide peroxide, carbantel lauryl Sul bisorbin, bisoprolol, bisorcic, bioxatin acetate, bispyrithione fate, carbaril, carbarSone, carbaspirin calcium, carbaZeran, magSulfeX, bithionol, bithionoloxide, bitipaZone, bitoterol, carbazochrome, carbazachrome Salicylate, carbazachrome bitoScantate, bleomycin, bluenSomycin, bofumustine, bolan Sulfonate, carbazocine, carbeniciltin, carbenicillin indanyl, diol dipropionate, bolasterone, bolazine, boldenone unde carbencillin phenyl, carbenoXolone, carbenzide, carbestrol, cylenate, bolenol, bolmantalate, bometolol, bopindolol, bor carbetapentane, carbidopa, carbimazole, carbinoxamine, naprine, bornaprolol, bornelone, botiacrine, boxidine, carbiphene, carbocloral, carbocysteine, carbofenotion, car brallobarbital, braZergoline, brefonalol, bremazocine, bre bol-fuSchin, carbomycin, carboplatin, carboprost, carbo quinar, bretylium tosylate, brindoXime, brivundine, brobac prost methyl, carboquone, carbromal, carbubarb, car tam, broclepride, brocresine, brocrinat, brodimoprim, bro burazepam, carbutamide, carbuterol, carcainium chloride, faromine, brofezil, brofoxine, brolaconazole, carebastine, carfentanil, carfimate, carisoprodol, carmanta brolamfetamine, bromacrylide, bromadoline, bromamid, dine, carmetizide, carmofur, carmustine, carnidazole, car bromazepam, bromchlorenone, bromebric acid, bromergu nitine, carocainide, caroverine, caroXaZone, carperidine, ride, brometenamine, bromfenac, bromheXine, bromindione, caperone, carphenazine, carpindolol, carpiramine, carpro bromisovalum, bromociclen, bromocriptine, bromodiphen fen, carpronium chloride, carsalam, cartazolate, carteolol, hydramine, bromofenofoS, bromopride, bromoxandide, bro carubicin, carumonam, carvedilol, carZenide, carZolamide, mperidol, bromperidol decanoate, brompheniramine, bro cathine, cathinone, cefaclor, cefadroxil, cefalonium, cefalo nopol, broparestrol, broperamole, bropirimine, ram, cefamandole, cefamandole naftate, cefaparole, cefatriz broquinaldol, broSotamide, broSuximide, brotianide, broti ine, cefazaflur, cefaZedone, cefazolin, cefbuperaZone, cef Zolam, brovanexine, brovincamine, broXaldine, broXaterol, canel, cefcanel daloxate, cefedrolor, cefempidone, cefepime, broxitalamic acid, broXuridine, broxyquinoline, bruceantin, cefetamet, cefetrizole, cefvitril, cefixime, cefimenoXime, brucine, bucainide, bucetin, buciclovir, bucillamine, bucin cefmepidium chloride, cefimetazole, cefminox, cefodizime, dolol, bucladesine, buclizine, buclosamide, bucloxic acid, cefonizid, cefoperaZone, ceforanide, cefotaxime, cefotetan, bucolome, bucricaine, bucromarone, bucrylate, bucumolol, cefotiam, cefoxazole, cefoXitin, cefpimizole, cefpiramide, budeSonide, budipine, budotitane, budralazine, bufenadrine, cefpirome, cefpodoxime, cefpodoxime proxetil, cefaui bufeniode, bufetolol, bufeXamac, bufezolac, buflomedil, nome, cefrotil, cefroXadine, cefSulodin, cefSumide, ceftazi bufogenin, buformin, bufrolin, bufuralol, bumadizone, dime, cefteram, cefteZole, ceftiofur, ceftiolene, ceftioxide, bumecaine, bumepidil, bumetanide, bumetrizole, bunaftine, ceftizoxime, ceftriaxone, cefuracetime, cefuroxime, bunamidine, bunamiodyl, bunaprolast, bunaZosin, buni cefuraxime axetil, cefurZonam, celiprolol, cephacetrile, trolol, bunolol, buparvaquone, bupicomide, bupivacaine, cephalexin, cephaloglycin, cephaloridine, cephalothin, bupranolol, buprenorphine, bupropion, buquineran, cephapirin, cephradine, cetaben, cetamolol, cetheXonium buquinolate, buquiterine, buramate, burodiline, buSpirone, chloride, cetiedil, cetirizine, cetocycline, cetohexazine, buSulfan, butabarbital, butacaine, butacetin, butaclamol, cetophenicol, cetotiamine, cetoxime, cetraxate, chaulmosul butadiaZamide, butafosfan, butalamine, but albital, butam fone, chendiol, chiniofon, chlophedianol, chloracy Zine, ben, butamirate, butamisole, butamoxane, butanediol cyclic chloral betaine, chloral hydrate, chloralose, chlorambucil, Sulfite, butanilicaine, butanixin, butanserin, butantrone, chloramine, chloramphenicol, chloramphenicol palmitate, butaperazine, butaprost, butaverine, butedronate, buterizine, chloramphenicol Succinate, chlorazanil, chlorbenzoxamine, butetamate, butethamine, buthiazide, butibufen, butidrine, chlorbetamide, chlorcyclizine, chlordantoin, chlordiazep butikacin, butilfenin, butinazocine, butinoline, butirosin, oxide, chlordimorine, chlorhexadol, chlorhexidine, chlo butixirate, butobendine, butoconazole, butocrolol, butocta rhexidine phosphanilate, chlorindanol, chlorisondamine mide, butofilolol, butonate, butopamine, butopiprine, buto chloride, chlormadinone acetate, chlormerodrin, chlormeZa prozine, butopyrammonium iodide, butorphanol, butoxam none, chlormidazole, chloronaphazine, chloroaZOdin, chlo ine, butoxylate, butriptyline, butropium bromide, butylated robutanol, chlorocreSol, chlorodihydroxyandrostenone, hydroxyanisole, butylated hydroxytoluene, butylparaben, chloroethyl mesylate, 5-chloro-3'-fluoro-2'3-dideoxyuri butynamine, buZepide metiodide, cabastine, cabergoline, dine, chloroguanide, chlorophenothane, chloroprednisone cadralazine, cafaminol, cafedrine, caffeine, calcifediol, cal acetate, chloroprocaine, chloropyramine, chloroquine, chlo US 2002/0102215 A1 Aug. 1, 2002

roSerpidine, chlorothen, chlorothiazide, chlorotriansene, dione, clormecaine, cloroperone, clorophene, cloroqualone, chloroxine, chloroxylenol, chlorozotocin, chlorphenesin, clorotepine, clorprenaline, clorSulon, clortermine, cloSantel, chlorphenesin carbamate, chlorpheniramine, chlorphenoc closiramine, clostebol, clothiapine, clothixamide, clotiaz tium amSonate, chlorphenoxamine, chlorphentermine, chlo epam, cloticasone propionate, clotioxone, clotrimazole, clo rproethazine, chlorproguanil, chlorpromazine, chlorpropam VOXamine, cloxacepride, cloxacillin, cloxacillin benzathine, ide, chlorprothixene, chlorquinaldol, chlortetracycline, cloxazolam, cloxestradiol, cloximate, cloxotestosterone, chlorthalidone, chlorthenoxazine, chlorZoaXaZone, chloecal cloxypendyl, cloxyquin, clozapine, cobamide, cocaine, ciferol, cholic acid, choline chloride, choline glycerophoS cocarboxylase, codeine, codoxime, cofisatin, cogaZOcine, phate, chromocarb, chromonar, ciadox, ciameXon, cianer colchicine, colestolone, colfenamate, colforsin, colterol, goline, cianidol, cianopramine, ciapilome, cicaprost, conessine, conorphone, copper gluconate, cormethasone cicarperOne, ciclactate, ciclafrine, ciclaZindol, cicletanine, acetate, corticosterone, cortisone acetate, cortisu Zol, corti ciclomenol, ciclonicate, ciclonium bromide, ciclopirox, Vazol, cortodoxone, cotarnine chloride, cotinine, cotrip ciclopramine, cicloprofen, cicloprolol, ciclosidomine, ciclo tyline, coumaphos, coumazoline, coumermycin, coumetarol, tizolam, ciclotropium bromide, cicloxilic acid, cicloxolone, creatinolfosfate, crisinatol, croconazole, cromakalim, cromi cicortonide, cicrotic acid, cidoxepin, cifenline, cifostodine, trile, cromolyn, cropropamide, croSpoVidone, crotamiton, ciglitaZone, ciheptolane, ciladopa, cilastatine, cilaZapril, crotetamide, crotoniazide, crufomate, cuprimyxin, cuproXo cilaZaprilat, cilobamine, cillofungin, ciloStamide, ciloStaZol, line, cyacetacide, cyamemazine, cyanocobalamine, cyclacil ciltoprazine, cimaterol, cimenoXin, cimepanol, cimetidine, lin, cyclandelate, cyclarbamate, cyclazocine, cyclazodone, cimetropium bromide, cimoxatone, cinchonine, cinchophen, cyclexanone, cyclindole, cycliramine, cyclizine, cyclobar cinecromen, cinepaXadil, cinepaZet, cinepazic acid, bital, cyclobendazole, cyclobenzaprine, cyclobutoic acid, cinepazide, cinfenine, cinfenoac, cinflumide, cingestol, cini cyclobutyrol, cyclofenil, cycloguanil, cloheximide, cyclo tapride, cinmetacin, cinnamaverine, cinnamedrine, cinnariz leucine, cyclomenol, cyclomethicone, cyclomethycaine, ine, cinnarizine clofibrate, cinnofuradione, cincotramide, cyclopentamine, cyclopenthiazide, cyclopentolate, cyclo cinodine, cinolazepam, cinoquidoX, cinoaxin, cinoxate, penazine, cyclophosphamide, cyclopregnol, cyclopyrro cinoXolone, cinooxopazide, cinperene, cinprazole, cinpro nium bromide, cycloSerine, cycloSporine, cyclothiazide, pazide, cinromide, cintaZone, cintriamide, cinperone, cyclovalone, cycotiamine, cycrimine, cyheptamide, cyhep ciprafamide, ciprafaZone, ciprefadol, ciprocinonide, ciprofi tropine, cynarine, cypenamine, cypothrin, cyprazepam, brate, ciprofloxacin, cipropride, ciprocquaZone, ciprostene, cyprenophine, cyprodenate, cyproheptadine, cyprolidol, ciramadol, cirazoline, cisapride, cisconazole, cismadinone, cyproduinate, cyproterone acetate, cyproximide, cystine, cisplatin, cistinexine, citalopram, citatepine, citenamide, cit cytarabine, dacarbazine, dacemazine, dacisteine, dacinomy enaZone, citicoline, citiolone, clamidoxic acid, clamox cin, dacuronium bromide, dagapamil, dalbraminol, daleda yguin, clanfenur, clanobutin, clantifen, clarithromycin, cla lin, daltroban, dametralast, damotepine, danazol, danitracen, Vulanic acid, claZolam, clazolimine, claZuril, clebopride, danosteine, danthron, dantrolene, dapiprazole, dapSone, clefamide, clemastine, clemeprol, clemizole, clenbuterol, daptomycin, darenzepine, darodipine, datelliptium chloride, clenpirin, cletoquine, clibucaine, clidafidine, clidanac, clidi dunorubicin, daZadrol, dazepinil, dazidamine, dazmegrel, num bromide, climaZolam, climbazole, climiqualine, clin dazolicine, dazopride, dazoquinast, dacoxiben, deanol ace damycin, clindamycin palmitate, clindamycin phosphate, glumate, deanol acetaminobenzoate, deaZauridine, debOX clinofibrate, clinolamide, cliquinol, clioxanide, clipoxamine, amet, debrisoquin, decamethonium bromide, decimenide, cliprofen, clobazam, clobenoside, clobenzepam, cloben decitropine, declaben, declenperone, decloxizine, decomi Zorex, clobenztropine, clobetasol propionate, clobetaSone nol, decoquinate, deditonium bromide, deferoxamine, butyrate, clobutinol, clobu Zarit, clocanfamide, cloca deflazacort, defosfamide, dehydroacetic acid, dehydroemet pramine, clociguanil, clocinizine, clocortolone acetate, clo ine, dehydro-7-methyltestosterone, delanterone, delapril, cortolone pivalate, clocoumarol, clodacaine, clodanolene, delergotrile, delfantrine, delmadinone acetate, delmetacin, clodazon, clodoxopone, clodronic acid, clofazimine, clofe delmopinol, delorazepam, deloxone, delprostenate, dem namic acid, clofenamide, clofenciclan, clofenetamine, brexine, demecarium bromide, demeclocycline, demecol clofenoxyde, clofenVinfos, clofeverine, clofeXamide, clof cine, demecycline, demegestone, demelverine, demexip eZone, clofibrate, clofibric acid, clofibride, clofilium phos tiline, democonazole, demoXepam, denaverine, phate, cloflucarban, clofoctol, cloforex, clofurac, clogestone denbufylline, denipride, denopamine, denpidaZone, denzi acetate, cloguanamil, clomacran, clomegestone acetate, mol, deoxyspergualin, depramine, deprodone, deprostil, clometacin, clometherone, clomethiazole, clometocillin, deptropine, derpanicate, desacetylcolchicine tartrate, desas clomifenoxide, clominorex, clomiphene, clomipramine, clo pidin, desiclovir, descinolone acetonide, deserpidine, mocycline, clomoxir, clonazepam, clonazoline, clonidine, desipramine, deslanoside, desmethylcolchicine, desmeth clonitaZene, clonitrate, clonixeril, clonixin, clopamide, clo ylmisonidazole, deSmethylmoramide, deSocriptine, penthixol, cloperastine, cloperidone, clopidogrel, clopidol, deSogeStrel, desomorphine, desonide, desoximetasone, des clopimozide, clopipazan, clopirac, cloponone, cloprednol, Oxycorticosterone acetate, desoxycorticosterone pivalate, cloprostenol, cloprothiazole, cloquinate, cloquinozine, clo desoxypyridoxine, detajmium bit artrate, detanosal, deter racetadol, cloranolol, cloraZepate, clorethate, clorexolone, enol, detomidine, detorubicin, detrothronine, devapamil, clorgiline, cloricromen, cloridarol, clorindanic acid, clorin dexamethasone, dexamethasone acefurate, dexamethasone US 2002/0102215 A1 Aug. 1, 2002 26 acetate, dexamethasone dipropionate, dexamethasone phos mycin, diprocqualone, diproteverine, diprotriozate, diproXa phate, dexamisole, dexbrompheniramine, dexchlorphe dol, dipyridamole, dipyrithione, dipyrocetyl, dipyrone, niramine, dexclamol, dexetimide, dexetoZoline, dexfenflu dirithromycin, disobutamide, disofenin, disogluSide, disopy ramine, deximafen, dexindoprofen, dexivacaine, ramide, disoxaril, distigmine bromide, disulergine, disulfa dexlofexidine, dexmedetomidine, deXOXadrol, deXpan mide, disulfiram, disuprazole, ditazole, ditercalinium chlo thenol, deXpropranolol, deXproxibutene, deXecoverine, dex ride, dithiazanine iodide, ditiocarb, ditiomustine, tilidine, dextroamphetamine, dextrofemine, dextromethor ditolamide, ditophal, divabuterol, dixanthogen, dizatrifone, phan, dextromoramide, dextrorphan, dextrothyroXine, dizocilpine, dobupride, dobutamine, docarpamine, docona deZaguanine, dezocine, diacerein, diacetamate, diacetolol, Zole, docusate, doliracetam, domaZoline, domiodol, domi diacetylmorphine, diamfenetide, diaminomethylphena phen bromide, domipizone, domoprednate, domoxin, dom Zinium chloride, diamocaine, diampromide, diamthazole, peridone, don, donetidine, dopamantine, dopamine, dianhydrogalactitol, diapamide, diarbarone, diathymoSul dopexamine, dopropidil, dogualast, dorastine, doreptide, fone, diatrizoic acid, diaveridine, diazepam, diaziquone, doSergoside, dotarizine, dotefonium bromide, dothiepin, diazoacetylglycine hydrazide, diazouracil, diaZOxide, doxacurium chloride, doxaminol, doxapram, doxaprost, dibekacin, dibemethine, dibenamine, dibenzepin, dibro doxazosin, doxefazepam, doxenitoin, doxepin, doxibetasol, impropamidine, dibromsalan, dibroSpidium chloride, doxifluridine, doxofylline, doxorubicin, doxpicomine, doxy dibucaine, dibuprol, dibupyrone, dibuSadol, dicarbine, cycline, doxylamine, dramedilol, draquinolol, deaZidox, dicarfen, dichlorallyl lawsone, dichlorisone acetate, dichlo dribendazole, drindene, drobuline, drocinonide, droclid rmeZanone, dichlorofluorimethane, dichloromethotrexate, inium bromide, drocode, drofenine, droloxifene, drometri dichlorophen, dichlorophenarsine, dichlorotetrafluoroet Zole, dromoStanolone, dromoStanolone propionate, dronab hane, dichloroxylenol, dichlorphenamide, dichlorVOS, dici inol, dropempine, droperidol, droprenilamine, dropropizine, ferron, dicirenone, diclaZuril, diclofenac, diclofensine, drotaverine, drotebanol, droxacin, droXicainide, droxicam, diclofurime, diclometide, diclonixin, dicloxacillin, dicobalt droxidopa, droxypropine, dulofibrate, duloZafone, duometa edetate, dicolinium iodide, dicreSulene, dicumarol, dicyclo cin, duoperone, dupracetam, durapatite, dyclonine, mine, didemnin, dideoxycytidine, didrovaltrate, dieldrin, dydrogesterone, dymanthine, dyphylline, ebastine, elbroti dienestrol, dienogest, diethadione, diethazine, diethylpro dine, ebSelen, ecastolol, echinomycin, echothiophate iodide, pion, diethylstilbestrol, diethylstilbestrol diphosphate, dieth ecipramidil, eclanamine, eclaZolast, econazole, ectylurea, ylstilbestrol dipropionate, diethylthiambutene, diethyltolua edelfosine, edetic acid, edetol, edifolone, edogestrone, mide, dietifen, difebarbamate, difemerine, difemetorex, edoxudine, edrophonicum chloride, efaroXan, efetozole, difenamizole, difencloxazine, difenoXimide, difenoxin, dife eflornithine, efloxate, efrotomycin, elantrine, elanzepine, tarSone, difeterol, diflorasone diacetate, difloxacin, diflua elderfield's pyrimidine mustard, elfazepam, ellagic acid, nine, diflucortolone, diflurcortolone pivalate, diflumidone, elliptinium acetate, elmustine, elnadipine, eltenac, eltopra diflunisal, difluprednate, diftalone, digalloyl trioleate, digi Zine, elucaine, elziverine, embramine, embutramide, eme toxin, digoxin, dihexyverine, dihydralazine, dihydroaZacy pronium bromide, emetine, emiglitate, emilium tosylate, tidine, dihydroergotamine, dihydrolenperone, dihydroStrep emopanil, emorfaZone, emylcamate, enalapril, enalaprilat, tomycin, dihydrotachysterol, dihydroxyfluoroprogestrone, enbucrilate, encainide, enciprazine, enclomiphene, diisopromine, diisopropanolamine, dilaZep, dilevalol, encyprate, endomide, endralazine, endrySone, enefeXine, dilimefone, diloxanide, diltiazem, dimabefylline, dimecam eneStebol, enfenamic acid, enflurane, eniclobrate, enilcona ine, dimecolonium iodide, dimecrotic acid, dimefadane, Zole, eniloSpirone, enisoprost, enocitabine, enolicam, enoxa dimefline, dimelazine, dimenorfan, dimenhydrinate, cin, enoXamast, enoXimone, enoXolone, eniprazole, enipro dimenoxadol, dimeheptanol, dimepranol, dimepregnen, line, enprazepine, enprofylline, enpromate, enproStil, dimeproZan, dimercaprol, dimeSna, dimeSone, dimetacrine, enrofloxacin, entSufon Sodium, enviomycin, enviradene, dimetamfetamine, dimethadione, dimethaminostyrylquino epalretat, epanolol, eperisone, ephedrine, epicainide, epicil line, dimethazan, dimethindene, dimethiodal, dimethiso lin, epicriptine, epieStriol, epimestrol, epinastine, epineph quin, dimethisterone, dimetholizine, dimethoxanate, dim rine, epinephryl borate, epipropidine, epirizole, epiroprim, ethylhydroxytestosterone, dimethylnorandrostadienone, epirubicin, epithiazide, epitioStanol, epoprostenol, epoStane, dimethylnortestosterone, dimethylstilbestrol, dimethyl, dim eprazinone, eproVafen, eproXindine, eproZinol, epsiprantel, ethylthiambutene, dimethyltubocurarinium chloride, eptaloprost, eptazocine, equilin, erdosteine, ergocalciferol, dimetipirium bromide, dimetofrine, dimetridazole, dimina ergoloid meSylates, ergonoVine, ergosterol, ergotamine, eri Zene, dimoxamine, dimoxaprost, dimoxyline, dimpylate, colol, erizepine, erocainide, erythrityl tetranitrate, erythro dinaline, dinaZafone, diniprofylline, dinitolmide, dinoprost, mycin, erythromycin acistrate, erythromycin ethylSuccinate, dinoprostone, dinsed, dioSmin, dioxadillol, dioxadrol, diox erythromycin propionate, erythrosine, eSaprazole, escu amate, dioxaphetyl butyrate, dioxethedrin, dioxifiedrine, lamine, eSeridine, esflurbiprofen, eSmolol, eSorubicin, esp dioxybenzone, dipenine bromide, diperodon, diphemanil roquin, estaZolam, estradiol, estradiol benzoate, estradiol methylsulfate, diphenadione, diphenan, diphenhydramine, cypionate, estradiol dipropionate, estradiol enanthate, estra diphendiol, diphenoxylate, diphenylpraline, diphoxazide, diol undecylate, estradiol Valerate, estramustine, estramus dipipanone, dipipoverine, dipliverin, diprafenone, diprenor tine phosphate, estrapronicate, estraZinol, estriol, estrofu phine, diprobutine, diprofene, diprogulic acid, diproleando rate, estrone, estrone hydrogen Sulfate, estropipate, US 2002/0102215 A1 Aug. 1, 2002 27 eSuprone, etabenzarone, etacepride, etafedrine, etafenone, florifenine, floSequinan, flotrenizine, floverine, floxacillin, etameStrol, etamiline, etamiphyllin, etamocycline, etanida floxacrine, floxuridine, fluacizine, fluialamide, fluianisone, Zole, etanterol, etaqualone, etasuline, etazepine, etazolate, fluazacort, flubanilate, flubendazole, flubepride, flucabril, etebenecid, eterobarb, eterSalate, ethacridine, ethacrynic flucetorex, flucindole, fluciprazine, flucloronide, flucona acid, ethambutol, ethamivan, ethamsylate, ethanolamine Zole, flucrylate, flucytosine, fludalanine, fludarabine phos oleate, ethaverine, ethchlorVynol, ethenZamide, ethazide, phate, fludazonium chloride, fludiazepam, fluidorex, fluidox ethidium chloride, ethinamate, ethinyl estradiol, ethiofoS, opone, fludrocortisone acetate, flufenamic acid, flufenisal, ethionamide, ethsterone, ethoheptazine, ethomoxane, etho flufosal, flufylline, fluindarol, fluindione, flumazenil, flume nam, ethopropazine, ethoSuximide, ethotoin, ethoxazene, cinol, flumedroXone-17-acetate, flumequine, flumeridone, ethoxazorutoSide, ethoXZolamide, ethyybenztropine, ethyl flumethasone, flumethasone pivalate,flumethiazide, flume biscoumacetate, ethyl carfluZepate, ethyl cartrizoate, ethyl tramide, flumeXadol, flumeZapine, fluminorex, flumizole, dibunate, ethyl dirazepate, ethylenediamine, ethyleStrenol, flumoxonide, flunamine, flunarizine, flunidazole, ethylhydrocupreine, ethyl loflazepate, ethylmethylthiam flunisolide, flunisolide acetate, flunitrazepan, flunixin, fluno butene, ethylmorphine, 9-ethyl-6-mercaptopurine, ethyl prost, flunoxaprofen, fluocinolone acetonide, fluocinonide, nitrite, ethylnorepinephrine, ethylparaben, ethylphenace flourcortin butyrate, fluocortolone, fluocortolone caproate, mide, ethylstibamine, ethynerone, ethynodiol diacetate, fluorescein, fluoresone, fluoroadenosine, 3-fluoroan ethypicone, etibendazole, eticlopride, eticyclidine, eti droStanol, fluorodopane, fluorohydroxyandrosterone, fluo docaine, etidronic acid, etifelmine, etifenin, etifoXine, etil rometholone, fluorometholone acetate, fluorosalan, 6-fluo amfetamine, etillefrine, etillefrine pivalate, etintidine, etiochl rOteStoSterOne propionate, fluorouracil, anolone, etipirium iodide, etiproston, etiracetam, etiroXate, 9-fluoroXotestenololactone, 9-fluoroXotestololacetone, flu etisazole, etisomicin, etisulergine, etizolam, etocarlide, etoc otracen, fluoxetine, fluoxymesterone, fluparoxan, flupen rylene, etodolac, etodroXZine, etofamide, etofenamate, tiXol, fluperamide, fluperlapine, fluperolone acetate, etofenproX, etofibrate, etoformin, etofuradine, etofylline, fluphenazine, fluphenazine enanthate, flupimazine, flupir etoglucid, etolorex, etolotifen, etoloxamine, etomidate, eto tine, flupranone, fluprazine, fluprednidene, fluprednisolone, midoline, etomoxir, etonitaZene, etoperidone, etoposide, fluprednisolone Valerate, fluprofen, flu profylline, flu.proqua etoprindole, etoprine, etorphine, etoSalamide, etoxadrol, Zone, fluproStenol, fluguaZone, fluradoline, flurandrenoline, etoxeridine, etozolin, etrabamine, etretinate, etryptamine, flurantel, flurazepam, flurbiprofen, fluretofen, flurithromy etymemazine, eucalyptol, eucatropine, eugenol, euprocin, cin, flurocitabine, flurofamide, flurogestone acetate, evandamine, Evans blue, exalamide, exametazine, flurothyl, fluroxene, flusoxolol, fluspiperone, fluspirilene, exaprolol, exepanol, exifone, exiproben, fallintolol, falli flutamide, flutazolam, flutemazepam, flutiazin, fluticaSone pamil, famiraprinium chloride, famotidine, famotine, fami propionate, flutizenol, flutonidine, flutoprazepam, flutroline, profaZone, fanetizole, fantridone, fazadinium bromide, faZ flutropium bromide, fluvoxamine, fluzinamide, fluZoperine, aribine, febantel, febarbamate, februpol, febuverine, folescutol, folic acid, fomidacillin, fominoben, fomocaine, feclemine, feclobu Zone, fedrilate, felbamate, felbinac, fonazine, fopiirtoline, forfenimex, formebolone, formetorex, felipyrine, felodipine, femoxetine, fenabutene, fenacetinol, formintrazole, formocortal, formoterol, fosarilate, foS fenaclon, fenadiazole, fenaptic acid, fenalamide, fenalcom azepam, foScarnet, foscolic acid, foSenazide, foSfocreatine, ine, fenamifuril, penamole, fenaperone, fenbendazole, fen fosfomycin, foSfonet, fosfosal, foSinapril, foSmenic acid, bencillin, fenbufen, fenbutrazate, fencamfamine, fenci foSmidomycin, forpirate, fostedil, fostriecin, fotemustine, butirol, fencleXonium metilsulfate, fenclofenac, fenclonine, fotreamine, frabuprofen, frentizole, fronepidil, froxiprost, fenclorac, fenlozic acid, fendiline, fendosal, feneritrol, ftaxilide, ftivazide, florafur, ftormetazine, ftorpropazine, fenestrel, fenethazine, fenethylline, fenetradil, fenflumizole, fubrogonium iodide, fuchsin, fumagillin, fumoXcillin, fupra fenfluramine, fenfluthrin, fengabine, fenharmane, fenimide, Zole, furacrinic acid, furafylline, furalazine, furaltadone, feniodium chloride, fenipentol, fenirofibrate, fenisorex, fen furaprofen, furazabol, furazolidone, furazolium chloride, metoZole, fenmetramide, fenobam, fenocinol, fenoctimine, furbucillin, furcloprofen, furegrelate, furethidine, furfeno fenofibrate, fenoldopam, fenoprofen, fenoterol, fenoverine, reX, furidarone, furmethoxadone, furobufen, furodazole, fenoxazoline, fenoxedil, fenozolone, fenpentadiol, femper furofenac, furomazine, furoSemide, furoStilbestrol, furSalan, ate, fenipalone, fenipramide, feniprane, fenpiverinium bro furSultiamine, furterene, furtrethonium iodide, fusidic acid, mide, fenprinast, fenproporex, fenproStalene, fenguizone, fuZlocillin, gabapentin, gabeXate, gabOXadol, galantamine, fenretinide, fenspiride, fentanyl, fentiazac, fenticlor, fenti gallamine triethodide, gallopamil, galoSemide, galtifenin, conazole, fentonium bromide, fenyripol, fepentolic acid, gampexine, gamolenic acid, ganciclovir, ganglefene, gapi fepitrizol, fepradinol, feprazone, fepromide, feprosidinine, comine, gapromidine, gefarnate, gemazocine, gemcadiol, ferriclate calcium, ferrotrenine, ferrous fumarate, ferrous gemeprost, gemfibrozil, gentamicin, gentian Violet, gepe gluconate, fetoxylate, feXicaine, feXinidazole, fezatione, frine, gepirone, geroquinol, gestaclone, gestadienol, feZolamine, fiacitabine, fibracillin, filenadol, filipin, fifexide, gestodene, geston.orone caproate, gestrinone, giparmen, flamenol, flavamine, flavodic acid, flavodil, flavone actic gitaloXin, gitoformate, glafenine, glaziovine, gliamilide, gli acid, flavoxate, flazalone, flecainide, flerobuterol, fleroxacin, bornuride, glibutimine, glicaramide, glicetanile, geroquinol, flesinoxan, flestolol, fletazepam, floctafenine, flomoxef, gestaclone, gestadienol, gestodene, geston.orone caproate, flopropione, florantyrone, flordipine, floredil, florfenicol, gestrinone, giparmen, gitaloxin, gitoformate, glafenine, US 2002/0102215 A1 Aug. 1, 2002 28 glaziovine, gliamilide, glibornuride, glibutimine, glicara idaverine, idaZOXan, idebenone, idenast, idoxuridine, idral mide, glicetanile, gliclazide, glicondamide, glidaZamide, fidine, idrocilamide, idropranolol, ifenprodil, ifosfamide, gliflumide, glimepiride, glipentide, glipizide, gliquidone, ifoxetine, ilmofoSine, iloprost, imafen, imanixil, imaZOdan, glisamuride, glisindamide, glisolamide, glisoxepide, glox imcarbofoS, imeXon, imiclopazine, imidazole Salicylate, aZone, gloXimonam, glucametacin, glucosamine, glucosul imidazopyrazole, imidecyl iodine, imidocarb, imidoline, famide, glucosulfone, glucurolactone, glucuronamide, imidurea, imiloxan, iminophendimide, imipenem, imi glunicate, glutamic acid, glutaral, glutarimide, glutaurine, pramine, imipraminoxide, imirestat, imolamine, imoxiterol, glutethimide, glyburide, glybuthiazol, glybuzole, glyceryl impacarzine, impromidine, improSulfan, imuracetam, ina monoStearate, glycidyl methacrylate, glycine, glyclopyra perisone, indacrinone, indalpine, indanazoline, indanidine, mide, glybiar.Sol, glycopyrrolate, glycyclamide, glyhexam indanorex, indapamide, indatraline, indacainide, indelox ide, glymidine, glyoctamide, glypinamide, glyprothiazol, azine, indenolol, indicine-N-oxide, indigotindisulfonic acid, glySobuzole, gold thiomalate, gold Sodium thiosulfate, gra indobufen, indocate, indocyanine green, indolapril, indol nisetron, griseofulvin, guabenXan, guacetisal, guafecainol, idan, indomethacin, indopanolol, indopine, indoprofen, guaiactamine, guaiapate, guaietolin, guaifenesin, guaimeSal, indoramin, indorenate, indoxole, indriline, inicarone, inoco guaisteine, guaithylline, guamecycline, guanabenz, guana terone, inosine, inosine dialdehyde, inositol niacinate, inp cline, guanadrel, guanaZodine, guanazole, guanclofine, roquone, intrazole, intriptyline, iobenzamic acid, iobutic guancydine, guanethidine, guanfacine, guanisoquin, guano acid, iocarmic acid, iocetamic acid, iodamide, iodecimol, clor, guanoctine, guanoXabenz, guanoxan, guanoxyfen, iodetryl, iodipamide, iodixanol, iodoalphionic acid, iodol, hadacidin, halazepam, halazone, halcinonide, halethazole, iodophthalein, iodoquinol, iodothiouracil, iodoxamic acid, halocortolone, halofantrine, halofenate, halofuginone, ioglicic acid, ioglucol, ioglucomide, ioglunide, ioglycamic halometasone, halonamine, halopemide, halopenium chlo acid, iogulamide, iohexol, iodlidonic acid, iolixanic acid, ride, haloperidol, haloperidol decanoate, haloperidone iomeglamic acid, iomeprol, iomorinic acid, iopamidol, acetate, haloprogesterone, haloprogin, halothane, halox iopanoic acid, iopentol, iophendylate, iophenoxic acid, aZolam, haloxon, halquinols, hedaquinium chloride, hep ioprocemic acid, iopromide, iopronic acid, iopydol, iopy ronicate, heptabarbital, heptaminol, heptaverine, heptola done, ioSarcol, ioSefamic acid, ioSeric acid, iosimide, iOSu mide, hepZidine, hetacillin, hetaflur, heteronium bromide, lamide, ioSumetic acid, iotasul, iotetric acid, iothalamic acid, hexachlorophene, hexacyclonate, hexacy prone, hexadiline, iotranic acid, iotrizoic acid, iotrolan, iotroXic acid, iOverSol, hexadimethrine bromide, hexafluorenium bromide, hexam ioXabrolic acid, iOXaglic acid, ioxitalamic acid, ioxotrizoic ethonium bromide, hexamidine, hexapradol, hexaprofen, acid, iOZomic acid, ipexidine, ipodic acid, ipragratine, ipra hexapropymate, heXasonium iodide, heXacarbacholine bro midil, ipratropium bromide, iprazochrome, ipriflavone, mide, hexedine, hexestrol, heXetidine, hexobarbital, heX iprindole, iprocinodine, iproclozide, iprocrolol, iprofenin, obendine, hexocyclium methylsulfate, hexoprenaline, heX iproheptine, iproniazid, iproidazole, iproplatin, iprotiazem, opyrronium bromide, hexylcaine, hexylene glycol, iproxamine, iprozilamine, ipsalazide, ipsapirone, iquin hexylresorcinol, histamine, histapyrrodine, homarylamine, damine, irindalone, irloxacin, irolapride, irSogladine, isam homatropine, homatropine methylbromide, homidium bro faZone, isamoltan, isamoxole, isaxonine, isbogrel, isepami mide, homochlorcyclizine, homofenazine, homoharringto cin, isoaminile, isobromindione, isobucaine, isobutamben, nine, homopipramol, homosalate, homotestosterone propi isocarboxazid, isoconazole, isocromil, isoetharine, isof onate, homprenorphine, hopantenic acid, hoquizil, eZolac, isoflupredone acetate, isoflurane, isoflurophate, iso hycanthone, hydracarbazine, hydralazine, hydrargaphen, leucine, isomazole, isomerol, isometamidium, isometha hydrobentizide, hydrochlorthiazide, hydrocodone, hydro done, isometheptene, isomylamine, isoniazid, isonixin, cortamate, hydrocortisone, hydrocortisone aceponate, isopraZone, isoprednidene, isoprofen, isoprofamide iodide, hydrocortisone acetate, hydrocortisone butyrate, hydrocor isopropicillin, isopropyl myristate, isopropyl palmitate, iso tisone cypionate, hydrocortisone-phosphate, hydrocortisone proterenol, isosorbide, isosorbide dinitrate, isosorbide Succinate, hydrocortisone Valerate, hydroflumethiazide, mononitrate, isospalglumic acid, isosulfan blue, isoSulpride, hydromadinone, hydromorphinol, hydromorphone, hydro isothipendyl, isotic, isotiquimide, isotretinoin, isoxaprolol, quinone, hydroxindasate, hydroxindasol, hydroxyOXocobal isoXepac, isoxicam, iSOXSuprine, isradipine, itanOXOne, itazi amin, hydroxy amphetamine, hydroxychloroquine, grel, itraconazole, itrocainide, ivermectin bib, ivoqualine, hydroxydimethandrostadienone, hydroxydione Succinate, josamycin, kainic acid, kalafungin, kanamycin, kebuZone, hydroxymethylandrostanone, 10-hydroxynorehisterone, keracyanin, ketamine, ketanserin, ketazocine, ketazolam, hydroxypethidine, hydroxyphenamate, hydroxyprocaine, kethoxal, ketipramine, ketobemidone, ketocaine, ketocainol, hydroxyprogeSerone, hydroxyprogesterone caproate, ketoconazole, ketoprofen, ketorfanol, ketorolac, ketotifen, hydroxypyridine tartrate, hydroxyStilbamidine, 7-hydrox ketotrexate, khellin, khelloSide, kitasamycin, labetalol, laci yteStololacetone, hydroxytestosterone propionate, hydrox dipine, lactalfate, lactose, lactulose, lamotrigine, lamtidine, ytetracaine, hydroxytoluic acid, hydroxyurea, hydroxy Zine, lanatoside, lapachol, lapinone, lapyrium chloride, lasalocid, hymecromone, hyoscyamine, hypericin, ibacitabine, iba laudexium methyl Sulfate, lauralkonium chloride, laureth, floxacin, ibazocine, ibopamine, ibrotamide, ibudilast, laurixamine, laurocapram, lauroguadine, laurolinium ibufenac, ibuprofen, ibuprofen piconol, ibuproxam, ibuterol, acetate, lauryl isoquinolinium, lefetamine, leflunomide, ibuverine, icazepam, icosipiramide, icotidine, idarubicin, leiopyrrole, lemidoSul, lenampicillin, leniquinsin, lenper US 2002/0102215 A1 Aug. 1, 2002 29 one, leptacline, lergotrile, letimide, letosteine, leucine, leu trenone, menbutone, menfegol, menglytate, menitrazepam, cinocaine, leucocianidol, leucovorin,levacecarnine, levallor menoctone, menogaril, menthol, meobentine, meparfynol, phan, levamfetamine, leVamisole, levdropropizine, mepazine, mepenZolate bromide, meperidine, mephenesin, levisoprenaline, levlofexidine, levobunolol, levocabastine, mephenoxalone, mephentermine, mephenyton, mephobar levocarnitine, levodopa, levofacetoperane, leVofenflu bital, mepindolol, mepiprazole, mepiroXol, mepitioStane, ramine, levofuraltadone, leVoglutamide, levomenol, mepivacaine, mepixanox, mepramidil, meprednisone, mep levomethadone, levomethadyl acetate, levomethorphan, robamate, meproscillarin, meproxitol, meprylcaine, levometiomeprazine, levomopranol, levomoramide, levo meptazinol, meguidox, meguinol, meguitazine, meralein, nantradol, levonordeprin, levonorgestrel, levophenacyl mor meralluride, merbarone, merbromin, mercaptamine, mer phan, levopropoxyphene, levopropylcillin, levopropyl captomerin, mercaptopurine, mercuderamide, mercufenol hexedrine, levoprotiline, levorin, levorphanol, chloride, mercumatilin, mercurobutol, mergocriptine, levothyroxine, leVOXadrol, leXofenac, libecillide, libenza merophan, merSalyl, mesabolone, meSalamine, meSecla pril, lidamidine, lidocaine, lidofenin, lidoflazine, lifibrate, ZOne, meSna, mesocarb.meso-hexestrol, mesoridazine, lilopristone, limaprost, lincomycin, lindane, linsidomine, mesipirenone,meStanolone, mesterolone, meStranol,meSu liothyronine, liroldine, lisinopril, lisuride, lithium carbonate, dipine, meSulergine,meSulfamide, meSulfen, meSuprine, lithium citrate, litracen, lividomycin, lixazinone, lobeline, metabromsalan, metacetamol, metaclazepam, metaglycodol, lobendazole, lobenzarit, lobuprofen, locicortone, lodax metahexamide, metamelfalan, metamfaZone, metamfepra aprine, lodaceZarlodinixil, lodiperone, lodoxamide, lodoxa mone, metampicillin, metanixin, metapramine, metaproter mide ethyl, lofemizole, lofendazam, lofentanil, lofepramine, enol, metaraminol, metaterol, metaxalone, metaZamide, lofexidine, loflucarban, lombazole, lomefloxacin, lometra metazide, metazocine, metbufen, meteneprost, metergoline, line, lomevactone, lomifylline, lomofungin, lomustine, lona metergotamine, metescufylline, meteSculletol, metethohep palene,lonaprofen, lonazolac, lonidamine, loperamide, lop tazine, metformin, methacholine chloride, methacycline, eramide oxide, lopirazepam, loprazolam, loprodiol, methadone, methadyl acetate, methallenestril, methallibure, lorajmine, lorapride, loratadine, lorazepam, lorbamate, methalthiazide, methamphetamine, methandriol, meth lorcainide, lorcinadol, lorglumide, lormetazepam, lorta androstenolone, methaniazide, methantheline bromide, lamine, lorZafone, losindole, loSulazine, lotifazole, lotrifen, methaphenilene, methapyrilene, methaqualone, metharbital, lotucaine, lovastatin, loxanast, loxapine, loxiglumide, loxo methastyridone, methazolamide, methdilazine, meth profen, loXtidine, lozilurea, lucanthone, lucartamide, luci enamine, methenolone acetate, methenolone enanthate, mycin, lufuradom, lupitidine, luprostiol, luxabendazole, metheptazine, methestrol, methetoin, methicillin, methima lyapolate Sodium, lycetamine, lydinycin, lymecycline, Zole, methiodal Sodium, methioguanine, methiomeprazine, lynestrenol, lySergide, lysine, mabuterol, maduramicin, methionine, methisaZone, methitural, methixene, methocar mafenide, mafoprazine, mafosfamide, magnesium citrate, bamol, methohexital, methopholine, methoSerpidine, meth magnesium gluconate, magnesium Salicylate, malathion, otrexate, methotrimeprazine, methoxamine, methoXSalen, malethamer, malic acid, malotilate, manidipine, manganese methoxyflurane, methoxyphedrine, methoxyphenamine, gluconate, mannitol, mannitol heXanitrate, mannomustine, methoxypromazine, methScopolamine bromide, methSuxim mannoSulfan, manoZodil, maprotiline, maridomycin, marip ide, methylclothiazide, N-methyladrealone hcl, methyl alco tiline, marOXepin, maytansine, maZaticol, mazindol, hol, methylatropine nitrate, methylbenactyzium bromide, maZipredone, mebanazine, mebendazole, mebenoside, methylbenzethonium, methylchromone, methyldesorphine, mebeverine, mebeZonium iodide, mebhydrolin, mebiquine, methyldihydromorphine, methyldopa, methyldopate, meth mebolazine, mebrofenin, mebutamate, mebutizide, ylene blue, methylphedrine, methylergonovine, methylfor mecamylamine, mecarbinate, mecetronium ethylsulfate, mamide, methyl nicotinate, 2-methyl-19-nortestosterone, mechlorethamine, meciadanol, mecinarone, meclizine, 2-methyl-11-oxoprogestrone, methyl palmoxirate, meth meclocycline, meclocycline Sulfosalicylate, meclofenamic ylparaben, methylphendiate, methylprednisolone, methyl acid, meclofenoxate, meclonazepam, mecloqualone, meclo prednisolone aceponate, methylprednisolone acetate, meth ralurea, meclorisone dibutyrate, mecloxamine, mecobal ylprednisolone hemisuccinate, methylprednisolone amin, mecrylate, mecysteine, medazepam, medaZomide, phosphate, methylprednisolone Suleptanate, methyl Salicy medetomidine, medibazine, medifoxamine, medorinone, late, methylstreptonigrin, 4-methyltestosterone, 7-methylt medorubicin, medrogestone, medronic acid, medroxalol, estosterone, 17-methyltestosterone, 7-methylteSosterone medroxyprogestrone, medroxyprogestrone acetate, medry propionate, methylthionosine, 16-methylthioprogestone, lamine, medrySone, mefeclorazine, mefenamic acid, mefeni methylthiouracil, methynodiol diacetate, methyprylon, dil, mefenidramium metilsulfate, mefenorex, mefeserpine, methySergide, metiamide, metiapine, metiaZinic acid, mefeXamide, mefloquine, mefruside, megalomicin, mege metibride, meticrane, metildigoxin, metindizate, metioprim, Strol acetate, meglitinide, megucycline, meglumine, meglu metioXate, metipiroX, metipranolol, metiprenaline, tol, meladrazine, melarSonyl, melarSoprol, melengestrol metitepine, metizoline, metkephamid, metochalcone, meto acetate, meletimide, melinamide, melitracen, melizame, cinium iodide, metoclopramide, metocurine iodide, meloxicam, melperone, melphalan, memantine, memotine, metofenazate, metogest, metolaZone, metomidate, metopi menabitan, menadiol, menadiol diphosphate, menadiol dis mazine, metopon, metoprine, metoprolol, metoquizine, ulfate, menadione, menadione Sodium bisulfite, menate metoSerpate, metoStilenol, metoxepin, metrafazoline, US 2002/0102215 A1 Aug. 1, 2002 30 metralindole, metrazifone, metrenperone, metribolone, met niclofolan, niclosamide, nicoboxil, nicoclonate, nicocodine, rifonate, metrifudil, metrizamide, metrizoic acid, metronida nicocortonide, nicodicodine, nicofibrate, nicofuranose, nico Zole, meturedepa, metyrapone, metyridine, metyrosine, furate, nicogrelate, nicomol, nicomorphine, nicopholine, mevastatin, meXafylline, mexazolam, meXenone, mexilet nicorandil, nicothiaZone, nicotinyl alcohol, nicOXamat, nic ine, mexiprostil, mexoprofen, meXrenoate, meZacopride, tiazem, nictindole, nodroxy Zone, nifedipine, nifenalol, meZepine, meZilamine, meZlocillin, mianserin, mibolerone, nifenaZone, niflumic acid, nifluridide, nifuradene, nifurald micinicate, miconazole, micronomicin, midaflur, midagli eZone, nifuralide, nifuratel, nifuratrone, nifurdazil, nifuret Zole, midalcipran, midamaline, midazogrel, midazolam, haZone, nifurfoline, nifurimide, nifurizone, nifurmazole, midecamycin, midodrine, mifentidine, mifepristone, mifo nifurmerone, nifuroquine, nifuroxazide, nifuroxime, nifurpi bate, miglitol, mikamycin, millacemide, milemperone, mili pone, nifurpirinol, nifurprazine, nifurcquinazole, nifurSemi pertine, miloxacin, milrinone, milverine, mimbane, Zone, nifurSol, nifurthiazole, nifurtimox, nifurtoinol, nifur minaprine, minaxolone, mindolilol, mindoperone, minepen Vidine, nifurzide, niguldipine, nihydraZone, nikethamide, tate, minocromil, minocycline, minoxidil, mioflazine, mipi nileprost, nilprazole, niludipine, nilutamide, nilvadipine, mazole, mirincamycin, miristalkonium chloride, miropro nimaZone, nimeSulide, nimetazepam, nimidane, nimodipine, fen, mirosamicin, misonidazole, misoprostol, mitindomide, nimorazole, nimustine, niometacin, niperotidine, nipradillol, mitobronitol, mitoclomine, mitoguaZone, mitolactol, mito niprofaZone, niridazole, nisbuterol, nisobamate, nisoldipine, mycin, mitonafide, mitopodozide, mitocquidone, mitotane, nisoxetine, nisterime acetate, nitarSone, nitaZOXanide, nithia mitotenamine, mitoxantrone, mitoZolomide, mivacurium mide, nitracrine, nitrafudam, nitralamine, nitramisole, chloride, mixidine, misoprostol, mitindomide, mitobronitol, nitraduaZone, nitrazepam, nitrefazole, nitrendipine, nitricho mitoclomine, mitoguaZone, mitolactol, mitomycin, mitona line, nitrochlofene, nitrocycline, nitrodan, nitrofurantoin, fide, mitopodozide, mitocquidone, mitotane, mitotenamine, nitrofuraZone, nitroglycerin, nitromerSol, nitromide, mitoxantrone, mitoZolomide, mivacurium chloride, mixi nitromifene, nitroScanate, nitroSulfathiazole, nitroXinil, dine, mizoribine, mobecarb, mobenzoxamine, mocimycin, nitroxoline, nivaZol, nivimeldone, nixylic acid, nizatidine, mociprazine, moclobemide, moctamide, modafinil, moda nizofenone, noberastine, nocloprost, nocodazole, nofecain line, mofebutaZone, mofloverine, mofoXime, molfarnate, ide, nogalamycin, nolinium bromide, nomegestrol, nomeli molinaZone, molindone, molracetam, molsidomine, dine, nomifensine, nonabine, nonaperone, nonapyrimine, mometasone furoate, monalazone disodium, monensin, nonoxynol-4, nonoxynol-9, noracymethadol, norbolethone, monobenzone, monoethanolamine, monometacrine, mono norbudrine, norcloStebol, norcodeine, nordazepam, norde phosphothiamine, monothioglycerol, monoxerutin, montire frin, nordinone, norepinephrine, norethandrolone, norethin lin, moperone, mopidamol, mopidralazine, moprolol, drone, norethindrone acetate, norethynodrel, noreximide, moduizone, morantel, moraZone, morclofone, morforex, norfenefrine, norfloxacin, norfloxacin Succinil, norflurane, moricizine, morinamide, morniflumate, morocromen, norgesterone, norgestimate, norgestomet, norgestrel, norg morOXydine, morpheridine, morphine, morSuximide, mot estrienone, norletimol, norlevorphanol, normethadone, apizone, motrazepam, motretinide, moveltipril, moxadolen, normethandrone, normorphine, norpipanone, nortestoster moxalactam, moxaprindine, moxastine, moxaverine, mox one propionate, nortetrazepam, nortriptyline, norvinister azocine, moxestrol, moxicoumone, moxipraquine, moxisy one, noSantine, noScapine, noSiheptide, novobiocin, noXip lyte, moxnidazole, moxonidine, mupirocin, murabutide, tiline, noxytiolin, nuclomedone, nuclotixine, nulfenoXole, murocainide, muzolimine, mycophenolic acid, my fadol, nuVenzepine, nylestriol, nylidrin, nyStatin, obidoxime, Ocilt myralact, myrophine, myrtecaine, nabaZenil, nabilone, nabi ide, ocrylate, octabenzone, octacaine, octafonium chloride, tan, naboctate, nabumetone, nadide, nadolol, nadoxolol, octamoxin, octamylamine, octanoic acid, octapinol, octast naepaine, nafamoStat, nafazatrom, nafcaproic acid, nafcillin, ine, octaverine, octaZamide, octenidine, octenidine Saccha nafenodone, nafenopin, nafetolol, nafimidone, nafiverine, rin, Octicizer, Octimibate, octorylene, octodrine, octopamine, naflocort, nafomine,nafoXadol, nafoxidine, nafronyl, nafta Octotiamine, Octoxynol-9, octriptyline, octrizole, ofloxacin, lofoS, naftaZone, naftifine, naftopidil, naftoxate, naftypra ofornine, oftasceine, Olaflur, olacquindox, oleanomycin, ole mide, nalbuphine, nalidixic acid, nalmefene, nalmexone, timol, oleyl alcohol, olivomycin a, Olmidine, olpimedone, nalorphine, naltrexone, naminterol, namoxyrate, nanapro olsalazine, oltipraz, olvanil, omeprazole, omidoline, Omo cin, nandrolone cyclotate, nandrolone decanoate, nan conazole, omonasteine, Onapristone, ondansetron, Ontianil, drolone phenpropionate, nanofin, nantradol, napacitadine, opiniazide, opipramol, oraZamide, orbutopril, orconazole, napamezole, naphazoline, naphthonone, naprodoxime, orestrate, Ormetoprim, ornidazole, ornipressin, ornithine, naproxen, naproXol, naranol, narasin, natamycin, naX ornoprostil, orotic acid, orotirelin, orpanoxin, orphenadrine, agolide, naxaprostene, nealbarbital, nebidrazine, nebivolol, ortetamine, OSalmid, OSmadizone, otilonium bromide, otim nebracetam, nedocromil, nefazodone, neflumozide, nefo erate Sodium, Ouabain, Oxabolone cipionate, OXabrexine, pam, neleZaprine, neoarSphenamine, neocinchophen, neo Oxaceprol, oxacillin, oxadimedine, oxaflozane, oxaflu mycin, neostigmine bromide, nequinate, neraminol, ner mazine, OXagrelate, Oxalinast, Oxaliplatin, OXamarin, OXam bacadol, neSapidil, neSOsteine, netilmicin, netobimin, etacin, oxamisole, Oxamniquine, Oxanamide, Oxandrolone, neutramycin, neXeridine, niacin, niacinamide, nialamide, OXantel, oxapadol, Oxapium iodide, Oxapropanium iodide, niaprazine, nibroxane, nicafenine, nicainoprol, nicametate, Oxaprotiline, Oxaprozin, oxarbazole, Oxatomide, oxazafone, nicarbazin, nicarpidine, nicergoline, niceritrol, niceverine, oxazepam, oxazidione, oxazolam, oxazorone, Oxcarba US 2002/0102215 A1 Aug. 1, 2002

