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WO2009074569A1 - Composés de ciblage et thérapeutiques avec un espaceur comprenant de la polyproline et microvésicules remplies de gaz comprenant lesdits composés - Google Patents

Composés de ciblage et thérapeutiques avec un espaceur comprenant de la polyproline et microvésicules remplies de gaz comprenant lesdits composés Download PDF

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Publication number
WO2009074569A1
WO2009074569A1 PCT/EP2008/067101 EP2008067101W WO2009074569A1 WO 2009074569 A1 WO2009074569 A1 WO 2009074569A1 EP 2008067101 W EP2008067101 W EP 2008067101W WO 2009074569 A1 WO2009074569 A1 WO 2009074569A1
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Prior art keywords
compound
moiety
peptide
gas
mmol
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PCT/EP2008/067101
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English (en)
Inventor
Edmund Marinelli
Christoph De Haen
Palaniappa Nanjappan
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Bracco International Bv
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Publication of WO2009074569A1 publication Critical patent/WO2009074569A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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
    • A61K49/22—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
    • 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
    • A61K49/227—Liposomes, lipoprotein vesicles, e.g. LDL or HDL lipoproteins, micelles, e.g. phospholipidic or polymeric
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the invention relates to new targeting or therapeutic compounds which can be incorporated into a composition of gas-filled microvesicles.
  • the invention further relates to gas-filled microvesicles for diagnostic and/or therapeutic use comprising said compounds and to their method of use. Background of the invention
  • a class of contrast agents particularly useful for ultrasound contrast imaging, includes suspensions of gas bubbles of nano- and/or micro-metric size dispersed in an aqueous medium.
  • gas bubbles are stabilized, for example by using emulsifiers, oils, thickeners or sugars, or by entrapping or encapsulating the gas or a precursor thereof in a variety of systems.
  • These stabilized gas bubbles are generally referred to in the art with various terminologies, such as, for instance, "microvesicles", “microspheres”, “microbubbles”, “microcapsules” or “microballoons”.
  • the term "microvesicles” is used to identify any of the above described stabilized gas-bubbles.
  • microvesicles can be associated (e.g. by inclusion in their boundary envelope) with therapeutic agents and/or with specific components which are capable to bind to a determined target within a patient's body (known as "targeting ligands").
  • targeting ligands include, for instance, peptides, proteins or antibodies, capable of binding to a specific organ or tissue such as, for instance, angiogenic or thrombolytic tissue.
  • a possible way to associate a targeting ligand or a therapeutic compound to the structure of a microvesicle is to bind it to suitable molecules which can be employed for the formation of the microvesicle's envelope.
  • the targeting or therapeutic moiety can be directly linked to the envelope-forming molecule or through a suitable spacer, in general an oligomeric or a polymeric spacer.
  • Sp spacer
  • Said component comprises a sequence of proline moieties and is suitably functionalized to bridge conventional moieties capable of being associated with an envelope of a gas-filled microvesicle (moiety "Mm”) with a targeting ligand or therapeutic agent (moiety "TL”), in a compound of formula Mm-Sp-TL.
  • Mm gas-filled microvesicle
  • TL targeting ligand or therapeutic agent
  • the polyproline spacer can be used either as such or in combination with other suitable spacer components, such as, for instance, hydrophilic polymers or hydrophilic polypeptides.
  • the use of a polyproline sequence employed as a spacer moiety "Sp" allows obtaining compounds of formula Mm-Sp-TL with a relatively effectively defined conformation.
  • the relatively high stiffness of the polyproline sequence provides an effective binding of the targeting ligand to the target site.
  • An aspect of the invention relates to a compound of formula : Mm-Sp-TL wherein:
  • Mm represents an amphiphilic moiety capable of being associated with an envelope of a gas-filled microvesicle
  • TL represents a targeting ligand or a therapeutic agent
  • Sp represents a spacer, bridging said moiety and said targeting ligand or therapeutic agent, which comprises a sequence of at least three proline or proline- like units of formula (I): where R represents hydrogen, a group of formula -NH-R ; a group of formula -
  • R can be in any of the 3, 4 or 5 position of the proline ring, in either cis or trans stereochemical orientation relative to the proline carbonyl group.
  • said sequence comprises from 5 to 25 proline or proline-like units of formula (I).
  • the above sequence is formed by proline units of formula (I) where R is hydrogen.
  • said amphiphilic moiety Mm comprises a phospholipid.
  • the spacer Sp further comprises a hydrophilic polymer, preferably a polyethyleneglycol.
  • the spacer Sp can be in the form of either orientation, such as 5 to 25 proline or proline-like units of (I) followed by a hydrophilic polymer or the orientation where the hydrophilic polymer is followed by the 5-to 25 proline or proline-like units of formula (I).
  • the hydrophilic polymer is connected to the Mm moiety, while the polyproline or polyproline-like sequence is connected to the TL moiety.
  • the spacer Sp further comprises a hydrophilic polypeptide, preferably comprising from 3 to 25 amino acids.
  • the aminoacids of said polypeptide are selected from the group consisting of lysine, arginine, aspartic acid, glutamic acid, serine, glycine and derivatives thereof.
