WO1998018498A2 - Ameliorations apportees a des agents diagnostiques et/ou therapeutiques ou les concernant - Google Patents

Ameliorations apportees a des agents diagnostiques et/ou therapeutiques ou les concernant Download PDF

Info

Publication number
WO1998018498A2
WO1998018498A2 PCT/GB1997/002958 GB9702958W WO9818498A2 WO 1998018498 A2 WO1998018498 A2 WO 1998018498A2 GB 9702958 W GB9702958 W GB 9702958W WO 9818498 A2 WO9818498 A2 WO 9818498A2
Authority
WO
WIPO (PCT)
Prior art keywords
agent
vector
microbubbles
gas
reporter
Prior art date
Application number
PCT/GB1997/002958
Other languages
English (en)
Other versions
WO1998018498A3 (fr
Inventor
Jo Klaveness
Pål RONGVED
Anders HØGSET
Helge Tolleshaug
Aslak Godal
Alan Cuthbertson
Dagfinn LØVHAUG
Magne Solbakken
Original Assignee
Marsden, John, Christopher
Nycomed Imaging As
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9622364.9A external-priority patent/GB9622364D0/en
Priority claimed from GBGB9622366.4A external-priority patent/GB9622366D0/en
Priority claimed from GBGB9622367.2A external-priority patent/GB9622367D0/en
Priority claimed from GBGB9700698.5A external-priority patent/GB9700698D0/en
Priority claimed from GBGB9700699.3A external-priority patent/GB9700699D0/en
Priority claimed from GBGB9708265.5A external-priority patent/GB9708265D0/en
Priority claimed from GBGB9711842.6A external-priority patent/GB9711842D0/en
Priority claimed from GBGB9711844.2A external-priority patent/GB9711844D0/en
Priority to JP10520191A priority Critical patent/JP2001502719A/ja
Priority to EP97910518A priority patent/EP0963209A2/fr
Priority to AU47870/97A priority patent/AU4787097A/en
Application filed by Marsden, John, Christopher, Nycomed Imaging As filed Critical Marsden, John, Christopher
Publication of WO1998018498A2 publication Critical patent/WO1998018498A2/fr
Publication of WO1998018498A3 publication Critical patent/WO1998018498A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058—Antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6925—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a microcapsule, nanocapsule, microbubble or nanobubble
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A61K49/0043—Fluorescein, used in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0497—Organic compounds conjugates with a carrier being an organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins

Definitions

  • This invention relates to diagnostic and/or therapeutically active agents, more particularly to diagnostic and/or therapeutically active agents incorporating moieties which interact with or have affinity for sites and/or structures within the body so that diagnostic imaging and/or therapy of particular locations within the body may be enhanced.
  • diagnostic agents for use in ultrasound imaging which are hereinafter referred to as targeted ultrasound contrast agents.
  • ultrasound imaging comprises a potentially valuable diagnostic tool, for example in studies of the vascular system, particularly in cardiography, and of tissue microvasculature .
  • contrast agents has been proposed to enhance the acoustic images so obtained, including suspensions of solid particles, emulsified liquid droplets, gas bubbles and encapsulated gases or liquids. It is generally accepted that low density contrast agents which are easily compressible are particularly efficient in terms of the acoustic backscatter they generate, and considerable interest has therefore been shown in the preparation of gas-containing and gas-generating systems .
  • Gas-containing contrast media are also known to be effective in magnetic resonance (MR) imaging, e.g. as susceptibility contrast agents which will act to reduce MR signal intensity.
  • Oxygen-containing contrast media also represent potentially useful paramagnetic MR contrast agents .
  • gases such as carbon dioxide may be used as negative oral contrast agents or intravascular contrast agents.
  • radioactive gases e.g. radioactive isotopes of inert gases such as xenon
  • scintigraphy for example for blood pool imaging .
  • Targeted ultrasound contrast agents may be regarded as comprising (i) a reporter moiety capable of interacting with ultrasound irradiation to generate a detectable signal; (ii) one or more vectors having affinity for particular target sites and/or structures within the body, e.g. for specific cells or areas of pathology; and (iii) one or more linkers connecting said reporter and vector (s), in the event that these are not directly joined.
  • the molecules and/or structure to which the agent is intended to bind will hereinafter be referred to as the target .
  • the target In order to obtain specific imaging of or a therapeutic effect at a selected region/structure in the body the target must be present and available in this region/structure.
  • the target may either be a defined molecular species (i.e. a target molecule) or an unknown molecule or more complex structure (i.e. a target structure) which is present in the area to be imaged and/or treated, and is able to bind specifically or selectively to a given vector molecule.
  • the vector is attached or linked to the reporter moiety in order to bind these moieties to the region/structure to be imaged and/or treated.
  • the vector may bind specifically to a chosen target, or it may bind only selectively, having affinty also for a limited number of other molecules/structures, again creating possible background problems.
  • US-A-5531980 is directed to systems in which the reporter comprises an aqueous suspension of air or gas microbubbles stabilised by one or more film- forming surfactants present at least partially in lamellar or laminar form, said surfactant (s) being bound to one or more vectors comprising "bioactive species designed for specific targeting purposes".
  • the microbubbles are not directly encapsulated by surfactant material but rather that this is incorporated in liquid- filled liposomes which stabilise the microbubbles.
  • lamellar or laminar surfactant material such as phospholipids present in such liposomes will inevitably be present in the form of one or more lipid bilayers with the lipophilic tails "back-to-back” and the hydrophilic heads both inside and outside (see e.g. Schneider, M. on “Liposomes as drug carriers: 10 years of research” in Drug targeting, Nyon, Swi tzerland, 3 -5 October 1984 , Buri, P. and Gumma, A. (Ed) , Elsevier, Amsterdam 1984) .
  • EP-A-0727225 describes targeted ultrasound contrast agents in which the reporter comprises a chemical having a sufficient vapour pressure such that a proportion of it is a gas at the body temperature of the subject.
  • This chemical is associated with a surfactant or albumin carrier which includes a protein-, peptide- or carbohydrate-based cell adhesion molecule ligand as vector.
  • the reporter moieties in such contrast agents correspond to the phase shift colloid systems described in WO-A-9416739 ; it is now recognised that administration of such phase shift colloids may lead to generation of microbubbles which grow uncontrollably, possibly to the extent where they cause potentially dangerous embolisation of, for example, the myocardial vasculature and brain (see e.g.
  • tissue-specific ultrasonic image enhancement may be achieved using acoustically reflective oligolamellar liposomes conjugated to tissue-specific ligands such as antibodies, peptides, lectins etc.
  • the liposomes are deliberately chosen to be devoid of gas and so will not have the advantageous echogenic properties of gas-based ultrasound contrast agents .
  • Further references to this technology e.g. in targeting to fibrin, thrombi and atherosclerotic areas are found in publications by Alkanonyuksel , H. et al . in J “ . Pharm . Sci . (1996) 85(5), 486-490; J “ . Am. Coll . Cardiol . (1996) 27(2) Suppl A,
  • the present invention is based on the finding that gas-containing and gas-generating diagnostic and/or therapeutic agents coupled to non-bioactive vectors are particularly useful targeting agents by virtue of their enhanced safety relative to conventional targeting agents, which may elicit undesirable and unwanted biological effects in the subject.
  • non- bioactive vectors may exhibit improved targeting efficacy as compared to bioactive vectors.
  • bioactive vectors usually will have to compete with endogenous ligands for the same binding site on the target molecule.
  • non- bioactive vectors will often bind to target molecules for which endogenous ligands do not exist, or alternatively non-bioactive vectors may bind at sites in a target molecule that is not involved in the biological function of the target molecule and for which natural ligands are not present in the body.
  • non-bioactive denotes two kinds of substances. First, it denotes a substance capable of interacting with or binding to a target molecule or target structure that is not normally involved in generating biological responses. Second, it denotes a substance capable of interacting with or binding to a given target without either giving or inhibiting the biological response normally elicited upon binding of a bioactive substance to the target, a bioactive substance being one which interacts with another molecule (i.e. a target) and generates a defined and measurable biological response.
  • receptors for transport proteins such as receptors for transferrin or lipoproteins
  • a moiety carried in the protein may be bioactive, but the apoprotein itself is not.
  • many cofactors, vitamins etc. are bioactive only after they have been carried into the cell, that is to say, they do not elicit a biological response simply upon binding to the carrier protein.
  • Vectors useful in accordance with the invention may, for example, be non-bioactive per se or may be non- bioactive at doses useful for diagnostic and/or therapeutic purposes.
  • a combination of otherwise bioactive vectors may be employed in such a way that a biological activity elicited by one type of vector is exactly counterbalanced by another kind or other kinds of vector molecules coupled to the same agent or otherwise present in the same diagnostic and/or therapeutic composition.
  • One advantageous embodiment of the invention is based on the additional finding that limited adhesion to targets is a highly useful property of diagnostic and/or therapeutically active agents, which property may be achieved using non-bioactive vectors giving temporary retention rather than fixed adhesion to a target.
  • agents rather than being fixedly retained at specific sites, may for example effectively exhibit a form of retarded flow along the vascular endothelium by virtue of their transient interactions with endothelial cells.
  • Such agents may thus become concentrated on the walls of blood vessels, in the case of ultrasound contrast agents providing enhanced echogenicity thereof relative to the bulk of the bloodstream, which is devoid of permanent structural features. They therefore may permit enhanced imaging of the capillary system, including the microvasculature, and so may facilitate distinction between normal and inadequately perfused tissue, e.g. in the heart, and may also be useful in visualising structures such as Kupffer cells, thrombi and atherosclerotic lesions or for visualising neo- vascularized and inflamed tissue areas.
  • the present invention is also well suited to image changes occurring to normal blood vessels which are situated in areas of tissue necrosis.
  • a targetable diagnostic and/or therapeutic agent e.g. an ultrasound contrast agent
  • a suspension in an aqueous carrier liquid e.g. an injectable carrier liquid
  • a reporter comprising gas-containing or gas-generating material and coupled to one or more vectors, characterised in that said vector or vectors are non- bioactive .
  • Vectors useful in accordance with the invention include proteins which bind to cell-surface proteoglycans , e.g. so that the contrast agent becomes concentrated on cell surfaces.
  • proteoglycans are large glycoproteins containing glucosaminoglycan side chains which, in many cases, are heparan sulphate. Binding to glucosoaminoglycans has not been shown to elicit a biological response.
  • one may isolate or synthesise the proteoglycan-binding (or, where appropriate, heparan sulphate-binding) part of the molecule for use as a vector, while avoiding any biological activity associated with other parts of the molecule.
  • non-bioactive monomeric or oligomeric vectors and the use of non-bioactive peptide vectors represent preferred aspects of this invention.
  • a further aspect of the present invention is for example where a vector or vectors is attached to the reporter or included non-covalently into the reporter in a manner where the said vector or vectors is not readily exposed to the targets or receptors.
  • Increased tissue specificity may therefore be achieved by applying an additional process to expose the vectors, e.g. the agent is exposed to external ultrasound to change the diffusibility of the moieties containing the vectors.
  • Any biocompatible gas may be present in the reporter of contrast agents according to the invention, the term "gas” as used herein including any substances (including mixtures) substantially or completely in gaseous (including vapour) form at the normal human body temperature of 37°C.
  • the gas may thus, for example, comprise air; nitrogen; oxygen; carbon dioxide; hydrogen; an inert gas such as helium, argon, xenon or krypton; a sulphur fluoride such as sulphur hexafluoride, disulphur decafluoride or trifluoromethylsulphur pentafluoride ; selenium hexafluoride; an optionally halogenated silane such as methylsilane or dimethylsilane; a low molecular weight hydrocarbon (e.g.
  • an alkane such as methane, ethane, a propane, a butane or a pentane, a cycloalkane such as cyclopropane, cyclobutane or cyclopentane, an alkene such as ethylene, propene, propadiene or a butene, or an alkyne such as acetylene or propyne; an ether such as dimethyl ether; a ketone; an ester; a halogenated low molecular weight hydrocarbon (e.g. containing up to 7 carbon atoms); or a mixture of any of the foregoing.
  • an alkane such as methane, ethane, a propane, a butane or a pentane
  • a cycloalkane such as cyclopropane, cyclobutane or cyclopentane
  • an alkene such as ethylene, propene, propadiene or a butene
  • biocompatible halogenated hydrocarbon gases may, for example, be selected from bromochlorodifluoromethane , chlorodifluoromethane , dichlorodifluoromethane , bromotrifluoromethane , chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane , chlorotrifluoroethylene , fluoroethylene, ethylfluoride, 1, 1-difluoroethane and perfluorocarbons , e.g.
  • perfluoroalkanes such as perfluoromethane , perfluoroethane, perfluoropropanes , perfluorobutanes (e.g. perfluoro-n-butane, optionally in admixture with other isomers such as perfluoro-iso- butane) , perfluoropentanes, perfluorohexanes and perfluoroheptanes ; perfluoroalkenes such as perfluoropropene, perfluorobutenes (e.g.
  • perfluorobut-2- ene and perfluorobutadiene; perfluoroalkynes such as perfluorobut-2-yne; and perfluorocycloalkanes such as perfluorocyclobutane , perfluoromethylcyclobutane , perfluorodimethylcyclobutanes, perfluorotrimethyl- cyclobutanes, perfluorocyclopentane, perfluoromethyl- cyclopentane, perfluorodimethylcyclopentanes , perfluorocyclohexane, perfluoromethylcyclohexane and perfluorocycloheptane .
  • halogenated gases include methyl chloride, fluorinated (e.g. perfluorinated) ketones such as perfluoroacetone and fluorinated (e.g. perfluorinated) ethers such as perfluorodiethyl ether.
  • perfluorinated gases for example sulphur hexafluoride and perfluorocarbons such as perfluoropropane , perfluorobutanes and perfluoropentanes, may be particularly advantageous in view of the recognised high stability in the bloodstream of microbubbles containing such gases.
  • the reporter may be in any convenient form, for example being any appropriate gas-containing or gas- generating ultrasound contrast agent formulation.
  • Representative examples of such formulations include microbubbles of gas stabilised (e.g. at least partially encapsulated) by a coalescence-resistant surface membrane (for example gelatin, e.g. as described in 0- A-8002365) , a filmogenic protein (for example an albumin such as human serum albumin, e.g.
  • a polymer material for example a synthetic biodegradable polymer as described in EP-A- 0398935 , an elastic interfacial synthetic polymer membrane as described in EP-A-0458745 , a microparticulate biodegradable polyaldehyde as described in EP-A-0441468 , a microparticulate N-dicarboxylic acid derivative of a polyamino acid - polycyclic imide as described in EP-A- 0458079, or a biodegradable polymer as described in O- A-9317718 or O-A-9607434 ) , a non-polymeric and non- polymerisable wall-forming material
  • gas-containing contrast agent formulations include gas-containing solid systems, for example microparticles (especially aggregates of microparticles) having gas contained therewithin or otherwise associated therewith (for example being adsorbed on the surface thereof and/or contained within voids, cavities or pores therein, e.g. as described in EP-A-0122624, EP-A- 0123235 , EP-A-0365467 , WO-A-9221382 , WO-A-9300930, WO-A-9313802 , WO-A-9313808 or WO-A- 9313809) .
  • the echogenicity of such microparticulate contrast agents may derive directly from the contained/associated gas and/or from gas (e.g. microbubbles) liberated from the solid material (e.g. upon dissolution of the microparticulate structure) .
  • Gas microbubbles and other gas-containing materials such as microparticles preferably have an initial average size not exceeding 10 ⁇ m (e.g. of 7 ⁇ m or less) in order to permit their free passage through the pulmonary system following administration, e.g. by intravenous injection.
  • phospholipid-containing compositions are employed in accordance with the invention, e.g. in the form of phospholipid-stabilised gas microbubbles
  • useful phospholipids include lecithins (i.e. phosphatidylcholines) , for example natural lecithins such as egg yolk lecithin or soya bean lecithin and synthetic or semisynthetic lecithins such as dimyristoylphosphatidylcholine , dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine; phosphatidic acids; phosphatidylethanolamines ; phosphatidylserines ; phosphatidylglycerols ; phosphatidylinositols ; cardiolipins; sphingomyelins ; fluorinated analogues of any of the foregoing; mixtures of any of the foregoing and mixtures with other lipids such as
  • phospholipids predominantly (e.g. at least 75%) comprising molecules individually bearing net overall charge, e.g. negative charge, for example as in naturally occurring (e.g. soya bean or egg yolk derived), semisynthetic (e.g. partially or fully hydrogenated) and synthetic phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, phosphatidic acids and/or cardiolipins, may be particularly advantageous.
  • naturally occurring e.g. soya bean or egg yolk derived
  • semisynthetic e.g. partially or fully hydrogenated
  • synthetic phosphatidylserines e.g. partially or fully hydrogenated
  • phosphatidylglycerols phosphatidylglycerols
  • phosphatidylinositols phosphatidic acids and/or cardiolipins
  • exemplary lipids which may be used to prepare gas-containing contrast agents include fatty acids, stearic acid, palmitic acid, 2-n-hexadecylstearic acid, oleic acid and other acid containing lipid structures. These lipid structures are considered particularly interesting when coupled by amide bond formation to amino acids containing one or more amino groups.
  • the resulting lipid modified amino acids e.g. dipalmitoyllysine, distearoyl-2 , 3 -diaminopropionic acid
  • a further extension of this invention relates to the synthesis of lipopeptide structures comprising a lipid reporter attached to a linker portion (e.g. PEG, polyamino acid, alkylhalide etc) the said linker being suitably functionalised for coupling to one or more vector molecules.
  • a linker portion e.g. PEG, polyamino acid, alkylhalide etc
  • the said linker being suitably functionalised for coupling to one or more vector molecules.
  • a positively charged linker element eg. two or more lysine residues
  • microbubbles carrying one or more reactive groups for non-specific modification of a multiplicity of receptor molecules located on cell surfaces.
  • Microbubbles comprising a thiol moiety can bind to cell surface receptors via disulphide exchange reactions. The reversible nature of this covalent bond means that bubble flow can be controlled by altering the redox environment.
  • activated' microbubbles of membranes comprising active esters such as N-hydroxysuccinimide esters can be used to modify amino groups found on a multiplicity of cell surface molecules.
  • gas-containing microparticulate materials which may be useful in accordance with the invention include carbohydrates (for example hexoses such as glucose, fructose or galactose; disaccharides such as sucrose, lactose or maltose; pentoses such as arabinose, xylose or ribose; - , ⁇ - and ⁇ -cyclodextrins ; polysaccharides such as starch, hydroxyethyl starch, amylose, amylopectin, glycogen, inulin, pulullan, dextran, carboxymethyl dextran, dextran phosphate, ketodextran, aminoethyldextran, alginates, chitin, chitosan, hyaluronic acid or heparin; and sugar alcohols, including alditols such as mannitol or sorbitol), inorganic salts (e.g.
  • X-ray contrast agents e.g. any of the commercially available carboxylic acid and non-ionic amide contrast agents typically containing at least one 2, 4 , 6-triiodophenyl group having substituents such as carboxyl, carbamoyl, N-alkylcarbamoyl , N- hydroxyalkylcarbamoyl , acylamino, N-alkylacylamino or acylaminomethyl at the 3- and/or 5 -positions, as in metrizoic acid, diatrizoic acid, iothalamic acid, ioxaglic acid, iohexol , iopentol, iopamidol , iodixanol, iopromide, metrizamide, iodipamide, meglumine iodipamide, meglumine acetrizo
  • the reporter may be made by any convenient process, for example by making gas-containing or gas- generating formulations.
  • Representative examples include the preparation of a suspension of gas microbubbles by contacting a surfactant with gas and mixing them in the presence of an aqueous carrier, as described in WO
  • a suitable process for attachment of the desired vector to the reporter comprises a surface modification of the preformed reporter with a suitable linker employing reactive groups on the surface of both the reporter and vector. It may be particularly advantageous physically to mix the reporter material with the vector- containing substance at any step of the process. Such a process will result in incorporation or an attachment of the vector to the reporter. An optional process step may remove the excess of vector not bound to the reporter by washing the gas-containing particles following separation, by for example, floatation.
  • a preferred aspect is the use of lipopeptide structures incorporating functional groups such as thiol, maleimide biotin etc. which can be premixed if desired with other reporter molecules before formation of gas-containing agents .
  • the attachment of vector molecules may be carried out using the linker reagents listed below.
  • Coupling of a reporter unit to the desired vectors may be achieved by covalent or non-covalent means, usually involving interaction with one or more functional groups located on the reporter and/or vectors.
  • functional groups located on the reporter and/or vectors.
  • chemically reactive functional groups include amino, hydroxyl, sulfhydryl, carboxyl, and carbonyl groups, as well as carbohydrate groups, vicinal diols, thioethers, 2-aminoalcohols , 2-aminothiols , guanidinyl, imidazolyl and phenolic groups.
  • Covalent coupling of reporter and vectors may therefore be effected using linking agents containing reactive moities capable of reaction with such functional groups .
  • N-Maleimide derivatives are also considered selective towards sulfhydryl groups, but may additionaly be useful in coupling to amino groups under certain conditions.
  • N-maleimides may be incorporated into linking systems for reporter-vector conjugation as described by Kitagawa, T. et al . in Chem . Pharm. Bull . (1981) 29, 1130 or used as polymer crosslinkers for bubble stabilisation as described by Kovacic, P. et al . in J. Am . Chem . Soc . (1959) 81, 1887.
  • Reagents such as 2-iminothiolane, e.g. as described by Traut, R. et al. in Biochemistry (1973) 12, 3266, which introduce a thiol group through conversion of an amino group, may be considered as sulfhydryl reagents if linking occurs through the formation of disulphide bridges .
  • reagents which introduce reactive disulphide bonds into either the reporter or the vector may be useful, since linking may be brought about by disulphide exchange between the vector and reporter;
  • examples of such reagents include Ellman's reagent (DTNB), 4,4'- dithiodipyridine, methyl-3-nitro-2-pyridyl disulphide and methyl -2 -pyridyl disulphide (described by Kimura, T. et al . in Analyt . Biochem . (1982) 122, 271) .
  • Examples of reactive moieties capable of reaction with amino groups include alkylating and acylating agents.
  • Representative amino-reactive acylating agents include : i) isocyanates and isothiocyanates, particularly aromatic derivatives, Which form stable urea and thiourea derivatives respectively and have been used for protein crosslinking as described by Schick, A.F. et al. in J " . Biol . Chem . (1961) 236, 2477; ii) sulfonyl chlorides, which have been described by Herzig, D.J. et al .
  • Carbonyl groups such as aldehyde functions may be reacted with weak protein bases at a pH such that nucleophilic protein side-chain functions are protonated.
  • Weak bases include 1 , 2-aminothiols such as those found in N-terminal cysteine residues, which selectively form stable 5-membered thiazolidine rings with aldehyde groups, e.g. as described by Ratner, S. et al . in J. Am. Chem . Soc . (1937) 59, 200.
  • Other weak bases such as phenyl hydrazones may be used, e.g. as described by Heitzman, H. et al . in Proc . Na tl . Acad . Sci . USA (1974) 71, 3537.
  • Aldehydes and ketones may also be reacted with amines to form Schiff's bases, which may advantageously be stabilised through reductive amination.
  • Alkoxylamino moieties readily react with ketones and aldehydes to produce stable alkoxamines, e.g. as described by Webb, R. et al . in Bioconjuga te Chem . (1990) 1, 96.
  • reactive moieties capable of reaction with carboxyl groups include diazo compounds such as diazoacetate esters and diazoacetamides, which react with high specificity to generate ester groups, e.g. as described by Herriot R.M. in Adv. Protein Chem . (1947) 3, 169.
  • Carboxylic acid modifying reagents such as carbodiimides , which react through O-acylurea formation followed by amide bond formation, may also usefully be employed; linking may be facilitated through addition of an amine or may result in direct vector-receptor coupling.
  • Useful water soluble carbodiimides include 1- cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide (CMC) and l-ethyl-3 - (3 -dimethylaminopropyl) carbodiimide (EDC) , e.g. as described by Zot, H.G. and Puett, D. in J " . Biol . Chem . (1989) 264, 15552.
  • CMC 1- cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide
  • EDC l-ethyl-3 - (3 -dimethylaminopropyl) carbodiimide