Zepine, oxdralazine, OXeladin, Oxendolone, OXepinac, oxet phenisonone, phenmetrazine, phenobarbital, phenobutiodil, acillin, oxethazaine, Oxetorone, Oxfendazole, Oxfenicine, phenolphtalein, phenolsulfonphthalein, phenomorphan, OXibendazole, Oxibetaine, oxiconazole, oxidopamine, phenoperidine, phenothiazine, phenothrin, phenoxyben oxidronic acid, oxifentorex, oxifungin, OXilorphan, oxi Zamine, phenoxypropazine, phenprobamate, phenprocou monam, OXindanac, OXiniacic acid, oxiperomide, oxirac mon, phenpromethamine, phensuXimide, phentermine, etam, OXiramide, oxisopred, OXisuran, OXitefonium bromide, phentolamine, phenylalanine, phenyl aminoSalicylate, phe oxitriptan, OXitriptyline, OXitropium bromide, OXmetidine, nylbutaZone, phenylrphrine, phenylethyl alcohol, phenylm OXOdipine, Oxogestone phenpropionate, oxolamine, OXolinic ercuric acetate, phenylmercuric borate, phenylmercuric acid, OXomemazine, OXonazine, Oxophenarsine, Oxoprostol, chloride, phenylmercuric nitrate, phenylmethylbarbituric oXpheneridine, Oxprenoate potassium, OXprenolol, OXtriph acid, phenylpropanolamine, phenylthilone, phenyltoloxam ylline, Oxybenzone, Oxybutynin, oxychlorosene, oxycin ine, phenyramidol, phenytoin, phetharbital, pholcodine, chophen, oxyclozanide, Oxycodone, Oxydipentonium chlo pholedrine, phosphoramide mustard, phoxim, phthalofyne, ride, Oxyfedrine, oxymesterone, Oxymetazoline, phthalysulfacetamide, phthalylsulfamethizole, phthalylsul oxymetholone, oxymorphone, oxypendyl, oxypertine, fathiazole, phySoStigmine, phytic acid, phytonadiol diphos OxyphenbutaZone, oxyphenonium bromide, Oxypurinol, phate, phytonadione, pibecarb, pibenzimol, pibecarb, piben Oxypyrronium bromide, oxyquinoline, Oxyridazine, oxySo Zimol, piberaline, picafibrate, picartamide, picenadol, nium iodide, Oxytetracycline, Oxytiocin, OZagrel, Ozolinone, picilorex, piclonidine, piclopastine, picloxydine, picoben pacrinolol, pactamycin, padimate, pafenolol, palatrigine, Zide, picodralazine, picolamine, piconol, picoperine, pico paldimycin, palmidrol, palmoxiric acid, pamabrom, pam prazole, picotamide, picotrin diolamine, picumast, pidolic aquine, pamatolol, pamidronic acid, pancuronium bromide, acid, pifarnine, pilfenate, pifeXole, piflutiXole, pifoXime, panidazole, panomifene, patenicate, panthenol, pantothenic piketoprofen, pildralazine, pilocarpine, pimoclone, pimefyl acid, panuramine, papaverine, papaveroline, parachlorophe line, pimelautde, pimetacin, pimethixene, pimetime, pime nol, paraflutizide, paraldehyde, paramethadione, parametha tremide, piminodine, pimobendan, pimondiazole, pimozide, Sone acetate, paranyline, parapenzolate bromide, para pinacidil, pinadoline, pinafide, pinaverium bromide, propamol, pararosaniline, pararosaniline embonate, pinazepam, pincainide, pindolol, pinolcaine, pinoxepin, paraXaZone, parbendazole, parconazole, pareptide, pare pioglitaZone, pipacycline, pipamazine, pipaperone, thoxycaine, pargeverine, pargolol, pargyline, paridocaine, pipazethate, pipebu Zone, pipecuronium bromide, pipemidic parodilol, paromomycin, paroxetine, paroxypropione, acid, pipenZolate bromide, pipequaline, piperacetazine, pip parSalmide, partricin, parvaquone, pasiniazid, paulomycin, eracillin, piperamide, piperazine, piperazinedione, piperi paXamate, paZelliptine, paZOxide, pcnu, pecilocin, pecocy dolate, piperilate, piperocaine, piperoxan, piperylone, pipo cline, pefloxacin, pelanSerin, pelretin, pelrinone, pemedolac, broman, pipoctanone, pipofeZine, pipOSulfan, pipotiazine pemerid, pemoline, pempidine, penamecillin, penbutolol, palmiate, pipOXizine, pipOXolan, pipradimadol, pipradol, pendecamaine, penfluridol, penflutizide, pengitoxin, peni pipramadol, pipratecol, piprinhydrinate, piprocurarium cillamine, penicillin procaine, penicillin, penimepicycline, iodide, piprofurol, piprozolin, piquindone, piquizil, pirac penimocycline, penirolol, penmesterol, penoctonium bro etam, pirandamine, pirarubicin, piraxelate, piraZmonam, mide, penprostene, pentabamate, pentacynium chloride, piraZolac, pirbenicillin, pirbuterol, pirdonium bromide, pentaerythritol tetranitrate, pentafluranol, pentagastrin, pent pirenoxine, piremperone, pirenzepine, pirepolol, piretanide, agestrone, pentalamide, pentamethonium bromide, pentam pirfenidone, piribedil, piridicillin, piridocaine, piridoxilate, ethylmelamine, pentamidine, pentamoxane, pentamustine, piridironic acid, pirifibrate, pirindazole, pirinixic acid, pirin pentapiperide, pentapiperium methylsulfate, pentaguine, iXil, piriprost, piriqualone, piriSudanol, piritramide, piritr pentazocine, pentetate calcium triSodium, pentetic acid, exim, pirlimycin, pirlindole, pirmagrel, pirmenol, pirnabine, penthienate bromide, penthrichloral, pentiapine maleate, piroctone, pirogliride, piroheptine, pirolate, pirolazamide, pentifylline, pentigetide, pentisomicin, pentisomide, pentizi piromidic acid, piroXantrone hcl, piroXicam, piroXicam cin done, pentobarbital, pentolinium tartrate, pentomone, pen namate, piroxicillin, piroXimone, pirozadil, pirprofen, topril, pentorex, pentosan polysulfate Sodium, pentostatin, pirquinozol, pirralkonium bromide, pirtenidine, pitenodil, pentoxifylline, pentrinitrol, pentylenetrazole, peplomycin, pitofenone, pituxate, pivampicillin, pivenfrine, pivopril, piv pepstatin, peraclopone, peradoxime, perafensine, peralo oxazepam, pizotyline, plafibride, plaunotol, pleuromulin, pride, peraquinsin, perastine, peratizole, perbufylline, per plicamycin, podilfen, podophylloxOXin, poldine methylsul fluiamine, perflunafene, pergolide, perhexilene, periciazine, fate, polidocanol, ploymyxin, polythiazide, ponalrestat, pon perimetazine, perindopril,perindoprilatperisoxal, perlapine, fibrate, porfiromycin, poskine, potassium guaiacolsulfonate, permethrin, perphenazine, persilic acid, petrichloral, peXan potassium nitrazepate, potassium Sodium tartrate, potassium tel, phanquone, phenacaine, phenacemide, phenacetin, Sorbate, potassium thiocyanate, practolol, prajmalium, prali phenact tropinium chloride, phenadoxone, phenaglycodol, doXime chloride, pramipexole, pramiracetam, pramiverine, phenamaZoline, phenampromide, phenarSone Sulfoxylate, pramoxime, prampine, pranolium chloride, pranoprofen, phenazocine, phenazopyridine, phencarbamide, phencyclid pranosal, prasterone, pravastatin, praXadine, prazepam, ine, phendimetrazine, phenelZine, pheneridine, phenesterin, prazepine, praZiquantel, praZitone, praZOcillin, praZosin, penethicillin, phenformin, phenglutarimide, phenicarbazide, preclamol, prednazate, prednazoline, prednicarbate, predni phenindamine, phenindione, pheniprazine, pheniramine, mustine, prednisolamate, prednisolone, prednisolone US 2002/0102215 A1 Aug. 1, 2002 32 acetate, prednisolone hemisuccinate, prednisolone phos recainam, reclazepam, relomycin, remoxipride, renanolone, phate, prednisolone Steaglate, prednisolone tebutate, pred rentiapril, repirinast, repromicin, reproterol, recimetol, res nisone, prednival, prednylidene, prefenamate, preg cinnamine, reserpine, resorantel, resorcinol, resorcinol nenolone, pregnenolone Succinate, premazepam, monoacetate, retelliptine, retinol, revenast, ribavirin, ribo prenalterol, prenisteine, prenoverine, prenoXciazine, preny flavin, riboflavin 5'-phosphate, riboprine, ribostamycin, lamine, pretamazium iodide, pretiadil, pribecaine, pridefine, ridazolol, ridiflone, rifabutin, rifamide, rifampin, rifamycin, prideperone, pridinol, prifelone, pri?inium bromide, prifuro rifapentine, rifaximin, rilapine, rilmazafone, rillmenidine, line, prilocaine, primaperone, primaquine, primidolol, rilopiroX, riloZarone, rimantadine, rimazolium metilsulfate, primidone, primycin, prinomide, pristinamycin, prizidilol, rimcazole, rimeXolone, rimiterol, rimoprogin, riodipine, rio proadifen, probarbital, probenecid, probicromil, probucol, prostil, ripazepam, risocaine, risperidone, ristianol, ristoce procainamide, procaine, procarbazine, procaterol, prochlor tin, ritanserin, ritiometan, ritodrine, ritropirronium bromide, perazine, procinolol, procinonide, proclonol, procodazole, ritroSulfan, robenidine, rocastine, rociverine, rodocaine, procyclidine, procymate, prodeconium bromide, prodili rodorubicin, rofelodine, roflurante, rokitamycin, roletamide, dine, prodipine, prodolic acid, profadol, profeXalone, profla rolgamidine, rolicyclidine, rolicyprine, rolipram, rollitetra Vine, proflazepam, progabide, progesterone, proglumetacin, cycline, rolodine, rolziracetam, romifenone, romifidine, proglumide, proheptazine, proligestone, proline, prolintane, ronactolol, ronidazole, ronifibrate, ronipamil, ronnel, ropi prolonium iodide, promazine, promegestone, promestriene, toin, ropivacaine, ropizine, roquinimex, rosaprostol, rosa promethazine, promolate, promoxolane, pronetalol, propac ramicin, rosaramicin butyrate, rosaramicin propionate, etamol, propafenone, propamidine, propanidid, pro roSoxacin, rosterolone, rotamicillin, rotoxamine, rotraxate, panocaine, propantheline bromide, proparacaine, propatyl roXarSone, roXatidine acetate, roXibolone, roXindole, nitrate, propazolamide, propendiazole, propentofylline, pro roXithromycin, roXolonium metilsulfate, roXoperone, penZolate, properidine, propetamide, propetandrol, propicil rufloxacin, rutamycin, rutin, ruvaZone, Sabeluzole, Saccha lin, propikacin, propinetidine, propiolactone, propiomazine, rin, Salacetamide, Salafibrate, Salantel, Salazodine, SalazOS propipocaine, propiram, propisergide, propiverine, Sulfadimedine, SalazOSulfamide, SalazOSulfathiazole, Sale propizepine, propofol, propoXate, propoxycaine, pro thamide, Salfluverine, Salicin, Salicyl alcohol, Salicylamide, poxyphene, propranolol, propyl docetrizoate, propylene gly Salicylanilide, Salicylic acid, Salinazid, Salinomycin, Salme col, propylene glycol monoStearate, propyl gallate, propyl fanol, Salmeterol, Salmisteine, Salprotoside, Salsalate, Salver hexedrine, propyliodone, propylparaben, propylthiouracil, ine, Sancycline, Sangivamycin, Saperconazole, Sarcolysin, propyperone, propyphenaZone, propyromazine bromide, Sarmazenil, Sarmoxicillin, Sarpicillin, Saterinone, Satranida produaZone, produinolate, prorenoate potassium, proroXan, Zole, SaVOXepin, Scarlet red, Scopafungin, Scopolamine, proscillaridin, prospidium chloride, prostalene, proSulpride, SeclaZone, Secnidazole, Secobarbital, Secoverine, Securinine, proSultiamine, proterguride, protheobromine, prothipendyl, Sedecamycin, SeganSerin, Seglitide, Selegiline, Selenium Sul prothixene, protiofate, protionamide, protirelin, protizinic fide, Selprazine, Sematilide, Semustine, Sepazonium chloride, acid, protokylol, protoveratine, protriptyline, proxazole, Seperidol, Sequifenadine, Serfibrate, Sergolexole, Serine, Ser proXibarbal, proxibutene, proXicromil, proxifeZone, proXor metacin, Serotonin, Sertaconazole, Sertraline, Setastine, Set phan, proxyphylline, proZapine, pseudoephedrine, psilocy aZindol, Setiptiline, Setoperone, Sevitropium mesilate, SeVof bine, pumiteba, puromycin, pyrabrom, pyran copolymer, lurane, Sevopramide, Siagoside, Sibutramine, Siccanin, pyrantel, pyrathiazine, pyrazinamide, pyrazofurin, pyricar Silandrone, Silibinin, Silicristin, Silidianin, Silver Sulfadiaz bate, pyridarone, pyridofylline, pyridoStigmine bromide, ine, Simetride, Simfibrate, SimtraZene, Simvastatin, Sinefun pyridoxine, pyrilamine, pyrimethamine, pyrimitate, pyrino gin, Sintropium bromide, Sisomicin, Sitalidone, Sitofibrate, line, pyrithione Zinc, pyrithyldione, pyritidium bromide, sitogluSide, Sodium benzoate, Sodium dibunate, Sodium pyritinol, pyronine, pyrophenindane, pyrovalerone, pyrox ethasulfate, Sodium formaldehyde Sulfoxylate, Sodium gen amine, pyrrobutamine, pyrrocaine, pyrroliphene, pyrrolni tisate, Sodium gualenate, Sodium nitrite, Sodium nitroprus trin, pyrvinium chloride, pytamine, quadazocine, quadrosi Side, Sodium oxybate, Sodium phenylacetate, Sodium pico lan, quatacaine, quazepam, quaZinone, quaZodine, fosfate, Sodium picosulfate, Sodium propionate, Sodium quaZolast, quifenadine, quillifoline, quinacainol, quinacillin, Stibocaptate, Sodium Stibogluconate, Sodium tetradecyl Sul quinacrine, quinaldine blue, quinapril, quinaprilat, fate, Sodium thiosulfate, Sofalcone, Solasulfone, Solpecainol, quinaZosin, quinbolone, quincarbate, quindecamine, quin Solypertine, Somantadine, Sopitazine, Sopromidine, Soqui donium bromide, quindoxin, quineStradol, quinestrol, nolol, Sorbic acid, Sorbinicate, Sorbinil, Sorbitan monolau quinethaZone, quinetolate, quine Zamide, quinfamide, rate, Sorbitan monooleate, Sorbitan monopalmitate, Sorbitan quingestanol acetate, quingestrone, quindine, quinine, monoStearate, Sorbitan trioleate, Sorbitan tristearate, Sorbi quinocide, quinpirole, quinterenol, quintiofoS, quinuclium tol, Sorndipine, Sotalol, Soterenol, Spaglumic acid, Sparfosic bromide, quinupramine, quipazine, quisultazine, race fem acid, SparSomycin, Sparteine, Spectinomycin, Spiclamine, ine, racemethionine, racemethorphan, racemetirosine, raclo Spiclomazine, Spiperone, Spiradoline, Spiramide, Spiramy pride, ractopamine, rafoxanide, ralitoline, raloxifene, ram cin, Spirapril, Spiraprilat, Spirendolol, Spirgetine, Spirilene, ciclane, ramefenaZone, ramipril, ramiprilat, ramixotidine, Spirofylline, Spirogermanium, Spiromustine, Spironolactone, ramnodignin, ranimustine, ranimycin, ranitidine, ranolazine, Spiroplatin, Spirorenone, Spirotriazine, SpiroXasone, SpiroX rathyronine, raZinodil, raZobazam, razoxane, reboxetine, atrine, SpiroXepin, Spizofurone, Stallimycin, Stanolone, Stan US 2002/0102215 A1 Aug. 1, 2002

Zolol, Stearic acid, Stearyl alcohol, Stearylsulfamide, Steffi testosterone enanthate, testosterone ketolaurate, testosterone mycin, Stenbolone acetate, Stepronin, Stercuronium iodide, phenylacetate, testosterone propionate, tetrabarbital, tetra Stevaladil, Stibamine glucoside, Stibophen, Stilbamidine, Stil benazine, tetracaine, tetrachloroethylene, tetracycline, tetra bazium iodide, Stilonium iodide, Stirimazole, Stiripentol, donium bromide, tetraethylammonium chloride, tetrahydro Stirocainide, Stirifos, Streptomycin, StreptonicOZid, Streptoni Zoline, tetramethrin, tetramisole, tetrandrine, tetrantoin, grin, Streptovarycin, Streptozocin, Strinoline, Strychnine, tetrazepam, tetriprofen, tetronasin 5930, tetroquinone, Styramate, Subathizone, Subendazole, Succimer, Succinyl tetroXoprim, tetrydamine, texacromil, thalicarpine, thalido choline chloride, Succinylsulfathiazole, Succisulfone, mide, thebacon, thebaine, thenalidine, thenium closylate, Suclofenide, Sucralfate, Sucrose octaacetate, SudeXanox, thenyldiamine, theobromine, theodrenaline, theofibrate, Sudoxicam, Sufentanil, Sufosfamide, Sufotidine, Sulazepam, theophylline, thiabendazole, thiacetarSamide, thialbarbital, Sulbactam, Sulbactam pivoxil, Sulbenicillin, Sulbenox, Sul thiambutosine, thiamine, thiamiprine, thiamphenicol, thia bentine, Sulbutiamine, Sulclamide, Sulconazole, Sulfabenz, mylal, thiaZeSim, thiazinamium chloride, thiazolsulfone, thi Sulfabenzamide, Sulfacarbamide, Sulfacecole, Sulfaceta ethyperazine, thihexinol methylbromide, thimerfonate, mide, Sulfachlorpyridazine, Sulfachrysoidine, Sulfaclomide, thimerosal, thiocarbanidin, thiocarZolamide, thiocolchio Sulfaclorazole, Sulfaclozine, Sulfacytine, Sulfadiazine, Sul Side, thiofuradene, thioguanine, thioguanine alpha-deoxyri fadicramide, Sulfadimethoxine, Sulfadoxine, Sulfaethidole, boside, thioguanine beta-deoxyriboside, thioguanosine, Sulfaguandide, Sulfaguanole, Sulfalene,Sulfaloxic acid, thiohexamide, thioinosine, thiopental, thiopropazate, thio SulfamaZone, Sulfamerazine, Sulfameter, Sulfamethazine, properazine, thioridazine, thiosalan, thiotepa, thiotetrabar Sulfamethizole, Sulfamethoxazole, Sulfamethoxypyridazine, bital, thiothixene, thiouracil, thiphenamil, thiphencillin, Sulfamethoxypyridazine acetyl, Sulfametomidine, Sulfame thiram, thonzonium bromide, thonZylamine, thozalinone, trole, Sulfamonomethoxine, Sulfamoxole, Sulfanil amide, threonine, thymidine, thymol, thymol iodide, thymopentin, Sulfanitran, Sulfaperin,Sulfaphenazole, Sulfaproxyline, Sul thyromedan, thyropropic acid, tiacrilast, tiadenol, tiafibrate, fapyridine, Sulfacquinoxaline, SulfarSphenamine, SulfaSala tiamenidine, tiametonium iodide, tiamulin, tianafac, tianep Zine, SulfaSomizole, SulfaSuccinamide, Sulfasymazine, Sul tine, tiapamil, tiapirinol, tiapride, tiaprofenic acid, tiaprost, fathiazole, Sulfathiourea, Sulfatolamide, Sulfatroxazole, tiaramide, tiazofurin, tiaZuril, tibalosin, tibenalast Sodium, Sulfatrozole, Sulfazamet, Sulfinalol, Sulfinpyrazone, Sulfiram, tibenzate, tibeZonium iodide, tibolone, tibric acid, tibrofan, Sulfisomidine, Sulfisoxazole, Sulfisoxazole, Sulfobromoph tic-mustard, ticabesone propionate, ticarbodine, ticarcillin, thalein, Sulfonethylmethane, Sulfonmethane, Sulfonterol, ticarcillin creSyl, ticlatone, ticlopidine, ticrynafen, tidiacic, Sulforidazine, Sulfoxone Sodium, Sullicrinat, Sulindac, Suli tiemoium iodide, tienocarbine, tienopramine, tienoXolol, Satin, Sulisobenzone, Sulmarin, Sulmazole, Sulmepride, tifemoxone, tiflamizole, tiflorex, tifluadom, tiflucarbine, Sulinidazole, Sulocarbilate, Suloctidil, Sulosemide, tiformin, tifurac, tigemonam, tigestol, tigloidine, tilbro Sulotroban, Suloxifen, Sulpiride, Sulprosal, Sulprostone, Sul quinol, tiletamine, tilidine, tiliquinol, tilisolol, tilmicosin, tamicillin, Sulthiame, Sultopride, Sultosilic acid, Sultropo tilomisole, tillorone, tilozepine, tilsuprost, timefurone, tim nium, Sulverapride, Sumacetamol, Sumatriptan, Sumetizide, egadine, timelotem, timepidium bromide, timiperone, Sunagrel, Suncillin, Supidimide, Suproclone, Suprofen, timobesone acetate, timofibrate, timolol, timonacic, timo Suramin, Suricainide, Suriclone, Suxemerid, SuXethonium prazole, tinabinol, tinazoline, tinidazole, tinisulpride, chloride, SuxibuZone, Symclosene, Symetine, Synephrine, tinofedrine, tinoridine, tiocarlide, tioclomarol, tioconazole, Syrisingopine, taclamine, tacrine, taglutimide, talampicillin, tioctilate, tiodaZosin, tiodonium chloride, tiomergine, talastine, talbutal, taleranol, talinolol, talipexole, tallisomy tiomesterone, tioperidone, tiopinac, tiopronin, tiopropamine, cin, talmetacin, talmetoprim, talniflumate, talopram, talos tioSpirone, tiotidine, tioxacin, tioXamast, tioxaprofen, tioxi alate, taloximine, talsupram, taltrimide, tameridone, tameti dazole, tioxolone, tipentosin, tipepidine, tipetropium bro cillin, tametraline, tamitinol, tamoxipen, tampramine, mide, tipindole, tipredane, tiprenolol, tiprinast, tipropidil, tandamine, taprostene, tartaric acid, tasuldine, taurocholic tiproStanide, tiprotimod, tiquinamide, tiquizium bromide, acid, taurolidine, tauromustine, tauroSelcholic acid, taurul tiratricol, tiropramide, tisocromide, tisopurine, tisoquone, tam, taxol, taZadolene, taZanolast, tazaburate, taZeprofen, tivandizole, tiXadil, tiXanox, tiXocortol pivalate, tizabrin, taZifylline, taZiprinone, tazolol, tebatizole, tebuduine, tianidine, tizolemide, tizoprolic acid, tobramycin, tobuterol, teclothiazide, teclozan, tedisamil, tefazoline, tefenperate, tocainide, tocamphyl, tocofenoxate, tocofibrate, tocopher tefludazine, teflurane, teflutiXol, tegafur, telenzepine, tema Solan, todralazine, tofenacin, tofetridine, tofisoline, tofiso floxacin, temarotene, temazepam, temefoS, temelastine, pam, tolamolol, tolaZamide, tolazoline, tolboxane, tolbuta temocillin, temodox, temozolomide, temurtide, tenamfe mide, tolciclate, toldimfoS, tolfamide, tolfenamic acid, tamine, tenilapine, teniloxazine, tenilsetam, teniposide, tolgabide, tolimidone, tolindate, toliodium chloride, tolip tenocyclidine, tenonitrozole, tenoxicam, tenylidone, teop rolol, tolmeSoxide, tolmetin, tolnaftate, tolnapersine, ranitol, teoprolol, tepirindole, tepoxalin, teraZosin, terbin tolnidamine, toloconium metilsulfate, tolonidine, tolonium afine, terbucromil, terbufibrol, terbuficin, terbuprol, terbuta chloride, toloxatone, toloxychlorinol, tolpadol, tolpentam line, terciprazine, terconazole, terfenadine, terfluranol, ide, tolperisone, toliprazole, tolpronine, tolpropamine, terguride, terizidone, ternidazole, terodiline, terofenamate, tolpyrramide, tolduinzole, tolrestat, toltraZuril, tolu fazepam, teroxalene, teroxirone, terpin hydrate, tertatolol, teSicam, tolycaine, tomelukast, tomoglumide, tomoxetine, tomox tesimide, testolactone, testosterone, testosterone cypionate, iprole, tonazocine, topiramate, toprilidine, tonazocine, topi US 2002/0102215 A1 Aug. 1, 2002 34 ramate, toprilidine, topterone, toguizine, toraSemide, toe cantril, VincofoS, Vinconate, Vincristine, Vindrburnol, Vin bafylline, toremifene, toSifen, toSufloxacin, toSulur, desine, Vindepidine, Vinformide, Vinglycinate, Vinorelbine, toyocamycin, toyomycin, traboxepine, tracazolate, tral Vinpocetine, Vinpoline, Vinrosidine, Vintiamol, Vintriptol, onide, tramadol, tramaZoline, trandolapril, tranexamic acid, Vinylbital, Vinylether, Vinzolidine, Viomycin, Viprostol, tranilast, transcainide, trantelinium bromide, tranylcyprom Vicqualine, Vicquidil, Virginiamycin factors, ViroXime, Visna ine, trapencaine, trapidil, traXanox, trazilitine, traZium eSi dine, Visnafylline, Vitamine, volazocine, warfarin, Xamot late, traZOdone, traZolopride, trebenzomine, trecadrine, tre erol, Xanoxic acid, Xanthinol niacinate, Xanthiol, Xantifi loxinate, trenbolone acetate, trengestone, trenizine, brate, Xantocillin, Xenalipin, Xenazoic acid, Xenbucin, troSulfan, trepibutone, trepipam, trepirium iodide, treptil Xenipentone, Xenthiorate, Xenygloxal, Xenyhexenic acid, amine, trequensin, trestolone acetate, trethinium tosilate, Xeny tropium bromide, Xibenolol, Xibornol, Xilobam, Ximo trethocanoic acid, tretinoin, tretoquinol, triacetin, triafungin, profen, Xinidamine, Xinomiline, Xipamide, Xipranolol, Xor triamcinolone, triamcinolone acetonide, triamcinolone phanol, Xylamidine, Xylazine, Xylocoumarol, Xylometazo acetonide-phosphate, triamcinolone benetonide, triamcino line, Xyloxemine, yohimbic acid, Zabicipril, Zacopride, lone diacetate, triamcinolone furetonide, triamcinolone Zafuleptine, Zaltidine, Zapizolam, Zaprinast, Zardaverine, hexacetonide, triampyZine, triamterene, triaZinate, triazi Zenazocine meSylate, Zepastine, Zeranol, Zetidoline, Zidapa quone, triazolam, tribendilol, tribenoside, tribromoethanol, mide, Zidometacin, Zidovudine, Zilantel, Zimeldine, Zimido tribromsalan, tribuZone, triacetamide, trichlormethiazide, ben, Zinc acetate, Zinc phenolsulfonate, Zinc undecylenate, trichlormethine, trichloroacetic acid, trichloroethylene, tric Zindotrine, Zindoxifene, Zinoconazole, Zinterol, ZinviroXime, ribine phosphate, triclabendazole, triclacetamol, triclazate, Zipeprol, Zocainone, Zofenopril, Zoficonazole, Zolamine, triclobisonicum chloride, triclocarban, triclodaZol, tri Zolazepam, Zolenzepine, Zolertine, Zolimidine, Zoliprofen, clofenol, piperazine, triclofoS, triclofylline, triclonide, tri Zoloperone, Zolpidem, Zomebazam, Zomepirac, Zometapine, closan, tricyclamol chloride, tridiheXethyl chloride, trien Zonisamide, Zopiclone, Zorubicin, Zotepine, ZOxazolamine, tine, triethylenemelamine, triethylenephosphoramide, Zuclomiphene, Zuclophenthixol, Zylofuramine. trifenagrel, trifeZolac, triflocin, triflubazam, triflumidate, tri 0147 The following non-limitative examples serve to fluomeprazine, trifluoperazine, trifluperidol, triflupro illustrate the invention. Confirmation of the microparticulate mazine, trifluridine, triflusal, trigeVolol, trihexyphenidyl, nature of products is performed using microScopy as triletide, triloStane, trimaZoSin, trimebutine, trimecaine, tri described in WO-A-9607434. Ultrasonic transmission mea medoxime bromide, trimeperidine, trimeprazine, trimetazi Surements may be made using a broadband transducer to dine, trimethadione, trimethamide, trimethaphan camsylate, indicate microbubble Suspensions giving an increased Sound trimethidinium methoSulfate, trimethobenzamide, trimetho beam attenuation compared to a Standard. Flow cytometric prim, trimetozine, trimetrexate, trimeXiline, trimipramine, analysis of products can be used to confirm attachment of trimoprostil, trimoxamine, trioxifene, trioxSalen, tripamide, macromolecules thereto. The ability of targeted triparanol, tripelennamine, tripotassium dicitratobismuthate, microbubbles to bind Specifically to cells expressing a target may be Studied in vitro by microScopy and/or using a flow triprolidine, tritioZine, tritoqualine, trityl cysteine, trixolane, chamber containing immobilised cells, for example employ trizoxime, trocimine, troclosene potassium, trofosfamide, ing a population of cells expressing the target Structure and troleandomycin, trolnitrate, tromantadine, tromethamine, a further population of cells not expressing the target. tropabazate, tropanserin, tropapride, tropatepine, tropen Radioactive, fluorescent or enzyme-labelled Streptavidin/ Ziline bromide, tropicamide, tropigline, tropiprine, tropod avidin may be used to analyse biotin attachment. ifene, trospectomycin, troSpium chloride, troXerutin, troXip ide, troXolamide, troXonium tosilate, troxypyrrolium EXAMPLE 1. tosilate, troxypyrrolium tosilate, truXicurium iodide, truXi picurium iodide, tryparsamide, tryptophan, tryptophane Adhesion of Poly-L-lysine-coated mustard, tuaminoheptane, tubercidine, tubocurarine chlo Phosphatidylserine-encapsulated Microbubbles to ride, tubulozole, tuclazepam, tulobutrol, tuVatidine, tybam Endothelial Cells ate, tylocrebin, tylosin, tyramine, tyropanic acid, tyrosine, 0148 Poly-L-lysine (8 mg) having a molecular weight of ubenimex, ubidecarenone, ubisindine, ufenamate, ufipra 115 kDa was dissolved in water (400 ul). Freshly redispersed Zole, uldazepam, ulobetaSol, undecoylium chloride, unde microbubbles of phosphatidylserine-encapsulated perfluo cyclenic acid, uracil mustard, urapidil, urea, uredepa, ure robutane (40 ul) were incubated in either water (400 All) or dofoS, urefibrate, urethane, uridine, urSodeoxycholic acid, the poly-L-lysine Solution for 15 minutes at room tempera urSucholic acid, Vadocaine, Valconazole, Valdetamide, Val ture. Zeta potential measurements confirmed that the poly dipromide, Valine, Valnoctamide, Valofane, Valperinol, Val L-lysine-coated microbubbles were positively charged while proate pivoxil, Valproic acid, Valpromide, Valtrate, Vanco the uncoated bubbles were negatively charged. A cell adhe Sion Study using human endothelial cells grown in culture mycin hcl, Vaneprim, Vanillin, Vanitolide, Vanyldisulfamide, dishes was performed with the above-described Vapi.prost, vecuronium bromide, Velnacrine maleate, ven microbubbles, the uncoated microbubbles being used as a lafaxine, Veradoline, Veralipride, Verapamil, Verazide, Ver control. Microscopy of the endothelial cells after incubation ilopam, Verofylline, Vesnarinone, Vetrabutine, Vidarabine, showed a much increased number of poly-L-lysine-coated Vidarabine phophate, vigabatrin, Viloxazine, Viminol, Vin microbubbles adhering to endothelial cells in comparison to barbital, vinblastine, Vinburnine, Vincamine, Vincanol, Vin the uncoated microbubbles. US 2002/0102215 A1 Aug. 1, 2002 35

EXAMPLE 2 45 Seconds, whereafter the Sample is put on a roller table. After centrifugation the infranatant is exchanged with 0.1M Gas-filled Microbubbles Comprising Sodium phosphate buffer having a pH of 7.5. The peptide Phosiphatidylserine and RGDC-Mal-PEGo-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 0149 a) Synthesis of Boc-NH-PEG3400-DSPE (t-butyl Washing procedure is then repeated. Carbamate Poly(Ethylene Glycol) Distearoylphosphatidyle thanolamine) 0159) f) Alternative Preparation of Gas-Filled 0150 DSPE (distearoylphosphatidylethanolamine) (31 Microbubbles Encapsulated by Phosphatidylserine and mg, Sygena Inc.) was added to a Solution of Boc-NH RGDC-Mal-PEG-DSPE (SEQ ID NO:1) PEG3400-SC (t-butyl carbamate poly(ethylene glycol)-suc 0160 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-PEGoo NMR. DSPE (SEQID NO: 1) dissolved in 0.1 M sodium phosphate buffer having a pH of 7.5 is added to the washed 0151 b) Synthesis of HN-PEGoo-DSPE (Amino-poly microbubbles, which are then placed on the roller table. The (Ethylene Glycol)-distearoylphosphatidylethanolamine) Washing procedure is repeated following incorporation of 0152 Boc-NH-PEGo-DSPE (167 mg) was stirred in 4 the RGDC-Mal-PEGo-DSPE (SEQ ID NO:1) into the M hydrochloric acid in dioxane (5 ml) for 2.5 hours at microbubble membranes. ambient temperature. The solvent was removed by rotary evaporation and the residue was taken up in chloroform (1.5 EXAMPLE 3 ml) and washed with water (2x1.5 ml). The organic phase was removed by rotary evaporation. TLC (chloroform/ Gas-Filled Microbubbles Encapsulated With methanol/water 13:5:0.8) gave the title product with Rf=0.6; Phosphatidylserine, Phosphatidylcholine and the Structure of the product, which was ninhydrin positive, Biotin-Amidocaproate-PEGoo-Ala-Cholesterol was confirmed by NMR. 0153 c) Synthesis of Mal-PEGo-DSPE (3-maleimi 0161 a) Synthesis of Z-Ala-Cholesterol (3-O-(Car dopropionate Poly(Ethylene Glycol)distearoyldhosphati bobenzyloxy-L-Alanyl)Cholesterol) dylethanolamine) 0162 Cholesterol (4 mmol), Z-alanine (5 mmol) and dimethylaminopyridine (4 mmol) were dissolved in dimeth 0154) A solution of N-Succinimidyl-3-maleimidopropi ylformamide/tetrahydrofuran (20 ml+5 ml) and dicyclo onate (5.6 mg, 0.018 mmol) in tetrahydrofuran (0.2 ml) is hexylcarbodiimide was added. The reaction mixture was added to HN-PEGoo-DSPE (65 mg, 0.012 mmol) dis Stirred at ambient temperature overnight. Dicyclohexylurea solved in tetrahydrofuran (1 ml) and 0.1 M sodium phos was filtered off and the solvent was rotary evaporated. The phate buffer pH 7.5 (2 ml). The reaction mixture is heated to residue was taken up in chloroform, undissolved dicyclo 30° C. and the reaction is followed to completion by TLC, hexylurea was filtered off and the solvent was removed by whereafter the Solvent is evaporated. rotary evaporation. The residue was placed on a column of O155 d) Synthesis of RGDC-Mal-PEG3400-DSPE (SEQ Silica gel, and Z-Ala-cholesterol was eluted with toluene/ ID NO:1) petroleum ether (20:2) followed by toluene/diethyl ether (20:2). The fractions containing the title compound were 0156) Mal-PEG3-DSPE (0.010 mmol) in 0.1 M combined and the Solvent was removed by rotary evapora Sodium phosphate buffer having a pH of 7.5 is added to the tion. The structure of the product was confirmed by NMR. peptide RGDC (SEQ ID NO:1) (0.010 mmol). The reaction mixture is heated to 37 C. if necessary and the reaction is 0163 b) Synthesis of Ala-Cholesterol (3-O-(L-Alan followed by TLC to completion, whereafter the solvent is yl)Cholesterol) removed. 0164 Z-Ala-cholesterol (0.48 mmol) is placed in tetrahy O157 e) Preparation of Gas-Filled Microbubbles Encap drofuran (20 ml) and glacial acetic acid (3 ml) and hydro sulated by Phosphatidylserine and RGDC-Mal-PEG genated in the presence of 5% palladium on charcoal for 2 DSPE (SEQ ID NO:1) hours. The reaction mixture is filtered and concentrated in 0158 To a mixture (5 mg) of phosphatidylserine (90-99.9 WCUO. mol %) and Mal-PEGo-DSPE (10-0.1 mol %) is added 0165) c) Synthesis of Boc-NH-PEG3400-Ala-Choles 5% propylene glycol-glycerol in water (1 ml). The disper terol sion is heated to not more than 80 C. for 5 minutes and then cooled to ambient temperature. The dispersion (0.8 ml) is 0166 Ala-cholesterol is added to a solution of Boc-NH then transferred to a vial (1 ml) and the head space is flushed PEGoo-SC (t-butyl carbamate poly(ethylene glycol)Suc with perfluorobutane. The Vial is Shaken in a cap-mixer for cinimidyl carbonate) in chloroform, followed by triethy US 2002/0102215 A1 Aug. 1, 2002 36 lamine. The suspension is stirred at 41 oO 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 0167 d) Synthesis of HN-PEG3400-Ala-Cholesterol tered off and the solvent is rotary evaporated. The title 0168 Boc-NH-PEGoo-Ala-cholesterol is stirred in 4M compound is purified by chromatography. hydrochloric acid in dioxane for 2.5 hours at ambient 0177 b) Preparation of Gas-Filled Microbubbles Encap temperature. The Solvent is removed by rotary evaporation sulated With Phosphatidylserine, Phosphatidylcholine, and the residue is taken up in chloroform and washed with water. The organic phase is rotary evaporated to dryness. Biotinamidocaproate-PEGoo-Ala-Cholesterol and Drug The crude product may be purified by chromatography. Cholesterol 0178 To a mixture (5 mg) of phosphatidylserine and 0169 e) Synthesis of Biotinamidocaproate-PEG3400 phosphatidylcholine (in total 90-99.9mol %) and biotinami Ala-Cholesterol docaproate-PEG3400-Ala-cholesterol (prepared as in 0170 A solution of biotinamidocaproate N-hydroxysuc Example 3) and drug-cholesterol (in total 10-0.31 mol%) is cinimide ester in tetrahydrofuran is added to HN-PEGoo added 5% propylene glycol-glycerol in water (1 ml). The Ala-cholesterol dissolved in tetrahydrofuran and 0.1 M dispersion is heated to not more than 80 C. for 5 minutes sodium phosphate buffer having a pH of 7.5 (2 ml). The and then cooled to ambient temperature. The dispersion (0.8 reaction mixture is heated to 30° C. and the reaction is ml) is transferred to a vial (1 ml) and the head space is followed to completion by TLC, whereafter the solvent is flushed with perfluorobutane. The vial is shaken in a cap evaporated. mixer for 45 Seconds whereafter the Sample is put on a roller 0171 f) Preparation of Gas-Filled Microbubbles Encap table. After centrifugation the infranatant is exchanged with sulated With Phosphatidylserine, Phosphatidylcholine and water and the Washing is repeated. Biotinamidocaproate-PEGoo-Ala-Cholesterol EXAMPLE 5 0172 To a mixture (5 mg) of phosphatidylserine and Gas-Filled Microbubbles Encapsulated With phosphatidylcholine (in total 90-99.9 mole) and biotinami Phosphatidylserine and docaproate-PEGoo-Ala-cholesterol (10-0.1 mole) is added 5% propylene glycol-glycerol in water (1 ml). The disper Thiolated-anti-CD34-Mal-PEGo-DSPE sion is heated to not more than 80 C. for 5 minutes and then 0179 a) Preparation of Thiolated Anti-CD34 Antibodies cooled to ambient temperature. The dispersion (0.8 ml) is 0180. Thiolation of anti-CD34 antibodies may be then transferred to a vial (1 ml) and the head space is flushed effected as described by Hansen, C. B. et al. (1995) Biochim. with perfluorobutane. The Vial is Shaken in a cap-mixer for Biophys. Acta 1239, 133-144. 45 Seconds, whereafter the Sample is put on a roller table. After centrifugation the infranatant is exchanged with water 0181 b) Preparation of Gas-Filled Microbubbles Encap and the Washing is repeated. sulated With Phosphatidylserine and Thiolated-anti-CD34 Mal-PEGo-DSPE 0173 g) Alternative Preparation of Gas-Filled Microbubbles Encapsulated With Phosphatidylserine, Phos 0182 To a mixture (5 mg) of phosphatidylserine (90 phatidylcholine and Biotinamidocaproate-PEGoo-Ala 99.9mol %) and Mal-PEGoo-DSPE (10-0.1 mol %, pre pared as in Example 2) is added 5% propylene glycol Cholesterol glycerol in water (1 ml). The dispersion is heated to not more 0174) To a mixture (5 mg) of phosphatidylserine and than 80 C. for 5 minutes and then cooled to ambient phosphatidylcholine is added 5% propylene glycol-glycerol temperature. The dispersion (0.8 ml) is transferred to a vial in water (1 ml). The dispersion is heated to not more than (1 ml) and the head space is flushed with perfluorobutane. 80 C. for 5 minutes and then cooled to ambient temperature. The Vial is shaken in a cap-mixer for 45 Seconds, whereafter The dispersion (0.8 ml) is then transferred to a vial (1 ml) the Sample is put on a roller table. After centrifugation the and the head space is flushed with perfluorobutane. The vial infranatant is exchanged with an appropriate buffer and is shaken in a cap-mixer for 45 Seconds, whereafter the coupling of the thiolated antibody to the microbubbles is Sample is put on a roller table. After centrifugation the performed, e.g. as described by Goundalkar, A., Ghose, T. infranatant is exchanged with water. Biotinamidocaproate and Mezei, M. in J. Pharm. Pharmacol. (1984)36465-66 or PEGoo-Ala-cholesterol dissolved in water is added to the Hansen, C. B. et al.(1995) Biochim. Biophys. Acta 1239 washed microbubbles, which are placed on a roller table for 133-144. The microbubbles are then placed on a roller table Several hours. The Washing procedure is repeated following for Several hours and are washed. Flow cytometric analysis incorporation of the biotinamidocaproate-PEG-Ala-cho of the resulting microbubbles (employing a fluorescently lesterol into the microbubble membranes. labeled secondary antibody) is used to confirm attachment of the anti-CD34 antibody to the bubbles. The ability of the EXAMPLE 4 bubbles to bind specifically to CD34-expressing cells is Studied by microScopy employing one population of cells Gas-Filled Microbubbles Comprising Phosphati dylserine, Phosphatidylcholine, Biotin-Amidoca expressing CD34 and one population that do not express proate-PEGoo-Ala-Cholesterol and Drug-Choles CD34. terol EXAMPLE 6 0175) a) Synthesis of Drug-Cholesterol Biotin Attached to Gas-Filled Microbubbles 0176 Cholesterol (4 mmol), a drug having an acid group 0183 Biotin may be attached to microbubbles in many and dimethylaminopyridine (4 mmol) are dissolved in dim different ways, e.g. in a similar way to that described by US 2002/0102215 A1 Aug. 1, 2002 37

Corley, P. and Loughrey, H. C. in (1994) Biochim. BiophyS. placed on a roller table during the reaction. After centrifu Acta 1195, 149-156. The resulting bubbles are analysed by gation the infranatant is exchanged with water and the flow cytometry, e.g. by employing fluorescent Streptavidin Washing is repeated. The functionality of the attached to detect attachment of biotin to the bubbles. Alternatively Streptavidin is analysed by binding, e.g. to fluorescently radioactive or enzyme-labelled Streptavidin/avidin is used to labeled biotin, biotinylated antibodies (detected with a fluo analyse biotin attachment. rescently labeled Secondary antibody) or biotinylated and fluorescence- or radioactively-labeled oligonucleotides. EXAMPLE 7 Analysis is performed by fluorescence microscopy or Scin tillation counting. Gas-Filled Microbubbles Encapsulated With 0191 d) Preparation of Gas-Filled Microbubbles Encap Distearoylphosphatidylserine and Biotin-DPPE sulated With Phosphatidylserine and Biotin Non-Covalently 0184) To distearoylphosphatidylserine (DSPS) (22.6 mg) Bound to Streptavidin-Succ-PEG3400-DSPE was added 4% propylene glycol-glycerol in water (4 ml). The dispersion was heated to not more than 80 C. for five 0192 Microbubbles from Example 8(c) are incubated in minutes and then cooled to ambient temperature. An acque 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 Gas-Filled Microbubbles Encapsulated With put on a roller table. After centrifugation for 7 minutes the Phosphatidylserine and Biotinylated infranatant was exchanged with water and the Washing was Oligonucleotide Non-Covalently Bound to repeated twice. Normal phase HPLC with an Evaporative Streptavidin-Succ-PEG-DSPE Light Scattering Detector confirmed that the membranes of the microbubbles contained 4 mol % biotin-DPPE. The 0193 a) Synthesis of Succ-PEG-DSPE mean particle diameter of the microbubbles was 4 um measured by Coulter Counter. Ultrasound transmission mea 0194 NH-PEG,00-DSPE (prepared as in Example 2) is surements using a 3.5 MHz broadband transducer showed carboxylated using Succinic anhydride, e.g. by a similar that a particle dispersion of <2 mg/ml gave a Sound beam method to that described by Nayar, R. and Schroit, A.J. in attenuation higher than 5 dB/cm. Biochemistry (1985) 24, 5967-71. 0.195 b) Preparation of Gas-Filled Microbubbles Encap EXAMPLE 8 sulated With Phosphatidylserine and Succ-PEGo-DSPE Gas-Filled Microbubbles Encapsulated With 0196) To a mixture (5 mg) of phosphatidylserine (90-99.9 Phosphatidylserine and Biotinylated Antibody mol %) and Succ-PEGo-DSPE (10-0.1 mol %) is added Non-covalently Bound to 5% propylene glycol-glycerol in water (1 ml). The disper Streptavidin-Succ-PEG-DSPE sion is heated to not more than 80 C. for 5 minutes and then cooled to ambient temperature. The dispersion (0.8 ml) is 0185) a) Synthesis of Succ-PEGo-DSPE transferred to a vial (1 ml) and the head space is flushed with perfluorobutane. The vial is shaken in a cap-mixer for 45 0186 NH-PEG,00-DSPE (prepared as in Example 2) is Seconds, whereafter the Sample is put on a roller table. After carboxylated using Succinic anhydride, e.g. by a similar centrifugation the infranatant is exchanged with water and method to that described by Nayar, R. and Schroit, A.J. in the Washing is repeated. Alternatively the microbubbles may Biochemistry (1985) 24, 59G7-71. be prepared as described in Example 20f). 0187 b) Preparation of Gas-Filled Microbubbles Encap 0197) c) Coupling of Streptavidin to Gas-Filled sulated With Phosphatidylserine and Succ-PEGo-DSPE Microbubbles Encapsulated With Phosphatidylserine and 0188 To a mixture (5 mg) of phosphatidylserine (90-99.9 Succ-PEGo-DSPE mol %) and Succ-PEGoo-DSPE (10-0.1 mol %) is added 0198 Streptavidin is covalently bound to Succ-PEG 5% propylene glycol-glycerol in water (1 ml). The disper DSPE in the microbubble membraness by standard coupling sion is heated to not more than 80 C. for 5 minutes and then methods using a water-Soluble carbodiimide. The sample is coooled to ambient temperature. The dispersion (0.8 ml) is placed on a roller table during the reaction. After centrifu transferred to a vial (1 ml) and the head space is flushed with gation the infranatant is exchanged with water and the perfluorobutane. The vial is shaken in a cap-mixer for 45 Washing is repeated. The functionality of the attached Seconds, whereafter the Sample is put on a roller table. After Streptavidin is analyzed by binding, e.g. to fluorescently centrifugation the infranatant is exchanged with water and labeled biotin, biotinylated antibodies (detected with a fluo the Washing is repeated. Alternatively the microbubbles may rescently labeled Secondary antibody) or biotinylated and be prepared as described in Example 20f). fluorescence- or radioactively-labeled oligonucleotides. 0189 c) Coupling of Streptavidin to Gas-Filled Analysis is performed by fluorescence microscopy or Scin Microbubbles Encapsulated With Phosphatidylserine and tillation counting. Succ-PEGo-DSPE 0199 d) Preparation of Gas-Filled Microbubbles Encap 0.190 Streptavidin is covalently bound to Succ-PEGoo sulated With Phosphatidylserine and a Biotinylated Oligo DSPE in the microbubble membranes by standard coupling nucleotide Non-Covalently Bound to Streptavidin-Succ methods using a water-Soluble carbodiimide. The Sample is PEG-DSPE US 2002/0102215 A1 Aug. 1, 2002 38