  • carboxylic acid modifying reagents include isoxazolium derivatives such as Woodwards reagent K; chloroformates such as p- nitrophenylchloroformate; carbonyldiimidazoles such as 1 , 1 ' -carbonyldiimidazole ; and N- carbalkoxydihydroquinolines such as N- (ethoxycarbonyl) - 2-ethoxy-l, 2-dihydroquinoline .
  • isoxazolium derivatives such as Woodwards reagent K
  • chloroformates such as p- nitrophenylchloroformate
  • carbonyldiimidazoles such as 1 , 1 ' -carbonyldiimidazole
  • N- carbalkoxydihydroquinolines such as N- (ethoxycarbonyl) - 2-ethoxy-l, 2-dihydroquinoline .
  • vicinal diones such as p-phenylenediglyoxal , which may be used to react with guanidinyl groups, e.g. as described by Wagner e ⁇ al . in Nucleic acid Res . (1978) 5, 4065; and diazonium salts, which may undergo electrophilic substitution reactions, e.g. as described by Ishizaka, K. and Ishizaka T. in J " . Immunol . (1960) 85, 163.
  • Bis-diazonium compounds are readily prepared by treatment of aryl diamines with sodium nitrite in acidic solutions.
  • functional groups in the reporter and/or vector may if desired be converted to other functional groups prior to reaction, e.g. to confer additional reactivity or selectivity.
  • methods useful for this purpose include conversion of amines to carboxylic acids using reagents such as dicarboxylic anhydrides; conversion of amines to thiols using reagents such as N-acetylhomocysteine thiolactone, S-acetylmercaptosuccinic anhydride, 2- iminothiolane or thiol -containing succinimidyl derivatives; conversion of thiols to carboxylic acids using reagents such as ⁇ -haloacetates; conversion of thiols to amines using reagents such as ethylenimine or 2-bromoethylamine ; conversion of carboxylic acids to amines using reagents such as carbodiimides followed by diamines ; and conversion of alcohols to thio
  • Vector-reporter coupling may also be effected using enzymes as zero-length crosslinking agents; thus, for example, transglutaminase, peroxidase and xanthine oxidase have been used to produce crosslinked products.
  • Reverse proteolysis may also be used for crosslinking through amide bond formation.
  • Non-covalent vector-reporter coupling may, for example, be effected by electrostatic charge interactions e.g. between a polylysinyl-functionalised reporter and a polyglutamyl-functionalised vector, through chelation in the form of stable metal complexes or through high affinity binding interaction such as avidin/biotin binding.
  • Polylysine, coated non- covalently to the negatively charged membrane surface can also increase non-specifically the affinity of a microbubble for a cell through charge interactions.
  • a vector may be coupled to a protein known to bind phospholipids.
  • a single molecule of phospholipid may attach to a protein such as a translocase, while other proteins may attach to surfaces consisting mainly of phospholipid head groups and so may be used to attach vectors to phospholipid microspheres; one example of such a protein is ⁇ 2-glycoprotein I (Chonn, A., Semple, S.C. and
  • a conjugate of a vector with such a phosphatidylserine-binding protein may therefore be used to attach the vector to phosphatidylserine-encapsulated microbubbles .
  • the amino acid sequence of a binding protein is known, the phospholipid-binding portion may be synthesised or isolated and used for conjugation with a vector, thus avoiding the biological activity which may be located elsewhere in the molecule.
  • phage libraries displaying small peptides could be used for such selection.
  • the selection may be made by simply mixing the microspheres and the phage display library and eluting the phages binding to the floating microspheres. If desired, the selection can be done under "physiological conditions" (e.g. in blood) to eliminate peptides which cross-react with blood components.
  • physiological conditions e.g. in blood
  • An advantage of this type of selection procedure is that only binding molecules that do not destabilize the microspheres should be selected, since only binding molecules attached to intact floating microspheres will rise to the top. It may also be possible to introduce some kind of "stress" during the selection procedure (e.g. pressure) to ensure that destabilizing binding moieties are not selected.
  • binding moieties identified in this way may be coupled (by chemical conjugation or via peptide synthesis, or at the DNA-level for recombinant vectors) to a vector molecule, constituting a general tool for attaching any vector molecule to the microspheres .
  • a vector which comprises or is coupled to a peptide, lipo-oligosaccharide or lipopeptide linker which contains a element capable of mediating membrane insertion may also be useful.
  • a peptide, lipo-oligosaccharide or lipopeptide linker which contains a element capable of mediating membrane insertion
  • Non-bioactive molecules consisting of known membrane insertion anchor/signal groups may also be used as vectors for certain applications, an example being the HI hydrophobic segment from the Na,K-ATPase - subunit described by Xie, Y. and Morimoto, T. in J " . Biol . Chem. (1995) 270(20), 11985-11991.
  • the anchor group may also be fatty acid(s) or cholesterol.
  • Coupling may also be effected using avidin or streptavidin, which have four high affinity binding sites for biotin. Avidin may therefore be used to conjugate vector to reporter if both vector and reporter are biotinylated. Examples are described by Bayer, E.A. and Wilchek, M. in Methods Biochem . Anal . (1980) 26, 1. This method may also be extended to include linking of reporter to reporter, a process which may encourage bubble association and consequent potentially increased echogenicity . Alternatively, avidin or streptavidin may be attached directly to the surface of reporter microparticles. Non-covalent coupling may also utilise the bifunctional nature of bispecific immunoglobulins .
  • bispecific IgG or chemically engineered bispecific F(ab)'2 fragments may be used as linking agents.
  • Heterobifunctional bispecific antibodies have also been reported for linking two different antigens, e.g. as described by Bode, C. et al . in J “ . Biol . Chem . (1989) 264, 944 and by Staerz, U.D. et al. in Proc . Na tl . Acad . Sci . USA (1986) 83, 1453.
  • any reporter and/or vector containing two or more antigenic determinants e.g. as described by Chen, Aa et al . in Am. J “ . Pathol . (1988) 130, 216) may be crosslinked by antibody molecules and lead to formation of multi-bubble cross-linked assemblies of potentially increased echogenicity.
  • Linking agents used in accordance with the invention will in general bring about linking of vector to reporter or reporter to reporter with some degree of specificity, and may also be used to attach one or more therapeutically active agents.
  • a PEG component as a stabiliser in conjunction with a vector or vectors or directly to the reporter in the same molecule where the PEG does not serve as a spacer.
  • the reporter unit will usually remain attached to the vectors.
  • the vector (often, a monoclonal antibody) is administered alone; subsequently, the reporter is administered, coupled to a moiety which is capable of specifically binding the vector molecule (when the vector is an antibody, the reporter may be coupled to an immunoglobulin-binding molecule, such as protein A or an anti-immunoglobulin antibody) .
  • an immunoglobulin-binding molecule such as protein A or an anti-immunoglobulin antibody
  • Ultrasound imaging modalities which may be used in accordance with the invention include two- and three- dimensional imaging techniques such as B-mode imaging (for example using the time-varying amplitude of the signal envelope generated from the fundamental frequency of the emitted ultrasound pulse, from sub-harmonics or higher harmonics thereof or from sum or difference frequencies derived from the emitted pulse and such harmonics, images generated from the fundamental frequency or the second harmonic thereof being preferred) , colour Doppler imaging and Doppler amplitude imaging, and combinations of the two latter with any of the modalities (techniques) above.
  • B-mode imaging for example using the time-varying amplitude of the signal envelope generated from the fundamental frequency of the emitted ultrasound pulse, from sub-harmonics or higher harmonics thereof or from sum or difference frequencies derived from the emitted pulse and such harmonics, images generated from the fundamental frequency or the second harmonic thereof being preferred
  • colour Doppler imaging and Doppler amplitude imaging and combinations of the two latter with any of the modalities (techniques) above.
  • the present invention accordingly provides a tool for therapeutic drug delivery in combination with vector-mediated direction of the product to the desired site.
  • therapeutic or “drug” is meant an agent having a beneficial effect on a specific disease in a living human or non-human animal.
  • combinations of drugs and ultrasound contrast agents have been proposed in, for example, WO-A-9428873 and WO-A-9507072 , these products lack vectors having affinity for particular sites and thereby show comparitively poor specific retention at desired sites prior to or during drug release .
  • Therapeutic compounds used in accordance with the present invention may be encapsulated in the interior of the microbubbles/microparticles or attached to or incorporated into the structure thereof.
  • the therapeutic compound may be linked to a part of the wall or matrix, for example through covalent or ionic bonds, or may be physically mixed into the encapsulating or matrix material, particularly if the drug has similar polarity or solubility to this material, so as to prevent it from leaking out of the product before it is intended to act in the body.
  • the release of the drug may be initiated merely by wetting contact with blood following administration or as a consequence of other internal or external influences, e.g. dissolution processes catalyzed by enzymes or the use of ultrasound.
  • the destruction of gas-containing microparticles using external ultrasound is a well known phenomenon in respect of ultrasound contrast agents, e.g. as described in WO-A-9325241; the rate of drug release may be varied depending on the type of therapeutic application, using a specific amount of ultrasound energy from the transducer.
  • the therapeutic may be covalently linked to the membrane or matrix surface using a suitable linking agent, e.g. as described herein.
  • a suitable linking agent e.g. as described herein.
  • one may initially prepare a phospholipid or lipopeptide or derivative thereof to which the drug is bonded through a biodegradable bond or linker, and then incorporate this derivative into the material used to prepare the reporter, as described above.
  • the product may initially be prepared without the therapeutic, which may then be coupled to or coated on the microbubbles or microparticles prior to use.
  • a therapeutic could be added to a suspension of microbubbles or microparticles in aqueous media and shaken in order to attach or adhere the therapeutic thereto.
  • Exemplary drug delivery systems suitable for use in the present compositions include any known therapeutic drugs or active analogues thereof containing thiol groups which are coupled to thiol containing microbubbles under oxidative conditions yielding disulphide bridges.
  • the drug/vector modified microbubbles are allowed to accumulate in the target tissue.
  • Administration of a reducing agent such as reduced glutathione then liberates the drug molecule from the targeted microbubble in the vicinity of the target cell increasing the local concentration of the drug and enhancing therapeutic effect.
  • the product may also be prepared without the therapeutic if desired.
  • the drug may then be coupled to or coated on the microbubbles prior to use.
  • a therapeutic could be added to a suspension of microbubbles in aqueous media and shaken in order to attach or adhere the therapeutic to the microbubbles.
  • Other drug delivery systems include vector modified phospholipid membranes doped with lipopeptide structures comprising a poly-L-lysine or poly-D-lysine chain in combination with a targeting vector.
  • the microbubble carrier is condensed with DNA or RNA via elecrostatic interaction with the polycation. This method has the advantage that the vector or vectors used for targeted delivery are not directly attached to the polysine carrier moiety.
  • the polylysine chain is also anchored more tightly in the microbubble membrane due to the presence of the lipid chains.
  • the use of ultrasound to increase the effectiveness of delivery is also considered useful.
  • free polylysine chains are firstly modified with drug or vector molecules then condensed onto the negative surface of targeted microbubbles.
  • drugs useful in accordance with the invention include antineoplastic agents such as vincristine, vinblastine, vindesine, busulfan, chlorambucil , spiroplatin, cisplatin, carboplatin, methotrexate, adriamycin, mitomycin, bleomycin, cytosine arabinoside, arabinosyl adenine, mercaptopurine, mitotane, procarbazine, dactinomycin (antinomycin D) , daunorubicin, doxorubicin hydrochloride, taxol, plicamycin, aminoglutethimide, estramustine, flutamide, leuprolide, megestrol acetate, tamoxifen, testolactone, trilostane, amsacrine (m-AMSA) , asparaginase (L-asparaginase) , etoposide, interferon a- 2
  • RNA RNA
  • DNA of natural or synthetic origin, including recombinant RNA and DNA. DNA encoding certain proteins may be used in the treatment of many different types of diseases .
  • tumor necrosis factor or interleukin-2 genes may be provided to treat advanced cancers; thymidine kinase genes may be provided to treat ovarian cancer or brain tumors; interleukin-2 genes may be provided to treat neuroblastoma, malignant melanoma or kidney cancer; and interleukin-4 genes may be provided to treat cancer.
  • Lipophilic derivatives of drugs linked to the microbubble wall through hydrophobic interactions may exhibit therapeutic effects as part of the microbubble or after release from the microbubble, e.g. by use of ultrasound.
  • a lipophilic group may be introduced for anchoring the drug to the membrane .
  • the lipophilic group should be introduced in a way that does not influence the in vivo potency of the molecule, or the lipophilic group may be cleaved releasing the active drug.
  • Lipophilic groups may be introduced by various chemical means depending on functional groups available in the drug molecule. Covalent coupling may be effected using functional groups in the drug molecule capable of reacting with appropriately functionalised lipophilic compounds.
  • lipophilic moieties include branched and unbranched alkyl chains, cyclic compounds, aromatic residues and fused aromatic and non-aromatic cyclic systems. In some instances the lipophilic moiety will consist of a suitably functionalised steroid, like cholesterol and related compounds .
  • functional groups particularly suitable for derivatisation include nucleophilic groups like amino, hydroxy and sulfhydryl groups.
  • Suitable processes for lipophilic derivatisation of any drug containing a sulfhydryl group, like captopril may include direct alkylation, e.g. reaction with an alkyl halide under basic conditions and thiol ester formation by reaction with an activated carboxylic acid.
  • derivatisation of any drug having carboxylic functions like atenolol and chlorambucil, include amide and ester formation by coupling of amines and alcohols, respectively, possesing requested physical properties.
  • a preferred aspect is attachment of cholesterol to a therapeutic compound by forming a degradable ester bond.
  • a preferred application of the present invention relates to angiogenesis, which is the formation of new blood vessels by branching from existing vessels.
  • the primary stimulus for this process may be inadequate supply of nutrients and oxygen (hypoxia) to cells in a tissue.
  • the cells may respond by secreting angiogenetic factors, of which there are many; one example is vascular endothelial growth factor. These factors initiate the secretion of proteolytic enzymes which break down the proteins of the basement membrane, as well as inhibitors which limit the action of these potentially harmful enzymes.
  • the combined effect of loss of attachment and signals from the receptors for angiogenetic factors is to cause the endothelial cells to move, multiply, and rearrange themselves, and finally to synthetise a basement membrane around the new vessels .
  • Tumors must initiate angiogenesis when they reach millimeter size in order to keep up their rate of growth.
  • angiogenesis is accompanied by characteristic changes in the endothelial cells and their environment, this process is a promising target for therapeutic intervention.
  • the transformations accompanying angiogenesis are also very promising for diagnosis, a preferred example being malignant disease, but the concept also shows great promise in inllammation and a variety of inflammation-related diseases. These factors are also involved in re-vascularisation of infarcted parts of the myocardium, which occurs if the stenosis is released within a short time.
  • angiogenesis mav be detected by the majority of the imaging modalities in use in medicine. Contrast -enhanced ultrasound may possess additional advantages, the contrast medium being microspheres which are restricted to the interior of blood vessels. Even if the target antigens are found on many cell types, the microspheres will attach exclusively to endothelial cells.
  • prodrugs may also be used in agents according to the invention.
  • drugs may be derivatised to alter their physicochemical properties and to adapt them for inclusion into the reporter; such derivatised drugs may be regarded as prodrugs and are usually inactive until cleavage of the derivatising group regenerates the active form of the drug.
  • Another alternative is to incorporate the prodrug, the prodrug-activating enzyme and the vector in the same microbubble in a system where the prodrug will only be activated after some external stimulus.
  • a stimulus may, for example, be a tumour-specific protease as described above, or bursting of the bubbles by external ultrasound after the desired targeting has been achieved.
  • Therapeutics may easily be delivered in accordance with the invention to diseased and necrotic areas including the heart and vasculature in general, and to the liver, spleen and kidneys and other regions such as the lymph system, body cavities or gastrointestinal system.
  • Products according to the present invention may be used for targeted therapeutic delivery either in vivo or in vi tro .
  • the products may be useful in in vi tro systems such as kits for diagnosis of different diseases or characterisation of different components in blood or tissue samples.
  • Similar techniques to those used to attach certain blood components or cells to polymer particles (e . g. monodisperse magnetic particles) in vi tro to separate them from a sample may be used in the present invention, using the low density of the reporter units in agents of the present invention to effect separation of the gas-containing material by floatation and repeated washing.
  • So-called zero-length linking agents which induce direct covalent joining of two reactive chemical groups without introducing additional linking material (e.g. as in amide bond formation induced using carbodiimides or enzymatically) may, if desired, be used in accordance with the invention, as may agents such as biotin/avidin systems which induce non-covalent reporter-vector linking and agents which induce hydrophobic or electrostatic interactions.
  • the linking agent will comprise two or more reactive moieties, e.g. as described above, connected by a spacer element.
  • a spacer element permits bifunctional linkers to react with specific functional groups within a molecule or between two different molecules, resulting in a bond between these two components and introducing extrinsic linker-derived material into the reporter- vector conjugate.
  • the reactive moieties in a linking agent may be the same (homobifunctional agents) or different (heterobifunctional agents or, where several dissimilar reactive moieties are present, heteromultifunctional agents) , providing a diversity of potential reagents that may bring about covalent bonding between any chemical species, either intramolecularly or intermolecularly .
  • extrinsic material introduced by the linking agent may have a critical bearing on the targeting ability and general stability of the ultimate product .
  • labile linkages e.g. containing spacer arms which are biodegradable or chemically sensitive or which incorporate enzymatic cleavage sites.
  • the spacer may include polymeric components, e.g. to act as surfactants and enhance bubble stability.
  • the spacer may also contain reactive moieties, e.g. as described above to enhance surface crosslinking, or it may contain a tracer element such as a fluorescent probe, spin label or radioactive material.
  • Contrast agents according to the present invention are therefore useful in all imaging modalities since contrast elements such as X-ray contrast agents, light imaging probes, spin labels or radioactive units may readily be incorporated in or attached to the reporter units .
  • Spacer elements may typically consist of aliphatic chains which effectively separate the reactive moieties of the linker by distances of between 5 and 30 A. They may also comprise macromolecular structures such as poly (ethylene glycols). Such polymeric structures, hereinafter referred to as PEGs, are simple, neutral polyethers which have been given much attention in biotechnical and biomedical applications (see e.g. Milton Harris, J.
  • PEGs Poly (ethylene glycol ) chemistry, biotechnical and biomedical applica tions" Plenum Press, New York, 1992
  • PEGs are soluble in most solvents, including water, and are highly hydrated in aqueous environments, with two or three water molecules bound to each ethylene glycol segment; this has the effect of preventing adsorption either of other polymers or of proteins onto PEG-modified surfaces.
  • PEGs are known to be nontoxic and not to harm active proteins or cells, whilst covalently linked PEGs are known to be non- immunogenic and non-antigenic .
  • PEGs may readily be modified and bound to other molecules with only little effect on their chemistry. Their advantageous solubility and biological properties are apparent from the many possible uses of PEGs and copolymers thereof, including block copolymers such as PEG-polyurethanes and PEG-polypropylenes .
  • Appropriate molecular weights for PEG spacers used in accordance with the invention may, for example, be between 120 Daltons and 20 kDaltons.
  • the major mechanism for uptake of particles by the cells of the reticuloendothelial system (RES) is opsonisation by plasma proteins in blood; these mark foreign particles which are then taken up by the RES.
  • the biological properties of PEG spacer elements used in accordance with the invention may serve to increase contrast agent circulation time in a similar manner to that observed for PEGylated liposomes (see e.g. Klibanov, A.L. et al . in FEBS Let ters (1990) 268, 235- 237 and Blume, G. and Cevc, G. in Biochim . Biophys . Acta (1990) 1029, 91-97).
  • Increased coupling efficiency to areas of interest may also be achieved using antibodies bound to the terminii of PEG spacers (see e.g. Maruyama, K. et al . in Biochim . Biophys . Acta (1995) 1234, 74-80 and Hansen, C.B. et al . in Biochim . Biophys . Acta (1995) 1239, 133-144) .
  • a PEG component as a stabiliser in conjunction with a vector or vectors or directly to the reporter in the same molecule where the PEG does not serve as a spacer .
  • Other representative spacer elements include structural-type polysaccharides such as polygalacturonic acid, glycosaminoglycans, heparinoids, cellulose and marine polysaccharides such as alginates, chitosans and carrageenans ; storage-type polysaccharides such as starch, glycogen, dextran and aminodextrans ; polyamino acids and methyl and ethyl esters thereof, as in homo- and co-polymers of lysine, glutamic acid and aspartic acid; and polypeptides, oligonucleotides and oligosaccharides, which may or may not contain enzyme cleavage sites.
  • structural-type polysaccharides such as polygalacturonic acid, glycosaminoglycans, heparinoids, cellulose and marine polysaccharides such as alginates, chitosans and carrageenans ; storage-type polysaccharides such as starch, glycogen, dextran and
  • spacer elements may contain cleavable groups such as vicinal glycol, azo, sulfone, ester, thioester or disulphide groups .
  • X and Z are selected from -0-, -S-, and -NR- (where R is hydrogen or an organic group) ; each Y is a carbonyl, thiocarbonyl, sulphonyl, phosphoryl or similar acid-forming group: m and n are each zero or 1; and R 1 and R 2 are each hydrogen, an organic group or a group -X.Y.(Z) m -, or together form a divalent organic group] may also be useful; as discussed in, for example, WO-A- 9217436 such groups are readily biodegraded in the presence of esterases, e.g. in vivo, but are stable in the absence of such enzymes. They may therefore advantageously be linked to therapeutic agents to permit slow release thereof .
  • Poly[N- (2-hydroxyethyl)methacrylamides] are potentially useful spacer materials by virtue of their low degree of interaction with cells and tissues (see e.g. Volfova, I., Rihova, B. and V.R. and Vetvicka, P. in J. Bioact . Comp . Polymers (1992) 7, 175-190) .
  • Work on a similar polymer consisting mainly of the closely related 2-hydroxypropyl derivative showed that it was endocytosed by the mononuclear phagocyte system only to a rather low extent (see Goddard, P., Williamson, I., Bron, J. , Hutchkinson, L.E., Nicholls, J. and Petrak, K. in J " . Bioct . Compa t . Polym . (1991) 6, 4-24.).
  • poymeric spacer materials include : i) copolymers of methyl methacrylate with methacrylic acid; these may be erodible (see Lee, P.I. in Pharm. Res . (1993) 10, 980) and the carboxylate substituents may cause a higher degree of swelling than with neutral polymers ; ii) block copolymers of polymethacrylates with biodegradable polyesters (see e.g. San Roman, J. and Guillen-Garcia, P. in Bioma terials (1991) 12, 236-241); iii) cyanoacrylates , i.e.
  • polymers of esters of 2- cyanoacrylic acid - these are biodegradable and have been used in the form of nanoparticles for selective drug delivery (see Forestier, F., Gerrier, P., Chaumard, C, Quero, A.M., Couvreur, P. and Labarre, C. in J " . Antimicrob . Chemoter. (1992) 30, 173-179) ; iv) polyvinyl alcohols, which are water-soluble and generally regarded as biocompatible (see e.g. Langer, R. in J. Control . Release (1991) 16, 53-60); v) copolymers of vinyl methyl ether with maleic anhydride, which have been stated to be bioerodible (see Finne, U.
  • Dacron R which are non-degradable but highly biocompatible; ix) block copolymers comprising biodegradable segments of aliphatic hydroxyacid polymers (see e.g. Younes, H., Nataf, P.R., Cohn, D., Appelbaum, Y.J., Pizov, G. and Uretzky, G. in Bioma ter. Artif . Cells Artif . Organs (1988) 16, 705-719) , for instance in conjunction with polyurethanes (see Kobayashi , H., Hyon, S.H. and Ikada, Y. in "Water-curable and biodegradable prepolymers" - J". Biomed . Mater. Res .
  • polyurethanes which are known to be well- tolerated in implants, and which may be combined with flexible "soft" segments, e.g. comprising poly(tetra methylene glycol), poly (propylene glycol) or poly (ethylene glycol)) and aromatic "hard” segments, e.g. comprising 4 , 4 ' -methylenebis (phenylene isocyanate) (see e.g. Ratner, B.D., Johnston, A.B. and Lenk, T.J. in J " . Biomed. Ma ter. Res : Applied Bioma terials (1987) 21, 59-90; Sa Da Costa, V. et al . in J.
  • DPPE dipalmitoylphosphatidylethanolamine
  • LC long chain Agents for protein modification
  • Targetable agents include the following:
  • Antibodies which can be used as vectors for a very wide range of targets, and which have advantageous properties such as very high specificity, high affinity (if desired) , the possiblity of modifying affinity according to need etc. Whether or not antibodies will be bioactive will depend on the specific vector/target combination. Both conventional and genetically engineered antibodies may be employed, the latter permitting engineering of antibodies to particular needs, e.g. as regards affinity and specificity. The use of human antibodies may be preferred to avoid possible immune reactions against the vector molecule.
  • a further useful class of antibodies comprises so-called bi- and multi-specific antibodies, i.e. antibodies having specificity for two Or more different antigens in one antibody molecule.
  • Such antibodies may, for example, be useful in promoting formation of bubble clusters and may also be used for various therapeutic purposes, e.g. for carrying toxic moieties to the target.
  • Various aspects of bispecific antibodies are described by McGuinness, B.T. et al . in Na t . Biotechnol . (1996) 14, 1149-1154; by George, A.J. et al . in J " . Immunol . (1994) 152, 1802-1811; by Bonardi et al . in Cancer Res . (1993) 53, 3015-3021; and by French, R.R. et al . in Cancer Res . (1991) 51, 2353-2361.
  • Non-bioactive binders of receptors for cell adhesion molecules, cytokines, growth factors or peptide hormones This category may include peptidic or non- peptidic non-bioactive vectors which will be neither agonists nor antagonists.
  • Oligonucleotides and modified oligonucleotides which bind DNA or RNA through Watson-Crick or other types of base-pairing DNA is usually only present in extracelluar space as a consequence of cell damage, so that such oligonucleotides, which will usually be non- bioactive, may be useful in, for example, targeting of necrotic regions, which are associated with many different pathological conditions.
  • Oligonucleotides may also be designed to bind to specific DNA- or RNA-binding proteins, for example transcription factors which are very often highly overexpressed or activated in tumour cells or in activated immune or endothelial cells.
  • Combinatorial libraries may be used to select oligonucleotides which bind specifically to any possible target molecules (from the examples of proteins to caffeine) and which therefore may be employed as vectors for targeting.
  • DNA-binding drugs may behave similarly to oligonuclotides , but may exhibit biological acitvity and/or toxic effects if taken up by cells.
  • Protease substrates/inhibitors are involved in many pathological conditions. Many substrates/inhibitors are non-peptidic but, at least in the case of inhibitors, are often bioactive.
  • Vector molecules may be generated from combinatorial libraries without necessarily knowing the exact molecular target, by functionally selecting (in vitro, ex vivo or in vivo) for molecules binding to the region/structure to be imaged.
  • Various small molecules including bioactive compounds known to bind to biological receptors of various kinds. Such vectors or their targets may be used for generate non-bioactive compounds binding to the same targets.
  • proteins or peptides which bind to glucosaminoglycan side chains eg heparan sulphate, including glucosoaminoglycan-binding portions of larger molecules, as binding to glucosoaminoglycans does not result in a biological response.
  • Proteoglycans are not found on red blood cells, which eliminates undesirable adsorption to these cells.
  • the following tables identify various vectors which may be targeted to particular types of targets and indicated areas of use for targetable diagnostic and/or therapeutic agents according to the invention which contain such vectors .
  • the vectors stated are bioactive it is understood that either: (1) non-bioactive analogs are used; (2) the vectors are used in doses too low to generate a biological response; or (3) the vectors are used in combinations in such a way that the resulting diagnostic and/or therapeutic composition gives no biological response.
  • Protein and peptide vectors - cell adhesion molecules etc Protein and peptide vectors - cell adhesion molecules etc .
  • Vectors comprising cytokines/growth factors/peptide hormones and fracrments thereof
  • Vectors comprising non-peptide agonists/antagonists or non-bioactive binders of receptors for cytokines/growth factors/peptide hormones/cell adhesion molecules
  • Receptors comprising DNA-binding drugs
  • Receptors comprising protease substrates
  • Receptors comprising protease inhibitors
  • targeted agents to bind specifically to cells expressing a target may be studied by microscopy and/or using a flow chamber containing immobilised cells, for example employing a population of cells expressing the target structure and a further population of cells not expressing the target.
  • Radioactive or fluorescent enzyme- labelled streptavidin/avidin may used to analyse biotin attachment .
  • Example 1 Gas-filled microbubbles encapsulated with phosphatidylserine , phosphatidylcholine and biotin- amidocaproate-PEG 3 ⁇ nn-Ala-cholesterol
  • Ala-cholesterol is added to a solution of Boc-NH-PEG 3400 - SC (t-butyl carbamate poly (ethylene glycol) - succinimidyl carbonate) (Shearwater) in chloroform, followed by triethylamine .
  • the suspension is stirred at 41 °C for 10 minutes.
  • the crude product is purified by chromatography .
  • Boc-NH-PEG 3400 -Ala-cholesterol is stirred in 4 M hydrochloric acid in dioxane for 2.5 hours at ambient temperature.
  • the solvent is removed by rotary evaporation and the residue is taken up in chloroform and washed with water.
  • the organic phase is rotary evaporated to dryness .
  • the crude product may be purified by chromatography.
  • Biotinamidocaproate-PEG 3400 -Ala- cholesterol dissolved in water is added the washed microbubbles, which are placed on a roller table for several hours. The washing procedure is repeated following incorporation of the biotinamidocaproate- PEG 3400 -Ala-cholesterol into the microbubble membranes.
  • Example 2 Gas-containing microparticles comprising phosphatidylserine. phosphatidylcholine. biotin- amidocaproate-PEG 3 , 100 -Ala-Cholesterol and drug-cholesterol
  • Cholesterol (4mmol) a drug having an acid group (see Example 2(b) for a list of cholesterol-derivatised drugs) and dimethylaminopyridine (4 mmol) are dissolved in dimethylformamide/tetrahydrofuran (20 ml + 5 ml) and dicyclohexylcarbodiimide is added. The reaction mixture is stirred at ambient temperature overnight. Dicyclohexylurea is filtered off and the solvent is rotary evaporated. The title compound is purified by chromatography .
  • Biotin may be attached to microbubbles in many different ways, e.g. in a similar way to that described by Corley, P. and Loughrey, H.C. in (1994) Biochim . Biophys . Acta 1195, 149-156.
  • the resulting bubbles are analysed by flow cytometry, e.g. by employing fluorescent streptavidin to detect attachment of biotin to the bubbles.
  • radioactive or enzyme-labelled streptavidin/avidin is used to analyse biotin attachment .
  • Example 4 -Gas- filled microbubbles encapsulated with 1.2-distearoyl-sn-Glycero-3- fPhospo-L-Serinel and biotin-DPPE
  • Example 5 Gas-filled microbubbles encapsulated with phosphatidylserine and biotinylated oligonucleotide non- covalently bound to streptavidin-Succ-PEG-DSPE
  • NH 2 -PEG 3400 -DSPE (prepared as in Preparation 1) is carboxylated using succinic anhydride, e.g. by a similar method to that described by Nayar, R. and Schroit, A.J. in Biochemis try (1985) 24, 5967-5971.
  • Streptavidin is covalently bound to succ-PEG 3400 -DSPE in the microbubbles by standard coupling methods using a water-soluble carbodiimide.
  • the sample is placed on a roller table during the reaction. After centrifugation the infranatant is exchanged with water and the washing is repeated.
  • the functionality of the attached streptavidin is analysed by binding, e.g. to fluorescently labeled biotin, biotinylated antibodies (detected with a fluorescently labeled secondary antibody) or biotinylated and fluorescence- or radioactively-labeled oligonucleotides. Analysis is performed by fluorescence microscopy or scintillation counting.
  • Microbubbles from (c) above are incubated in a solution containing a biotinylated oligonucleotide.
  • the oligonucleotide-coated bubbles are washed as described above. Binding of the oligonucleotide to the bubbles is detected e.g. by using fluorescent-labeled oligonucleotides for attachment to the bubbles, or by hybridising the attached oligonucleotide to a labeled (fluorescence or radioactivity) complementary oligonucleotide.
  • the functionality of the oligonucleotide-carrying microbubbles is analysed, e.g.
  • an oligonucleotide complementary to ribosomal DNA (of which there are many copies per haploid genome) and an oligonucleotide complementary to an oncogene (e.g. ras of which there is one copy per haploid genome) are used.
  • Example 6 The peptide FNFRLKAGOKIRFGAAAWEPPRARI attached to gas-filled microbubbles encapsulated with phosphatidylserine
  • the peptide FNFRLKAGOKIRFGAAAWEPPRARI comprising phosphatidylserine-binding and heparin-binding sections, is synthesised.
  • the peptide is added to preformed phosphatidylserine-encapsulated perfluorobutane microbubbles and thoroughly mixed.
  • Example 7 Gas-filled microbubbles encapsulated with phosphatidylserine and inactivated human thrombin-Succ- PEG 400 ⁇ DSPE
  • Human thrombin is inactivated by incubation with a 20 % molar excess of D-Phe-L-Pro-L-Arg-chloromethyl ketone in 0.05 M HEPES buffer, pH 8.0, at 37 °C for 30 minutes.
  • Example 5(a) To a mixture (5 mg) of phosphatidylserine (90-99.9 mol%) and Succ-PEG 3400 -DSPE (10 - 0.1 mol%, prepared as in Example 5(a)) is added 5% propyleneglycol -glycerol in 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 head space is flushed with perfluorobutane . The vial is shaken in a cap-mixer for 45 seconds, whereafter the sample is put on a roller table. After centrifugation the infranatant is exchanged with water and the washing is repeated. Alternatively the microbubbles may be prepared as described in Preparation 1(f).
  • Inactivated human thrombin is covalently bound to succ- PEG 3400 -DSPE in the microbubbles from (b) above by standard coupling methods using a water-soluble carbodiimide.
  • the sample is placed on a roller table during the reaction. After centrifugation the infranatant is exchanged with water and the washing is repeated.
  • Example 8 Gas-containing microparticles comprising polymer from ethylidene bis (16-hydroxyhexadecanoate) and adipoyl chloride and biotin-amidocaproate-Ala covalently attached to the polymer a) Synthesis of Z-Ala-polymer (3 -0- (carbobenzyloxy-L- alanyl) -polymer)
  • the polymer is prepared from ethylidene bis (16- hydroxyhexadecanoate) and adipoyl chloride as described in WO-A- 9607434 , and a polymer fraction with molecular weight 10000 is purified using gel permeation chromatography (GPC) .
  • GPC gel permeation chromatography
  • 10 g of the material (corresponding to 1 mmol OH groups), Z-alanine (5 mmol) and dimethylaminopyridine (4 mmol) are dissolved in dry dimethylformamide/tetrahydrofuran and dicyclohexylcarbodiimide is then added.
  • the reaction mixture is stirred at ambient temperature overnight.
  • Dicyclohexylurea is filtered off and the solvent is removed using rotary evaporation.
  • the product is purified by chromatography, fractions containing the ti tle compound are combined and the solvent is removed using rotary evaporation.
  • the structure of the product is
  • Z-Ala-polymer (0.1 mmol) is stirred in toluene/tetrahydrofuran and glacial acetic acid (15% of the total volume) and hydrogenated in the presence of 5 % palladium on charcoal for 2 hours. The reaction mixture is filtered and concentrated in vacuo .
  • a solution of biotinamidocaproate N-hydroxysuccinimide ester in tetrahydrofuran is added to H 2 N-Ala-polymer dissolved in a mixture of tetrahydrofuran and dimethylformamide and 0.1 M sodium phosphate buffer having a pH of 7.5.
  • the reaction mixture is heated to 30 °C and stirred vigorously; the reaction is followed by TLC to completion. The solvent is evaporated and the crude product is used without further purification.
  • streptavidin conjugated to horseradish peroxidase was added to both suspensions and the tubes were rotated for 1 hour to allow for reaction.
  • the microspheres were then washed three times, resuspended in 100 mM citrate-phosphate buffer (pH 5) containing 0.1 mg/ml phenylenediamine dihydrochloride and 0.01% hydrogen peroxide, and rotated for 10 minutes. Development of a yellow-green colour was indicative of the presence of enzyme. The following results were obtained:
  • the agent from Example 7, comprising phosphatidylserine- encapsulated microbubbles having inactivated human thrombin-Succ-PEG 3400 -DSPE incorporated into the encapsulating membrane is lyophilised from 0.01 M phosphate buffer, pH 7.4.
  • the product is redispersed in sterile water and injected intravenously into a patient with suspected venous thrombosis in a leg vein.
  • the leg is examined by standard ultrasound techniques.
  • the thrombus is located by increased contrast as compared with surrounding tissue.
  • Example 11 Adhesion of poly-L-lysine-coated phosphatidylserine-encapsulated microbubbles to endothelial cells
  • Poly-L-lysine (8 mg, Sigma P-1274 lot no. 14H5546) having a molecular weight of 115 kDa was dissolved in water (400 ⁇ l) .
  • a cell adhesion study using human endothelial cells grown in culture dishes (Type CRL 1730) was performed with the above-described microbubbles, the uncoated microbubbles being used as a control. Microscopy of the endothelial cells after incubation showed a much increased number of poly-L-lysine-coated microbubbles adhering to endothelial cells in comparison to the uncoated microbubbles .
  • Example 12 Preparation and biological evaluation of gas-containing microbubbles of DSPS 'doped' with a lipopeptide consisting of a heparin sulphate binding peptide (KRKR) and a fibronectin peptide fWOPPRARI) .
  • KRKR heparin sulphate binding peptide
  • fWOPPRARI fibronectin peptide
  • the lipopeptide was synthesised on a ABI 433A automatic peptide synthesiser starting with Fmoc-Ile-Wang resin (Novabiochem) on a 0.1 mmol scale using 1 mmol amino acid cartridges. All amino acids and palmitic acid were preactivated using HBTU before coupling.
  • DSPS vanti, 4.5 mg
  • lipopeptide from a 0.5 mg
  • DSPS 0.8 mL
  • a solution of 1.4% propylene glycol/2.4% glycerol was added to each vial.
  • the mixture was warmed to 80°C for 5 minutes (vials shaken during warming) .
  • the samples were cooled to room temperature and the head space flushed with perfluorobutane gas.
  • the vials were shaken in a cap mixer for 45 s and the microbubbles rolled overnight.
  • Bubbles were washed several times with deionised water and analysed by Coulter counter (Size: 1-3 micron (87 %), 3-5 micron (11.5%)) and acoustic attenuation (frequency max att . : 3.5 MHz) .
  • the microbubbles were stable at 120 mm Hg .
  • MALDI mass spectral analysis was used to confirm incorporation into DSPS microbubbles as follows; ca . 0.05-0.1 mL of microbubble suspension was transferred to a clean vial and 0.05-0.1 mL methanol added. The suspension was sonicated for 30 s and the solution analysed by MALDI MS. Positive mode gave M+H at 2200, expected for lipopeptide, 2198.
  • the human endothelial cell line ECV 304 derived from a normal umbilical cord (ATCC CRL-1998) was cultured in 260 mL Nunc culture flasks (chutney 153732) in RPMI 1640 medium (Bio Whittaker) to which L-Glutamine 200 mM,
  • Penicillin/ Streptomycin (10.000 U/mL and 10.000 mcg/mL) and 10% Fetal Bovine Serum (Hyclone Lot no. AFE 5183) were added.
  • the cells were subcultured with a split ratio of 1:5 to 1:7 when reaching confluence.
  • Cover-glasses, 22mm in diameter were sterilised and placed on the bottom of 12 well culture plates (Costar) before cells in 0,5 mL complete medium with serum was added on top.
  • the chamber consists of a groove carved into a glass plate upon which the cover slip with cells was placed with the cells facing the groove forming a flow channel.
  • Ultrasound microbubbles from section b) were passed from a reservoir held at 37 degree Celsius through the flow chamber and back to the reservoir using a peristaltic pump. The flow rate was adjusted to simulate physiological relevant shear rates .
  • the flow chamber was placed under a microscope and the interaction between the microspheres and cells viewed directly. A camera mounted on the microscope was connected to a colour video printer and a monitor.
  • a 22 kg mongrel dog was anaesthetized with pentobarbital and mechanically ventilated.
  • the chest was opened by a midline sternotomy, the anterior pericardium was removed, and a 30 mm gelled silicone rubber spacer was inserted between the heart and a P5-3 transducer of an ATL HDI-3000 ultrasound scanner.
  • the scanner was set for intermittent short axis imaging once in each end-systole by delayed EGC triggering.
  • a net volume of 2 mL of microbubbles from b) were injected as a rapid intravenous bolus. 3 seconds later, the imaged right ventricle was seen to contain contrast material, another 3 seconds later, the left ventricle was also filled, and a transient attenuation shadow that obscured the view of the posterior parts of the left ventricle was observed. A substantial increase in brightness of the myocardium was seen, also in the portions of the heart distal to the left ventricle when the attenuation shadow subsided. After passage of the inital bolus, the ultrasound scanner was set to continuous, high frame rate high output power imaging, a procedure known to cause destruction of utrasound contrast agent bubbles in the imaged tissue regions.
  • the scanner was adjusted back to its initial setting.
  • the myocardium was then darker, and closer to the baseline value. Moving the imaged slice to a new position resulted in re-appearance of contrast effects, moving the slice back to the initial position again resulted in a tissue brightness again close to baseline.
  • a net volume of 2 mL microbubbles prepared in an identical manner to b) above with the exception that no lipopeptide was included in the preparation was injected, using the same imaging procedure as above.
  • the myocardial echo enhancement was far less intense and of shorter duration than observed in case 1.
  • At the completion of the left ventricular attenuation phase there was also almost complete loss of myocardial contrast effects, and a myocardial echo increases in the posterior part of the left ventricle as in case 1 was not observed.
  • Example 13 Targeted gas-containing microbubbles of DSPS coated non-covalently with polylysine and a fusion peptide comprising a PS binding component and a Fibronectin peptide sequence
  • the peptide was synthesised on an ABI 433A automatic peptide synthesiser starting with Fmoc-Ile-Wang resin (Novabiochem) on a 0.1 mmol scale using 1 mmol amino acid cartridges. All amino acids were preactivated using HBTU before coupling. The simultaneous removal of peptide from the resin and side-chain protecting groups was carried out in TFA containing 5% phenol , 5% EDT and 5% H 2 0 for 2 hours giving a crude product yield of 302 mg .
  • DSPS (5 mg, Avanti) was weighed into a clean vial along with poly-L-lysine (Sigma, 0.2 mg) and peptide from a) above (0.2 mg) .
  • To the vial was added 1.0 mL of a solution of 1.4% propylene glycol/ 2.4% glycerol. The mixture was warmed to 80°C for 5 minutes. The sample was cooled to room temperature and the head space flushed with perfluorobutane gas. The vials were shaken in a cap mixer for 45 s and the microbubbles centrifuged at 1000 rpm for 3 minutes . Following extensive washing with water, PBS and water the final solution was examined for polylysine and peptide content using MALDI MS.
  • the spacer element contained within the PS binding/Fibronectin fusion peptide can also be replaced with other spacers such as PEG 200o or P°ly alanine (-AAA-) . It is also envisaged that a form of pre-targeting may be employed, whereby the DSPS binding/Fibronectin fragment fusion peptide is firstly allowed to associate with cells via the fibronectin peptide binding. This is followed by administration of PS microbubbles which then bind to the PS binding peptide .
  • Example 14 Gas containing microbubbles of DSPS covalently modified with CD71 FITC-labelled anti- transferrin receptor antibody and 'doped' with a lipopeptide with affinity for endothelial cells.
  • This example is directed at the preparation of multiple vector targeted ultrasound agents .
  • the lipopeptide shown below was synthesised on a ABI 433A automatic peptide synthesiser starting with a Rink amide resin on a 0.1 mmol scale using 1 mmol ammo acid cartridges .
  • DSPS vanti, 4.5 mg
  • lipopeptide from a) 0.5 mg
  • PE-PEG 2000 -Maleimide from example 2 0.5 mg
  • the mixture was warmed to 80°C for 5 minutes then filtered through a 4.5 micron filter.
  • the sample was cooled to room temperature and the head space flushed with perfluorobutane gas .
  • the vials were shaken in a cap mixer for 45 s and the microbubbles washed three times with distilled water.
  • FITC labelled CD71 anti-transferrin receptor Ab 100 ⁇ g/ mL, Becton Dickinson
  • 0.7 mL in PBS was modified with Traut's reagent (0.9 mg, Pierce) at room temperature for 1 h.
  • Excess reagent was separated from modified protein on a NAP-5 column (Pharmacia) .
  • Fig. 1 of the accompanying drawing represents the flow cytometric comparison of negative control microbubbles of DSPS (left curve) with bubbles conjugated with CD71 FITC- labelled anti-transferrin antibody (filled curve, right), showing that 92% of the population fluoresce. Incorporation into the microbubbles of lipopeptide was confirmed by MALDI mass spectrometry as described in example 12 b) .