0200 Microbubbles from Example 9(c) are incubated in 0209) c) Preparation of Thiolated-anti-E-selectin Anti a Solution containing a biotinylated oligonucleotide. The bodies oligonucleotide-coated bubbles are washed as described above. Binding of the oligonucleotide to the bubbles is 0210. Thiolation of anti-E-selectin antibodies may be detected e.g. by using fluorescent-labeled oligonucleotides effected as described by Hansen, C. B. et al. in (1995) for attachment to the bubbles, or by hybridising the attached Biochim. Biophys. Acta 1239, 133-144. oligonucleotide to a labeled (fluorescence or radioactivity) 0211 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-PEG-DSPE, Thiolated-anti-ICAM-1-Mal hybridising the bubbles with immobilized DNA-containing PEGo-DSPE, Thiolated-anti-E-Selectin-Mal-PEGoo Sequences complementary to the attached oligonucleotide. DSPE AS examples, an oligonucleotide complementary to riboso mal DNA (of which there are many copies per haploid 0212 To a mixture (5 mg) of phosphatidylserine (90-99.9 genome) and an oligonucleotide complementary to an onco mol%) and Mal-PEGo-DSPE (10-0.1 mol %, prepared as gene (e.g. ras of which there is one copy per haploid in Example 2) is added 5% propylene glycol-glycerol in genome) may be used. water (1 ml). The dispersion is heated to not more than 80 C. for 5 minutes and is then cooled to ambient temperature. The dispersion (0.8 ml) is transferred to a vial (1 ml) and the EXAMPLE 10 head space is flushed with perfluorobutane. The vial is Shaken in a cap-mixer for 45 seconds, whereafter the Sample Gas-Filled Microbubbles Encapsulated With is put on a roller table. After centrifugation the infranatant is Phosphatidylserine and Folate-PEG-Succ-DSPE eXchanged with an appropriate buffer, and coupling of the 0201 a) Preparation of 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, 0202 Folate-PEG-Succ-DSPE is synthesised as A., Ghose, T. and Mezei, M. in J. Pharm. Pharmacol. (1984) described by Lee, R.J. and Low, P. S. in (1995) Biochimica. 36, 465-466 or by Hansen, C. B. et al. in (1995) Biochim. Biophysica. Acta 1233, 134-144. Biophys. Acta 1239, 133-144. The microbubbles are placed 0203 b) Preparation of Gas-Filled Microbubbles Encap on a roller table for several hours and are then washed. sulated With Phosphatidylserine and Folate-PEG-Succ DSPE EXAMPLE 12 0204) To a mixture (5 mg) of phosphatidylserine (90-99.9 The Peptide mol %) and folate-PEG-DSPE (10-0.1 mol%) is added 5% FNFRLKAGOKIRFGAAAWEPPRARI (SEQ ID propylene glycol-glycerol in water (1 ml). The dispersion is NO:2) Attached to Gas-Filled Microbubbles heated to not more than 80 C. for 5 minutes and is then Encapsulated With Phosphatidylserine cooled to ambient temperature. The dispersion (0.8 ml) is transferred to a vial (1 ml) and the head space is flushed with 0213) The peptide FNFRLKAGOKIRFGAAAWEP perfluorobutane. The vial is shaken in a cap-mixer for 45 PRARI (SEQ ID NO:2), comprising phosphatidylserine Seconds, whereafter the Sample is put on a roller table. After binding and heparin-binding Sections, is Synthesised. The centrifugation the infranatant is exchanged with water and peptide is added to preformed phosphatidylserine-encapsu the Washing is repeated. Alternatively the microbubbles are lated perfluorobutane microbubbles and thoroughly mixed. prepared as described in Example 20e) or 20f). Analysis of folate attachment may for example be done by microscopic EXAMPLE 13 study of the binding of the folate-containing microbubbles to cells expressing different levels of folate receptors. Fibronectin Covalently Bound to Gas-Filled Microbubbles Encapsulated With EXAMPLE 11 Phosphatidylserine and Phosphatidylethanolamine Gas-Filled Microbubbles Encapsulated With Phos 0214) a) Microbubbles Preparation phatidylserine and Thiolated-anti-CD34-Mal 0215 DSPS (25 mg) and DSPE (5.0 mg) were weighed PEGo-DSPE, Thiolated-anti-ICAM-1-Mal into a clean vial and 5 ml of a solution of 1.4% propylene PEGoo-DSPE and Thiolated-anti-E-Selectin-Mal glycol/2.4% glycerol was added. The mixture was warmed PEG-DSPE to 80° C. for 5 minutes. The sample was cooled to room temperature and the head Space was flushed with perfluo 0205) a) Preparation of Thiolated-anti-CD34 Antibodies robutane gas. The Vials were Shaken in a cap mixer for 45 0206. Thiolation of anti-CD34 antibodies may be Seconds and the microbubbles were twice washed with effected as described by Hansen, C. B. et al. in (1995) distilled water then resuspended in 0.1 M Sodium borate Biochim. Biophys. Acta 1239, 133-144. buffer, pH 9. 0207 b) Preparation of Thiolated-anti-ICAM-1 Antibod 0216 b) Modification of Fibronectin ies 0217 Fibronectin (1.0 mg) in 5 ml 0.01 M Hepes buffer, 0208. Thiolation of anti-ICAM-1 antibodies may be pH 8, was added to 0.1 mmol of the crosslinker SDBP. The effected as described by Hansen, C. B. et al. in (1995) mixture was incubated on ice for 2 hours. Biochim. Biophys. Acta 1239, 133-144. 0218 c) Microbubble Modification. US 2002/0102215 A1 Aug. 1, 2002 39

0219. 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 0220 d) In vitro Aanalysis. Gas-Filled Microbubbles Encapsulated With 0221) The microbubbles were tested in the in vitro assay Phosphatidylserine and Inactivated Human detailed in Example 21. A gradual accumulation of Thrombin-Succ-PEGo-DSPE microbubbles binding to the cells was observed. 0227 a) Inactivation of Human Thrombin EXAMPLE 1.4 0228 Human thrombin was inactivated by incubation with a 20% molar excess of D-Phe-L-Pro-L-Arg-chlorom Gas-filled Microbubbles Encapsulated With Phos ethyl ketone in 0.05 N. HEPES buffer, pH 8.0, at 37° C. for phatidylserine, and 3.3-FN-(N',N'-Dimethylaminoet 30 minutes. hane)Carbamoylcholesterol 0229 b) Preparation of Gas-Filled Microbubbles Encap 0222 a) Synthesis of 3B-N-(N',N'-Dimethylaminoethan sulated With Phosphatidylserine and Succ-PEGo-DSPE e)Carbamoylcholesterol (DC-chol) (Farhood, H., Gao. X, 0230. To a mixture (5 mg) of phosphatidylserine (90-99.9 Barsoum, J. and Huang. L. Anal. Biochem. 225. 89-93 mol %) and Succ-PEGo-DSPE (10-0.1 mol %, prepared (1995)) as in Example 9(a)) was added 5% propylene glycol 0223) To a stirred solution of 2-dimethylaminoethy glycerol in water (1 ml). The dispersion was heated to not lamine (19.40 mg, 24:1, 0.22 mmol) and triethylamine (310 more than 80 C. for 5 minutes and was then cooled to l, 2.23 mmol) in dichloromethane (3 ml) at room tempera ambient temperature. The dispersion (0.8 ml) was trans ture was slowly added a solution of cholesteryl chlorofor ferred to a vial (1 ml) and the head space was flushed with mate (100 mg, 0.22 mmol) in 1,4-dioxane. When the reac perfluorobutane. The Vial was shaken in a cap-mixer for 45 tion was completed, the mixture was evaporated to dryneSS Seconds, whereafter the Sample was put on a roller table. and the residue was purified by flash chromatography After centrifugation the infranatant was eXchanged with (CHCl/MeOH, 4:1). A white solid was obtained, yield 105 water and the washing was repeated. Alternatively the mg (95%). The structure was verified by NMR and MALDI. microbubbles may be prepared as described in Example 2(f). 0224 b) Preparation of Microbubble Dispersion 0231 c) Preparation of Gas-Filled Microbubbles Encap 0225 Monolayer-encapsulated microbubbles containing sulated With Phosphatidylserine and Inactivated Human perfluorobutane are made from a mixture of 90% phosphati Thrombin-Succ-PEGo-DSPE dylserine and 10% (DC-chol) by weighing DSPS (4.5 mg) 0232) Inactivated human thrombin was covalently bound and (DC-chol) (0.5 mg) into a 2 ml vial. 0.8 ml propylene to Succ-PEGo-DSPE in the microbubbles from Example glycol/glycerol (4%) in water was added. The Solution was 16(b) by Standard coupling methods using a water-Soluble heated at 80 C. for 5 minutes and shaken. The solution was carbodiimide. The Sample was placed on a roller table then cooled to ambient temperature and the headspace was during the reaction. After centrifugation the infranatant was flushed with perfluorobutane. The vial was shaken on a eXchanged with water and the Washing was repeated. cap-mixer at 4450 oscillations/minute for 45 seconds and put on a roller table. The Sample was washed by centrifuging EXAMPLE 1.7 at 2000 rpm for 5 minutes. The infranatant was removed by a Syringe and distilled water was added to the same Volume. Gas-Filled Microbubbles Having Methotrexate and The headspace was again flushed with perfluorobutane and Prodrug-Activating Enzyme Attached the Sample was kept on a roller table until a homogeneous 0233 a) Methotrexate Attached via a Peptide Linker to appearance was obtained. The Washing procedure was Gas-Filled Micrububbles repeated again. 0234 Methods for attaching aminoacids to the anticancer EXAMPLE 1.5 drug methotrexate (MTX) are well described in the literature (see e.g. Huennekens, F. M. (1994), TIBTECH 12, 234-239 and references therein). Instead of a single amino acid a Gas-Filled Microbubbles Encapsulated With peptide may be attached to MTX using the Same technology. Phosphatidylserine and WEPPRARI-PE (SEQ ID Such a peptide may constitute a linker for the attachment of NO:3) MTX to the Surface of microbubbles. One class of Such 0226 Phosphatidylethanolamine (PE) is reacted with an linkers comprises peptides of the general structure (MTX)- equimolar amount of the crosslinker N-hydroxySuccinim F-K/R-X-R-Z-C where X is any amino acid and Z is a idyl-2,3-dibromopropionate in a 1:1 mixture of dioxane and hydrophobic amino acid. A Specific example of Such a linker 0.02 M HEPES buffer, pH 8.0. Following incubation for 2 is (MTX)-F-K-L-R-L-C (SEQID NO:4). The SH-group in hours on ice, an equimolar amount of the heparin-binding the Cys-residue is employed for attachment of the MTX peptide WEPPRARI (SEQ ID NO:3) is added, the pH is peptide to the microbubbles (e.g. composed of phosphati brought to 9 by the addition of 0.2 M disodium tetraborate, dylserine and Mal-PEG-DSPE) using standard technology, and the incubation is continued for 2 hours at room tem e.g. as in Example 2. A linker of this kind is expected to be US 2002/0102215 A1 Aug. 1, 2002 40 cleaved by the enzyme cathepsin B which often is Selec EXAMPLE 1.9 tively overexpressed outside and on the Surface of tumour cells (Panchal, R. G. et al. (1996), Nat. Biotechnol. 14, Gas-Filled Microbubbles Encapsulated With 852-856). Thus, the potential prodrug (MTX)-F-K/R-X-R Phosphatidylserine, would be liberated Selectively in tumours. This prodrug can Thiolated-anti-CEA-Mal-PEGo-DSPE and the further be activated to the active drug MTX by the action of Anticancer Prodrug carboxypeptidases, either present endogeneously in the N-trifluoroacetyl-adriamycin-14-valerate tumour or targeted to the tumour e.g. by tumour-associated antibodies (see below). 0241 a) Preparation of thiolated anti-CEA antibodies Thiolation of anti-CEA antibodies may be effected as 0235 b) Prodrug-Activating Enzyme Covalently described by Hansen, C. B. et al. in (1995) Biochim. Attached to the Surface of Gas-Filled Microbubbles Biophys. Acta 1239 133-144. 0236 An example of a prodrug-activating enzyme is carboxypeptidase A (CPA), which may be conjugated to the 0242 b) Preparation of Gas-Filled Microbubbles Encap Surface of microbubbles encapsulated by, for example, a sulated With Phosphatidylserine, Thiolated-anti-CEA-Mal mixture of phosphatidylserine and phosphatidylethanola PEG-DSPE and the Anticancer Prodrug N-trifluoro mine, e.g. by using a 3400 Da poly(ethylene glycol) chain acetyl-adriamycin-14-Valerate bearing an N-hydroxySuccinimide group at both ends (Per 0243 To a mixture (5 mg) of phosphatidylserine (90-99.9 ron, M. J. and Page, M., Br. J. Cancer 73, 281-287); the mol %), Mal-PEGoo-DSPE (10-0.1 mol %, prepared as in microbubbles may be prepared by standard methods. Example 2) and the anticancer prodrug N-trifluoroacetyl 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 (1995) Biochim. Biophys. Acta 1239 133-144. The Gas-Filled Microbubbles Encapsulated With Phos microbubbles are placed on a roller table for several hours phatidylserine, Thiolated-anti-CEA-Mal-PEGoo and are then washed. DSPE and the Anticancer Prodrug. 3',5'-O-Dipami toyl-5-Fluoro-2'-Deoxyuridine EXAMPLE 2.0 0237) a) Preparation of Thiolated Anti-CEA Antibodies 0238. Thiolation of anti-CEA antibodies may be effected Method of Use as described by Hansen, C. B. et al. in (1995) Biochim. 0244 An agent comprising phosphatidylserine-encapsu Biophys. Acta 1239, 133-144. lated microbubbles having inactivated human thrombin 0239 b) Preparation of Gas-Filled Microbubbles Encap Succ-PEGoo-DSPE incorporated into the encapsulating sulated With Phosphatidylserine, Thiolated-anti-CEA-Mal membrane is lyophilised from 0.01 M phosphate buffer, pH PEG-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 bosis in a leg vein. The leg is examined by Standard 0240 To a mixture (5 mg) of phosphatidylserine (90-99.9 ultrasound techniques. The thrombus is located by increased mol %), Mal-PEGoo-DSPE (10-0.1 mol %, prepared as in contrast as compared with Surrounding tissue. Example 2) and the anticancer prodrug 3',5'-O-dipamitoyl 5-fluoro-2'-deoxyuridine (Mori, A. et al. (1995) Cancer EXAMPLE 21 Chemother: Pharmacol. 35, 447-456) is added 5% propylene glycol-glycerol in water (1 ml). The dispersion is heated to Preparation and Biological Evaluation of not more than 80 C. for 5 minutes and is then cooled to Gas-Containing Microbubbles of DSPS Doped ambient temperature. The dispersion (0.8 ml) is transferred With a Lipopeptide Comprising a Heparin Sulphate to a vial (1 ml) and the head space is flushed with perfluo robutane. The Vial is shaken in a cap-mixer for 45 seconds, Binding Peptide (KRKR) (SEQ ID NO:5) and a whereafter the Sample is put on a roller table. After cen Fibronectin Peptide (WOPPRARI) (SEQ ID NO:6) trifugation the infranatant is exchanged with an approperiate 0245. This example is directed at the preparation of buffer, and coupling of the antibody to the microbubble is targeted microbubbles comprising multiple peptidic vectors performed, e.g. as described by Goundalkar, A., Ghose, T. arranged in a linear Sequence. and Mezei, M. in J. Pharm. Pharmacol. (1984)36 465-466 or by Hansen, C. B. et al. in (1995) Biochim. Biophys. Acta 0246) a) Synthesis of a Lipopeptide Consisting of a 1239 133-144. The microbubbles are placed on a roller table Heparin Sulphate Binding Peptide (KRKR) (SEQID NO:5) for Several hours and are then washed. and Fibronectin Peptide (WOPPRARI) (SEQ ID NO:6) US 2002/0102215 A1 Aug. 1, 2002 41

(SEQ ID NO: 7) O ~s N NH2 NH

O O O H H N N N N H H O O

HN HN N NH2 NH O NH2

O O O O H H H N N N N N OH N N N N N H H H H O O O O O e NH S. NH NH 1. NH2 1. NH2

0247 The lipopeptide was synthesised on an ABI 433A 0249 DSPS (4.5 mg) and lipopeptide from (a) (0.5 mg) automatic peptide Synthesiser Starting with Fmoc-Ile-Wang were weighed into each of two vials and 0.8 ml of a solution resin on a 0.1 mmol Scale using 1 mmol amino acid of 1.4% propylene glycol/2.4% glycerol was added to each cartridges. All amino acids and palmitic acid were preacti vial. The mixtures were warmed to 80 C. for 5 minutes vated using HBTU before coupling. The simultaneous (vials shaken during warming). The samples were cooled to removal of peptide from the resin and Side-chain protecting room temperature and the head Spaces flushed with perfluo groups was carried out in TFA containing 5% phenol, 5% robutane gas. The Vials were Shaken in a cap mixer for 45 EDT, 5% anisole and 5% HO for 2 hours, giving a crude seconds and rolled overnight. The resulting microbubbles product yield of 150 mg. Purification by preparative HPLC were washed Several times with deionised water and analy sed by Coulter counter size: 1-3 micron (87%), 3-5 micron of a 40 mg aliquot of crude material was carried out using (11.5%) and acoustic attenuation (frequency at maximum a gradient of 70 to 100% B over 40 minutes (A=0.1% attenuation: 3.5 MHz). The microbubbles were stable at 120 TFA/water and B=MeOH) at a flow rate of 9 ml/min. After mm Hg. MALDI mass Spectral analysis was used to confirm lyophilisation, 16 mg of pure material were obtained (ana incorporation of lipopeptide into DSPS microbubbles as lytical HPLC, gradient 70-100% B where B=MeOH, follows: ca. 0.05-0.1 ml of microbubble Suspension was A=0.01% TFA/water: detection-UV 260 and fluorescence, transferred to a clean vial and 0.05-0.1 ml methanol was Ex280, Em350 product retention time=19.44 minutes). added. The Suspension was Sonicated for 30 Seconds and the Further product characterisation was carried out using solution was analysed by MALDI MS. Positive mode gave MALDI mass spectrometry: expected M--H at 2198, found M+H at 2200 (expected for lipopeptide, 2198). at 2199 0250) c) In vitro Study of Gas-Filled Microbubbles of 0248 b) Preparation of Gas-Filled Microbubbles of DSPS doped With a Multiple-Specific Lipopeptide Con DSPS Doped With a Multiple-Specific Lipopeptide Con sisting of a Heparin Sulphate-Binding Peptide (KRKR) sisting of a Heparin Sulphate Binding Peptide (KRKR) (SEQ ID NO:5) and Fibronectin Peptide (WOPPRARI) (SEQ ID NO:5) and Fibronectin Peptide (WOPPRARI) (SEQ ID NO:6): Binding to Endothelial Cells Under Flow (SEQ ID NO:6) Conditions US 2002/0102215 A1 Aug. 1, 2002 42

0251) 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 above the plates. When the cells reached confluence the Preparation of Gas-Filled Microbubbles Encapsuled coverslips were placed in a custom-made flow chamber With DSPS Comprising Thiolated consisting of a groove carved into a glass plate upon which Anti-CD34-MAL-PEG-PE the cover Slip with cells was placed, with the cells facing the groove, So as to form a flow channel. Microbubbles prepared 0257) a) Preparation of Gas-Filled Microbubbles Encap as in (b) were passed from a reservoir held at 37° C. through Suled With DSPS and PE-PEG2000-Mal the flow chamber and back to the reservoir using a peristaltic 0258 DSPS (4.5 mg, 3.9 mmol) and PE-PEG-Mal pump. The flow rate was adjusted to Simulate physiologi from Example 50 (0.5 mg) were weighed into a clean vial cally relevant shear rates. The flow chamber was placed and 1 ml of a solution of 1.4% propylene glycol/2.4% under a microScope and the interaction between the glycerol was added. The mixture was warmed to 80° C. for microbubbles and cells was viewed directly. A camera 5 minutes then filtered through a 4.5 micron filter. The mounted on the microScope was connected to a colour video Sample was cooled to room temperature and the head Space printer and a monitor. A gradual accumulation of was flushed with perfluorbutane gas. The vials were shaken microbubbles on the cells took place at a rate dependent on in a cap mixer for 45 Seconds and the resulting microbubbles the flow rate. On further increasing the flow rate, cells were washed three times with distilled water. started to become detached from the coverslip, but the microbubbles remained bound to the cells. Control bubbles 0259 b) Thiolation of Anti-CD34 Antibodies not carrying the vector did not adhere to the endothelial cells 0260 To 0.3 mg of anti-CD34 antibody dissolved in 0.5 and disappeared from the chamber under minimal flow ml phosphate buffered saline (PBS), pH7, was added 0.3 mg conditions. Traut's reagent and the Solution was stirred at room tem 0252 d) In vivo Experiment in Dog perature for 1 hour. ExceSS reagent was separated from the 0253) Case 1) modified protein on a NAP-5 column. 0254. A 22 kg mongrel dog was anaesthetised with 0261 c) Conjugation of Thiolated Anti-CD34 Antibody pentobarbital and mechanically ventilated. The chest was to Gas-Filled Microbubbles Encapsuled With DSPS and opened by a midline Sternotomy, the anterior pericardium Comprising DSPE-PEGooo-MAL was removed, and a 30 mm gelled Silicone rubber Spacer 0262 0.5 ml of the thiolated antibody praparation from was inserted between the heart and a P5-3 transducer of an (b) was added to an aliquot of microbubbles from (a) and the ATL HDI-3000 ultrasound Scanner. The Scanner was set for conjugation reaction was allowed to proceed for 30 minutes intermittent Short axis imaging once in each end-Systole by on a roller table. Following centifugation at 2000 rpm for 5 delayed EGC triggering. A net Volume of 2 ml of minutes the infranatant was removed. The microbubbles microbubbles from (b) was injected as a rapid intravenous were washed a further three times with water. bolus; 3 Seconds later, the imaged right ventricle was seen to contain contrast material, and another 3 Seconds later the left 0263 d) Detection of the Antibody Encapsulated in the Ventricle was also filled and a transient attenuation Shadow Microbubbles Using a FITC-Conjugated Secondary Anti which obscured the view of the posterior parts of the left body Ventricle was observed. Substantial increases in brightness 0264. To the microbubble suspension from (c) was added were seen in the myocardium and, when the attenuation 0.025 mL FITC-conjugated goat-anti-mouse antibody. The shadow subsided, in the portions of the heart distal to the left mixture was incubated in the dark at room temperature for Ventricle. After passage of the inital bolus, the ultrasound 30 minutes on a roller table and was then centrifuged at 2000 Scanner was Set to continuous, high frame rate, high output rpm for 5 minutes. The infranatant was then removed and the power imaging, a procedure known to cause destruction of microbubbles were washed a further three times with water. ultrasound contrast agent microbubbles in the imaged tissue Flow cytometric analysis of the microbubble suspension regions. After a few Seconds, the Scanner was adjusted back showed that 98% of the population was fluorescent. to its initial Setting. The myocardium was then darker, and closer to the baseline value. Moving the imaged slice to a EXAMPLE 23 new position resulted in re-appearance of contrast effects, moving the slice back to the initial position again resulted in Preparation of Gas-Filled Microbubbles Encapsuled a tissue brightness close to baseline. With DSPS Comprising Thiolated 0255 Case 2) Comparative Anti-CD62-MAL-PEGooo-PE 0256 A net volume of 2 ml microbubbles prepared in an 0265 An identical procedure to that described in identical manner to (b) above with the exception that no Example 22 was used to prepare microbubbles comprising lipopeptide was included in the preparation was injected, anti-CD62 antibodies. US 2002/0102215 A1 Aug. 1, 2002 42

EXAMPLE 24 0276 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-PEGooo-PE before coupling. The Simultaneous removal of peptide from 0266 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% HO 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 PEGooo-PE and Thiolated-anti-ICAM-1-Mal material was obtained (analytical HPLC, gradient 20 to 50% PEG-00-PE B where B=0.1% TFA/acetonitrile, A=0.01% TFA/water: 0267. This example is directed to the preparation of detection-UV 214 and 260 nm product retention time= microbubbles comprising multiple antibody vectors for tar 12.4 minutes). Further product characterization was carried geted ultrasound imaging. out using MALDI mass spectrometry: expected M-H at 0268 a) Preparation of Gas-Filled Microbubbles Encap 2856, found at 2866. sulated With DSPS and PE-PEGo-Mal 0277 b) Preparation of Gas-Filled Microbubbles Com 0269) DSPS (4.5 mg) and PE-PEG -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 0278 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 0270 b) Thiolation of Anti-CD62 and Anti-ICAM-1 temperature and the head Space was flushed with perfluo Antibodies robutane gas. The Vials were Shaken in a cap mixer for 45 0271 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 0272 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-PEGo-Mal resulting solution for polylysine and PS-binding/fibronectin 0273 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 0274 The PEG spacer length may be varied to include longer (e.g. PEGaloo and PEGsooo) or shorter (e.g. PEGoo Poly-L-lysine 786,914, 790,919, or PEG soo) 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 0279 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 PEGooo or polyalanine (-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 0275) 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 2002/0102215 A1 Aug. 1, 2002 43

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-en room temperature and the head Space was flushed with dothelin-1 Peptide (biotin-D-Trp-Leu-Asp-Ile-Ile perfluorobutane gas. The vial was shaken in a cap-mixer for Trp.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 0280 This example is directed at the preparation of tic attenuation. targeted ultrasound microbubbles whereby streptavidin is 0289 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). 0290 To the microbubble preparation from (d) was added 0281 a) Synthesis of Biotin-PEG-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 0282) 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 PEG-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 Bioti 0283) b) Synthesis of Biotinylated Endothelin-1 Peptide nyl-endothelin-1 Peptide (Biotin-D-Trp-Leu-Asp (Biotin-D-Trp-Leu-Asp-Ile-Ile-Trp.OH) (SEQ ID NO:9) Ile-Ile-Trp.OH) (SEQ ID NO:9) and Biotinyl-fibrin 0284. The peptide was synthesised on a ABI 433A auto anti-polymerant Peptide (biotin matic peptide Synthesiser starting with Fmoc-Trp(Boc)- GPRPPERHOS.NH) (SEQ ID NO:10) Wang resin on a 0.1 mmol Scale using 1 mmol amino acid 0291 a) Preparation of Biotin-Containing Microbubbles cartridges. All amino acids were preactivated using HBTU before coupling. The Simultaneous removal of peptide from 0292 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% HO 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 0293 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= Streptavidin and a Mixture of Biotinyl-endothelin-1 (Biotin 12.6 minutes). Further product characterization was carried D-Trp-Leu-Asp-Ile-Ile-Trp.OH) (SEQ ID NO:9) and Bioti out using MALDI mass spectrometry: expected M-H at nyl-fibrin-anti-polymerant Peptide (Biotin-GPRPPERHO 1077, found at 1077. S.NH) (SEQ ID NO:10) 0285 c) Synthesis of Biotinyl-fibrin-anti-polymerant 0294 The procedure detailed in Example 27 was fol Peptide (Biotin-GPRPPERHOS.NH)(SEQ ID NO:10) lowed. 0286 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 0287 d) Preparation of Multiple-Specific Gas-Filled Peptide/Fibronectin Peptide/RGD Peptide and Microbubbles Encapsulated With Phosphatidylserine and Fluorescein Biotin-PEG-b-Alanine Cholesterol 0295) a) Synthesis of a Lipopeptide Containing the RGD 0288 DSPS (4.5 mg) and biotin-PEGoo-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-Lys Acetyl-Arg-Gly-Asp US 2002/0102215 A1 Aug. 1, 2002 44

(SEQ ID NO: 11)

N

NH2 N------O Lys (fluorescein)Gly'OH

0296. The lipopeptide was synthesised as described in 0299 c) Preparation of Multiple-Specific Gas-Filled Example 21(a) using commercially available amino acids Microbubbles of DSPS Functionalised With a Heparin Sul and polymers. The lipopeptide was cleaved from the resin in phate-Binding Peptide, a fibronectin Peptide, Acetyl-RGD TFA containing 5% water, 5% phenol and 5% EDT for 2 Peptide and Fluorescein hours. Following evaporation in vacuo the crude product was precipitated and triturated with diethyl ether. Purifica 0300 DSPS (4 mg., 3.9 mmol), lipopeptide from (a) (0.5 tion by preparative HPLC of a 40 mg aliquot of crude mg, 0.2 mmol) and lipopeptide from (b) (0.5 mg) were material was carried out using a gradient of 60 to 100% B weighed into each of two vials and 0.8 ml of a solution of over 40 minutes (A=0.1% TFA/water and B=0.1% TFA/ 1.4% propylene glycol/2.4% glycerol was added to each acetonitrile) at a flow rate of 9 ml/min. After lyophilisation vial. The mixtures were warmed to 80 C. for 5 minutes 10 mg of pure material (analytical HPLC, gradient 60-100% (vials shaken during warming). The samples were cooled to B where B=0.1% TFA/acetonitrile, A=0.01% TFA/water: room temperature and the head Spaces were flushed with detection-UV 260 product retention time=20-22 min perfluorobutane gas. The Vials were Shaken in a cap mixer utes). Further product characterisation was carried out using for 45 seconds and then rolled overnight. The microbubbles MALDI mass spectrometry: expected M--H at 1922, found So obtained were washed several times with deionised water at 1920. and analysed by MALDI mass spectrometry as described in 0297 b) Synthesis of a Lipopeptide Containing a Heparin Example 21(b). The microbubbles were investigated by Sulphate-Binding Sequence and a Fibronectin Peptide microScopy and were Seen to have a range of sizes between 0298 Synthesis and purification were carried out as 1 and 5 microns. Furthermore the microbubbles were fluo described in Example 21 (a). reScent. US 2002/0102215 A1 Aug. 1, 2002 45

EXAMPLE 30 utes). Further product characterisation was carried out using MALDI mass spectrometry: expected M+H at 2369, found Gas-Filled Microbubbles Comprising DSPS at 2373. Covalently Modified With CD71 FITC-Labelled Anti-Transferrin Receptor Antibody and Doped 0305 b) Preparation of Gas-Filled Microbubbles Com With a Lipopeptide With Affinity for Endothelial prising DSPS Doped With a Endothelial Cell-Binding Cells Lipopeptide and PE-PEGooo-Mal 0301 This example is directed at the preparation of 0306 DSPS (4.5 mg) and lipopeptide from (a) (0.5 mg) multiple vector targeted ultrasound agents. along with PE-PEGo-Mal from Example 50 (0.5 mg) 0302) a) Synthesis of an Endothelial Cell Binding were weighed into a clean vial and 1 ml of a solution of 1.4% Lipopeptide: 2-n-hexadecylstearyl-Lys-Leu-Ala-Leu-Lys propylene glycol/2.4% glycerol was added. The mixture was Leu-Ala-Leu-Lys-Ala-Leu-Lys-Ala-Ala-Leu-Lys-Leu-Ala warmed to 80 C. for 5 minutes and then filtered through a NH. (SEQ ID NO:12) 4.5 micron filter. The Sample was cooled to room tempera 0303. The lipopeptide shown below was synthesised on a ture and the head Space was flushed with perfluorobutane ABI 433A automatic peptide synthesiser starting with a Rink gas. The Vial was shaken in a cap mixer for 4% Seconds and amide resin on a 0.1 mmol Scale using 1 mmol amino acid the resulting microbubbles were washed three times with cartridges. distilled water.

H O H Null Null N Nulls N O H O H O H

N N A v. A v. H. H. H. H. H. H. H. H. v. v. V. A N N

O H O H O H O H O H O

Nulls N Nulls N Nulls N Nulls N Nulls N Nulls N N-H E H H O H O SN O H O SN O > O

A v.