  • Example 15 Preparation of Transferrin/Avidin coated ⁇ a.- filled microbubbles for targeted ultrasound imaging.
  • This example is directed to the preparation of microbubbles containing multiple protein vectors for targeted ultrasound/therapy.
  • the lipid structure shown above was synthesised on a ABI 433A automatic peptide synthesiser starting with Fmoc- Cys (Trt) -Wang resin (Novabiochem) on a 0.25 mmol scale using 1 mmol amino acid cartridges. All amino acids and palmitic acid were preactivated using HBTU coupling chemistry .
  • DSPS (Avanti, 4.5 mg) and the lipid structure from a) above (0.5 mg) were weighed into a clean vial and 0.8 mL of a solution containing 1.4% propylene glycol/ 2.4% glycerol in water added. The mixture was warmed to 80°C for 5 minutes (vials shaken during warming) and filtered while still hot through a 40 micron filter. The samples were cooled to room temperature and the head space flushed with perfluorobutane gas. The vials were shaken in a cap mixer for 45 s and the microbubbles placed on roller table overnight. Bubbles were washed several times with deionised water and analysed for thiol group incorporation using Ellmans Reagent.
  • Example 16 Preparation of functionalised gas-filled microbubbles for targeted ultrasound imaging.
  • This example is directed to the preparation of microbubbles having a reactive group on the surface for non-specific targeting, principally utilising disulphide exchange reactions to effect binding to a multiplicity of cellular targets.
  • DSPS vanti, 5.0 mg
  • thiol containing lipid structure from example 15 a 1.0 mg
  • the mixture was warmed to 80°C for 5 minutes (vials shaken during warming) and filtered while still hot through a 40 micron filter.
  • the samples were cooled to room temperature and the head space flushed with perfluorobutane gas.
  • the vials were shaken in a cap mixer for 45 s and the microbubbles placed on roller table overnight. Bubbles were washed several times with deionised water and analysed for thiol group incorporation using Ellmans
  • Example 17 Gas-containing microbubbles of DSPS comprising a lipopeptide for endothelial cell targeting and a captopril containing molecule. This example is directed to the preparation of ultrasound agents for combined targeting and therapeutic applications .
  • the structure shown above was synthesised using a manual nitrogen bubbler apparatus starting with Fmoc protected Rink Amide MBHA resin (Novabiochem) on a 0.125 mmol scale. All amino acids were purchased from Novabiochem and palmitic acid from Fluka. 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 preactivation . Captopril (Sigma) dissolved in DMF was introduced on the solid- phase using DBU as base.
  • the lipopeptide was synthesised on a ABI 433A automatic peptide synthesiser starting with Rink amide resin
  • DSPS (Avanti, 4.5 mg) , product from a) (0.5 mg) and product from b) (0.5 mg) were weighed into a vial and 1.0 mL of a solution of 1.4% propylene glycol/ 2.4% glycerol was added to each vial. The mixture was warmed to 80°C for 5 minutes (vials shaken during warming) . The samples were cooled to room temperature and the head space flushed with perfluorobutane gas. The vials were firstly shaken in a cap mixer for 45 s then rolled for 1 h followed by extensive washing with deionised water. No detectable levels of starting material was found in the final wash solution as evidenced by MALDI MS . MALDI mass spectral analysis was used to confirm incorporation of the products from section a) and b) into the microbubbles as described in example 12 b) .
  • Example 18 Preparation of gas-containing microbubbles of DSPS loaded with a lipopeptide comprising a helical peptide with affinity for cell membranes and the peptide antibiotic polymixin B sulphate.
  • This example is directed at the preparation of targeted microbubbles comprising multiple peptidic vectors having a combined targeting and a therapeutic application.
  • DSPS (Avanti, 5.0 mg) , lipopeptide from a) (0.3 mg)and polymixin B sulphate (Sigma, 0.5 mg) was weighed into a clean vial and 1.0 mL of a solution of 1.4% propylene glycol/ 2.4% glycerol added. The mixture was sonicated for 3-5 mins, warmed to 80°C for 5 minutes then filtered through a 4.5 micron filter. The mixture was cooled to room temperature and the head space flushed with perfluorobutane gas. The vial was shaken in a cap mixer for 45 s and the microbubbles centrifuged at 1000 rpm for 3 minutes.
  • microbubbles were washed in water until no polymixin B sulphate or lipopeptide could be detected in the infranatant by MALDI-MS. Microscopy showed that the size distribution of the bubble population was between 1-8 micron as desired. To the washed bubbles (ca. 0.2 mL) was added methanol (0.5 mL) and the mixture placed in a sonic bath for 2 min. The resulting clear solution, following analysis by MALDI-MS, was found to contain both lipopeptide and polymixin B sulphate (expected 1203, found 1207).
  • Example 19 Preparation of multiple-specific gas- containing microbubbles of DSPS 'doped' with a lipopeptide comprising a IL-1 receptor binding sequence and modified with a branched structure containing the drug methotrexate .
  • This example is directed at the preparation of targeted microbubbles comprising a non-bioactive vector for targeting and a component for drug delivery.
  • the lipopeptide was synthesised on a ABI 433A automatic peptide synthesiser starting with Fmoc-Ala-Wang resin (Novabiochem) on a 0.1 mmol scale using 1 mmol amino acid cartridges. All amino acids and palmitic acid were preactivated using HBTU before coupling.
  • the methotrexate structure was synthesised on a ABI 433A automatic peptide synthesiser starting with Fmoc- Cys(Trt) Tentagel resin on a 0.1 mmol scale.
  • the simultaneous removal of product from the resin and deprotection of protecting groups was carried out in TFA containing 5% EDT and 5% H 2 0 for 2 hours giving a crude product yield of 160 mg .
  • DSPS (Avanti, 4.5 mg) and thiol containing lipopeptide from example 15 a) (0.5 mg) and lipopeptide from a) (0.2 mg) above were weighed into a clean vial and 1.0 mL of a solution of 1.4% propylene glycol/ 2.4% glycerol added. The mixture was sonicated for 3-5 mins, warmed to 80°C for 5 minutes then filtered through a 4.5 micron filter. The mixture was cooled to room temperature and the head space flushed with perfluorobutane gas. The vials were shaken in a cap mixer for 45 s and the microbubbles centrifuged at 1000 rpm for 3 minutes following which the infranatant was discarded.
  • methotrexate structure from b) above (0.5 mg) was dissolved in PBS pH 8.0. The solution was then added to the thiol containing bubbles from c) and disulphide bond formation allowed to proceed for 16 h. Following extensive washing with PBS and water the bubbles were analysed by microscopy and MALDI MS.
  • the disulphide bond linking the methotrexate structure to the microbubble may be reduced in vivo liberating the free drug molecule.
  • This in combination with a tumour specific vector is a drug delivery system.
  • a physiologically relevant reducing agent such as glutathione may be used to bring about drug release.
  • Example 20 Preparation of microbubbles encapsulated with DSPS and unctionalised with a thrombi-tar ⁇ eting lipopeptide and the thrombolytic enzyme tissue plasminogen activator.
  • This example is directed at the preparation of thrombus targeted US agents comprising a therapeutic thromolytic agent .
  • the lipopeptide was synthesised on a ABI 433 A automatic peptide synthesiser starting with Rink amide resin (Novabiochem) on a 0.1 mmol scale using 1 mmol amino acid cartridges. All ammo acids and palmitic acid were preactivated using HBTU before coupling. The simultaneous removal of peptide from the resin and side-chain protecting groups was carried out in TFA containing 5% phenol, 5% EDT, 5% anisole and 5% H 2 0 for 2 h giving a crude product yield of 80 mg . Purification by preparative HPLC (Vydac 218TP1022 column) of a 20 mg aliquot of the crude material was carried out. After lyophilization 6 mg of pure material was obtained. The product was characterized by MALDI mass spectrometry and analytical HPLC.
  • a solution of 0.1 mL of ammonim carbonate buffer containing 0.1 mg of t-PA (Sigma) was made up to 0.2 mL by the addition of water.
  • To this solution was added 0.4 mg of Sulpho-SMPB (Pierce) dissolved in 0.05 mL DMSO.
  • the protein solution was left standing at room temperature for 45 min then purification carried out on a Superdex 200 column. The product was eluted in PBS and the modified protein fraction collected.
  • DSPS (Avanti, 5.0 mg) was weighed into a clean vial along with 0.5 mg of the lipopeptide from a) and 0.5 mg of the thiol containing lipopeptide from example 15 a) . To this was added 1.0 mL of a solution of 1.4% propylene glycol/ 2.4% glycerol and the mixture sonicated for 2 min then warmed to 80°C for 5 minutes. Immediately following warming the solution was filtered through a 4 micron filter. The sample was cooled to room temperature and the head space flushed with perfluorobutane gas . The vials were shaken in a cap mixer for 45 s and the microbubbles washed 2 times with deionised water.
  • Example 21 Preparation of gas-containing microbubbles encapsuled with DSPS comprising thiolated anti-CD34-MAL-
  • DSPS Advanti, 4,5 mg
  • PE-PEG 2000 -maleimide from example x (0,5 mg) were weighed into a clean vial and 1 mL of a solution of 1.4% propylene glyco/2.4% glycerol added. The mixture was warmed to 80°C for 5 minutes then filtered through a 4.5 micron filter. The sample was cooled to room temperature and the head space flushed with perfluorbutane gas. The vials were shaken in a cap mixer for 45 s and the microbubbles washed three times with destilled water.
  • 0.5mL of the thiolated antibody praparation from b) was added to an aliqout of microbubbles from a) and the conjugation reaction allowed to proceed for 30 min on a roller table. Following centifugation at 2000 rpm for 5 min the infranatant was removed. The microbubbles were washed a further three times with water.
  • Example 22 Preparation of gas-containing microbubbles of DSPS loaded with a lipopeptide comprising a helical peptide with affinity for cell membranes
  • This example is directed at the preparation of targeted microbubbles comprising a non-bioactive peptidic vector for targeting of cell membrane structures. a) Synthesis of a lipopeptide comprising a helical peptide with affinity for cell membranes
  • the lipopeptide was synthesised on a ABI 433A automatic peptide synthesiser starting with Rink amide resin (Novabiochem) on a 0.2 mmol scale using lmmol amino acid cartridges. All amino acids and 2-n-hexadecylstearic acid were preactivated using HBTU before coupling.
  • the simultaneous removal of lipopeptide from the resin and side-chain protecting groups was carried out in TFA containing 5% H 2 0 for 2 hours giving a crude product yield of 520 mg.
  • Purification by preparative HPLC (Vydac 218TP1022 column) of a 30 mg aliqout of crude material was carried out using a gradient of 90 to 100 % B over
  • DSPS vanti, 4.5 mg and lipopeptide from a) (0.5 mg) was weighed into a clean vial and 1.0 ml of a solution of 1.4% propylene glycol/ 2.4% glycerol added. The mixture was sonicated for 3-5 mins, warmed to 80°C for 5 minutes then filtered through a 4.5 mm filter. The mixture was cooled to room temperature and the head space flushed with perfluorobutane gas. The vials were shaken in a cap mixer for 45 s and the microbubbles centrifuged at 1000 rpm for 3 minutes. The bubbles were washed in water until no lipopeptide could be detected by MALDI-MS.
  • Example 23 Preparation of gas-containing microbubbles of DSPS loaded with a lipopeptide comprising a non- bioactive interleukin 1 receptor binding peptide
  • This example is directed at the preparation of targeted microbubbles comprising a non-bioactive peptidic vector for targeting at the IL-1 recptor which does not induce signal tranduction or prevent IL-1 binding.
  • the lipopeptide was synthesised on a ABI 433A automatic peptide synthesiser starting with Fmoc-Ala-Wang resin (Novabiochem) on a 0.1 mmol scale using 1 mmol amino acid cartridges. All amino acids and palmitic acid were preactivated using HBTU before coupling.
  • the simultaneous removal of lipopeptide from the resin and side-chain protecting groups was carried out in TFA containing 5% H 2 0, 5% anisole, 5 % phenol and 5% EDT for 2 hours giving a crude product yield of 150 mg.
  • DSPS vanti, 4.5 mg
  • lipopeptide from a 0.5 mg
  • the mixture was sonicated for 3-5 mins , warmed to 80°C for 5 minutes then filtered through a 4.5 micron filter.
  • the mixture was cooled to room temperature and the head space flushed with perfluorobutane gas.
  • the vials were shaken in a cap mixer for 45 s and the microbubbles centrifuged at 1000 rpm for 3 minutes. The bubbles were washed in water until no lipopeptide could be detected by MALDI-MS. To the washed bubbles (ca.
  • Example 24 Preparation of PFP containing microbuhhl p of DSPC. DSPS and endothelial cell binding lipopeptide for targeted ultrasound imaging.
  • the headspace was again flushed with perfluorobutane and the sample was kept on a table roller until a homogenous appearance was obtained. The washing procedure was repeated. The resulting ultrasound contrast agent was confirmed by Coulter counter, acoustic attenuation measurements and resistance to external pressure. The microbubbles were tested in the in vitro assay as detailed in example 12. A gradual accumulation of bubbles binding to the cells was observed.
  • Example 25 Preparation of SF f containing microbubbles of DSPC. DSPS and endothelial cell binding lipopeptide for targeted ultrasound imaging.
  • DSPC DSPS (3:1) (5mg/ ml) in propyleneglycol/glycerol (4% in water) was added 0.5 mg of the lipopeptide from example 17 b) .
  • the mixture was heated to 80°C for 5 minutes and shaken.
  • the solution was then cooled to ambient temperature and the headspace was flushed with SF 6 gas.
  • the vial was shaken on a Capmix (Espe Capmix) for 45 seconds and placed on a roller table for 5 min.
  • the sample was centrifuged (Juan MR 14.11) at 2000 rpm for 5 minutes and the infranatant removed and replaced with destilled water.
  • the headspace was again flushed with perfluorobutane and the sample was kept on a table roller until a homogenous appearance was obtained. The washing procedure was repeated. The resulting ultrasound contrast agent was confirmed by Coulter counter, acoustic attenuation measurements and resistance to external pressure.
  • Example 26 Preparation of PFB containing microbubbles of DSPG and endothelial cell binding lipopeptide for targeted ultrasound imaging.
  • Example 27 Preparation of PFP containing microbubbles of DSPG and endothelial cell binding lipopeptide for targeted ultrasound imaging.
  • the headspace was again flushed with perfluorobutane and the sample was kept on a table roller until a homogenous appearance was obtained. The washing procedure was repeated. The resulting ultrasound contrast agent was confirmed by Coulter counter, acoustic attenuation measurements and resistance to external pressure. The microbubbles were tested in the in vitro assay as detailed in example 12. A gradual accumulation of bubbles binding to the cells was observed.
  • Example 28 Preparation of SF 6 containing microbubbles of DSPG and endothelial cell binding lipopeptide for targeted ultrasound imaging.
  • the headspace was again flushed with perfluorobutane and the sample was kept on a table roller until a homogenous appearance was obtained. The washing procedure was repeated. The resulting ultrasound contrast agent was confirmed by Coulter counter, acoustic attenuation measurements and resistance to external pressure.
  • Example 29 Targeted gas -containing microbubbles of DSPS coated non-covalently with polylysine
  • DSPS (5 mg, Avanti) was weighed into a clean vial along with poly-L-lysine (Sigma, 0.2 mg) .
  • poly-L-lysine (Sigma, 0.2 mg) was added to the vial.
  • To the vial was added 1.0 ml of a solution of 1.4% propylene glycol/ 2.4% glycerol.
  • the mixture was warmed to 80°C for 5 minutes .
  • the sample was cooled to room temperature and the head space flushed with perfluorobutane gas .
  • the vials were shaken in a cap mixer for 45 s and the microbubbles centrifuged at 1000 rpm for 3 minutes.
  • the lipopeptide was synthesised on a ABI 433 A automatic peptide synthesiser starting with Rink amide resin (Novabiochem) on a 0.1 mmol scale using 1 mmol amino acid cartridges. All amino acids and palmitic acid were preactivated using HBTU before coupling. The simultaneous removal of peptide from the resin and side-chain protecting groups was carried out in TFA containing 5% phenol, 5% EDT, 5% anisole and 5% H 2 0 for 2 h giving a crude product yield of 80 mg. Purification by preparative HPLC (Vydac 218TP1022 column) of a 20 mg aliquot of the crude material was carried out. After lyophilization 6 mg of pure material was obtained. The product was characterized by MALDI mass spectrometry and analytical HPLC.
  • DSPS vanti, 4.5 mg
  • lipopeptide from a 1.0 mg
  • the mixture was warmed to 80°C for 5 minutes then the sample filtered through a 4 micron filter. After cooling to room temperature the head space was flushed with perfluorobutane gas.
  • the vials were shaken in a cap mixer for 45 s and the microbubbles washed extensively with deionised water. The bubbles were analysed by microscopy Coulter counter. MALDI-MS was used to confirm the presence of lipopeptide as previously described.
  • Example 31 Gas-filled microbubbles of DSPS comprising a lipopeptide consisting of a heparin sulphate binding peptide (KRKR) and a fibronectin peptide (WOPPRARI) for targeting and a lipopeptide containing atenolol for therapeutic applications
  • KRKR heparin sulphate binding peptide
  • WOPPRARI fibronectin peptide
  • the reaction mixture was poured onto water saturated with potassium hydrogensulphate and organic material was extracted into ethyl acetate. The organic phase was washed with water and brine, dried (Na 2 S0 4 ) and filtered to give 0.6 g of crude material.
  • the product was purified by chromatography (silica, CHCl 3 /MeOH/AcOH 85:10:5). The solution was concentrated and the residue was taken up in glacial acetic acid and lyophilised. Yield 415 mg (56%) , white solid. The structure was confirmed by : H and 13 C NMR analysis.
  • the structure shown above was synthesised using a manual nitrogen bubbler apparatus starting with Fmoc protected Rink Amide MBHA resin (Novabiochem) on a 0.125 mmol scale, using amino acids from Novabiochem, palmitic acid from Fluka and the compound from a) . Coupling was carried out using standard TBTU/HOBt/DIEA protocols. Simultaneous removal of the peptide from the resin and deprotection of side-chain protecting groups was carried out in TFA containing 5% EDT and 5% water for 2h.
  • a solution of 1.4% propylene glycol / 2.4% glycerol (1.0 ml) was added to a mixture of DSPD (Avanti, 5.0 mg) , product from Example 12 a) (0.5 mg) and product from b) (0.5 mg) in a vial.
  • the mixture was sonicated for 5 min and then heated at 80 °C for 5 min (vial was shaken during warming) .
  • the solution was filtered and cooled. Head space was flushed with perfluorobutane gas and the vial was shaken in a cap mixer for 45s followed by extensive washing with deionised water.
  • Example 32 Gas-filled microbubbles encapsulated with DSPS and a compound containing folic acid for diagnostic applications
  • a solution of 1.4% propylene glycol / 2.4% glycerol (1.0 ml) was added t a mixture of DSPS (Avanti, 4.5 mg) and product from a) (0.5 mg) in a vial.
  • Dilute ammonia (to pH 8) and DMSO (40 ⁇ l) were added and the mixture was sonicated for 5 min and then heated at 80 °C for 5 min (vial was shaken during warming) .
  • the solution was filtered and cooled. Head space was flushed with perfluorobutane gas and the vial was shaken in a cap mixer for 45s followed by extensive washing with deionised water.
  • microbubbles were tested in the in vitro assay as detailed in example 12. A gradual accumulation of bubbles binding to the cells was observed.
  • Example 33 Gas-filled microbubbles of phosphatidylserine comprising biotinamide-PEG- ⁇ -Ala- Cholesterol and a cholesteryl ester of chlorambucil for diagnostic and therapeutic applications
  • a solution of 1.4% propylene glycol / 2.4% glycerol (1.0 ml) was added to a mixture of DSPS (Avanti, 5 mg) and product from c) (0.5 mg) and d) (0.5 mg) in a vial.
  • the mixture was sonicated for 5 min and then heated at 80 °C for 5 min (vial was shaken during warming) and cooled. Head space was flushed with perfluorobutane gas and the vial was shaken in a cap mixer for 45s followed by extensive washing with deionised water.
  • MALDI mass spectrometry showed no detectable level of compound from c and d) in the final wash solution.
  • Example 34 Flotation of endothelial cells by micro bubbles with vectors that specifically bind to the endothelial cells
  • the human endothelial cell line ECV 304 derived from a normal umbilical cord (ATCC CRL-1998) was cultures in Nunc culture flasks (chutney 153732) in RPMI 1640 medium (Bio Whitaker) to which L-Glutamine 200 mM, Penicillin/Streptomycin (10.000 U/ml and 10.00 mcg/ml) and 10% Fetal Calf Serum (Hyclone Lot no AFE 5183) were added. The cells were subcultured following trypsination with a split ratio of 1:5 to 1:7 when reaching confluence. 2 mill . cells from trypsinated confluent cultures were added to each set of 5 centrifuge tubes .
  • control microbubbles or bubbles carrying the vector including WQPPARI (example 12) , or the endothelial cell binding peptide vector (example 14) were added at 2, 4, 6 ,8 or 10 mill bubbles per tube.
  • the cells at the bottom of the tubes after centrifugation at 400 g for 5 minutes were counted with a Coulter counter. It was found the 4 or more microbubbles binding to a cell did bring the cells to top of the fluid in the centrifugation tube. All cells were floated by the endothelial cell binding peptide vector and about 50 % with the WQPPARI vector.
  • This example is directed at the preparation of targeted microbubbles for gene transfer.
  • DSPS 4,5 mg
  • lipopeptide from 17 b 0.5 mg
  • DSPS lipopeptide from 17 b
  • 0.8 ml propyleneglycol/glycerol 4%) in water was added.
  • the solution was heated at 80°C for 5 minutes and shaken.
  • the solution was then cooled to ambient temperature and the headspace flushed with perfluorobutane .
  • the vials were shaken on a Capmix (Espe Capmix, 4450 oscillations/min) for 45 seconds and put on a roller table for 5 minutes.
  • the content of the vials were mixed and the sample washed by centrifugation at 2000 rpm for 5 minutes.
  • the infranatant was removed and the same volume of distilled water added.
  • the washing procedure was repeated once.
  • Poly-L-lysine HBr (Sigma, 20.6 mg) was dissolved in 2 mL water then an aliquot (0.4 mL) made up to 2 mL water. To 1.2 mL of the diluted poly-L-lysine solution was added 0.12 mL of the DSPS-lipopeptide bubble suspension. Following incubation excess polylysine was removed by extensive washing with water.
  • Endothelial cells (ECV 304) were cultured in 6 well plates to a uniform subconfluent layer.
  • a transfection mixture consisting of 5 ⁇ g DNA (an Enhanced Green Fluorescent Protein vector from CLONTECH) and 50 ⁇ l of microbubble suspension from a) in RPMI medium at a final volume of 250 ⁇ l was prepared. The mixture was left standing for 15 min at room temperature then 1 mL of complete RPMI medium added. The medium was removed from the cell culture dish, and the DNA-microbubble mixture added to the cells. The cells were incubated in a cell culture incubator (37 °C) .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Nanotechnology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Radiology & Medical Imaging (AREA)
  • Immunology (AREA)
  • Acoustics & Sound (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention a pour objet des agents diagnostiques et/ou thérapeutiquement actifs pouvant être ciblés, par exemple des agents de contraste utilisés en échographie, qui comprennent une suspension dans un liquide porteur aqueux d'un rapporteur contenant un matériau contenant ou produisant du gaz, ledit rapporteur étant associé ou lié à un ou plusieurs vecteurs non bioactifs.
PCT/GB1997/002958 1996-10-28 1997-10-28 Ameliorations apportees a des agents diagnostiques et/ou therapeutiques ou les concernant WO1998018498A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU47870/97A AU4787097A (en) 1996-10-28 1997-10-28 Improvements in or relating to diagnostic/therapeutic agents
EP97910518A EP0963209A2 (fr) 1996-10-28 1997-10-28 Ameliorations apportees a des agents diagnostiques et/ou therapeutiques ou les concernant
JP10520191A JP2001502719A (ja) 1996-10-28 1997-10-28 改良された診断／治療用薬剤

Applications Claiming Priority (19)

Application Number Priority Date Filing Date Title
GBGB9622364.9A GB9622364D0 (en) 1996-10-28 1996-10-28 Improvements in or relating to diagnostic/therapeutic agents
GBGB9622367.2A GB9622367D0 (en) 1996-10-28 1996-10-28 Improvements in or relating to diagnostic/therapeutic agents
GB9622364.9 1996-10-28
GBGB9622366.4A GB9622366D0 (en) 1996-10-28 1996-10-28 Improvements in or relating to diagnostic/therapeutic agents
GBGB9700698.5A GB9700698D0 (en) 1997-01-15 1997-01-15 Improvements in or relating to diagnostic/therapeutics agents
GBGB9700699.3A GB9700699D0 (en) 1997-01-15 1997-01-15 Improvements in or relating to diagnostic/therapeutic agents
GBGB9708265.5A GB9708265D0 (en) 1997-04-24 1997-04-24 Contrast agents
US4926797P 1997-06-06 1997-06-06
US4926597P 1997-06-06 1997-06-06
US4926497P 1997-06-06 1997-06-06
GBGB9711842.6A GB9711842D0 (en) 1997-06-06 1997-06-06 Improvements in or relating to diagnostic/therapeutic agents
GB9708265.5 1997-06-06
GB9711842.6 1997-06-06
GB9622366.4 1997-06-06
GB9700698.5 1997-06-06
GBGB9711844.2A GB9711844D0 (en) 1997-06-06 1997-06-06 Improvements in or relating to diagnostic/therapeutic agents
GB9622367.2 1997-06-06
GB9700699.3 1997-06-06
GB9711844.2 1997-06-06