0304 All amino acids and 2-m-hexadecylstearic acid 0307 c) Thiolation of FITC-Labelled Anti-Transferrin were preactivated using HBTU before coupling. The simul Receptor Antibody taneous removal of peptide from the resin and Side-chain 0308 FITC-labelled CD71 anti-transferrin receptor Ab protecting groups was carried out in TFA containing 5% (100 mg/ml in PBS, 0.7ml) was reacted with Traut's reagent EDT and 5% HO for 2 hours, giving a crude product yield (0.9 mg) at room temperature for 1 hour. Excess reagent was of 150 mg. Purification by preparative HPLC of a 40 mg separated from modified protein on a NAP-5 column. aliquot of crude material was carried out using a gradient of 0309 d) Conjugation of Thiolated FITC-Labelled Anti 90 to 100% B over 50 minutes (A=0.1% TFA/water and Transferrin Receptor Antibody to Gas-Filled Microbubbles B=MeOH) at a flow rate of 9 ml/min. After lyophilisation, Comprising DSPS Doped With an Endothelial Cell-Bind 10 mg of pure material was obtained (analytical HPLC, ing Lipopeptide and DSPE-PEGooo-Mal gradient 90-100% B where B=MeOH, A=0.01%TFA/water: 0310. A 0.5 ml aliquot of the protein fraction (2 ml in detection-UV 214 nm product retention time=23 min total) from (c) above was added to the microbubbles from US 2002/0102215 A1 Aug. 1, 2002 46

(b) and the conjugation reaction was allowed to proceed for tivation. Captopril dissolved in DMF was introduced on the 10 minutes on a roller table. Following centrifugation at solid-phase using DBU as base. Simultaneous removal of 1000 rpm for 3 minutes the protein solution was removed and the conjugation repeated twice more with 1 ml and 0.5 the peptide from the resin and deprotection of Side-chain ml aliquots of protein solution respectively. The bubbles protecting groups was carried out in TFA containing 5% were then washed four times in distilled water and a Sample EDT, 5% water and 5% ethyl methyl sulphide for 2 hours. analysed for the presence of antibody by flow cytometry and An aliquot of 10 mg of the crude material was purified by microscopy. A fluorescent population of >92% was observed preparative liquid chromatography using a gradient of 70 to (see FIG. 1). 100% B over 60 minutes (A=0.1% TFA/water and B=0.1% 0311 Incorporation of lipopeptide into the microbubbles TFA/acetonitrile) at a flow rate of 10 ml/min. After lyophi was confirmed by MALDI mass spectrometry as described lisation a yield of 2 mg of pure material was obtained in Example 21 (b). (analytical HPLC, gradient 70-100% B over 20 minutes, EXAMPLE 31 A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow rate 1 ml/min., detection UV 214 nm, retention time 26 minutes). Gas-Filled Microbubbles Comprising DSPS, a Further characterisation was carried out using MALDI maSS Lipopeptide for Endothelial Cell Targeting and a Captopril-Containing Molecule Spectrometry, giving M--H at 1265 as expected. 0312 This example is directed to the preparation of ultrasound agents for combined targeting and therapeutic applications. 0315 b) Synthesis of a Lipopeptide With Affinity for 0313 a) Synthesis of a Lipopeptide Functionalised With Endothelial Cells: Dipalmitoyl-Lys-Lys-Lys-Aca-Ile-Arg Captopril Arg-Val-Ala-Arg-Pro-Pro-Leu-NH. (SEQ ID NO:14)

O

H NH2

O O O H N N N us Null H NH, O O

O O OH NH2 HN ^^^~us

0314. The structure shown above was synthesised using a manual nitrogen bubbler apparatus Starting with Fmoc protected Rink Amide MBHA resin on a 0.125 mmol scale. Coupling was carried out using standard TBTU/HOBt/DIEA protocols. Bromoacetic acid was coupled through the Side chain of Lys as a Symmetrical anhydride using DIC preac US 2002/0102215 A1 Aug. 1, 2002 47

NH2

O H H N N N N-1N1\ H H O O

NH2 "N. NH NH

O NH2 N O O H H O N Ruls Rul N N Jr. H O

l NH NH

HN 1s NH HN ls NH

0316 The lipopeptide was synthesised on a ABI 433A contents were extensively washed with deionised water. No automatic peptide Synthesiser Starting with Rink amide resin detectable level of starting material was found in the final on a 0.1 mmol Scale using 1 mmolamino acid cartridges. All wash solution as evidenced by MALDI MS. MALDI mass amino acids and palmitic acid were preactivated using Spectral analysis was used to confirm incorporation of the HBTU before coupling. The simultaneous removal of pep products from (a) and (b) into the microbubbles as described tide from the resin and Side-chain protecting groups was in Example 21(b). carried out in TFA containing 5% phenol, 5% EDT and 5% H20 for 2 hours, giving a crude product yield of 160 mg. 0319 d) In vitro Study of Gas-Filled Microbubbles Com Purification by preparative HPLC of a 35 mg aliquot of prising DSPS, a Lipopepitcle for Endothelial Cell Targeting crude material was carried out using a gradient of 70 to and a Captopril-Containing Molecule for Therapeutic Appli 100% B over 40 minutes (A=0.1% TFA/water and cations B=MeOH) at a flow rate of 9 ml/min. After lyophilisation, 0320 The in vitro assay decribed in Example 21(c) was 20 mg of pure material was obtained (analytical HPLC, used to examine cell binding under flow conditions. A gradient 70-100% B where B=MeOH, A=0.01%TFA/water: gradual accumulation of microbubbles on the cells took detection-UV 214 and 260 nm product retention time=16 place, depending on the flow rate. On further increasing the minutes). Further product characterisation was carried out flow rate cells started to become detached from the cover using MALDI mass spectrometry: expected M--H at 2050, slip, but the microbubbles remained bound to the cells. found at 2055. Control microbubbles not carrying the vector did not adhere 0317 c) Preparation of Gas-Filled Microbubbles Com to the endothelial cells and disappeared from the chamber prising DSPS, a Lipopeptide for Endothelial Cell Targeting under minimal flow conditions. and a Captopril-Containing Molecule for Drug Delivery EXAMPLE 32 0318 DSPS (4.5 mg), product from (a) (0.5 mg) and product from (b) (0.5 mg) were weighed into a vial and 1.0 Preparation of Gas-Filled Microbubbles Comprising ml of a Solution of 1.4% propylene glycol/2.4% glycerol was DSPS Loaded with a Lipopeptide Comprising a added. The mixture was warmed to 80 C. for 5 minutes Helical Peptide With Affinity for Cell Membranes (vial shaken during warming). The sample was cooled to and the Peptide Antibiotic Polymixin B Sulphate room temperature and the head Space was flushed with 0321) This example is directed to the preparation of perfluorobutane gas. The vial was firstly Shaken in a cap targeted microbubbles comprising multiple peptidic vectors mixer for 45 seconds then rolled for 1 hour, whereafter the having a combined targeting and therapeutic application. US 2002/0102215 A1 Aug. 1, 2002 48

0322 a) Synthesis of a Lipopeptide Comprising a Helical lation was in the desired range of 1-8 micron. To the washed Peptide With Affinity for Cell Membranes: Hexadecyl bubbles (ca. 0.2 ml) was added methanol (0.5 ml), and the Stearyl-Lys-Leu-Ala-Leu-Lys-Leu-Ala-Leu-Lys-Ala-Leu mixture was placed in a Sonicator bath for 2 minutes. The Lys-Ala-Ala-Leu-Lys-Leu-Ala-NH. (SEQ ID NO:12) resulting clear Solution, on analysis by MALDI-MS, was found to contain both lipopeptide and polymixin B Sulphate 0323 This is prepared as described in Example 30(a). (expected 1203, found 1207). 0324 b) Preparation of Multiple-Specific Gas-Filled Microbubbles EXAMPLE 33 0325 DSPS (5.0 mg), lipopeptide from (a)(0.3 mg) and Preparation of Gas-Filled Microbubbles Comprising polymixin B Sulphate (0.5 mg) were weighed into a clean DSPS Doped With a Lipopeptide Comprising a vial and 1.0 ml of a solution of 1.4% propylene glycol/2.4% IL-1 Receptor-Binding Sequence and Modified glycerol was added. The mixture was Sonicated for 3-5 With a Branched Structure Containing the Drug minutes, warmed to 80 C. for 5 minutes and then filtered Methotrexate through a 4.5 micron filter. The mixture was cooled to room 0326. This example is directed to the preparation of temperature and the head Space was flushed with perfluo targeted microbubbles comprising multiple vectors for tar robutane gas. The Vial was Shaken in a cap-mixer for 45 geted/therapeutic applications. Seconds and the resulting microbubbles were centrifuged at 1000 rpm for 3 minutes. The microbubbles were washed 0327) a) Synthesis of a Lipopeptide Comprising an Inter with water until no polymixin B Sulphate or lipopeptide leukin-1 Receptor-Binding Peptide: Dipalmitoyl-Lys-Gly could be detected in the infranatant by MALDI-MS. Micros Asp-Trp-Asp-Gln-Phe-Gly-Leu-Trp-Arg-Gly-Ala-Ala.OH copy showed that the size distribution of the bubble popu (SEQ ID NO:15)

-O - O H ,O 4N NH

| H-N Y N-H N-H

| O | O | O

Nulls N Nulls N Nulls O E H O H O H

2 N-H US 2002/0102215 A1 Aug. 1, 2002 49

0328. The lipopeptide was synthesised on a ABI 433A 0332 c) Preparation of Multiple-Specific Gas-filled automatic peptide Synthesiser Starting with Fmoc-Ala-Wang Microbubbles resin on a 0.1 mmol Scale using 1 mmol amino acid 0333 DSPS (4.5 mg), thiol-containing lipopeptide from cartridges. Example 64(a) (0.5 mg) and lipopeptide from (a) (0.2 mg) 0329 All amino acids and palmitic acid were preacti were weighed into a clean vial and 1.0 ml of a Solution of vated using HBTU before coupling. The simultaneous 1.4% propylene glycol/2.4% glycerol was added. The mix removal of lipopeptide from the resin and Side-chain pro ture was Sonicated for 3-5 minsutes, warmed to 80 C. for 5 tecting groups was carried out in TFA containing 5% HO, minutes and then filtered through a 4.5 micron filter. The 5% anisole, 5% phenol and 5% EDT for 2 hours, giving a mixture was cooled to room temperature and the head Space crude product yield of 150 mg. Purification by preparative was flushed with perfluorobutane gas. The Vial was Shaken HPLC of a 30 mg aliquot of crude material was carried out in a cap mixer for 45 Seconds and the resulting microbubbles using a gradient of 90 to 100% B over 40 minutes (A=0.1% were centrifuged at 1000 rpm for 3 minutes, whereafter the TFA/water and B=MeOH) at a flow rate of 9 milmin. After infranatant was discarded. lyophilisation, 4 mg of pure material was obtained (analyti 0334 d) Conjugation of Methotrexate Branched Struc cal HPLC, gradient 90-100% B over 20 minutes where ture to Thiolated Microbubbles B=MeOH, A=0.01% TFA/water: Detection-UV 214 0335) The methotrexate structure from (b) above (0.5 nim-product retention time=23 minutes). Further product mg) was dissolved in PBS, pH 8.0. The solution was then characterisation was carried out using MALDI maSS Spec added to the thiol-containing microbubbles from (c) and trometry: expected M+H at 2083, found at 2088. disulphide bond formation was allowed to proceed for 16 0330 b) Synthesis of a Branched Methotrexate Core hours. Following extensive washing with PBS and water the Structure Containing a Thiol Moiety bubbles were analysed by microscopy and MALDI MS.

HN N N rS COOH

H H CulNH2 N N N H O O

HN N N COOH COOH O rS H N HN COOH CCulNH2 N O Yst H O O

COOH

0331. The methotrexate structure was synthesised on an 0336. The disulphide bond linking the methotrexate ABI 433A automatic peptide synthesiser starting with Fmoc structure to the microbubbles may be reduced in vivo to Cys(Trt) Tentagel resin on a 0.1 mmol scale. The simulta liberate the free drug molecule, so that such microbubbles in combination with a tumour Specific vector comprise a drug neous removal of product from the resin and deprotection of delivery System. A physiologically acceptable reducing protecting groups was carried out in TFA containing 5% agent Such as glutathione may be used to bring about drug EDT and 5% HO for 2 hours, giving a crude product yield release. of 160 mg. Purification by preparative HPLC of a 30 mg aliquot of crude material was carried out using a gradient of EXAMPLE 34 10 to 30% B over 40 minutes (A=0.1% TFA/water and B=0.1%TFA/acetonitrile) and a flow rate of 9 ml/min. After Preparation of Gas-Filled Microbubbles Coated lyophilisation of the pure fractions, 9 mg of pure material With Poly-L-lysine Complexed to was obtained (analytical HPLC, gradient 5-50% B where Fluorescein-Labeled DNA Fragments from Plasmid B=0.1% TFA/acetonitrile, A=0.01% TFA/water: detection pBR322 UV 214 nm-product retention time=9.5 minutes). Further 0337 This example is directed to the preparation of product characterisation was carried out using MALDI maSS microbubbles for gene therapy/anti-Sense applications. Spe spectrometry: expected M--H at 1523, found at 1523. cific targeting may be achieved by further doping of US 2002/0102215 A1 Aug. 1, 2002 50 microbubble membranes with vector-modified lipid struc pH 8). The solution was made up to a total of 0.6 ml by tures as described in Example 21. addition of water and the pH was adjusted to 8. Complexing was allowed to proceed for 1 hour, after which 0.05 mL of 0338 a) Preparation of DSPS-Encapsulated Gas-Filled the polylysine-DNA solution was added to the microbubble Microbubbles Suspension from (a) above. After 1 hour microscopy was 0339) DSPS (4.5 mg) was weighed into a clean vial. 1.0 used to show that the bubbles were fluorescent, confirming ml of a Solution of 1.4% propylene glycol/2.4% glycerol was the presence of DNA. added and the mixture was Sonicated for 2 minutes and then warmed to 80° C. for 5 minutes. Immediately following EXAMPLE 35 warming the Solution was filtered through a 4 micron filter. The Sample was cooled to room temperature and the head Preparation of Gas-Filled Microbubbles Containing Space was flushed with perfluorobutane gas. The Vial was a Branched Core Peptide Comprising a Shaken in a cap mixer for 45 Seconds. The resulting Dabsylated-Atherosclerotic Plague-Binding microbubbles were then washed once with deionised water Sequence and RGDS and the infranatant was discarded. The microbubbles were 0342. This example is directed to the preparation of then resuspended in 0.5 ml water. microbubbles having a thiol group on the Surface for modi 0340 b) Preparation of Poly-L-lysine/DNA Complex and fication with thiol-containing vectors for targeting/drug Loading of DSPS-Encapsulated Microbubbles delivery and drug release. 0341) To 1 mg of poly-L-lysine (70-150 kD) in a clean 0343 a) Synthesis of the Branched Peptide Dabsyl-Tyr vial was added 0.1 ml of a fluorescein-labeled digest of Ara-Ala-Leu-Val-Asp-Thr-leu-Lys-Lys (NH-Arg-Gly-Asp plasmid pBR322 dissolved in TE buffer (10 mM tris-HCl, Ser)-Gly-Cys.OH (SEQ ID NO:16)

OH

O Nulls N1 > H

O O O H H H N N OH s P O R O R COOH > Nshi HO O Issus ~'s NH s l O YCOOH O US 2002/0102215 A1 Aug. 1, 2002

0344) The peptide was synthesised on an ABI 433A graphed (methylene chloride/methanol/water, 40:8:1). The automatic peptide Synthesiser starting with Fmoc-Cys(Trt)- product was obtained as a yellow gum (112 mg, 94%), and Tentagel resin on a 0.1 mmol Scale using 1 mmolamino acid structure was verified by NMR and MALDI-MS. cartridges. All amino acids were preactivated using HBTU before coupling. The Simultaneous removal of peptide from 0352 b) Binding of Fluorescein-Conjugated Streptavidin the resin and Side-chain protecting groups was carried out in to Gas-Filled Microbubbles TFA containing 5% phenol, 5% EDT and 5% HO for 2 0353 Gas-filled microbubbles were prepared by mixing hours, giving a crude product yield of 160 mg. Purification DSPS and biotin-PEG-acyl-phosphatidylethanolamine by preparative HPLC of a 30 mg aliquot of crude material as described in previous examples. The microbubble Sus was carried out using a gradient of 10 to 60% B over 40 pension was divided into 0.2 ml aliquots and fluorescein minutes (where A=0.1% TFA/water and B=acetonitrile) at a conjugated Streptavidin was added as shown in the table flow rate of 9 ml/min. After lyophilisation, 2.5 mg of pure below. The samples were incubated on a roller table for 15 material was obtained (analytical HPLC, gradient 10-50% B or 30 minutes at ambient temperature before removal of over 20 minutes where B=0.1% TFA/acetonitrile and excess protein by washing in PBS. The samples were A=0.01% TFA/water: detection-UV 214 and 435 analysed by flow cytometry and Coulter Counter. The results nim-product retention time=21 minutes). Further product are Summarized in the table below. characterisation was carried out using MALDI maSS Spec trometry: expected M+H at 2070, found at 2073. 0354) Results: 0345 b) Preparation of Thiol-Containing Gas-Filled Microbubbles Added Particle 0346) These were prepared as described in Example Streptavidin Incubation % median Aliquot (mg/200:1 time (amb. Fluorescent diameter 64(a). O. sample) temp.) particles (microns) 0347 c) Oxidative Coupling of Thiolated Microbubbles 1. O 2.O With Multiple-Specific Peptide via Disulphide Bond For 2 O 12 (foam) mation 3 O.2 30 min 7.8 3.9 (3 x 10) mmol 0348. The infranatant from the microbubbles from (b) 4 2 30 min 26.2 4.2 above was discarded and replaced with a Solution of dabsyl (3 x 10) mmol peptide from (a) (1 mg) in 0.7 ml dilute ammonia Solution 5 1O 15 min 30.5 la (1.5 x 107) mmol (pH 8). To this was added 0.2 ml of a stock solution 6 2O 30 min 97.9 5.2 containing 6 mg of potassium ferricyanate dissolved in 2 ml (3 x 107) mmol of water. The vial was placed on a roller table and thiol 7 40 15 min 96.7 5.1 oxidation allowed to proceed for 2 hours. The bubbles were (6 x 107) mmol then washed extensively with water until the infranatant was 8 DSPS 2O 15 min O6 3.7 free of the dabsyl-peptide as evidenced by HPLC and control (3 x 107) mmol MALDI MS. Detection of microbubble-bound peptide was carried out by reduction of the disulphide bond using the 0355 c) Conjugation of Streptavin-Coated Microbubbles water Souble reducing agent tris-(2-carboxyethyl)-phos With the Oligonucleotide Biotin-GAAAGG phine. Following reduction, the infranatant was found to TAGTGGGGTCGTGTGCCGG (SEQ ID NO:17) and contain free dabsyl-peptide as evidenced by HPLC and Biotinylated Fibrin-anti-polymerant Peptide Biotin-GPRP MALDIMS PERHOS (SEQ ID NO:10) 0349 Other physiologically acceptable reducing agents 0356. The particles from aliquot no. 6 above were cen Such as reduced glutathione may also be useful for initiating trifuged and the Supernatant was replaced with 1 ml PBS release. buffer, pH 7.5, containing 0.2 mg of biotin-GAAAGG TAGTGGGGTCGTGTGCCGG (SEQ ID NO:17) and 0.2 EXAMPLE 36 mg of biotin-GPRPPERHQS (SEQ ID NO:10) (prepared as Preparation of Gas-Filled Microbubbles Encapsu in Example 27(b) and (c)). After incubation for 24 hours the lated With DSPS and Biotin-PEG-acyl-phos particles were washed extensively with PBS and water. phatidylethanolamine and Functionalised With 0357. Other biotinylated vectors or therapeutic agents Streptavidin, Oligonucleotide Biotin-GAAAGG may be conjugated to Streptavidin- or avidin-coated TAGTGGGGTCGTGTGCCGG (SEQ ID NO:17) microbubbles using this procedure. and Biotinylated Fibrin-anti-polymerant Peptide (Biotin-GPRPPERHOS.NH)(SEQ ID NO:10) EXAMPLE 37 0350 a) Synthesis of Biotin-PEGoo-acyl-phosphatidyl Preparation of Gas-Filled Microbubbles Ethanolamine Encapsulated With DSPS and Functionalised With 0351. A mixture of dipalmitoyl phosphatidyl ethanola a Thrombi-Targeting Lipopeptide and the mine, (21.00 mg, 0.03 mmol), biotin-PEG-CO-NHS, (100 Thrombolytic Enzyme Tissue Plasminogen mg, 0.03 mmol) and triethylamine (42 ul, 0.30 mmol) in a Activator solution of chloroform/methanol (3:1) was stirred at room 0358. This example is directed at the preparation of temperature for 2 hours. After evaporation of the Solvents thrombus targeted ultrasound contrast agents comprising a under reduced pressure, the residue was flash chromato therapeutic thromolytic agent. US 2002/0102215 A1 Aug. 1, 2002 52

0359 a) Synthesis of a Lipopeptide With Affinity for thiol-containing lipopeptide from Example 64(a). To this Thrombi (Dipalmitoyl-Lys-Asn-Asp-Gly-Asp-Phe-Glu was added 1.0 ml of a solution of 1.4% propylene glycol/ Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln-NH) (SEQ ID NO:18) 2.4% glycerol and the mixture was Sonicated for 2 minutes

COOH COOH O O

N Rul N H H H O YCONH, O O COOH

COOH O O

N 1. O O O H H H E i N N N .. N N NH2 H H E N O O N COOH CONH2

OH

0360 The lipopeptide was synthesised on an ABI 433 A and then warmed to 80° C. for 5 minutes. Immediately automatic peptide Synthesiser Starting with Rink amide resin following warming, the Solution was filtered through a 4 on a 0.1 mmol Scale using 1 mmolamino acid cartridges. All micron filter. The Sample was cooled to room temperature amino acids and palmitic acid were preactivated using and the head Space flushed with perfluorobutane gas. The HBTU before coupling. The simultaneous removal of pep Vial was shaken in a cap mixer for 45 Seconds and the tide from the resin and Side-chain protecting groups was resulting microbubbles were washed twice with deionised carried out in TFA containing 5% phenol, 5% EDT, 5% water. The infranatant was discarded and replaced with a 1 aniSole and 5% H2O for 2 hours, giving a crude product ml aliquot of the protein solution from (b) above. The yield of 80 mg. Purification by preparative HPLC of a 20 mg conjugation reaction was allowed to proceed for 1 hour. The aliquot of the crude material was carried out. After lyophi microbubbles were centrifuged and the infranatant was lisation, 6 mg of pure material was obtained. The product exchanged with a further 1 ml of protein solution. The was characterised by MALDI mass spectrometry and ana incubation Step was repeated until all protein Solution was lytical HPLC. used up. The microbubbles were then washed extensively 0361 b) Modification of Tissue Plasminogen Activator with water and analysed by Coulter counter. The With Sulpho-SMPB microbubbles were tested in the flow chamber assay described in Example 21(c). Microbubbles modified with 0362. A solution of 0.1 ml of ammonim carbonate buffer protein were found to bind in higher numbers than those containing 0.1 mg of t-PA was made up to 0.2 ml by the comprising either lipopeptide/DSPS or DSPS alone. addition of water. To this solution was added 0.4 mg of Sulpho-SMPB (dissolved in 0.05 ml DMSO. The protein 0365. The targeting/therapeutic/ultrasound activities of Solution was left Standing at room temperature for 45 these microbubbles be evaluated in models of both in vitro minutes, whereafter purification was carried out on a Super and in Vivo thrombogenisis. dex 200 column. The product was eluted in PBS and the modified protein fraction was collected. EXAMPLE 38 0363 c) Preparation of Gas-Filled Microbubbles Encap Preparation of Gas-Filled Microbubbles Comprising sulated With DSPS/Thrombi-Binding Lipopeptide and DSPS Loaded With a Lipopeptide Comprising a Thiol-Containing Lipoeptide and Conjugation of Modified Helical Peptide With Affinity for Cell Membranes TiSSue Plasminogen Activator 0366. This example is directed to the preparation of 0364 DSPS (5.0 mg) was weighed into a clean vial along targeted microbubbles comprising a peptidic vector for with 0.5 mg of the lipopeptide from (a) and 0.5 mg of the targeting of cell membrane Structures. US 2002/0102215 A1 Aug. 1, 2002 53

0367 a) Synthesis of a Lipopeptide Comprising a Helical tide could be detected by MALDI-MS. Coulter counter, Peptide With Affinity for Cell Membranes (SEQ ID NO:12) acoustic attenuation and preSSure Stability Studies were

| Nulls N Nulls N Nulls N N n O E h O h O E h O

N N M. V. M. V. H H H H H H H. H. V / V / N N

O | O | O | O | O | O ----N-N N --- N Nulls--N-sul-N-lN N N --- H " . . . " . . . . H

N M V H. H.

0368. The lipopeptide was synthesised on an ABI 433A performed. To an aliquot of the washed bubbles (ca. 0.2 ml) automatic peptide Synthesiser Starting with Rink amide resin was added methanol (0.5 ml), and the mixture was placed in on a 0.2 mmol Scale using 1 mmolamino acid cartridges. All a Sonicator bath for 2 minutes. The resulting clear Solution, amino acids and 2-m-hexadecylstearic acid were preacti- on analysis by MALDI-MS, was found to contain the vated using HBTU before coupling. The simultaneous lipopeptide. removal of lipopeptide from the resin and Side-chain pro- 0371 c) In vitro and in vivo Tests tecting groups was carried out in TFA containing 5% HOfor 2 hours, giving a crude product yield of 520 mg. Purification 0372 The microbubbles had similar characteristics in by preparative HPLC of a 30 mg aliqout of crude material vitro and in vivo as was found for the microbubbles made in was carried out using a gradient of 90 to 100% B over 40 Example 21. minutes (A=0.1% TFA/water and B=MeOH) at a flow rate of 9 ml/min. After lyophilisation, 10 mg of pure material was EXAMPLE 39 obtained (analytical HPLC, gradient 90-100% B over 20 minutes where B=MeOH, A=0.01% TFA/water: detection- Gas-Filled Microbubbles Encapsulated With UV 214 nm-product retention time=23 minutes). Further Phosphatidylserine and a Biotinylated Lipopeptide product characterisation was carried out using MALDI maSS spectrometry: expected M--H at 2369, found at 2375. 0373) a) Synthesis of Lipopeptide Dipalmitoyl-lysinyl tryptophanyl-lysinyl-lysinyl-lysinyl(biotinyl)-glycine (SEQ 0369 b) Preparation of Gas-Filled Microbubbles ID NO:19) 0370 DSPS (4.5 mg) and lipopeptide from (a)(0.5 mg) 0374. The lipopeptide was synthesised on an ABI 433A were weighed into a clean vial and 1.0 ml of a Solution of automatic peptide Synthesiser Starting with Fmoc-Gly-Wang 1.4% propylene glycol/2.4% glycerol was added. The mix- resin on a 0.1 mmol Scale using immol amino acid car ture was Sonicated for 3-5 minutes, warmed to 80° C. for 5 tridges. All amino acids and palmitic acid were preactivated minutes and then filtered through a 4.5 mm filter. The using HBTU before coupling. The simultaneous removal of mixture was cooled to room temperature and the head Space peptide from the resin and Side-chain protecting groups was was flushed with perfluorobutane gas. The Vial was Shaken carried out in TFA containing 5% phenol, 5% EDT, 5% in a cap mixer for 45 Seconds and the resulting microbubbles aniSole and 5% HO for 2 hours, giving a crude product were centrifuged at 1000 rpm for 3 minutes. The yield of 150 mg. Purification by preparative HPLC of a 40 microbubbles were then washed with water until no lipopep- mg aliqout of crude material was carred out using a gradient US 2002/0102215 A1 Aug. 1, 2002 54 of 70 to 100% B over 40 minutes (A=0.1% TFA/water and confirm incorporation of lipopeptide into DSPS B=MeOH) at a flow rate of 9 ml/min. After lyophilisation. microbubbles as follows: ca. 50-100 ml of microbubbles 14 mg of pure material (analytical HPLC, gradient 70-100% were transferred to a clean vial and 50-100 ml water were B where B=MeOH, A=0.01% TFA/water: detection-UV added. The mixture was Sonicated for 30 seconds and 260 and fluorescence, Ex280, Em350 product retention spotted onto a clean target disc (1 ml+0.5 ml ACH matrix). time=22 minutes). Further product characterisation was car Positive mode gave M+H at 1474, expected for lipopeptide ried out using MALDI mass spectrometry: expected M--H at at 1478. 1478, found at 1471. 0375 b) Preparation of Gas-Filled Microbubbles Com EXAMPLE 40 prising DSPS Doped With the Biotinylated Lipopeptide Sequence from (a) Preparation of Multiple-Specific Gas-Filled 0376 DSPS (4.5 mg) and lipopeptide from (a) (0.5 mg, Microbubbles Comprising DSPS Loaded With a 0.2 mmol) were weighed into each of two vials, and 0.8 ml Lipopeptide Comprising a Non-Bioactive of a Solution of 1.4% propylene glycol/2.4% glycerol was Interleukin-1 Receptor-Binding Peptide added to each vial. The mixtures were warmed to 80 C. for 0377 This example is directed to the preparation of 5 minutes (vials shaken during warming). The samples were targeted microbubbles comprising a non-bioactive peptidic cooled to room temperature and the head Spaces were vector for targeting at the IL-1 recptor which does not induce flushed with perfluorobutane gas. The vials were shaken in Signal tranduction or prevent IL-1 binding. a cap mixer for 45 Seconds and then rolled overnight. The resulting microbubbles were washed several times with 0378 a) Synthesis of a Lipopeptide Comprising a non deionised water and analysed by Coulter counter and acous Bioactive Interleukin-1 Receptor-Binding Peptide (SEQ ID tic attenuation. MALDI mass spectral analysis was used to NO:15)

H O H O H 4N NH

| H-N N-H

H Y o1 N-H O |Null O |Nulls O |Nulls O |Nulls O | susO O E H O H E H O H O H

H-N O %YN-H US 2002/0102215 A1 Aug. 1, 2002

0379 The lipopeptide was synthesised on an ABI 433A EXAMPLE 42 automatic peptide Synthesiser Starting with Fmoc-Ala-Wang resin on a 0.1 mmol Scale using 1 mmol amino acid Preparation of Sulphur Hexafluoride-Containing 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 tecting groups was carried out in TFA containing 5% H2O, 0383) To 0.8 ml of a solution containing DSPC:DSPS 5% anisole, 5% phenol and 5% EDT for 2 hours, giving a (3:1) (5mg/ml) in propylene glycol/glycerol (4% in water) 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 Sulphur hexafluoride gas. The TFA/water and B=MeOH) at a flow rate of 9 ml/min. After Vial was shaken on a cap-mixer for 45 Seconds and placed lyophilisation, 4 mg of pure material was obtained (analyti on a roller table for 5 minutes. The Sample was centrifuged cal HPLC, gradient 90-100% B over 20 minutes where at 2000 rpm for 5 minutes and the infranatant was removed B=MeOH, A=0.01% TFA/water: Detection-UV 214 and replaced with distilled water. The headspace was again nim-product retention time=23 minutes). Further product flushed with Sulphur hexafluoride and the Sample was kept characterisation was carried out using MALDI maSS Spec on a roller table until a homogenous appearance was trometry: expected M+H at 2083, found at 2088. obtained. The Washing procedure was repeated. 0380 b) Preparation of Gas-Filled Microbubbles 0384. The resulting ultrasound contrast agent was con firmed by Coulter counter, acoustic attenuation measure 0381 DSPS (4.5 mg) and lipopeptide from (a) (0.5 mg) ments and resistance to external pressure. were weighed into a clean vial and 1.0 ml of a Solution of 1.4% propylene glycol/2.4% glycerol was added. The mix EXAMPLE 43 ture was Sonicated for 3-5 mins, warmed to 80° C. for 5 minutes and then filtered through a 4.5 micron filter. The Preparation of Gas-Filled Microbubbles Comprising mixture was cooled to room temperature and the head Space DSPG and Endothelial Cell-Binding Lipopeptide was flushed with perfluorobutane gas. The vials were shaken for Targeted Ultrasound Imaging in a cap mixer for 45 Seconds and the resulting microbubbles 0385) To 0.8 ml of a solution containing DSPG (Smg/ml) were centrifuged at 1000 rpm for 3 minutes. The in propylene glycol/glycerol (4% in water) was added 0.5 microbubbles were then washed with water until no lipopep mg of the lipopeptide from Example 31(b). The mixture was tide could be detected by MALDI-MS. To the washed heated to 80° C. for 5 minutes and shaken. The solution was microbubbles (ca. 0.2 ml) was added methanol (0.5 ml), and then cooled to ambient temperature and the headspace was the mixture was placed in a Sonicator bath for 2 minutes. The flushed with perfluorobutane. The vial was shaken on a resulting clear Solution, on analysis by MALDI-MS, was cap-mixer for 45 Seconds and placed on a roller table for 5 found to contain lipopeptide (expected 2083, found 2088). minutes. The sample was centrifuged at 2000 rpm for 5 minutes and the infranatant was removed and replaced with distilled water. The headspace was again flushed with per EXAMPLE 41 fluorobutane and the Sample was kept on a roller table until a homogenous appearance was obtained. The Washing pro Preparation of Perfluoropropane-Filled cedure was repeated. The resulting ultrasound contrast agent Microbubbles Comprising DSPC, DSPS and was characterised by Coulter counter analysis, acoustic Endothelial Cell-Binding Lipopeptide for Targeted attenuation measurements and resistance to external pres Ultrasound Imaging sure. The microbubbles were tested in the in vitro assay as 0382 To 0.8 ml of a solution containing DSPC:DSPS detailed in Example 21: a gradual accumulation of (3:1) (5mg/ml) in propylene glycol/glycerol (4% in water) microbubbles binding to the cells was observed. was added 0.5 mg of the lipopeptide from Example 31(b). EXAMPLE 44 The mixture was heated to 80° C. for 5 minutes and shaken. The Solution was then cooled to ambient temperature and the Preparation of Perfluoropropane-Filled headspace was flushed with perfluoropropane. The Vial was Microbubbles Comprising DSPG and Endothelial Shaken on a cap-mixer for 45 Seconds and placed on a roller Cell Binding Lipopeptide for Targeted Ultrasound table for 5 minutes. The sample was centrifuged at 2000 rpm Imaging for 5 minutes and the infranatant was removed and replaced 0386 To 0.8 ml of a solution containing DSPG (5 mg/ml) with distilled water. The headspace was again flushed with in propylene glycol/glycerol (4% in water) was added 0.5 perfluoropropane and the Sample was kept on a roller table mg of the lipopeptide from Example 31(b). The mixture was until a homogeneous appearance was obtained. The Washing heated to 80° C. for 5 minutes and then shaken. The Solution procedure was repeated. The resulting ultrasound contrast was then cooled to ambient temperature and the headspace agent was characterised by Coulter counter analysis, acous was flushed with perfluoropropane. The Vial was shaken on tic attenuation measurements and resistance to external a cap-mixer for 45 Seconds and placed on a roller table for pressure. The microbubbles were tested in the in vitro assay 5 minutes. The sample was centrifuged at 2000 rpm for 5 as detailed in Example 21. A gradual accumulation of minutes and the infranatant was removed and replaced with microbubbles binding to the cells was observed. distilled water. The headspace was again flushed with per US 2002/0102215 A1 Aug. 1, 2002 56 fluorobutane and the Sample was kept on a roller table until washing with water, PBS and water, the final Solution was a homogeneous appearance was obtained. The Washing examined for polylysine content using MALDI MS. No procedure was repeated. The resulting ultrasound contrast polypeptide material was observed in the final wash Solu agent was characterised by Coulter counter analysis, acous tion. Acetonitrile (0.5 ml) was then added and the tic attenuation measurements and resistance to external microbubbles were Sonicated until all bubbles had burst. pressure. The microbubbles were tested in the in vitro assay Analysis of the resulting Solution for polylysine was again as detailed in Example 21: a gradual accumulation of carried out using MALDI MS. The results were as follows: microbubbles binding to the cells was observed. EXAMPLE 45 MALDI expected MALDI found Preparation of Sulphur Hexafluoride-Containing Poly-L-lysine 786,914, 1042, 117O 790,919, 1048, 1177 Microbubbles Comprising DSPG and Endothelial Cell-Binding Lipopeptide for Targeted Ultrasound Imaging EXAMPLE 47 0387 To 0.8 ml of a solution containing DSPG (5mg/ml) Preparation of Functionalised Gas-Filled in propylene glycol/glycerol (4% in water) was added 0.5 Microbubbles for Targeted Ultrasound Imaging mg of the lipopeptide from Example 31(b). The mixture was (SEQ ID NO:20) heated to 80 C. for 5 minutes and shaken. The solution was 0389. This example is directed to the preparation of then cooled to ambient temperature and the headspace was microbubbles having a reactive group on the Surface for flushed with Sulphur hexafluoride gas. The vial was shaken non-specific targeting, principally utilising disulphide on a cap-mixer for 45 Seconds and placed on a roller table eXchange reactions to effect binding to a multiplicity of for 5 minutes. The sample was centrifuged at 2000 rpm for cellular targets. 5 minutes and the infranatant was removed and replaced 0390 a) Synthesis of a Thiol-Functionalised Lipid Mol with distilled water. The headspace was again flushed with ecule (SEQ ID NO:20)

O

H NH2

SH O O O H H N Null N N--- N OH O O O

NH2

Sulphur hexafluoride and the sample was kept on a roller 0391 The lipid structure shown above was synthesised table until a homogeneous appearance was obtained. The on an ABI 433A automatic peptide Synthesiser Starting with Washing procedure was repeated. The resulting ultrasound Fmoc-Cys(Trt)-Wang resin on a 0.25 mmol scale using 1 contrast agent was characterised by Coulter counter analy mmol amino acid cartridges. All amino acids and palmitic sis, acoustic attenuation measurements and resistance to acid were preactivated using HBTU coupling chemistry. The external pressure. Simultaneous removal of peptide from the resin and depro EXAMPLE 46 tection of Side-chain protecting groups was carried out in TFA containing 5% EDT and 5% HO for 2 hours, giving a Targeted Gas-Filled Microbubbles Comprising crude product yield of 250 mg. Purification by preparative DSPS Coated Non-Covalently With Polylysine HPLC of a 40 mg aliquot of crude material was carried out 0388 DSPS (5 mg) was weighed into a clean vial along using a gradient of 90 to 100% B over 50 minutes (A=0.1% with poly-L-lysine (0.2 mg). To the vial was added 1.0 ml of TFA/water and B=MeOH) at a flow rate of 9 ml/min. After a solution of 1.4% propylene glycol/2.4% glycerol. The lyophilisation, 24 mg of pure material was obtained (ana mixture was warmed to 80 C. for 5 minutes. The sample lytical HPLC, gradient 70-100% B where B=0.1% TFA/ was cooled to room temperature and the head Space flushed acetonitrile, A=0.01% TFA/water: detection-UV 214 with perfluorobutane gas. The vial was shaken in a cap mixer nim-product retention time=23 minutes). Further product for 45 seconds and the resulting microbubbles were centri characterisation was carried out using MALDI maSS Spec fuged at 1000 rpm for 3 minutes. Following extensive trometry: expected M+H at 1096, found at 1099. US 2002/0102215 A1 Aug. 1, 2002 57

0392 b) Preparation of Gas-Filled Microbubbles Com tide from the resin and Side-chain protecting groups was prising DSPS Doped With a Thiol-Containing Lipid Struc carried out in TFA containing 5% phenol, 5% EDT, 5% ture anisole and 5% HO for 2 hours, giving a crude product 0393 DSPS (4.5 mg) and the lipid structure from (a) yield of 80 mg. Purification by preparative HPLC of a 20 mg above (0.5 mg, 0.4 mmol) were weighed into a clean vial and aliquot of the crude material was carried out. After lyophi 0.8 ml of a solution containing 1.4% propylene glycol/2.4% lisation, 6 mg of pure material were obtained. The product glycerol in water was added. The mixture was warmed to 80° C. for 5 minutes (vial shaken during warming) and was characterised by MALDI mass spectrometry and ana filtered while still hot through a 40 mm filter. The sample lytical HPLC. was cooled to room temperature and the head Space was flushed with perfluorobutane gas. The Vial was Shaken in a 0396 b) Preparation of Thromi-Targeting Ultrasound cap mixer for 45 Seconds and then placed on roller table Microbubbles overnight. The resulting microbubbles were washed several 0397) DSPS (4.5 mg) and lipopeptide from (a) (1.0 mg) times with deionised water and analysed for thiol group were weighed into a vial and 0.8 ml of a solution of 1.4% incorporation using Ellmans Reagent. propylene glycol/2.4% glycerol was added. The mixture was warmed to 80 C. for 5 minutes and then filtered through a EXAMPLE 48 4 micron filter. After cooling to room temperature the head Preparation of Gas-Filled Microbubbles Comprising Space was flushed with perfluorobutane gas. The Vial was DSPS Doped With a Thrombus-Binding Shaken in a cap mixer for 45 Seconds and the resulting Lipopeptide microbubbles were washed extensively with deionised water. The microbubbles were characterised by microscopy 0394) a) Synthesis of a Lipopeptide With Affinity for and Coulter counter analysis. MALDI-MS was used to Thrombi (Dipalmitoyl-Lys-Asn-Asp-Gly-Asp-Phe-Glu confirm the presence of lipopeptide as described in previous Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln-NH) (SEQ ID NO:18) examples.