Publications (2)

Publication Number Publication Date
WO1998018498A2 true WO1998018498A2 (fr) 1998-05-07
WO1998018498A3 WO1998018498A3 (fr) 1998-07-16

Family

ID=27581684

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1997/002958 WO1998018498A2 (fr) 1996-10-28 1997-10-28 Ameliorations apportees a des agents diagnostiques et/ou therapeutiques ou les concernant

Country Status (2)

Country Link
EP (1) EP0963209A2 (fr)
WO (1) WO1998018498A2 (fr)

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999020312A1 (fr) * 1997-10-21 1999-04-29 Nycomed Imaging As Imagerie ultrasonique avec agent de contraste cible sur le systeme microvasculaire et medicament vasodilatateur
WO1999053963A1 (fr) * 1998-04-22 1999-10-28 Marsden, John, Christopher Ameliorations apportees a des agents de contraste ou en rapport avec ces agents
WO1999055383A2 (fr) * 1998-04-28 1999-11-04 Nycomed Imaging As Perfectionnements relatifs a des agents diagnostiques/therapeutiques
WO2000002588A1 (fr) * 1998-07-13 2000-01-20 The Board Of Regents Of The University Of Nebraska Compositions destinees a l'administration ciblee specifique de site de medicaments et procede d'utilisation
EP0977597A4 (fr) * 1996-09-11 2000-02-09 Imarx Pharmaceutical Corp Procedes ameliores d'imagerie diagnostique utilisant un agent de contraste et un vasodilatateur
EP1017425A1 (fr) * 1996-09-11 2000-07-12 IMARx PHARMACEUTICAL CORP. Procedes ameliores d'imagerie diagnostique utilisant un agent de contraste et un vasodilatateur coronarien
WO2001017566A2 (fr) * 1999-09-08 2001-03-15 Institut für Diagnostikforschung GmbH an der Freien Universität Berlin Agents de contraste de type l-selectine
US6245747B1 (en) 1996-03-12 2001-06-12 The Board Of Regents Of The University Of Nebraska Targeted site specific antisense oligodeoxynucleotide delivery method
US6548048B1 (en) 1998-04-28 2003-04-15 Amersham Health As Lipopeptide stabilized microbubbles as diagnostic/therapeutic agents
WO2005044313A2 (fr) * 2003-11-06 2005-05-19 Amersham Health As Composes pharmaceutiques
US6984373B2 (en) 2000-12-23 2006-01-10 Dyax Corp. Fibrin binding moieties useful as imaging agents
US7109167B2 (en) 2000-06-02 2006-09-19 Bracco International B.V. Compounds for targeting endothelial cells, compositions containing the same and methods for their use
EP1944312A1 (fr) 2003-03-03 2008-07-16 Dyax Corporation Peptides qui lient spécifiquement le récepteur HGF (CMET) et utilisations associées
EP2014310A2 (fr) 2002-03-01 2009-01-14 Dyax Corporation Peptides de liaison KDR et VEGF/KDR et leur utilisation pour le diagnostic et la thérapie
EP2147684A1 (fr) 2008-07-22 2010-01-27 Bracco Imaging S.p.A Agents de diagnostics sélectifs contre les métalloprotéases
EP2281580A2 (fr) 2003-01-13 2011-02-09 Bracco Imaging S.p.A Peptides à libération de gastrine (GRP) marqués
EP2359864A2 (fr) 2005-12-09 2011-08-24 Bracco Suisse SA Conjugués vecteur de ciblage-phospholipide
US8263739B2 (en) 2000-06-02 2012-09-11 Bracco Suisse Sa Compounds for targeting endothelial cells, compositions containing the same and methods for their use
US8642010B2 (en) 2002-03-01 2014-02-04 Dyax Corp. KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy
US9056138B2 (en) 2002-03-01 2015-06-16 Bracco Suisse Sa Multivalent constructs for therapeutic and diagnostic applications
US9545457B2 (en) 1998-01-14 2017-01-17 Lantheus Medical Imaging, Inc. Preparation of a lipid blend and a phospholipid suspension containing the lipid blend
US9789210B1 (en) 2016-07-06 2017-10-17 Lantheus Medical Imaging, Inc. Methods for making ultrasound contrast agents
US10022460B2 (en) 2014-12-31 2018-07-17 Lantheus Medical Imaging, Inc. Lipid-encapsulated gas microsphere compositions and related methods
US10471163B2 (en) 2013-09-13 2019-11-12 The General Hospital Corporation Activatable fibrin-binding probes
US10588988B2 (en) 2016-05-04 2020-03-17 Lantheus Medical Imaging, Inc. Methods and devices for preparation of ultrasound contrast agents

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8623822B2 (en) 2002-03-01 2014-01-07 Bracco Suisse Sa KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy
US7794693B2 (en) 2002-03-01 2010-09-14 Bracco International B.V. Targeting vector-phospholipid conjugates

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4232755A1 (de) * 1992-09-26 1994-03-31 Schering Ag Mikropartikelpräparationen aus biologisch abbaubaren Mischpolymeren
WO1994028873A1 (fr) * 1993-06-11 1994-12-22 Unger Evan C Nouveaux systemes d'administration de medicaments
WO1995003357A1 (fr) * 1993-07-23 1995-02-02 Massachusetts Institute Of Technology Particules biodegradables
WO1995003356A1 (fr) * 1993-07-23 1995-02-02 Massachusetts Institute Of Technology Nanoparticules et microparticules de copolymeres multibloc hydrophiles-hydrophobes non lineaires
WO1995007072A2 (fr) * 1993-09-09 1995-03-16 Schering Aktiengesellschaft Principes actifs et gas contenant des microparticules
WO1995015118A1 (fr) * 1993-11-30 1995-06-08 Imarx Pharmaceutical Corp. Microspheres gazeuses pour application topique et sous-cutanee
US5534241A (en) * 1993-07-23 1996-07-09 Torchilin; Vladimir P. Amphipathic polychelating compounds and methods of use
EP0727225A2 (fr) * 1995-02-14 1996-08-21 Sonus Pharmaceuticals, Inc. Compositions et méthodes pour l'imagerie ultrasonique dirigée
WO1996040277A2 (fr) * 1995-06-07 1996-12-19 Brown University Research Foundation Microparticules polymeres sechees par pulverisation contenant des agents d'imagerie
WO1996041647A1 (fr) * 1995-06-08 1996-12-27 Barnes-Jewish Hospital D/B/A Systeme de liaison a des sites specifiques, procede et composition d'imagerie
WO1998000172A2 (fr) * 1996-06-28 1998-01-08 Board Of Regents Of The University Of Nebraska Compositions et methodes modifiant la biodistribution d'agents biologiques

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4232755A1 (de) * 1992-09-26 1994-03-31 Schering Ag Mikropartikelpräparationen aus biologisch abbaubaren Mischpolymeren
WO1994028873A1 (fr) * 1993-06-11 1994-12-22 Unger Evan C Nouveaux systemes d'administration de medicaments
WO1995003357A1 (fr) * 1993-07-23 1995-02-02 Massachusetts Institute Of Technology Particules biodegradables
WO1995003356A1 (fr) * 1993-07-23 1995-02-02 Massachusetts Institute Of Technology Nanoparticules et microparticules de copolymeres multibloc hydrophiles-hydrophobes non lineaires
US5534241A (en) * 1993-07-23 1996-07-09 Torchilin; Vladimir P. Amphipathic polychelating compounds and methods of use
WO1995007072A2 (fr) * 1993-09-09 1995-03-16 Schering Aktiengesellschaft Principes actifs et gas contenant des microparticules
WO1995015118A1 (fr) * 1993-11-30 1995-06-08 Imarx Pharmaceutical Corp. Microspheres gazeuses pour application topique et sous-cutanee
EP0727225A2 (fr) * 1995-02-14 1996-08-21 Sonus Pharmaceuticals, Inc. Compositions et méthodes pour l'imagerie ultrasonique dirigée
WO1996040277A2 (fr) * 1995-06-07 1996-12-19 Brown University Research Foundation Microparticules polymeres sechees par pulverisation contenant des agents d'imagerie
WO1996041647A1 (fr) * 1995-06-08 1996-12-27 Barnes-Jewish Hospital D/B/A Systeme de liaison a des sites specifiques, procede et composition d'imagerie
WO1998000172A2 (fr) * 1996-06-28 1998-01-08 Board Of Regents Of The University Of Nebraska Compositions et methodes modifiant la biodistribution d'agents biologiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
A.L. KLIBANOV ET AL.: "TARGETING OF ULTRASOUND CONTRAST MATERIAL. AN IN VITOR FEASIBILTY STUDY." ACTA RADIOLOGICA, vol. 38, no. 412, 1997, page 113-120 XP002063479 *
V.R. MUZYKANTOV: "IMMUNOTARGETING OF STREPTAVIDIN TO THE PULMONARY ENDOTHELIUM" JOURNAL OF NUCLEAR MEDICINE, vol. 35, no. 8, August 1994, NEW YORK US, pages 1358-1365, XP002063478 *

Cited By (56)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6245747B1 (en) 1996-03-12 2001-06-12 The Board Of Regents Of The University Of Nebraska Targeted site specific antisense oligodeoxynucleotide delivery method
EP1323434A3 (fr) * 1996-09-11 2004-03-24 Imarx Pharmaceutical Corp. Procédés améliorés d'imagerie diagnostique utilisant un agent de contraste et un vasodilatateur
EP1323434A2 (fr) * 1996-09-11 2003-07-02 Imarx Pharmaceutical Corp. Procédés améliorés d'imagerie diagnostique utilisant un agent de contraste et un vasodilatateur
EP1017425A4 (fr) * 1996-09-11 2002-03-06 Imarx Pharmaceutical Corp Procedes ameliores d'imagerie diagnostique utilisant un agent de contraste et un vasodilatateur coronarien
EP0977597A4 (fr) * 1996-09-11 2000-02-09 Imarx Pharmaceutical Corp Procedes ameliores d'imagerie diagnostique utilisant un agent de contraste et un vasodilatateur
EP0977597A1 (fr) * 1996-09-11 2000-02-09 Imarx Pharmaceutical Corp. Procedes ameliores d'imagerie diagnostique utilisant un agent de contraste et un vasodilatateur
EP1017425A1 (fr) * 1996-09-11 2000-07-12 IMARx PHARMACEUTICAL CORP. Procedes ameliores d'imagerie diagnostique utilisant un agent de contraste et un vasodilatateur coronarien
WO1999020312A1 (fr) * 1997-10-21 1999-04-29 Nycomed Imaging As Imagerie ultrasonique avec agent de contraste cible sur le systeme microvasculaire et medicament vasodilatateur
US6811766B1 (en) 1997-10-21 2004-11-02 Amersham Health As Ultrasound imaging with contrast agent targeted to microvasculature and a vasodilator drug
US9545457B2 (en) 1998-01-14 2017-01-17 Lantheus Medical Imaging, Inc. Preparation of a lipid blend and a phospholipid suspension containing the lipid blend
WO1999053963A1 (fr) * 1998-04-22 1999-10-28 Marsden, John, Christopher Ameliorations apportees a des agents de contraste ou en rapport avec ces agents
WO1999055383A2 (fr) * 1998-04-28 1999-11-04 Nycomed Imaging As Perfectionnements relatifs a des agents diagnostiques/therapeutiques
US6548048B1 (en) 1998-04-28 2003-04-15 Amersham Health As Lipopeptide stabilized microbubbles as diagnostic/therapeutic agents
AU763191B2 (en) * 1998-04-28 2003-07-17 Amersham Health As Improvements in or relating to diagnostic/therapeutic agents
WO1999055383A3 (fr) * 1998-04-28 2000-07-06 Nycomed Imaging As Perfectionnements relatifs a des agents diagnostiques/therapeutiques
WO2000002588A1 (fr) * 1998-07-13 2000-01-20 The Board Of Regents Of The University Of Nebraska Compositions destinees a l'administration ciblee specifique de site de medicaments et procede d'utilisation
WO2001017566A2 (fr) * 1999-09-08 2001-03-15 Institut für Diagnostikforschung GmbH an der Freien Universität Berlin Agents de contraste de type l-selectine
WO2001017566A3 (fr) * 1999-09-08 2001-12-20 Diagnostikforschung Inst Agents de contraste de type l-selectine
US8263739B2 (en) 2000-06-02 2012-09-11 Bracco Suisse Sa Compounds for targeting endothelial cells, compositions containing the same and methods for their use
US7109167B2 (en) 2000-06-02 2006-09-19 Bracco International B.V. Compounds for targeting endothelial cells, compositions containing the same and methods for their use
US7820621B2 (en) 2000-06-02 2010-10-26 Bracco International B.V. Compounds for targeting endothelial cells, compositions containing the same and methods for their use
US7884183B2 (en) 2000-06-02 2011-02-08 Bracco Suisse Sa Compounds for targeting endothelial cells, compositions containing the same and methods for their use
US6984373B2 (en) 2000-12-23 2006-01-10 Dyax Corp. Fibrin binding moieties useful as imaging agents
EP2301587A2 (fr) 2002-03-01 2011-03-30 Dyax Corporation Peptides de liaison KDR et VEGF/KDR et leur utilisation pour le diagnostic et la thérapie
US9629934B2 (en) 2002-03-01 2017-04-25 Dyax Corp. KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy
US9056138B2 (en) 2002-03-01 2015-06-16 Bracco Suisse Sa Multivalent constructs for therapeutic and diagnostic applications
EP2014310A2 (fr) 2002-03-01 2009-01-14 Dyax Corporation Peptides de liaison KDR et VEGF/KDR et leur utilisation pour le diagnostic et la thérapie
US8642010B2 (en) 2002-03-01 2014-02-04 Dyax Corp. KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy
EP2281580A2 (fr) 2003-01-13 2011-02-09 Bracco Imaging S.p.A Peptides à libération de gastrine (GRP) marqués
EP2500040A1 (fr) 2003-01-13 2012-09-19 Bracco Imaging S.p.A Peptides à libération de gastrine (GRP) marqués
US9000124B2 (en) 2003-03-03 2015-04-07 Dyax Corp. Peptides that specifically bind HGF receptor (cMet) and uses thereof
US9845340B2 (en) 2003-03-03 2017-12-19 Dyax Corp. Peptides that specifically bind HGF receptor (cMet) and uses thereof
EP2949658A2 (fr) 2003-03-03 2015-12-02 Dyax Corp. Peptides qui lient spécifiquement le récepteur HGF (CMET) et utilisations associées
US8044175B2 (en) 2003-03-03 2011-10-25 Dyax Corp. Peptides that specifically bind HGF receptor (CMET) and uses thereof
EP1944312A1 (fr) 2003-03-03 2008-07-16 Dyax Corporation Peptides qui lient spécifiquement le récepteur HGF (CMET) et utilisations associées
US7785566B2 (en) 2003-11-06 2010-08-31 Ge Healthcare, Inc. Pharmaceutical compounds
WO2005044313A3 (fr) * 2003-11-06 2006-05-11 Amersham Health As Composes pharmaceutiques
WO2005044313A2 (fr) * 2003-11-06 2005-05-19 Amersham Health As Composes pharmaceutiques
JP2007510643A (ja) * 2003-11-06 2007-04-26 ジーイー・ヘルスケア・アクスイェ・セルスカプ 医薬化合物
EP2359864A2 (fr) 2005-12-09 2011-08-24 Bracco Suisse SA Conjugués vecteur de ciblage-phospholipide
EP2147684A1 (fr) 2008-07-22 2010-01-27 Bracco Imaging S.p.A Agents de diagnostics sélectifs contre les métalloprotéases
US10471163B2 (en) 2013-09-13 2019-11-12 The General Hospital Corporation Activatable fibrin-binding probes
US11395856B2 (en) 2014-12-31 2022-07-26 Lantheus Medical Imaging, Inc. Lipid-encapsulated gas microsphere compositions and related methods
US10022460B2 (en) 2014-12-31 2018-07-17 Lantheus Medical Imaging, Inc. Lipid-encapsulated gas microsphere compositions and related methods
US10583207B2 (en) 2014-12-31 2020-03-10 Lantheus Medical Imaging, Inc. Lipid-encapsulated gas microsphere compositions and related methods
US10588988B2 (en) 2016-05-04 2020-03-17 Lantheus Medical Imaging, Inc. Methods and devices for preparation of ultrasound contrast agents
US10220104B2 (en) 2016-07-06 2019-03-05 Lantheus Medical Imaging, Inc. Methods for making ultrasound contrast agents
US10583208B2 (en) 2016-07-06 2020-03-10 Lantheus Medical Imaging, Inc. Methods for making ultrasound contrast agents
US9913919B2 (en) 2016-07-06 2018-03-13 Lantheus Medical Imaging, Inc. Methods for making ultrasound contrast agents
US11266750B2 (en) 2016-07-06 2022-03-08 Lantheus Medical Imaging, Inc. Methods for making ultrasound contrast agents
US11266749B2 (en) 2016-07-06 2022-03-08 Lantheus Medical Imaging, Inc. Methods for making ultrasound contrast agents
US11344636B2 (en) 2016-07-06 2022-05-31 Lantheus Medical Imaging, Inc. Methods for making ultrasound contrast agents
US9789210B1 (en) 2016-07-06 2017-10-17 Lantheus Medical Imaging, Inc. Methods for making ultrasound contrast agents
US11529431B2 (en) 2016-07-06 2022-12-20 Lantheus Medical Imaging, Inc. Methods for making ultrasound contrast agents
US11857646B2 (en) 2016-07-06 2024-01-02 Lantheus Medical Imaging, Inc. Methods for making ultrasound contrast agents
US11925695B2 (en) 2016-07-06 2024-03-12 Lantheus Medical Imaging, Inc. Methods for making ultrasound contrast agents

Also Published As

Publication number Publication date
WO1998018498A3 (fr) 1998-07-16
EP0963209A2 (fr) 1999-12-15

Similar Documents

Publication Publication Date Title
AU733477B2 (en) Improvements in or relating to diagnostic/therapeutic agents
AU733495B2 (en) Improvements in or relating to diagnostic/therapeutic agents
EP0973552B1 (fr) Ameliorations apportees a des agents diagnostiques et/ou therapeutiques ou les concernant
WO1998018498A2 (fr) Ameliorations apportees a des agents diagnostiques et/ou therapeutiques ou les concernant
EP0991427A2 (fr) Ameliorations apportees a des agents diagnostiques et/ou therapeutiques ou les concernant
US7109167B2 (en) Compounds for targeting endothelial cells, compositions containing the same and methods for their use
AU777304B2 (en) Novel methods of imaging and treatment with targeted compositions
US8257683B2 (en) Multicomponent assemblies having enhanced binding properties for diagnosis and therapy
JP4215820B2 (ja) 診断的および治療的使用のための新規な標的化組成物
US8263739B2 (en) Compounds for targeting endothelial cells, compositions containing the same and methods for their use
US20090162293A1 (en) Methods and compositions for ultrasound imaging of apoptosis
MXPA99003867A (en) Improvements in or relating to diagnostic/therapeutic agents
JP2001502719A (ja) 改良された診断／治療用薬剤
JP2002515889A (ja) 改良された診断／治療用薬剤
CZ149599A3 (cs) Diagnostický a/nebo léčebný prostředek

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US US US UZ VN YU ZW AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH KE LS MW SD SZ UG ZW AT BE CH DE DK ES FI FR GB GR IE IT

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
CFP Corrected version of a pamphlet front page

Free format text: ADD INID NUMBER (63) "RELATED BY CONTINUATION (CON) OR CONTINUATION-IN-PART (CIP) TO EARLIER APPLICATION" WHICH WAS INADVERTENTLY OMITTED FROM THE FRONT PAGE

ENP Entry into the national phase in:

Ref country code: JP

Ref document number: 1998 520191

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1997910518

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1997910518

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1997910518

Country of ref document: EP

NENP Non-entry into the national phase in:

Ref country code: CA