COOH

Nii

COOH N -NH O O O H H H O N N NH2 H H

COOH CONH2

OH

0395. The lipopeptide was synthesised on an ABI 433 A EXAMPLE 49 automatic peptide Synthesiser Starting with Rink amide resin Preparation of Transferrin-Coated Gas-Filled on a 0.1 mmol Scale using 1 mmolamino acid cartridges. All Microbubbles for Targeted Ultrasound Imaging amino acids and palmitic acid were preactivated using 0398 a) Synthesis of a Thiol-Functionalised Lipid Mol HBTU before coupling. The simultaneous removal of pep ecule (SEQ ID NO:20) US 2002/0102215 A1 Aug. 1, 2002 58

O

H NH2

S O O H H N Nulls N N--- N OH H H H - O O O

0399. The lipid structure shown above was synthesised a Sephadex 200 column using PBS as eluent. The protein on an ABI 433A automatic peptide Synthesiser Starting with fraction was collected and Stored at 4 C. prior to use. Fmoc-Cys(Trt)-Wang resin on a 0.25 mmol scale using 1 mmol amino acid cartridges. All amino acids and palmitic 04.04 d) Microbubble Conjuration With Transferrin acid were preactivated using HBTU before coupling. The 04.05) To the thiol-containing microbubbles from (b) was Simultaneous removal of peptide from the resin and depro added 1 ml of the modified transferrin protein solution from tection of Side-chain protecting groups was carried out in (c). After adjusting the pH of the Solution to 9 the conjuga TFA containing 5% EDT and 5% HO for 2 hours, giving a tion reaction was allowed to proceed for 2 hours at room crude product yield of 250 mg. Purification by preparative temperature. Following extensive Washing with deionised HPLC of a 40 mg aliquot of crude material was carried out water the microbubbles were analysed by Coulter counter using a gradient of 90 to 100% B over 50 minutes (A=0.1% (97% between 1 and 5 mm) and fluorescence microscopy TFA/water and B=MeOH) at a flow rate of 9 ml/min. After (highly fluorescent microbubbles). lyophilisation, 24 mg of pure material was obtained (ana EXAMPLE 50 lytical HPLC, gradient 70-100% B where B=0.1% TFA/ acetonitrile, A=0.01% TFA/water: detection-UV 214 Gas-Filled Microbubbles Comprising DSPS nim-product retention time=23 minutes). Further product Incorporating PE-PEGooo-Mal Conjugated to characterisation was carried out using MALDI maSS Spec Thiolated Trypsin Fluorescein trometry: expected M+H at 1096, found at 1099. 0406) a) Synthesis of Boc-NH-PEGo-DSPE (t-butyl Carbamate Poly(Ethylene Glycol)Distearoylphosphati 0400 b) Preparation of Gas-Filled Microbubbles Com dylethanolamine) prising DSPS Doped With a Thiol-Containing Lipid Struc ture 04.07 DSPE (31 mg) was added to a solution of Boc NH-PEG-SC (150 mg) in chloroform (2 ml), followed 04.01 DSPS (4.5 mg) and lipid structure from (a) above by triethylamine (33 ul). The mixture was stirred at 41° C. (0.5 mg, 0.4 mmol) were weighed into a clean vial and 0.8 for 10 minutes until the starting material had dissolved. The ml of a Solution of 1.4% propylene glycol/2.4% glycerol was Solvent was rotary evaporated and the residue was taken up added. The mixture was warmed to 80 C. for 5 minutes in acetonitrile (5 ml). The resulting dispersion was cooled to (vial shaken during warming) and filtered while still hot 4 C. and centrifuged, whereafter the solution was filtered through a 40 mm filter. The sample was cooled to room and evaporated to dryneSS. The Structure of the resulting temperature and the head Space was flushed with perfluo product was confirmed by NMR. robutane gas. The Vial was Shaken in a cap mixer for 45 Seconds and then placed on roller table overnight. The 0408 b) Synthesis of HN-PEGo-DSPE (Amino-poly resulting microbubbles were washed several times with (Ethylene Glycol)-distearoylphosphatidylethanolamine) deionised water and analysed for thiol group incorporation 04.09 Boc-NH-PEGooo-DSPE (167 mg) was stirred in 4 using Ellmans Reagent. M hydrochloric acid in dioxane (5 ml) for 2.5 hours at 0402 c) Modification of Transferrin With Fluorescein ambient temperature. The solvent was removed by rotary NHS and Sulpho-SMPB evaporation and the residue was taken up in chloroform (1.5 ml) and washed with water (2x1.5 ml). The organic phase 0403) To 4 mg of transferrin (Holo, human) in PBS (1 ml) was evaporated in vacuo. TLC analysis (chloroform/metha was added 0.5 ml DMSO solution containing 1 mg Sulpho nol/water 13:5:0.8) gave a single ninhydrin positive spot SMPB and 0.5 mg fluorescein-NHS. The mixture was stirred with Rf=0.6; confirmation of the structure was obtained by for 45 minutes at room temperature and then passed through NMR. US 2002/0102215 A1 Aug. 1, 2002 59

0410 c) Synthesis of Mal-PEGooo-DSPE (3-maleimi then charged on a SephadeX 200 column and the product was dopropionate Poly(Ethylene Glycol)Distearoylphosphati eluted with PBS. The protein fraction containing maximum dylethanolamine) fluorescent intensity was collected in a total Volume of 6 ml. 0411 A Solution of N-Succinimidyl-3-maleimidopropi 0418 g) Conjugation of Microbubbles With Thiolated, onate (5.6 mg, 0.018 mmol) in tetrahydrofuran (0.2 ml) was Fluorescein-Labelled Trypsin added to HN-PEG--DSPE (65 mg, 0.012 mmol) dis 0419 Microbubbles from (d) were incubated on a roller solved in tetrahydrofuran (1 ml) and 0.1 M sodium phos table in 1 ml of protein solution from (f) above. The phate buffer pH 7.5 (2 ml). The mixture was warmed to 30° conjugation was allowed to proceed at pH 7.3-7.8 for 10 C. and the reaction was followed to completion by TLC, minutes before centrifugation and removal of the infrana whereafter the Solvent was removed in vacuo. The title tant. The process was repeated a further three times, after material was purified on a flash Silica column using 80:20 which the bubbles were washed four times with water to chloroform: methanol as eluent. The structure of the pure remove unconjugated protein. product was confirmed by NMR and mass spectrometry. 0420 D. Bubbles contained active enzyme as evi 0412 d) Preparation of Gas-Filled Microbubbles of denced by the cleavage of an Arg-pNA derivative in DSPS Doped' With PE-PEG-Mal PBS. 0413 DSPS (4.5 mg) and PE-PEGooo-Mal from (c) 0421 E. Analysis of the bubbles by Coulter and mea above (0.5 mg) were weighed into a clean vial and 1 ml of Surement of echogenicity was carried out. a Solution of 1.4% propylene glycol/2.4% glycerol was 0422) Bubbles were Pressure Stable (see FIG. 2) added. The mixture was warmed to 80 C. for 5 minutes and then filtered through a 4.5 mm filter. The sample was cooled to room temperature and the head Space was flushed with FEK-O22-O15 perfluorobutane gas. The Vial was shaken in a cap mixer for Total O.83 45 seconds and the resulting microbubbles were washed concentration three times with distilled water. Diameter 1-3 mm 40 Diameter 3-5 mm 28 0414) e) Preparation of Fluorescein-Labelled Trypsin Diameter 5-7 mm 13 Freq of max Atten. 3.3 0415) To 5 mg of trypsin in PBS (1 ml) was added 0.2 ml Atten at 2. Mhz. 4.9 DMSO solution containing 1 mg of fluorescein-NHS. The Atten at 3.5 Mhz. 7.8 mixture was stirred for 45 minutes at room temperature. A Atten at 5.0 MHz, 7.2 Sephadex 200 column was then charged with the modified protein mixture and product was eluted at a flow rate of 1 ml/min using PBS. The protein fraction (5 ml) was collected and stored at 4 C. EXAMPLE 51 0416 f) Preparation of Thiolated, Fluorescein-Labelled Gas-Filled Microbubbles Comprising DSPS and a Trypsin Captopril-Containing Molecule for Diagnostic and Therapeutic Applications 0417. To the protein fraction from (e) was added 1 mg of Traut's reagent and the mixture Stirred at room temperature 0423 a) Synthesis of a Lipopeptide Functionalised With for a further 1 hour. 4 ml of the Trauts-modified product was Captopril (SEQ ID NO:13)

~~~~ NH2

O O O H H N N N N NH2 H O

NH2 "N^. ~us S O O) US 2002/0102215 A1 Aug. 1, 2002 60

0424 The structure shown above was synthesised by the ethyl acetate, washed with brine and dried (Na2SO). The manual “bubbler method starting with Fmoc-protected Rink Solution was filtered and concentrated. The dark residue was Amide MBHA resin on a 0.125 mmol scale. Coupling was chromatographed (silica, hexane/ethyl acetate 7:3) to give carried out using standard TBTU/HOBt/DIEA protocol. 2.25 g (34%) of a colourless oil. "H (300 MHz) and 'C Bromoacetic acid was coupled through the Side-chain of LyS NMR (75 MHz) spectra were in accordance with the struc as a Symmetrical anhydride using DIC preactivation. Cap ture. topril dissolved in DMF was introduced on the solid phase using DBU as base. Simultaneous removal of the peptide 0430 ii) Synthesis of Methyl 4-2-hydroxy-3-(1-meth from the resin and deprotection of Side-chain protecting ylethyl)aminopropoxyphenylacetate groups was carried out in TFA containing 5% EDT, 5% water and 5% ethyl methylsulphide for 2 hours. An aliquot 0431) A mixture of methyl 4-(2,3-epoxy)propoxyphe of 10 mg of the crude material was purified by preparative nylacetate (2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 liquid chromatography using a gradient of 70 to 100% B mol) and water (1.35 ml, 74.7 mmol) was stirred at room over 60 minutes (A=0.1% TFA/water and B=0.1% TFA/ temperature overnight. The reaction mixture was concen acetonitrile) at a flow rate of 10 ml/min. After lyophilisation, trated (rotavapor) and the oily residue was dissolved in a yield of 2 mg of pure material was obtained (analytical chloroform and dried (NaSO). Filtration and concentration HPLC, gradient 70-100% B over 20 minutes, A=0.1% gave quantitative yield of a yellow oil that was used in the TFA/water and B=0.1% TFA/acetonitrile, flow rate 1 ml/min, detection UV 214 nm, retention time 26 minutes). next Step without further purification. The Structure was Further characterisation was carried out using MALDI maSS verified by H and 'C NMR analysis. Spectrometry, giving M--H at 1265 as expected. 0432 iii) Synthesis of 4-2-hydroxy-3-(1-methylethy 0425 b) Preparation of Gas-Filled Microbubbles Com l)aminopropoxyphenylacetic Acid Hydrochloride prising DSPS and a Compound Containing Captopril 0433) A solution of methyl 4-2-hydroxy-3-(1-methyl 0426 A Solution of 1.4% propylene glycol/2.4% glycerol ethyl)aminopropoxyphenylacetate (563 mg, 2.00 mmol) in (1.0 ml) was added to a mixture of DSPS (4.5 mg) and 6M hydrochloric acid (15 ml) was heated at 100° C. for product from (a) (0.5 mg) in a vial. The mixture was Sonicated for 5 minutes and then warmed to 80 C. for 5 4-hours. The reaction mixture was concentrated (rotavapor) minutes (vial was Shaken during warming). The Vial was and the residue was taken up in water and lyophilised. "H then cooled and the head Space was flushed with perfluo and 'C NMR spectra were in accordance with the strucure robutane gas. The Vial was Shaken in a cap mixer for 45 and MALDI mass spectrometry gave a M+H at 268 as Seconds and the resulting microbubbles were extensively expected. washed with deionised water. MALDI mass spectrometry 0434) iv) Synthesis of N-Boc-4-2-hydroxy-3-(1-meth showed no detectable level of compound from (a) in the final wash Solution. Incorporation of captopril-containing ylethyl)aminopropoxyphenylacetic Acid lipopeptide into the microbubbles was confirmed by 0435 A solution of the 4-2-hydroxy-3-(1-methylethy MALDI-MS as follows: ca. 50 ul of microbubbles were l)aminopropoxyphenylacetic acid hydrochloride (2.0 transferred to a clean vial containing ca. 100 ul of 90% methanol. The mixture was Sonicated for 30 seconds and mmol) in water (2 ml) was added to a Solution of Sodium analysed by MALDI mass Spectrometry, giving a M-H peak bicarbonate (0.60 g, 7.2 mmol) in water/dioxane (2:1, 15 corresponding to lipopeptide from (a). ml). A solution of di-tert-butyl dicarbonate (0.48 g, 2.2 mmol) in dioxane (5 ml) was added. Progress of the reaction EXAMPLE 52 was monitored by TLC analysis (silica, CHCl/MeOH/ AcOH 85:10:5), and portions of di-tert-butyl dicarbonate Gas-Filled Microbubbles Comprising DSPS and a were added until conversion was complete. The reaction Vector With Affinity for Adrenergic Receptors for Diagnostic and Therapeutic Applications mixture was poured onto water Saturated with potassium hydrogen Sulphate and organic material was extracted into 0427 a) Synthesis of a Protected Atenolol Derivative ethyl acetate. The organic phase was washed with water and Suitable for Solid Phase Coupling brine, dried (Na2SO) and filtered to give 0.6 g of crude 0428 i) Synthesis of Methyl 4-(2,3-epoxy)propoxyphe material. The product was purified by chromatography nylacetate (silica, CHCl/MeOH/AcOH 85:10:5). The solution was concentrated and the residue was taken up in glacial acetic 0429. A mixture of methyl 4-hydroxyphenylacetate (4.98 acid and lyophilised. Yield 415 mg (56%), white solid. The g, 0.030 mol), epichlorohydrin (23.5 ml, 0.30 mol) and structure was confirmed by H and 'C NMR analysis. pyridine (121 ul, 1.5 mmol) was stirred at 85°C. for 2 hours. The reaction mixture was cooled and exceSS epichlorohydrin 0436 b) Synthesis of a Lipopeptide Functionalised With was distilled off (rotavapor). The residue was taken up in Atenolol (SEQ ID NO:21) US 2002/0102215 A1 Aug. 1, 2002 61

NH2

---O N--H O i H NH2 HN O---

0437. The structure shown above was synthesised by the MALDI mass spectrometry showed no detectable level of manual bubbler method starting with Fmoc-protected Rink compound from (b) in the final wash Solution. Incorporation Amide MBHA resin on a 0.125 mmol scale, using the of atenolol-containing lipopeptide into the microbubbles compound from (a). Coupling was carried out using stan was confirmed by MALDI-MS as follows: ca. 50 ul of dard TBTU/HOBt/DIEA protocols. Simultaneous removal microbubbles were transferred to a clean vial containing ca. of the peptide from the resin and deprotection of Side-chain 100 ul of 90% methanol. The mixture was Sonicated for 30 protecting groups was carried out in TFA containing 5% seconds and analysed by MALDI-MS (ACH-matrix), giving EDT and 5% water for 2 hours. Crude material was pre a M+H peak at 1259 corresponding to lipopeptide (b). cipitated from ether and purified by preparative liquid chro matography using a gradient of 70 to 100% B over 60 minutes (A=0.1% TFA/water and B=0.1%TFA/acetonitrile) 0440 d) In vitro Analysis at a flow rate of 10 ml/min. After lyophilisation, a yield of 38 mg of pure material was obtained (analytical HPLC, gradient 70-100% B over 20 minutes, A=0.1% TFA/water 0441 The microbubbles were tested in the in vitro assay and B=0.1% TFA/acetonitrile, flow rate 1 ml/minute, detec as detailed in Example 21. A gradual accumulation of tion UV 214 nm, retention time 25 minutes). Further char microbubbles binding to the cells was observed. acterisation was carried out using MALDI mass spectrom etry (ACH matrix), giving M+H at 1258, expected 1257. EXAMPLE 53 0438 c) Preparation of Gas-Filled Microbubbles Com prising DSPS and a Lipopeptide Containing Atenolol Gas-Filled Microbubbles Comprising DSPS and a Lipopeptide Consisting of a Heparin 0439 A solution of 1.4% propylene glycol/2.4% glycerol Sulphate-Binding Peptide (KRKR) (SEQ ID NO:5) and a Fibronectin Peptide (WOPPRARI) (SEQ ID (1.0 ml) was added to a mixture of DSPS (4.5 mg) and NO:6) for Targeting and a Lipopeptide Containing product from (b) (0.5 mg) in a vial. The mixture was Atenolol for Therapeutic Application Sonicated for 5 minutes, heated at 80° C. for 5 minutes (vial was shaken during warming) and then cooled. The head Space was flushed with perfluorobutane gas and the vial was 0442 a) Synthesis of a Lipopeptide Consisting of a Shaken in a cap mixer for 45 Seconds, whereafter the Heparin Sulphate-Binding Peptide (KRKR) (SEQ ID NO:5) contents were extensively washed with deionised water. and a Fibronectin Peptide (WOPPRARI) (SEQ ID NO:6) US 2002/0102215 A1 Aug. 1, 2002 62

(SEO ID NO: 7) N NH2 s NH HN HN

O O O H H N N N N N H i H i H O O O

NH2 NH2 HN O

O O O H H N O N N NH2 1N1 ns NH HN 5 H O O O '',

e NH 6 /

0443) The lipopeptide was synthesised on an ABI 433A in ethyl acetate, washed with brine and dried (NaSO). 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% EDT, 5% anisole and 5% HO for 2 hours, giving a crude 0447 ii) Synthesis of Methyl 4-2-hydroxy-3-(1-meth product yield of 150 mg. Purification by preparative HPLC ylethyl)aminopropoxyphenylacetate of a 40 mg aliquot of crude material was carried out using 0448. A mixture of methyl 4-(2,3-epoxy)propoxyphe a gradient of 70 to 100% B over 40 minutes (A=0.1% nylacetate (2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 TFA/water and B=MeOH) at a flow rate of 9 ml/min. After mol) and water (1.35 ml, 74.7 mmol) was stirred at room lyophilisation, 16 mg of pure material was obtained (ana temperature overnight. The reaction mixture was concen lytical HPLC, gradient 70-100% B where B=MeOH, trated (rotavapor) and the oily residue was dissolved in A=0.01% TFA/water: detection-UV 260 and fluorescence, chloroform and dried (NaSO). Filtration and concentration Ex280, Em350 -product retention time=19.44 minutes). gave quantitative yield of a yellow oil that was used in the Further product characterisation was carried out using next Step without further purification. The Structure was MALDI mass spectrometry: expected M--H at 2198, found verified by H and 'C NMR analysis. at 2199 0449) iii) Synthesis of 4-2-hydroxy-3-(1-methylethy 0444 b) Synthesis of a Protected Atenolol Derivative l)aminopropoxyphenylacetic Acid Hydrochloride Suitable for Solid Phase Coupling 0450 A solution of methyl 4-2-hydroxy-3-(1-methyl 04:45 i) Synthesis of Methyl 4-(2,3-epoxy)propoxyphe ethyl)aminopropoxyphenylacetate (563 mg, 2.00 mmol) in nylacetate 6M hydrochloric acid (15 ml) was heated at 100° C. for 4 0446. A mixture of methyl 4-hydroxyphenylacetate (4.98 hours. The reaction mixture was concentrated (rotavapor) g, 0.030 mol), epichlorohydrin (23.5 ml, 0.30 mol) and and the residue was taken up in water and lyophilised. "H pyridine (121 ul, 1.5 mmol) was stirred at 85°C. for 2 hours. and 'C NMR spectra were in accordance with the strucure The reaction mixture was cooled, and exceSS epichlorohy and MALDI mass spectrometry gave a M+H at 268 as drin was distilled off (rotavapor). The residue was taken up expected. US 2002/0102215 A1 Aug. 1, 2002 63

0451 iv) Synthesis of N-Boc-4-2-hydroxy-3-(1-meth 25 minutes). Further characterisation was carried out using ylethyl)aminopropoxyphenylacetic Acid MALDI mass spectrometry (ACH matrix), giving M+H at 0452. A solution of the 4-2-hydroxy-3-(1-methylethy 1258, expected 1257. l)aminopropoxyphenylacetic acid hydrochloride (2.0 0455 d) Preparation of Gas-Filled Microbubbles Com mmol) in water (2 ml) was added to a Solution of Sodium prising DSPS and a Lipopeptide Consisting of a Heparin bicarbonate (0.60 g, 7.2 mmol) in water/dioxane (2:1, 15 Sulphate-Binding Peptide (KRKR) (SEQ ID NO:5), a ml). A solution of di-tert-butyl dicarbonate (0.48 g, 2.2 Fibronectin Peptide (WOPPRARI) (SEQ ID NO:6) and a mmol) in dioxane (5 ml) was added. Progress of the reaction Lipopeptide Containing Atenolol was monitored by TLC analysis (silica, CHCl/MeOH/ 0456. A solution of 1.4% propylene glycol/2.4% glycerol AcOH 85:10:5), and portions of di-tert-butyl dicarbonate (1.0 ml) was added to a mixture of DSPS (5.0 mg), product were added until conversion was complete. The reaction from (a) (0.5 mg) and product from (c) (0.5 mg) in a vial. mixture was poured onto water Saturated with potassium The mixture was Sonicated for 5 minutes and then heated at hydrogen Sulphate and organic material was extracted into 80° C. for 5 minutes (vial was shaken during warming). The ethyl acetate. The organic phase was washed with water and Solution was filtered and cooled. The head Space was flushed brine, dried (Na2SO) and filtered to give 0.6 g of crude with perfluorobutane gas and the vial was shaken in a cap material. The product was purified by chromatography mixer for 45 Seconds, whereafter the contents were exten (silica, CHCl/MeOH/AcOH 85:10:5). The solution was sively washed with deionised water. Incorporation of concentrated and the residue was taken up in glacial acetic atenolol-containing lipopeptide into the microbubbles was acid and lyophilised. Yield 415 mg (56%), white solid. The confirmed by MALDI-MS as follows: ca. 50 ul of structure was confirmed by H and 13C NMR analysis. microbubbles were transferred to a clean vial containing ca. 0453 c) Synthesis of a Lipopeptide Functionalised With 100 ul of 90% methanol. The mixture was Sonicated for 30 Atenolol (SEQ ID NO:21) seconds and analysed by MALDI-MS (ACH matrix), giving

O

NH2

O O O H H N N --~~~~ N N NH2 H O O---

0454. The structure shown above was synthesised by the two M-H peaks at 2202 and 1259, corresponding to lipopep manual bubbler method starting with Fmoc-protected Rink tide (a) and to lipopeptide (c) respectively. Amide MBHA resin on a 0.125 mmol scale, using appro 0457 e) In vitro Analysis priate amino acids, palmitic acid and the compound from 0458. The microbubbles were tested in the in vitro assay (a). Coupling was carried out using standard TBTU/HOBt/ as detailed in example 21. A gradual accumulation of DIEA protocols. Simultaneous removal of the peptide from microbubbles binding to the cells was observed. the resin and deprotection of Side-chain protecting groups was carried out in TFA containing 5%. EDT and 5% water EXAMPLE 54 for 2 hours. Crude material was precipitated from ether and Gas-Filled Microbubbles Comprising DSPS and a purified by preparative liquid chromatography using a gra Lipophilic Derivative of Atenolol With Affinity for dient of 70 to 100% B over 60 minutes (A=0.1% TFA/water Adrenergic Receptors for Diagnostic and and B=0.1% TFA/acetonitrile) at a flow rate of 10 ml/min. Therapeutic Applications After lyophilisation, a yield of 38 mg of pure material was 0459 a) Synthesis of N-hexadecyl-4-2-hydroxy-3-F(1- obtained (analytical HPLC, gradient 70-100% B over 20 methyethyl)aminopropoxyphenylacetamide minutes, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, 0460) i) Synthesis of Methyl 4-(2,3-epoxy)propoxyphe flow rate 1 ml/minute, detection UV 214 nm, retention time nylacetate US 2002/0102215 A1 Aug. 1, 2002

0461) A mixture of methyl 4-hydroxyphenylacetate (4.98 and hexadecylamine (60 mg, 0.25 mmol) in DMF (5 ml) was g, 0.030 mol), epichlorohydrin (23.5 ml, 0.30 mol) and cooled to 0° C. HOBt (39 mg, 0.25 mmol) and N-(3- pyridine (121 ul, 1.5 mmol) was stirred at 85°C. for 2 hours. dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride The reaction mixture was cooled and exceSS epichlorohydrin (water soluble carbodiimide) (48 mg, 0.25 mmol) were was distilled off (rotavapor). The residue was taken up in added. The reaction mixture was stirred at 0° C. for 1 hour ethyl acetate, washed with brine and dried (NaSO). The Solution was filtered and concentrated. The dark residue was and then at room temperature overnight. The reaction mix chromatographed (silica, hexane/ethyl acetate 7:3) to give ture was poured onto water (25 ml) containing Sodium 2.25 g (34%) of a colourless oil. "H (300 MHz) and 'C carbonate (2.5 g) and Sodium chloride (4.0 g). Precipitated NMR (75 MHz) spectra were in accordance with the struc material was filtered off, washed with water and taken up in ture. chloroform. The chloroform phase was washed with 5% 0462 ii) Synthesis of Methyl 4-2-hydroxy-3-(1-meth sodium carbonate and water and dried (NaSO). The solu ylethyl)aminopropoxyphenylacetate tion was filtered and concentrated to give 150 mg of yellow 0463 A mixture of methyl 4-(2,3-epoxy)propoxyphe white crude material. The product was purified by column nylacetate (2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 chromatography (silica, chloroform/methanol 95.5) to give mol) and water (1.35 ml, 74.7 mmol) was stirred at room 118 mg (80%) of white material. The structure was verified temperature overnight. The reaction mixture was concen by H (500 MHz) and 'C (125 MHz) NMR. The product trated (rotavapor) and the oily residue was dissolved in was further characterised by MALDI mass spectrometry, chloroform and dried (Na2SO). Filtration and concentration giving a M+Na peak at 614 as expected. gave quantitative yield of a yellow oil that was used in the next Step without further purification. The Structure was 0470 vi) Synthesis of N-hexadecyl-4-2-hydroxy-3-(1- verified by H and 'C NMR analysis. methyl-ethyl)aminopropoxyphenylacetamide 0464) iii) Synthesis of 4-2-hydroxy-3-(1-methylethy 0471) To a solution of N'-Boc-N-hexadecyl-4-2-hy l)aminopropoxyphenylacetic Acid Hydrochloride droxy-3-(1-methyl-ethyl)aminopropoxyphenylacetamide 0465) A solution of methyl 4-2-hydroxy-3-(1-methyl (10 mg) in dichloromethane (9 ml) was added trifluoroacetic ethyl)aminopropoxyphenylacetate (563 mg, 2.00 mmol) in acid (1 ml). The reaction mixture was stirred for 2 hours at 6M hydrochloric acid (15 ml) was heated at 100° C. for 4 room temperature. TLC (silica, chloroform/methanol 95:5) hours. The reaction mixture was concentrated (rotavapor) showed complete conversion of Starting material. Solvents and the residue was taken up in water and lyophilised. "H were evaporated off and the residue was taken up in water/ and 'C NMR spectra were in accordance with the strucure acetonitrile and lyophilised to give a quantitative yield of and MALDI mass spectrometry gave a M+H at 268 as white solid material. The structure was verified by "H (500 expected. MHz) and 'C (125 MHz) NMR analysis and further char 0466 iv) Synthesis of N-Boc-4-2-hydroxy-3-(1-meth acterised by MALDI mass spectrometry, giving M--H at 492 ylethyl)aminopropoxyphenylacetic Acid and M+Na at 514 as expected. 0467 A solution of the 4-2-hydroxy-3-(1-methylethy 0472 b) Preparation of Gas-filled Microbubbles Com l)aminopropoxyphenylacetic acid hydrochloride (2.0 prising DSPS and N-hexadecyl-4-2-hydroxy-3-(1-methyl mmol) in water (2 ml) was added to a Solution of Sodium ethyl)aminopropoxyphenylacetamide for Diagnostic and bicarbonate (0.60 g, 7.2 mmol) in water/dioxane (2:1, 15 Therapeutic Applications ml). A solution of di-tert-butyl dicarbonate (0.48 g, 2.2 mmol) in dioxane (5 ml) was added. Progress of the reaction 0473 A Solution of 1.4% propylene glycol/2.4% glycerol was monitored by TLC analysis (silica, CHCl/MeOH/ (1.0 ml) was added to a mixture of DSPS (4.5 mg) and AcOH 85:10:5), and portions of di-tert-butyl dicarbonate N-hexadecyl-4-2-hydroxy-3-(1-methylethyl)aminopro were added until conversion was complete. The reaction poxyphenylacetamide (0.5 mg) in a vial. The mixture was mixture was poured onto water Saturated with potassium Sonicated for 5 minutes and then heated at 80° C. for 5 hydrogen Sulphate and organic material was extracted into minutes (vial was shaken during warming). The Solution was ethyl acetate. The organic phase was washed with water and filtered and cooled. The head space was flushed with per brine, dried (Na2SO) and filtered to give 0.6 g of crude fluorobutane gas and the Vial was shaken in a cap mixer for material. The product was purified by chromatography 45 Seconds, whereafter the contents were extensively (silica, CHCl/MeOH/AcOH 85:10:5). The solution was washed with deionised water. Incorporation of compound concentrated and the residue was taken up in glacial acetic from (a) into the microbubbles was confirmed by MALDI acid and lyophilised. Yield 415 mg (56%), white solid. The MS as follows; ca. 50 ul of microbubbles were transferred structure was confirmed by H and 'C NMR analysis. to a clean vial containing ca. 100 ul of 90% methanol. The 0468 v) Synthesis of N'-Boc, N-hexadecyl-4-2-hy mixture was Sonicated for 30 Seconds and analysed by droxy-3-(1-methylethyl)aminopropoxyphenylacetamide MALDI-MS, giving a M+H peak at 492 corresponding to 0469 A solution of N-Boc-4-2-hydroxy-3-(1-methyl N-hexadecyl-4-2-hydroxy-3-(1-methylethyl)aminopro ethyl)aminopropoxyphenylacetic acid (92 mg, 0.25 mmol) poxyphenylacetamide. US 2002/0102215 A1 Aug. 1, 2002 65

EXAMPLE 55 acetonitrile, flow rate 1.0 ml/minute, giving a product peak with retention time 27 minutes detected at UV 368 nm. Gas-Filled Microbubbles Encapsulated With DSPS and a Compound Containing Folic Acid for 0476 b) Preparation of Gas-Filled Microbubbles Com Diagnostic Applications prising DSPS and a Lipopeptide Containing Folic Acid 0474 a) Synthesis of a Lipopeptide Containing Folic 0477. A solution of 1.4% propylene glycol/2.4% glycerol Acid (1.0 ml) was added to a mixture of DSPS (4.5 mg) and

(SEO ID NO: 22) O

NH NH2

O O O

N N N N NH2 H H O O

NH HN 2 NR O OH

N O - H O O O N Nn N R= H and/or 2 N N NH2 H O O

HO N O H O N 2 ls N N NH2 H

0475. The structure shown above was synthesised by the product from (a) (0.5 mg) in a vial. Dilute ammonia (to pH manual bubbler method starting with Fmoc-protected Rink 8) and DMSO (40 ul) were added and the mixture was Amide MBHA resin on a 0.125 mmol scale, using appro Sonicated for 5 minutes and then heated at 80° C. for 5 priate amino acids, palmitic acid and folic acid. Coupling minutes (vial was shaken during warming). The Solution was was carried out using standard TBTU/HOBt/DIEA proto filtered and cooled. The head space was flushed with per fluorobutane gas and the Vial was shaken in a cap mixer for cols. Simultaneous removal of the peptide from the resin and 45 Seconds, whereafter the contents were extensively deprotection of Side-chain protecting groups was carried out washed with deionised water. Incorporation of Structure in TFA containing 5% EDT and 5% water for 2 hours. Crude from (a) into the bubbles was confirmed by MALDI-MS as material was precipitated from ether and analysed by follows: ca. 50 ul of microbubbles were transferred to a MALDI mass spectrometry, giving a M--H peak correspond clean vial containing ca. 100 ul of 90% methanol. The ing to the structure at 1435, expected 1430. The material was mixture was Sonicated for 30 Seconds and analysed by further characterised by analytical HPLC, gradient 70-100% MALDI-MS (ACH matrix), giving a M+H peak at 1238 B over 20 minutes, A=0.1% TFA/water and B=0.1 TFA/ corresponding to structure from (a). US 2002/0102215 A1 Aug. 1, 2002

0478 c) In vitro Analysis 0485. A mixture of methyl 4-hydroxyphenylacetate (4.98 0479. The microbubbles were tested in the in vitro assay g, 0.030 mol), epichlorohydrin (23.5 ml, 0.30 mol) and as detailed in Example 21. A gradual accumulation of pyridine (121ul, 1.5 mmol) was stirred at 85°C. for 2 hours. microbubbles binding to the cells was observed. The reaction mixture was cooled and exceSS epichlorohydrin was distilled off (rotavapor). The residue was taken up in EXAMPLE 56 ethyl acetate, washed with brine and dried (Na2SO). The Solution was filtered and concentrated. The dark residue was Gas-Filled Microbubbles Comprising DSPS and a chromatographed (silica, hexane/ethyl acetate 7:3) to give Cholesterol Ester of Chlorambucil for Diagnostic 2.25 g (34%) of a colourless oil. "H (300 MHz) and 'C and Therapeutic Applications NMR (75 MHz) spectra were in accordance with the struc 0480) a) Synthesis of Cholesterol 4-F4-Fbis(2-chloroet ture. hyl)aminolphenylbutanoate DIC (170 ul, 1.10 mmol) was added to a solution of chlorambucil (669 mg, 2.20 mmol) in 0486 ii) Synthesis of Methyl 4-2-hydroxy-3-(1-meth dry dichloromethane (15 ml). The mixture was stirred at ylethyl)aminopropoxyphenylacetate room temperature for 0.5 hour and added to a solution of 0487. A mixture of methyl 4-(2,3-epoxy)propoxyphe cholesterol (387 mg, 1.00 mmol) and DMAP (122 mg, 1.00 nylacetate (2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 mmol) in dichloromethane (10 ml). The reaction mixture was stirred overnight and then poured onto 5% sodium mol) and water (1.35 ml, 74.7 mmol) was stirred at room bicarbonate. The phases were separated and the organic temperature overnight. The reaction mixture was concen phase was washed with brine and dried (MgSO). The trated (rotavapor) and the oily residue was dissolved in Solution was filtered and concentrated and the product was chloroform and dried (NaSO). Filtration and concentration purified by column chromatography (Silica, chloroform) to gave quantitative yield of a yellow oil that was used in the give 560 mg (83%) of colouless oil. The product was next Step without further purification. The Structure was characterised by MALDI mass spectrometry, giving M--H at verified by H and 'C NMR analysis. 674 as expected. Further characterisation was carried out using H (500 MHz) and 'C (125 MHz) NMR analysis, 0488 iii) Synthesis of 4-2-hydroxy-3-(1-methylethy giving spectra in accordance with the Structure. l)aminopropoxyphenylacetic Acid Hydrochloride 0481 b) Preparation of Gas-Filled Microbubbles Com 0489. A solution of methyl 4-2-hydroxy-3-(1-methyl prising DSPS and a Cholesterol Ester of Chlorambucil for ethyl)aminopropoxyphenylacetate (563 mg, 2.00 mmol) in Diagnostic and/or Therapeutic Applications 6M hydrochloric acid (15 ml) was heated at 100° C. for 4 hours. The reaction mixture was concentrated (rotavapor) 0482 Asolution of 1.4% propylene glycol/2.4% glycerol and the residue was taken up in water and lyophilised. "H (1.0 ml) was added to a mixture of DSPS (4.5 mg) and product from (a) (0.5 mg) in a vial. The mixture was and CNMR spectra were in accordance with the structure Sonicated for 5 minutes and then heated at 80 C. for 5 and MALDI mass spectrometry gave a M+H at 268 as minutes (vial was shaken during warming) and cooled. The expected. head Space was flushed with perfluorobutane gas and the Vial 0490 iv) Synthesis of N-Boc-4-2-hydroxy-3-(1-meth was shaken in a cap mixer for 45 Seconds, whereafter the ylethyl)aminopropoxyphenylacetic Acid contents were extensively washed with deionised water. MALDI mass spectrometry showed no detectable level of 0491) A solution of the 4-2-hydroxy-3-(1-methylethy compound from (a) in the final wash Solution. Incorporation l)aminopropoxyphenylacetic acid hydrochloride (2.0 of chlorambucil cholesteryl ester into the bubbles was mmol) in water (2 ml) was added to a Solution of Sodium confirmed by MALDI-MS as follows: ca. 50 ul of bicarbonate (0.60 g, 7.2 mmol) in water/dioxane (2:1, 15 microbubbles were transferred to a clean vial containing ca. ml). A solution of di-tert-butyl dicarbonate (0.48 g, 2.2 100 ul of 90% methanol. The mixture was Sonicated for 30 mmol) in dioxane (5 ml) was added. Progress of the reaction seconds and analysed by MALDI-MS, giving a M+H peak was monitored by TLC analysis (silica, CHCl/MeOH/ at 668 corresponding to Structure from AcOH 85:10:5), and portions of di-tert-butyl dicarbonate were added until conversion was complete. EXAMPLE 57 0492. The reaction mixture was poured onto water satu Gas-Filled Microbubbles Comprising DSPS and a rated with potassium hydrogen Sulphate and organic mate Lipopeptide Containing Atenolol and a Cholesterol rial was extracted into ethyl acetate. The organic phase was Derivative of Chlorambucil for Diagnostic and washed with water and brine, dried (NaSO) and filtered to Therapeutic Applications give 0.6 g of crude material. The product was purified by chromatography (silica, CHCl/MeOH/AcOH 85:10:5). The 0483 a) Synthesis of a Protected Atenolol Derivative Solution was concentrated and the residue was taken up in Suitable for Solid Phase Coupling glacial acetic acid and lyophilised. Yield 415 mg (56%), 0484) i) Synthesis of Methyl 4-(2,3-epoxy)propoxyphe white solid. The structure was confirmed by H and 13C nylacetate NMR analysis. US 2002/0102215 A1 Aug. 1, 2002 67

0493 b) Synthesis of a Lipopeptide Functionalised With Atenolol (SEQ ID NO:21)

O

~ss NH2

O O O H N --~~~~ N sus N NH2 H O O-l

0494. The structure shown above was synthesised by the 0497 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 0498 Asolution of 1.4% propylene glycol/2.4% glycerol (a). Coupling was carried out using standard TBTU/HOBt/ (1.0 ml) was added to a mixture of DSPS (5.0 mg), product DIEA protocols. Simultaneous removal of the peptide from from (b) (0.5 mg) and product from (c) (0.5 mg) in a vial. the resin and deprotection of Side-chain protecting groups The mixture was Sonicated for 5 minutes and then warmed was carried out in TFA containing 5% EDT and 5% water for to 80° C. for 5 minutes (vial was shaken during warming). 2 hours. Crude material was precipitated from ether and The solution was filtered and cooled. The head space was purified by preparative liquid chromatography using a gra flushed with perfluorobutane gas and the Vial was Shaken in a cap mixer for 45 Seconds, whereafter the contents were dient of 70 to 100% B over 60 minutes (A=0.1% TFA/water extensively washed with deionised water. Incorporation of and B=0.1% TFA/acetonitrile) at a flow rate of 10 ml/min. compounds (b) and (c) into the microbubbles was confirmed After lyophilisation, a yield of 38 mg of pure material was by MALDI-MS as follows: ca. 50 ul of microbubbles were obtained (analytical HPLC, gradient 70-100% B over 20 transferred to a clean vial containing ca. 100 ul of 90% minutes, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, methanol. The mixture was Sonicated for 30 seconds and flow rate 1 ml/minute, detection UV 214 nm, retention time analysed by MALDI-MS (ACH-matrix), giving a M+H peak 25 minutes). Further characterisation was carried out using corresponding to lipopeptide (b) and cholesteryl ester (c). e) MALDI mass spectrometry (ACH matrix), giving M+H at In vitro analysis The microbubbles were tested in the in vitro 1258, expected 1257. assay as detailed in Example 21. A gradual accumulation of 0495 c) Synthesis of Cholesterol 4-4-bis(2-chloroethy microbubbles binding to the cells was observed. l)aminophenylbutanoate EXAMPLE 58 0496 DIC (170 ul, 1.10 mmol) was added to a solution of chlorambucil (669 mg, 2.20 mmol) in dry dichlo Gas-Filed Microbubbles Comprising DSPS and a romethane (15 ml). The mixture was stirred at room tem Lipopeptide Containing Atenolol for Cell Targeting perature for 0.5 hour and added to a solution of cholesterol and a Lipophilic Thiol Ester of Captopril for (387 mg, 1.00 mmol) and DMAP (122 mg, 1.00 mmol) in Therapeutic Use dichloromethane (10 ml). The reaction mixture was stirred 0499 a) Synthesis of a Protected Atenolol Derivative overnight and then poured onto 5% sodium bicarbonate. The Suitable for Solid Phase Coupling phases were separated and the organic phase was washed with brine and dried (MgSO). The solution was filtered and 0500 i) Synthesis of Methyl 4-(2,3-epoxy)propoxyphe concentrated and the product was purified by column chro nylacetate matography (silica, chloroform) to give 560 mg (83%) of 0501) A mixture of methyl 4-hydroxyphenylacetate (4.98 colouless oil. The product was characterised by MALDI g, 0.030 mol), epichlorohydrin (23.5 ml, 0.30 mol) and mass spectrometry, giving M-H at 674 as expected. Further pyridine (121ul, 1.5 mmol) was stirred at 85°C. for 2 hours. characterisation was carried out using H (500 MHz) and The reaction mixture was cooled and exceSS epichlorohydrin 'C (125 MHz) NMR analysis, giving spectra in accordance was distilled off (rotavapor). The residue was taken up in with the structure. ethyl acetate, washed with brine and dried (Na2SO). The US 2002/0102215 A1 Aug. 1, 2002 68

Solution was filtered and concentrated. The dark residue was hydrogen Sulphate and organic material was extracted into chromatographed (silica, hexane/ethyl acetate 7:3) to give ethyl acetate. The organic phase was washed with water and 2.25 g (34%) of a colourless oil. "H (300 MHz) and 'C NMR (75 MHz) spectra were in accordance with the struc brine, dried (NaSO) and filtered to give 0.6 g of crude ture. material. The product was purified by chromatography (silica, CHCl/MeOH/AcOH 85:10:5). The solution was 0502 ii) Synthesis of Methyl 4-2-hydroxy-3-(1-meth concentrated and the residue was taken up in glacial acetic ylethyl)aminopropoxyphenylacetate acid and lyophilised. Yield 415 mg (56%), white solid. The 0503) A mixture of methyl 4-(2,3-epoxy)propoxyphe structure was confirmed by H and 'C NMR analysis. nylacetate (2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 mol) and water (1.35 ml, 74.7 mmol) was stirred at room 0508 b) Synthesis of a Lipopeptide Functionalised With temperature overnight. The reaction mixture was concen Atenolol (SEQ ID NO:21)

O

~ss, NH2

O O O H --~~~~ N sus N Nulls NH2 H H E O l O l NH2 HN O-1 trated (rotavapor) and the oily residue was dissolved in 0509. The structure shown above was synthesised by the chloroform and dried (Na2SO). Filtration and concentration manual bubbler method starting with Fmoc-protected Rink gave quantitative yield of a yellow oil that was used in the Amide MBHA resin on a 0.125 mmol scale, using appro next Step without further purification. The Structure was priate amino acids, palmitic acid and the compound from verified by H and 'C NMR analysis. (a). Coupling was carried out using standard TBTU/HOBt/ 0504 iii) Synthesis of 4-2-hydroxy-3-(1-methylethy DIEA protocols. Simultaneous removal of the peptide from l)aminopropoxyphenylacetic Acid Hydrochloride the resin and deprotection of Side-chain protecting groups 0505) A solution of methyl 4-2-hydroxy-3-(1-methyl was carried out in TFA containing 5% EDT and 5% water for ethyl)aminopropoxyphenylacetate (563 mg, 2.00 mmol) in 2 hours. Crude material was precipitated from ether and 6M hydrochloric acid (15 ml) was heated at 100° C. for 4 purified by preparative liquid chromatography using a gra hours. The reaction mixture was concentrated (rotavapor) dient of 70 to 100% B over 60 minutes (A=0.1% TFA/water and the residue was taken up in water and lyophilised. "H and B=0.1% TFA/acetonitrile) at a flow rate of 10 ml/min. and 'C NMR spectra were in accordance with the strucure After lyophilisation, a yield of 38 mg of pure material was and MALDI mass spectrometry gave a M+H at 268 as obtained (analytical HPLC, gradient 70-100% B over 20 expected. minutes, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, 0506 iv) Synthesis of N-Boc-4-2-hydroxy-3-(1-meth flow rate 1 ml/minute, detection UV 214 nm, retention time ylethyl)aminopropoxyphenylacetic Acid 25 minutes). Further characterisation was carried out using 0507 A solution of the 4-2-hydroxy-3-(1-methylethy MALDI mass spectrometry (ACH matrix), giving M+H at l)aminopropoxyphenylacetic acid hydrochloride (2.0 1258, expected 1257. mmol) in water (2 ml) was added to a Solution of Sodium 0510) c) Synthesis of Cholanic Acid Thiol Ester of Cap bicarbonate (0.60 g, 7.2 mmol) in water/dioxane (2:1, 15 topril ml). A solution of di-tert-butyl dicarbonate (0.48 g, 2.2 mmol) in dioxane (5 ml) was added. Progress of the reaction 0511) A mixture of 5-3-cholanic acid (361 mg, 1.00 was monitored by TLC analysis (silica, CHCl/MeOH/ mmol) and DIC (77 ul, 0.50 mmol) in dichloromethane (5 AcOH 85:10:5), and portions of di-tert-butyl dicarbonate ml) was stirred for 10 minutes and then added to a solution were added until conversion was complete. The reaction of captopril (130 mg, 0.600 mmol) and DBU (180 ul, 1.20 mixture was poured onto water Saturated with potassium mmol) in dichloromethane (10 ml). The reaction mixture US 2002/0102215 A1 Aug. 1, 2002 was Stirred overnight and then poured onto dilute hydro (MgSO). After filtration and evaporation the crude product chloric acid. Chloroform (30 ml) was added. The phases was chromatographed (silica, chloroform/methanol 99:1) to were separated and the organic phase was washed with water give 1.63 g (97%) of white solid. The structure was con and brine and dried (MgSO). After filtration and concen firmed by H NMR (500 MHz). tration, the crude material was chromatographed (silica, 0518) b) Synthesis of Cholesteryl B-Alaninate Hydro chloroform/methanol/acetic acid 95:4:1). The product was chloride lyophilised from a acetonitrile/water/ethanol mixture. Yield 137 mg (49%) of off-white solid. The structure was verified 0519) 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. B-alaninate hydrochloride. The structure was confirmed by 0512 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 0520 c) Biotin-PEGoo-BB-Ala-Cholesterol Therapeutic Use 0521. To a solution of cholesteryl B-alaninate hydrochlo 0513. 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-PEGoo-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 0522 d) Synthesis of cholesterol 4-4-bis(2-chloroethy poration of compounds from (b) and (c) into the l)aminophenylbutanoate microbubbles was confirmed by MALDI-MS as follows: ca. 50 ul of microbubbles were transferred to a clean vial 0523) 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 Sonicated for 30 seconds and analysed by MALDI-MS romethane (15 ml). The mixture was stirred at room tem (ACH-matrix), giving peaks according to structures from (b) perature for 0.5 hour and added to a solution of cholesterol and (c) respectively. (387 mg, 1.00 mmol) and DMAP (122 mg, 1.00 mmol) in dichloromethane (10 ml). The reaction mixture was stirred 0514 e) In vitro Analysis overnight and then poured onto 5% sodium bicarbonate. The 0515. The microbubbles were tested in the in vitro assay phases were separated and the organic phase was washed as detailed in Example 21. A gradual accumulation of with brine and dried (MgSO). The solution was filtered and microbubbles binding to the cells was observed. concentrated and the product was purified by column chro matography (silica, chloroform) to give 560 mg (83%) yield EXAMPLE 59 of colouless oil. The product was characterised by MALDI mass spectrometry, giving M-H at 674 as expected. Further Gas-Filled Microbubbles Comprising Phosphatidylserine and characterisation was carried out using H (500 MHz) and Biotinamide-PEG-f-Ala-Cholesterol and a 'C (125 MHz) NMR analysis, giving spectra in accordance Cholesterol Ester of Chlorambucil for Diagnostic with the structure. and Therapeutic Applications 0524) e) Preparation of Gas-Filled Microbubbles 0516) a) Synthesis of Cholesterol N-Boc-f-alaninate 0525) A solution of 1.4% propylene glycol/2.4% glycerol 0517 DIC (510 ul) was added to a solution of Boc-?3 (1.0 ml) was added to a mixture of DSPS (5 mg) and Ala-OH (1.25 g, 6.60 mmol) in dichloromethane (15 ml) products from (c) (0.5 mg) and (d) (0.5 mg) in a vial. The under an inert atmosphere. The reaction mixture was Stirred mixture was Sonicated for 5 minutes and then heated at 80 for 30 minutes and then transferred to a flask containing a C. for 5 minutes (vial was shaken during warming) and solution of cholesterol (1.16 g, 3.00 mmol) and DMAP (367 cooled. The head space was flushed with perfluorobutane mg, 3.00 mmol) in dichloromethane (15 ml). The reaction gas and the vial was Shaken in a cap mixer for 45 seconds, mixture was stirred for 2 hours and then poured onto an whereafter the contents were extensively washed with deio aqeous Solution of potassium hydrogen Sulphate. After phase nised water. MALDI mass spectrometry showed no detect Separation the aqueous phase was extracted with chloro able level of compound from (c) or (d) in the final wash form. The combined organic phases were washed with Solution. Incorporation of compounds from (c) and (d) into aqueous potassium hydrogen Sulphate and water and dried the microbubbles was confirmed by MALDI-MS as follows: US 2002/0102215 A1 Aug. 1, 2002 70 ca. 50 ul of microbubbles were transferred to a clean vial MALDI mass spectrometry (ACH matrix), giving M+H at containing ca. 100 ul of 90% methanol. The mixture was 1315, expected 1314. Sonicated for 30 seconds and analysed by MALDI-MS (ACH-matrix), giving M-H peaks corresponding to com 0528 b) Preparation of Gas-Filled Microbubbles Com pounds from (c) and (d). prising DSPS and a Lipopeptide Containing a Derivative of EXAMPLE 60 Bestatin for Diagnostic and Therapeutic Applications Gas-Filled Microbubbles Comprising DSPS and a 0529) A solution of 1.4% propylene glycol/2.4% glycerol Lipopeptide Containing a Derivative of Bestatin for (1.0 ml) was added to a mixture of DSPS (4.5 mg) and Diagnostic and Therapeutic Applications product from (a) (0.5 mg) in a vial. The mixture was Sonicated for 5 minutes and then heated at 80° C. for 5 0526 a) Synthesis of a Lipopeptide Containing a Deriva minutes (vial was shaken during warming) and cooled. The tive of Bestatin (SEQ ID NO:23) head Space was flushed with perfluorobutane gas and the Vial

O

~ss H NH2

O O O H H N Nulls N Null NH2 H E H E O O

O OH O NH2 NH2 HN

O i H

0527 The structure shown above was synthesised by the was shaken in a cap mixer for 45 Seconds and the contents manual bubbler method starting with Fmoc-protected Rink were extensively washed with deionised water. MALDI Amide MBHA resin on a 0.125 mmol scale, using appro mass spectrometry showed no detectable level of compound priate amino acids and palmitic acid. Coupling was carried from (b) in the final wash solution. Incorporation of out using standard TBTU/HOBt/DIEA protocols. Simulta atenolol-containing lipopeptide into the microbubbles was neous removal of the peptide from the resin and deprotection confirmed by MALDI-MS as follows: ca. 50 ul of of Side-chain protecting groups was carried out in TFA microbubbles were transferred to a clean vial containing ca. 100 ul of 90% methanol. The mixture was Sonicated for 30 containing 5% EDT and 5% water for 2 hours. Crude seconds and analysed by MALDI-MS (ACH-matrix), giving material was precipitated from ether and purified by pre a M+H peak at 1320, expected at 1314, corresponding to parative liquid chromatography using a gradient of 70 to lipopeptide from (a). 100% B over 60 minutes (A=0.1% TEA/water and B=0.1% TFA/acetonitrile) at a flow rate of 10 ml/min. After lyophi lisation, a yield of 12 mg of pure material was obtained 0530 c) In vitro Analysis (analytical HPLC, gradient 70-100% B over 20 minutes, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow rate 0531. The microbubbles were tested in the in vitro assay 1 ml/minute, detection UV 214 nm, retention time 25 as detailed in Example 21. A gradual accumulation of minutes). Further characterisation was carried out using microbubbles binding to the cells was observed. US 2002/0102215 A1 Aug. 1, 2002 71

EXAMPLE 61 MALDI mass spectrometry showed no detectable level of compound from (a) in the final wash Solution. Incorporation of chlorambucil-containing lipopeptide into the bubbles was Gas-Filled Microbubbles Comprising DSPS and a Lipopeptide Containing Chlorambucil for confirmed by MALDI-MS as follows: ca. 50 ul of Diagnostic and Therapeutic Applications microbubbles were transferred to a clean vial containing ca. 100 ul of 90% methanol. The mixture was Sonicated for 30 seconds and analysed by MALDI-MS (ACH-matrix), giving 0532 a) Synthesis of a Lipopeptide Containing Chloram a M+H peak at 1300, expected at 1294 and a M--Na peak at bucil (SEQ ID NO:24) 1324, expected 1317. ~ss,O

O O

--~~~~ NH i

I O r Cl

0533. The structure shown above was synthesised by the 0536 c) In vitro Analysis manual bubbler method starting with Fmoc-protected Rink Amide MBHA resin on a 0.125 mmol scale, using appro 0537) The microbubbles were tested in the in vitro assay priate amino acids and palmitic acid. Coupling was carried as detailed in Example 21. A gradual accumulation of out using standard TBTU/HOBt/DIEA protocol. Chloram microbubbles binding to the cells was observed. bucil was coupled through the Side-chain of Lys as a Symmetrical anhydride using DIC preactivation. Simulta EXAMPLE 62 neous removal of the peptide from the resin and deprotection of Side-chain protecting groups was carried out in TFA Gas-Filled Microbubbles Comprising DSPS, a containing 5% EDT, 5% water and 5% ethyl methylsulphide Lipopeptide Containing Atenolol and a Lipophilic for 2 hours. An aliqout of 10 mg of the crude material was Derivative of Captopril for diagnostic and purified by preparative liquid chromatography using a gra Therapeutic Applications dient of 70 to 100% B over 60 minutes (A=0.1% TFA/water 0538 a) Synthesis of a Protected Atenolol Derivative and B=0.1% TFA/acetonitrile) at a flow rate of 10 ml/min. Suitable for Solid Phase Coupling After lyophilisation, a yield of 30 mg of pure material was 0539 i) Synthesis of Methyl 4-(2,3-epoxy)propoxyphe obtained (analytical HPLC, gradient 70-100% B over 20 nylacetate minutes, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow rate 1 ml/minute, detection UV 214 nm retention time 0540 A mixture of methyl 4-hydroxyphenylacetate (4.98 26.5 minutes). Further characterisation was carried out using g, 0.030 mol), epichlorohydrin (23.5 ml, 0.30 mol) and MALDI mass spectrometry, giving M+H at 1295, expected pyridine (121ul, 1.5 mmol) was stirred at 85°C. for 2 hours. 1294. The reaction mixture was cooled, and exceSS epichlorohy drin was distilled off (rotavapor). The residue was taken up 0534 b) Preparation of Gas-Filled Microbubbles Com in ethyl acetate, washed with brine and dried (NaSO). The prising DSPS and a Lipopeptide Containing Chlorambucil Solution was filtered and concentrated. The dark residue was for Diagnostic and Therapeutic Applications chromatographed (silica, hexane/ethyl acetate 7:3) to give 0535 A solution of 1.4% propylene glycol/2.4% glycerol 2.25 g (34%) of a colourless oil. "H (300 MHz) and 'C (1.0 ml) was added to a mixture of DSPS (4.5 mg) and NMR (75 MHz) spectra were in accordance with the struc product from (a) (0.5 mg) in a vial. The mixture was ture. Sonicated for 5 minutes and then heated at 80 C. for 5 minutes (vial was shaken during warming) and cooled. The 0541) ii) Synthesis of Methyl 4-2-hydroxy-3-(1-meth head Space was flushed with perfluorobutane gas and the Vial ylethyl)aminopropoxyphenylacetate was shaken in a cap mixer for 45 Seconds, whereafter the 0542. A mixture of methyl 4-(2,3-epoxy)propoxyphe contents were extensively washed with deionised water. nylacetate (2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 US 2002/0102215 A1 Aug. 1, 2002 72 mol) and water (1.35 ml, 74.7 mmol) was stirred at room 0547 b) Synthesis of a Lipopeptide Functionalised With temperature overnight. The reaction mixture was concen Atenolol (SEQ ID NO:21)

O

~~~~ NH2

O O H H N N i: NH i NH2

H O-lOH trated (rotavapor) and the oily residue was dissolved in 0548. The structure shown above was synthesised by the chloroform and dried (Na2SO). Filtration and concentration manual bubbler method starting with Fmoc-protected Rink gave quantitative yield of a yellow oil that was used in the Amide MBHA resin on a 0.125 mmol scale, using appro next Step without further purification. The Structure was priate amino acids, palmitic acid and the compound from verified by H and "c NMR analysis. (a). Coupling was carried out using standard TBTU/HOBt/ 0543) iii) Synthesis of 4-2-hydroxy-3-(1-methylethy DIEA protocols. Simultaneous removal of the peptide from l)aminopropoxyphenylacetic Acid Hydrochloride the resin and deprotection of Side-chain protecting groups was carried out in TFA containing 5% EDT and 5% water for 0544) A solution of methyl 4-2-hydroxy-3-(1-methyl 2 hours. Crude material was precipitated from ether and ethyl)aminopropoxyphenylacetate (563 mg, 2.00 mmol) in purified by preparative liquid chromatography using a gra 6M hydrochloric acid (15 ml) was heated at 100° C. for 4 hours. The reaction mixture was concentrated (rotavapor) dient of 70 to 100% B over 60 minutes (A=0.1% TFA/water and the residue was taken up in water and lyophilised. "H and B=0.1% TFA/acetonitrile) at a flow rate of 10 ml/min. and 'C NMR spectra were in accordance with the strucure After lyophilisation, a yield of 38 mg of pure material was and MALDI mass spectrometry gave a M+H at 268 as obtained (analytical HPLC, gradient 70-100% B over 20 expected. minutes, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow rate 1 ml/minute, detection UV 214 nm, retention time 0545) iv) Synthesis of N-Boc-4-2-hydroxy-3-(1-meth 25 minutes). Further characterisation was carried out using ylethyl)aminopropoxyphenylacetic Acid MALDI mass spectrometry (ACH matrix), giving M+H at 0546 A solution of the 4-2-hydroxy-3-(1-methylethy 1258, expected 1257. l)aminopropoxyphenylacetic acid hydrochloride (2.0 0549 c) Synthesis of N-(S)-3-hexadecylthio-2-methyl mmol) in water (2 ml) was added to a Solution of Sodium propionylproline bicarbonate (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 0550 DIEA (188 ul, 1.10 mmol) was added to a solution mmol) in dioxane (5 ml) was added. Progress of the reaction of 1-iodohexadecane (176 mg, 0.500 mmol), captopril (120 was monitored by TLC analysis (silica, CHCl/MeOH/ mg, 0.550 mmol) and DBU (165ul, 1.10 mmol) in tetrahy AcOH 85:10:5), and portions of di-tert-butyl dicarbonate drofuran (5 ml). The mixture was heated at 70° C. for 2 were added until conversion was complete. The reaction hours and then concentrated. The residue was poured onto mixture was poured onto water Saturated with potassium water Saturated with potassium hydrogen Sulphate and hydrogen Sulphate and organic material was extracted into organic material was extracted into chloroform. The organic ethyl acetate. The organic phase was washed with water and phase was washed with water and dried (MgSO). The brine, dried (Na2SO) and filtered to give 0.6 g of crude product was purified by chromatography (silica, CHCl/ material. The product was purified by chromatography MeOH/AcOH 85:10:5) and lyophilised to give 105 mg (silica, CHCl/MeOH/AcOH 85:10:5). The solution was (48%) of white solid material. The structure was verified by concentrated and the residue was taken up in glacial acetic "H (500 Mhz) and 'C (125 Mhz) NMR analysis and further acid and lyophilised. Yield 415 mg (56%), white solid. The characterised by MALDI mass spectrometry, giving M-H in structure was confirmed by H and 'C NMR analysis. negative mode at m/z 440 as expected. US 2002/0102215 A1 Aug. 1, 2002

0551 d) Preparation of Gas-Filled Microbubbles Com 6M hydrochloric acid (15 ml) was heated at 100° C. for 4 prising DSPS, a Lipopeptide Containing Atenolol and a hours. The reaction mixture was concentrated (rotavapor) Lipophilic Derivative of Captopril for Diagnostic and Thera and the residue was taken up in water and lyophilised. "H peutic Applications and 'C NMR spectra were in accordance with the strucure and MALDI mass spectrometry gave a M+H at 268 as 0552. 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 products from (b) (0.5 mg) and (c) in a vial. The mixture was 0561 d) Synthesis of N-Boc-4-2-hydroxy-3-(1-methyl Sonicated for 5 minutes and then heated at 80 C. for 5 ethyl)aminopropoxyphenylacetic Acid minutes (vial was shaken during warming) and cooled. The 0562) A solution of the 4-2-hydroxy-3-(1-methylethy head Space was flushed with perfluorobutane gas and the Vial l)aminopropoxyphenylacetic acid hydrochloride (2.0 was shaken in a cap mixer for 45 Seconds, whereafter the mmol) in water (2 ml) was added to a Solution of Sodium contents were extensively washed with deionised water. bicarbonate (0.60 g, 7.2 mmol) in water/dioxane (2:1, 15 MALDI mass spectrometry showed no detectable level of ml). A solution of di-tert-butyl dicarbonate (0.48 g, 2.2 compound from (b) or (c) in the final wash Solution. Incor mmol) in dioxane (5 ml) was added. Progress of the reaction poration of compounds (b) and (c) into the microbubbles was monitored by TLC analysis (silica, CHCl/MeOH/ was confirmed by MALDI-MS as follows: ca. 50 ul of AcOH 85:10:5), and portions of di-tert-butyl dicarbonate microbubbles were transferred to a clean vial containing ca. were added until conversion was complete. The reaction 100 ul of 90% methanol. The mixture was Sonicated for 30 mixture was poured onto water Saturated with potassium seconds and analysed by MALDI-MS (ACH-matrix), giving hydrogen Sulphate and organic material was extracted into M+H peaks corresponding to structures (b) and (c) respec ethyl acetate. The organic phase was washed with water and tively. brine, dried (Na2SO) and filtered to give 0.6 g of crude 0553 e) In vitro Analysis material. The product was purified by chromatography (silica, CHCl/MeOH/AcOH 85:10:5). The solution was 0554. 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. EXAMPLE 63 0563) e) Synthesis of Cholesteryl N-Boc-B-alaninate 0564) DIC (510 ul) was added to a solution of Boc-?3 Gas-Filled Microbubbles Comprising DSPS and a Ala-OH (1.25 g, 6.60 mmol) in dichloromethane (15 ml) Cholesterol Derivative of Atenolol for Diagnostic under an inert atmosphere. The reaction mixture was Stirred and Therapeutic Applications for 30 minutes and then transferred to a flask containing a solution of cholesterol (1.16 g, 3.00 mmol) and DMAP (367 0555 a) Synthesis of Methyl 4-(2,3-epoxy)propoxy mg, 3.00 mmol) in dichloromethane (15 ml). The reaction phenylacetate mixture was stirred for 2 hours and then poured onto an 0556. A mixture of methyl 4-hydroxyphenylacetate (4.98 aqeous Solution of potassium hydrogen Sulphate. After phase g, 0.030 mol), epichlorohydrin (23.5 ml, 0.30 mol) and Separation the aqueous phase was extracted with chloro pyridine (121 ul, 1.5 mmol) was stirred at 85°C. for 2 hours. form. The combined organic phases were washed with The reaction mixture was cooled, and exceSS epichlorohy aqueous potassium hydrogen Sulphate and water and dried drin was distilled off (rotavapor). The residue was taken up (MgSO). After filtration and evaporation the crude product in ethyl acetate, washed with brine and dried (Na2SO). The was chromatographed (silica, chloroform/methanol 99:1) to Solution was filtered and concentrated. The dark residue was give 1.63 g (97%) of white solid. The structure was con chromatographed (silica, hexane/ethyl acetate 7:3) to give firmed by H NMR (500 MHz). 2.25 g (34%) of a colourless oil. "H (300 MHz) and 'C 0565 f) Synthesis of Cholesterol B-Alaninate Hydrochlo NMR (75 MHz) spectra were in accordance with the struc ride ture. 0566. A solution of compound from (a) (279 mg, 0.500 0557 b) Synthesis of Methyl 4-2-hydroxy-3-(1-meth mmol) in 1M hydrochloric acid in 1,4-dioxane (5 ml) was ylethyl)aminopropoxyphenylacetate Stirred at room temperature for 4 hours. The reaction mixture 0558 A mixture of methyl 4-(2,3-epoxy)propoxyphe was concentrated to give a quantitative yield of cholesteryl nylacetate (2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 B-alaninate hydrochloride. The structure was confirmed by mo) and water (1.35 ml, 74.7 mmol) was stirred at room 1H NMR (500 MHz) analysis and by MALDI mass spec temperature overnight. The reaction mixture was concen trometry, giving a M+Na peak at 482, expected 481. trated (rotavapor) and the oily residue was dissolved in chloroform and dried (Na2SO). Filtration and concentration 0567 g) Synthesis of Cholesterol N-Boc-4-2-hydroxy gave quantitative yield of a yellow oil that was used in the 3-(1-methylethyl)aminopropoxyphenylacetyl-f-alaninate. next Step without further purification. The Structure was 0568 To a solution of N-Boc-4-2-hydroxy-3-(1-meth verified by H and 'C NMR analysis. ylethyl)aminopropoxyphenylacetic acid (55 mg, 0.15 0559 c) Synthesis of 4-2-hydroxy-3-(1-methylethy mmol) and cholesteryl B-alaninate hydrochloride (74 mg, l)aminopropoxyphenylacetic Acid Hydrochloride 0.15 mmol) in DMF (5 ml) was added DIEA (26 ml, 0.15 mmol). HOBt (23 mg, 0.15 mmol) and water-soluble car 0560 A solution of methyl 4-2-hydroxy-3-(1-methyl bodiimide (WSC) (29 mg, 0.15 mmol) were added. The ethyl)aminopropoxyphenylacetate (563 mg, 2.00 mmol) in reaction mixture was stirred at room temperature overnight US 2002/0102215 A1 Aug. 1, 2002 74 and then poured onto water (25 ml) containing Sodium EXAMPLE 64 carbonate (2.5 g) and Sodium chloride (4.0 g). Precipitated material was extracted into chloroform. The organic phase Preparation of Multiple-Sepecific was washed with water and dried (MgSO). After filtration Transferrin/Avidin-Coated Gas-Filled Microbubbles and concentration, crude material (132 mg) was purified by for Targeted Ultrasound Imaging column chromatography (silica, chloroform/methanol/acetic acid, 95:4:1). Pooled fractions were concentrated, taken up 0575. This example is directed to the preparation of in glacial acetic acid and lyophilised. Yield 83 mg (69%), microbubbles containing vectors for targeted ultrasound/ yellow-white solid. Structure was confirmed by H NMR therapy. analysis. 0569 h) Synthesis of Cholesterol 4-2-hydroxy-3-(1- 0576) a) Synthesis of a Thiol-Functionalised Lipid Mol methylethyl)aminopropoxyphenylacetyl-3-alaninate Trif ecule: Dipalmitoyi-Lys-Lys-Lys-Aca-CyS.OH (SEQ ID luoroacetate NO:20)

O

--~~~~ H NH2

O O O H H N N sus N N--- N 1-on H H H O O O

0570) To a solution of N-Boc-4-2-hydroxy-3-(1-meth 0577. The lipid structure shown above was synthesised ylethyl)aminopropoxyphenylacetyl-3-alaninate (40 mg, on an ABI 433A automatic peptide Synthesiser Starting with Fmoc-Cys(Trt)-Wang resin on a 0.25 mmol scale using 1 0.05 mmol) in dry dichloromethane (4 ml) was added mmol amino acid cartridges. All amino acids and palmitic trifluoroacetic acid (2 ml). The reaction mixture was stirred acid were preactivated using HBTU coupling chemistry. The for 2 hours and then concentrated. The product was lyophi Simultaneous removal of peptide from the resin and depro lised from a acetonitrile/water mixture to give a quantitative tection of Side-chain protecting groups was carried out in yield of white-yellow material. The product was character TFA containing 5% EDT and 5% HO for 2 hours, giving a ised by MALDI mass spectrometry giving M+H at 708 as crude product yield of 250 mg. Purification by preparative expected. HPLC of a 40 mg aliquot of crude material was carried out using a gradient of 90 to 100% B over 50 minutes (A=0.1% 0571) i) Preparation of Gas-Filled Microbubbles Com TFA/water and B MeOH) at a flow rate of 9 ml/min. After prising DSPS and a Cholesterol Derivative of Atenolol for lyophilisation, 24 mg of pure material was obtained (ana Diagnostic and Therapeutic Applications lytical HPLC, gradient 70-100% B where B=0.1% TFA/ acetonitrile, A=0.01% TFA/water: detection-UV 214 0572 A Solution of 1.4% propylene glycol/2.4% glycerol nim-product retention time=23 minutes). Further product (1.0 ml) was added to a mixture of DSPS (4.5 mg) and characterisation was carried out using MALDI maSS Spec product from (h) (0.5 mg) in a vial. The mixture was trometry: expected M+H at 1096, found at 1099. Sonicated for 5 minutes and then heated at 80 C. for 5 0578 b) Preparation of Gas-Containing Microbubbles minutes (vial was shaken during warming) and cooled. The Comprising DSPS Doped With a Thiol-Containing Lipid head Space was flushed with perfluorobutane gas and the Vial Structure was shaken in a cap mixer for 45 Seconds, whereafter the 0579 DSPS (4.5 mg) and the lipid structure from (a) contents were extensively washed with deionised water. above (0.5 mg) were weighed into a clean vial and 0.8 ml of MALDI mass spectrometry showed no detectable level of a Solution containing 1.4% propylene glycol/2.4% glycerol compound from (b) in the final wash Solution. Incorporation in water was added. The mixture was warmed to 80 C. for 5 minutes (vial Shaken during warming) and filtered while of compound from (h) into the microbubbles was confirmed still hot through a 40 micron filter. The sample was cooled by MALDI mass spectrometry. to room temperature and the head Space was flushed with 0573) j) In vitro Analysis perfluorobutane gas. The Vial was shaken in a cap mixer for 45 seconds and then placed on aroller table overnight. The 0574. The microbubbles were tested in the in vitro assay resulting microbubbles were washed several times with as detailed in Example 21. A gradual accumulation of deionised water and analysed for thiol group incorporation bubbles binding to the cells was observed. using Ellmans Reagent. US 2002/0102215 A1 Aug. 1, 2002

0580 c) Modification of Transferrin and Avidin With 0589) c) Ultrasonic Treatment Fluorescein-NHS and Sulpho-SMPB 0590. After 15 minutes incubation, selected wells were 0581) To a mixture of 2 mg of transferrin (Holo, human) exposed to continious wave ultrasound of 1 MHz, 0.5 and 2 mg of avidin in PBS (1 ml) was added 0.5 ml of a W/cm, for 30 seconds. DMSO solution containing 1 mg Sulpho-SMPB and 0.5 mg 0591 d) Incubation and Examination fluorescein-NHS. The mixture was stirred for 45 minutes at 0592. The cells were further incubated in the cell culture room temperature then passed through a SephadeX 200 incubator (37° C) for approximately 4.5 hours. The medium column using PBS as eluent. The protein fraction was containing DNA-microbubbles was then removed by aspi collected and stored at 4 C. prior to use. ration, and 2 ml complete RPMI medium was added. The cells were incubated for 40-70 hours before examination. 0582 d) Microbubble Conjugation With Modified Trans Most of the medium was then removed and the cells were ferrin/Aidin examined by fluorescence microScopy. The results were 0583. To the thiol-containing microbubbles from (b) was compared to the results from control experiments where added 1 ml of the modified transferrin/avidin protein solu DNA or DNA-polylysine were added to the cells. tion from (c). After adjusting the pH of the solution to 9, the conjugation reaction was allowed to proceed for 2 hours at EXAMPLE 66 room temperature. Following extensive Washing with deio Flotation of Endothelial Cells by Microbubbles nised water the microbubbles were analysed by Coulter With Vectors That Specifically Bind to the counter (81% between 1 and 7 micron) and fluorescence microscopy (highly fluorescent microbubbles were Endothelial Cells observed). 0593. This experiment was carried out to show that the present invention can be used for Separation of cells to which EXAMPLE 65 the microbubbles are targeted. The human endothelial cell line ECV304, derived from a normal umbilical cord (ATCC Gene Transfer by Gas-Filled Microbubbles CRL-1998) was cultured in Nunc culture flasks (chutney 153732) in RPMI 1640 medium to which L-glutamine (200 0584) This example is directed at the preparation of mM), penicillin/streptomycin (10,000 U/ml and 10.00 targeted microbubbles for gene transfer. ug/ml) and 10% fetal calf serum were added. The cells were Subcultured following trypsination with a split ratio of 1:5 to 0585) 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 0586) 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 HBr (20.6 mg) was dissolved in 2 ml water, then an aliquot EXAMPLE 67 (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 Distearoylphosphatidylserine 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 0587 b) Transfection of Cells 0594) a) Synthesis of 4'-(3,4-dimethyl-5-isoxazolyl)sul famoylsuccinanilic Acid 0588) Endothelial cells (ECV 304) were cultured in 6 well plates to a uniform Subconfluent layer. A transfection 0595) 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 Fluorescent Protein vector from CLONTECH) and 50 ul of mmol) and 4-dimethylaminopyridine (122 mg, 1.00 mmol). microbubble suspension from (a) in RPMI medium at a final The reaction mixture was stirred at 80° C. for 2 hours and volume of 250 ul was prepared. The mixture was left then concentrated. The residue was taken up in 5% aqueous Standing for 15 minutes at room temperature then 1 ml of Sodium bicarbonate solution and extracted with ethyl complete RPMI medium was added. The medium was acetate. The aqueous Solution was acidified with dilute removed from the cell culture dish and the DNA-mi hydrochloric acid and organic material was extracted into crobubble mixture was added to the cells. The cells were ethyl acetate. The organic phase was washed with dilute incubated in a cell culture incubator (37° C). hydrochloric acid, water and brine, treated with active US 2002/0102215 A1 Aug. 1, 2002 76 charcoal and dried (MgSO). The solution was filtered and acterisation was carried out using MALDI mass spectrom concentrated to give 280 mg (76%) of white solid. The etry, giving a M+H at m/z 1359, expected 1356. structure was verified by H (300 MHz) and 'C (75 MHz) NMR spectroscopy. Further characterisation was carried out 0598 c) Preparation of Gas-Filled Microbubbles Com using MALDI mass spectrometry (ACH matrix), giving a prising the Compound From (b) M+Na peak at m/z 390 and a M--K peak at m/z 406 as 0599 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 0596) b) Synthesis of a Lipopeptide Functionalised With product from (b) (0.5 mg) in a vial. The mixture was Sulfisoxazole (SEQ ID NO:25) Sonicated for 5 minutes and then heated at 80° C. for 5

--~~~~ NH NH NH NH N

O O

NH2

O

NH2

O O \/ / N O SnH o1

NH NH

O

0597. The structure shown above was synthesised on a minutes (vial was shaken during warming) and cooled. The manual nitrogen bubbler apparatus Starting with Fmoc head Space was flushed with perfluorobutane gas and the Vial protected Rink Amide BMHA resin on a 0.125 mmol scale, was Shaken in a cap mixer for 45 Seconds followed by using appropriate amino acids, palmitic acid and the com extensive washing with deionised water. MALDI mass pound from (a). Coupling was carried out using Standard Spectrometry showed no detectable level of compound from TBTU/HOBt/DIEA protocols. Simultaneous removal of the peptide from the resin and deprotection of Side-chain pro (b) in the final wash Solution. Incorporation of isoxazole tecting groups was carried out in TFA containing 5% EDT containing lipopeptide into the microbubbles was confirmed and 5% water for 2 hours. Crude material was precipitated by MALDI-MS as follows: ca. 50 ul of microbubbles were from ether. The product was analysed by analytical HPLC, transferred to a clean vial containing ca. 100 ul of 90% gradient 70-100% B over 20 minutes, A=0.1% TFA/water methanol. The mixture was Sonicated for 30 seconds and and B=0.1% TFA/acetonitrile, flow rate 1 ml/minute, detec analysed by MALDI-MS (ACH-matrix), giving a m+H peak tion UV 214 nm, retention time 27 minutes). Further char at m/z 1359 corresponding to lipopeptide (b). US 2002/0102215 A1 Aug. 1, 2002 77

SEQUENCE LISTING

NUMBER OF SEQ ID NOS: 31 SEQ ID NO 1 LENGTH 4 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence:RGDC-Mal-PEG3400-DSPE

<400 SEQUENCE: 1 Arg Gly Asp Cys 1

SEQ ID NO 2 LENGTH 25 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence Peptide comprising phosphatidylserine-binding and heparin-binding sections

<400 SEQUENCE: 2 Phe Asn. Phe Arg Lieu Lys Ala Gly Glin Lys Ile Arg Phe Gly Ala Ala 1 5 10 Ala Trp Glu Pro Pro Arg Ala Arg Ile 2O 25

SEQ ID NO 3 LENGTH 8 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence Heparin-binding peptide

<400 SEQUENCE: 3 Trp Glu Pro Pro Arg Ala Arg Ile 1 5

SEQ ID NO 4 LENGTH 6 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence Linker sequence NAME/KEY: MOD RES LOCATION: (1) OTHER INFORMATION: MTX-phenylalanine

<400 SEQUENCE: 4 Phe Lys Lieu Arg Lieu. Cys 1 5

SEQ ID NO 5 LENGTH 4 TYPE PRT ORGANISM: Artificial Sequence FEATURE: US 2002/0102215 A1 Aug. 1, 2002 78

-continued <223> OTHER INFORMATION: Description of Artificial Sequence Heparin sulphate binding peptide

<400 SEQUENCE: 5 Lys Arg Lys Arg 1

<210> SEQ ID NO 6 &2 11s LENGTH 8 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence:Fibronectin peptide

<400 SEQUENCE: 6 Trp Gln Pro Pro Arg Ala Arg Ile 1 5

<210 SEQ ID NO 7 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Lipopeptide consisting of a heparin sulphate binding peptide and a fibronectin peptide <221 NAME/KEY: MODRES <222> LOCATION: (1) <223> OTHER INFORMATION: Dipalmitoyl-lysine <400 SEQUENCE: 7 Lys Lys Arg Lys Arg Trp Gln Pro Pro Arg Ala Arg Ile 1 5 10

<210 SEQ ID NO 8 <211& LENGTH 24 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence:Fibronectin peptide sequence

<400 SEQUENCE: 8 Phe Asn. Phe Arg Lieu Lys Ala Gly Glin Lys Ile Arg Phe Gly Gly Gly 1 5 10 15 Gly Trp Gln Pro Pro Arg Ala Ile 2O

<210 SEQ ID NO 9 &2 11s LENGTH 6 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Biotinylated endothelin-1 peptide <221 NAME/KEY: MODRES <222> LOCATION: (1) <223> OTHER INFORMATION: Biotin-D-Trp <400 SEQUENCE: 9 US 2002/0102215 A1 Aug. 1, 2002 79

-continued

Trp Lieu. Asp Ile Ile Trp 1 5

<210> SEQ ID NO 10 &2 11s LENGTH 10 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Biotinylated fibrin-anti-polymerant peptide <221 NAME/KEY: MODRES <222> LOCATION: (1) <223> OTHER INFORMATION: Biotinylated-Gly <221 NAME/KEY: MODRES <222> LOCATION: (10) &223> OTHER INFORMATION AMIDATION

<400 SEQUENCE: 10 Gly Pro Arg Pro Pro Glu Arg His Glin Ser 1 5 10

<210> SEQ ID NO 11 &2 11s LENGTH 5 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Lipopeptide containing RGD sequence and fluorescein reporter group <221 NAME/KEY: MODRES <222> LOCATION: (1) <223> OTHER INFORMATION: Dipalmitoyl-Lys <221 NAME/KEY: MODRES <222> LOCATION: (4) <223> OTHER INFORMATION: Acetyl-RGD-K-fluorescein side chain

<400 SEQUENCE: 11 Lys Lys Lys Lys Gly 1 5

<210> SEQ ID NO 12 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence:Endothelial cell binding lipopeptide <221 NAME/KEY: MODRES <222> LOCATION: (1) <223> OTHER INFORMATION: 2-n-hexadecyl stearyl-Lys <221 NAME/KEY: MOD RES <222> LOCATION: (18) &223> OTHER INFORMATION AMIDATION

<400 SEQUENCE: 12 Lys Lieu Ala Lieu Lys Lieu Ala Lieu Lys Ala Lieu Lys Ala Ala Lieu Lys 1 5 10 15

Leu Ala

<210> SEQ ID NO 13 <211& LENGTH 4 &212> TYPE PRT US 2002/0102215 A1 Aug. 1, 2002 80

-continued <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Lipopeptide functionalised with captopril <221 NAME/KEY: MODRES <222> LOCATION: (1) <223> OTHER INFORMATION: Dipalmitoyl-Lys <221 NAME/KEY: MODRES <222> LOCATION: (4) <223> OTHER INFORMATION: Amide linked via side chain to captopril <221 NAME/KEY: MODRES <222> LOCATION: (4) &223> OTHER INFORMATION AMIDATION

<400 SEQUENCE: 13 Lys Lys Lys Lys 1

<210> SEQ ID NO 14 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Lipopeptide with an affinity for endothelial cells <221 NAME/KEY: MODRES <222> LOCATION: (1) <223> OTHER INFORMATION: Dipalmitoyl-Lys <221> NAME/KEY: MOD RES <222> LOCATION: (4) <223> OTHER INFORMATION: Acp <221 NAME/KEY: MODRES <222> LOCATION: (13) &223> OTHER INFORMATION AMIDATION

<400 SEQUENCE: 14 Lys Lys Lys Xala Ile Arg Arg Val Ala Arg Pro Pro Leu 1 5 10

<210 SEQ ID NO 15 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Lipopeptide comprising an interleukin-1 receptor binding peptide <221 NAME/KEY: MOD RES <222> LOCATION: (1) <223> OTHER INFORMATION: Dipalmitoyl-Lys <400 SEQUENCE: 15 Lys Gly Asp Trp Asp Glin Phe Gly Lieu Trp Arg Gly Ala Ala 1 5 10

<210> SEQ ID NO 16 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <221 NAME/KEY: MOD RES <222> LOCATION: (1) <223> OTHER INFORMATION: Dabsyl-Tyr <221 NAME/KEY: MODRES US 2002/0102215 A1 Aug. 1, 2002 81

-continued <222> LOCATION: (10) <223> OTHER INFORMATION: RGDS chain linked via NH2 group of lysine <223> OTHER INFORMATION: Description of Artificial Sequence:Branched core peptide comprising a dabsylated atherosclerotic plague-binding sequence and RGDS

<400 SEQUENCE: 16 Tyr Arg Ala Lieu Val Asp Thr Lieu Lys Lys Gly Cys 1 5 10

<210 SEQ ID NO 17 &2 11s LENGTH 25 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide <221 NAME/KEY: misc feature <222> LOCATION: (1) <223> OTHER INFORMATION: Biotinylated <400 SEQUENCE: 17 gaaagg tagt ggggtogtgt gcc.gg 25

<210> SEQ ID NO 18 &211's LENGTH 15 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Lipopeptide with affinity for thrombi <221 NAME/KEY: MOD RES <222> LOCATION: (1) <223> OTHER INFORMATION: Dipalmitoyl-Lys <221 NAME/KEY: MODRES <222> LOCATION: (15) &223> OTHER INFORMATION AMIDATION

<400 SEQUENCE: 18 Lys Asn Asp Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Lieu Glin 1 5 10 15

<210 SEQ ID NO 19 &2 11s LENGTH 6 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Lipopeptide <221 NAME/KEY: MOD RES <222> LOCATION: (1) <223> OTHER INFORMATION: Dipalmitoyl-Lys <221 NAME/KEY: MOD RES <222> LOCATION: (5) <223> OTHER INFORMATION: Biotinylated-Lys <400 SEQUENCE: 19 Lys Trp Llys Lys Lys Gly 1 5

<210> SEQ ID NO 20 &2 11s LENGTH 5 US 2002/0102215 A1 Aug. 1, 2002 82

-continued

&212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Thiol-functionalised lipid molecule <221 NAME/KEY: MODRES <222> LOCATION: (1) <223> OTHER INFORMATION: Dipalmitoyl-Lys <221 NAME/KEY: MODRES <222> LOCATION: (4) <223> OTHER INFORMATION: Acp <400 SEQUENCE: 20 Lys Lys Lys Xala Cys 1 5

<210> SEQ ID NO 21 <211& LENGTH 4 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Lipopeptide functionalised with atenolol <221 NAME/KEY: MODRES <222> LOCATION: (1) <223> OTHER INFORMATION: Dipalmitoyl-Lys <221 NAME/KEY: MOD RES <222> LOCATION: (4) <223> OTHER INFORMATION: Lysine with side chain linked via amide bond to attenolol <221 NAME/KEY: MODRES <222> LOCATION: (4) &223> OTHER INFORMATION AMIDATION

<400 SEQUENCE: 21 Lys Lys Lys Lys 1

<210> SEQ ID NO 22 <211& LENGTH 4 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Lipopeptide containing folic acid <221 NAME/KEY: MOD RES <222> LOCATION: (1) <223> OTHER INFORMATION: Dipalmitoyl-Lys <221 NAME/KEY: MODRES <222> LOCATION: (4) &223> OTHER INFORMATION AMIDATION <221 NAME/KEY: MODRES <222> LOCATION: (4) <223> OTHER INFORMATION: Lysine with side chain linked via amide bond to folic acid

<400 SEQUENCE: 22 Lys Lys Lys Lys 1

<210> SEQ ID NO 23 <211& LENGTH 4 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE US 2002/0102215 A1 Aug. 1, 2002 83

-continued <223> OTHER INFORMATION: Description of Artificial Sequence Lipopeptide containing a derivative of bestatin <221 NAME/KEY: MODRES <222> LOCATION: (1) <223> OTHER INFORMATION: Dipalmitoyl-Lys <221 NAME/KEY: MOD RES <222> LOCATION: (4) &223> OTHER INFORMATION AMIDATION <221 NAME/KEY: MODRES <222> LOCATION: (4) <223> OTHER INFORMATION: Lysine with side chain linked via amide bond to derivative of bestatin

<400 SEQUENCE: 23 Lys Lys Lys Lys 1

<210> SEQ ID NO 24 <211& LENGTH 4 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Lipopeptide containing chlorambucil <221 NAME/KEY: MOD RES <222> LOCATION: (1) <223> OTHER INFORMATION: Dipalmitoyl-Lys <221 NAME/KEY: MODRES <222> LOCATION: (4) &223> OTHER INFORMATION AMIDATION <221 NAME/KEY: MODRES <222> LOCATION: (4) <223> OTHER INFORMATION: Lysine with side chain linked via amide bond to chlorambucil

<400 SEQUENCE: 24 Lys Lys Lys Lys 1

<210> SEQ ID NO 25 <211& LENGTH 4 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Lipopeptide functionalised with sulfisoxazole <221 NAME/KEY: MOD RES <222> LOCATION: (1) <223> OTHER INFORMATION: Dipalmitoyl-Lys <221 NAME/KEY: MODRES <222> LOCATION: (4) &223> OTHER INFORMATION AMIDATION <221 NAME/KEY: MODRES <222> LOCATION: (4) <223> OTHER INFORMATION: Lysine with side chain linked via amide bond to sulfisoxazole

<400 SEQUENCE: 25 Lys Lys Lys Lys 1

<210> SEQ ID NO 26 US 2002/0102215 A1 Aug. 1, 2002 84

-continued

&2 11s LENGTH 9 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Atherosclerotic plague-binding peptide

<400 SEQUENCE: 26 Tyr Arg Ala Lieu Val Asp Thr Lieu Lys 1 5

<210 SEQ ID NO 27 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Atherosclerotic plague-binding peptide

<400 SEQUENCE: 27 Tyr Ala Lys Phe Arg Glu Thir Lieu Glu Asp Thr Arg Asp Arg Met Tyr 1 5 10 15

<210> SEQ ID NO 28 &2 11s LENGTH 17 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence Atherosclerotic plague-binding peptide

<400 SEQUENCE: 28 Arg Ala Lieu Val Asp Thr Glu Phe Lys Val Lys Glin Glu Ala Gly Ala Lys 1 5 10 15

<210 SEQ ID NO 29 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Thrombus binding peptide

<400 SEQUENCE: 29 Asn Asp Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Lieu Glin 1 5 10

<210 SEQ ID NO 30 <211& LENGTH 4 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Thrombus binding peptide

<400 SEQUENCE: 30 Gly Pro Arg Gly 1

<210> SEQ ID NO 31 &2 11s LENGTH 13 US 2002/0102215 A1 Aug. 1, 2002

-continued

&212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Platelet binding peptide

<400 SEQUENCE: 31 Pro Leu Tyr Lys Lys Ile Ile Lys Lys Lieu Lieu Glu Ser 1 5 10

1. A targetable diagnostic and/or therapeutically active 12. An agent as claimed in claim 11 wherein the vector or agent comprising a Suspension in an aqueous carrier liquid vectors are Selected from ligands for cell adhesion proteins of a reporter comprising gas-filled microbubbles Stabilised and cell adhesion proteins which have corresponding ligands by monolayers of film-forming Surfactant, Said agent further on endothelial cell Surfaces. comprising at least one vector. 13. An agent as claimed in any of the preceding claims 2. An agent as claimed in claim 1 wherein the gas wherein the vector or vectors are sited Such that they are not comprises air, nitrogen, oxygen, carbon dioxide, hydrogen, readily exposed to the target. an inert gas, a Sulphur fluoride, Selenium hexafluoride, a low 14. An agent as claimed in any of the preceding claims molecular weight hydrocarbon, a ketone, an ester, a halo wherein the vector is covalently coupled or linked to the genated low molecular weight hydrocarbon or a mixture of reporter. any of the foregoing. 15. An agent as claimed in any of claims 1 to 13 wherein 3. An agent as claimed in claim 2 wherein the gas the vector is coupled or linked to the reporter through comprises a perfluorinated ketone, perfluorinated ether or electroStatic charge interactions. perfluorocarbon. 16. An agent as claimed in any of claims 1 to 13 wherein 4. An agent as claimed in claim 2 wherein the gas the vector is coupled or linked to the reporter by means of comprises Sulphur hexafluoride or a perfluoropropane, per avidin-biotin and/or streptavidin-biotin interactions. fluorobutane or perfluoropentane. 17. An agent as claimed in any of the preceding claims 5. An agent as claimed in any of the preceding claims which further contains moieties which are radioactive or are wherein the film-forming Surfactant material comprises a effective as X-ray contrast agents, light imaging probes or non-polymeric and non-polymerisable wall-forming Surfac Spin labels. tant material, a polymer Surfactant material or a phospho 18. An agent as claimed in any one of the preceding lipid. claims further comprising a therapeutic compound. 6. An agent as claimed in claim 5 wherein at least 75% of 19. An agent as claimed in claim 18 wherein Said thera the film-forming Surfactant material comprises phospholipid peutic compound is an antineoplastic agent, blood product, molecules individually bearing net overall charge. biological response modifier, antifungal agent, hormone or 7. An agent as claimed in claim 6 wherein at least 75% of hormone analogue, Vitamin, enzyme, antiallergic agent, tis the film-forming Surfactant material comprises one or more Sue factor inhibitor, platelet inhibitor, coagulation protein phospholipids Selected from phosphatidylserines, phosphati target inhibitor, fibrin formation inhibitor, fibrinolysis pro dylglycerols, phosphatidylinositols, phosphatidic acids and moter, antiangiogenic, circulatory drug, metabolic potentia cardiolipins. tor, antitubercular, antiviral, Vasodilator, antibiotic, antiin 8. An agent as claimed in claim 7 wherein at least 80% of flammatory, antiprotozoan, antirheumatic, narcotic, opiate, Said phospholipids comprise phosphatidylserines. cardiac glycoside, neuromuscular blocker, Sedative, local 9. An agent as claimed in any of the preceding claims anaesthetic, general anaesthetic or genetic material. wherein the film-forming Surfactant material comprises a 20. An agent as claimed in claim 18 or claim 19 wherein lipopeptide. Said therapeutic compound is covalently coupled or linked to 10. An agent as claimed in any of the preceding claims the reporter through disulphide groups. wherein the vector is selected from antibodies; cell adhesion 21. An agent as claimed in claim 18 or claim 19 wherein molecules, cell a adhesion molecule receptors, cytokines, a lipophilic or lipophilically-derivatised therapeutic com growth factors, peptide hormones and pieces thereof; non pound is linked to the Surfactant monolayerS Stabilising the peptide agonists/antagonists and non-bioactive binders of gas-filled microbubbles of the reporter through hydrophobic receptors for cell adhesion molecules, cytokines, growth interactions. factors and peptide hormones, oligonucleotides and modi 22. A combined formulation comprising: fied oligonucleotides, DNA-binding drugs, protease Sub Strates/inhibitors, molecules generated from combinatorial i) a first administrable composition comprising a pre libraries, and Small bioactive molecules. targeting vector having affinity for a Selected target; and 11. An agent as claimed in any of the preceding claims ii) a Second administrable composition comprising an wherein the Vector or vectors have affinity for targets at a agent as claimed in any of the preceding claims, Said level such that the agent interacts with but does not fixedly agent comprising a vector having affinity for Said bind to Said targets. pre-targeting Vector. US 2002/0102215 A1 Aug. 1, 2002 86

23. A combined formulation as claimed in claim 22 generating an ultrasound, magnetic resonance, X-ray, radio wherein Said pre-targeting vector is a monoclonal antibody. graphic or light image of at least a part of Said body. 24. A combined formulation comprising: 31. A method as claimed in claim 30 which comprises the i) a first administrable composition comprising an agent Steps: as claimed in any of claims 1 to 21; and i) administering to said body a pre-targeting vector having ii) a Second administrable composition comprising a affinity for a Selected target; and thereafter Substance capable of displacing or releasing Said agent ii) administering an agent as claimed in any of claims 1 to from its target. 21, Said agent comprising a vector having affinity for 25. A combined formulation comprising: Said pre-targeting Vector. 32. A method as claimed in claim 31 wherein said i) a first administrable composition comprising an agent pre-targeting vector is a monoclonal antibody. as claimed in claim 20; and 33. A method as claimed in claim 30 which comprises the ii) a Second administrable composition comprising a Steps: reducing agent capable of reductively cleaving the disulphide groups coupling or linking the therapeutic i) administering to Said body an agent as claimed in any compound and reporter in the agent of Said first admin of claims 1 to 21; and thereafter istrable composition. ii) administering a Substance capable of displacing or 26. A proceSS for the preparation of a targetable diagnostic releasing Said agent from its target. and/or therapeutically active agent as defined in claim 1 34. A method as claimed in any of claims 30 to 33 wherein which comprises either coupling or linking at least one Said agent further comprises a therapeutic compound. vector to a reporter comprising gas-filled microbubbles 35. A method as claimed in claim 34 wherein said Stabilised by monolayers of film-forming Surfactant or gen therapeutic compound is covalently coupled or linked to the erating gas-filled reporter microbubbles using film-forming reporter through disulphide groups, and a composition com Surfactant having at least one vector attached thereto. prising a reducing agent capable of reductively cleaving Said 27. A proceSS as claimed in claim 26 wherein a therapeutic disulphide groups is Subsequently administered. compound is also combined with the reporter. 36. A method for in vitro investigation of targeting by an 28. A proceSS as claimed in claim 27 wherein a therapeutic agent as defined in any of claims 1 to 21 wherein cells compound containing thiol groups is linked to thiol group expressing a target are fixedly positioned in a flow chamber, containing Surfactant monolayerS Stabilising the gas-filled a Suspension of Said agent in a carrier liquid is passed microbubbles of the reporter by reaction under oxidative through said chamber, and binding of said agent to said cells conditions So as to generate disulphide groups. is examined. 29. Use of an agent as claimed in any of claims 1 to 21 37. A method as claimed in claim 36 wherein the flow rate as a targetable ultrasound contrast agent. of carrier liquid is controlled to Simulate shear rates encoun 30. A method of generating enhanced images of a human tered in vivo. or non-human animal body which comprises administering to Said body an agent as claimed in any of claims 1 to 21 and