  • the hydrophilic polypeptide is connected to the Mm moiety, while the polyproline or polyproline-like portion of the spacer is connected to the TL moiety.
  • the moiety TL is associated with the moiety Sp through a covalent bond.
  • the moiety TL is associated with the moiety Sp through a non-covalent bond.
  • Another aspect of the invention relates to gas-filled microvesicles comprising a compound of formula Mm-Sp-TL as above defined.
  • the compounds of the invention can be effectively introduced in the structure of the microvesicles and microvesicles comprising compounds of the inventions possess advantageous binding activity and echogenicity.
  • a further aspect of the invention relates to a method for ultrasound imaging which comprises administering a composition comprising a gas-filled microvesicle including said compound of formula Mm-Sp-TL to a patient and subjecting said patient to ultrasound irradiation.
  • FIGS 1 to 15 illustrate some reaction schemes for the preparation of compounds of formula Mm-Sp-TL
  • arylene identifies a bifunctional homocyclic or heterocyclic aromatic compound, preferably monocyclic, including, for instance, phenylene (-C 6 H 4 -) and pyridylene (-NC 5 H 3 -).
  • gas-filled microvesicles includes any structure comprising bubbles of gas of micronic or nanometric size surrounded by an envelope or layer (including film-forming layers) of a stabilizing material.
  • the term includes what is known in the art as gas-filled liposomes, microbubbles, microspheres, microballoons or microcapsules.
  • the stabilizing material can be any material typically known in the art including, for instance, surfactants, lipids, sphingolipids, oligolipids, phospholipids, proteins, polypeptides, carbohydrates, and synthetic or natural polymeric materials.
  • compositions which, upon reconstitution with an aqueous carrier in the presence of a gas, will produce a suspension of gas-filled microvesicles.
  • Said compositions typically include any of the above-cited stabilizing materials in dry powdered form (e.g. freeze-dried or spray-dried) capable of forming gas-filled microvesicles upon shaking an aqueous suspension thereof in the presence of a gas.
  • microbubbles includes aqueous suspensions in which the bubbles of gas are bounded at the gas/liquid interface by a very thin envelope (film) involving a stabilizing amphiphilic material disposed at the gas to liquid interface (sometimes referred to in the art as an "evanescent" envelope).
  • Microbubble suspensions can be prepared by contacting a suitable precursor thereof, such as powdered amphiphilic materials (e.g. freeze-dried preformed liposomes or freeze-dried or spray-dried phospholipid solutions) with air or other gas and then with an aqueous carrier, while agitating to generate a microbubble suspension which can then be administered, preferably shortly after its preparation.
  • aqueous suspensions of gas microbubbles are disclosed, for instance, in US 5,271,928, US 5,445,813, US 5,413,774, US 5,556,610, 5,597,549, US 5,827,504 and WO 04/069284, which are here incorporated by reference in their entirety.
  • microballoons or “microcapsules” include suspensions in which the bubbles of gas are surrounded by a solid material envelope of a lipid or of natural or synthetic polymers. Examples of microballoons and of the preparation thereof are disclosed, for instance, in US 5,711,933 and US 6,333,021.
  • association when referred in particular to the moiety "Mm” capable of associating or being associated with an envelope of a gas-filled microvesicle, includes any suitable chemical or physical interaction which may allow said moiety to aggregate with, incorporate into or bind to the envelope surrounding gas-filled microvesicles. Said association includes either the association of a single molecule or of a molecule included in supermolecular structures, such as, for instance, micelles.
  • association may be covalent or non-covalent.
  • association of the Mm moiety to the envelope includes the contribution of said moiety to the formation of the stabilizing envelope; in these preferred embodiments said moiety Mm is an envelope-forming moiety.
  • envelope-forming moiety includes any moiety which is capable of participating in the formation of the stabilizing envelope of gas-filled microvesicles. Said moiety is preferably an amphiphilic material, preferably comprising a phospholipid.
  • Non-covalent association includes intermolecular interactions among two or more molecules which do not involve a covalent bond such as, for instance, ionic or electrostatic interactions, dipole-dipole interactions, hydrogen bonding, hydrophilic or hydrophobic interactions, van der Waal's forces and combinations thereof.
  • Non-covalent interactions further include interaction between moieties of an affinity binding pair, such as, for instance, the interaction between avidin or streptavidin and biotin; protein A or G binding and Fc- region of immunoglobulin; oligonucleotides and complementary sequences, e.g.
  • targeting ligand includes any compound, moiety or residue having, or being capable of promoting, a targeting activity towards tissues and/or receptors in vivo.
  • Targets with which a targeting ligand may be associated include tissues such as, for instance, myocardial tissue (including myocardial cells and cardiomyocytes), membranous tissues (including endothelium and epithelium), laminae, connective tissue (including interstitial tissue) or tumors; blood clots; and receptors such as, for instance, cell-surface receptors for peptide hormones, neurotransmitters, antigens, complement fragments, immunoglobulins and cytoplasmic receptors for steroid hormones.
  • targeted gas-filled microvesicle includes any gas-filled microvesicle comprising at least one targeting ligand in its formulation.
  • intermediate of a targeted gas-filled microvesicle includes any gas-filled microvesicle which can be converted into a targeted gas-filled microvesicle.
  • Such intermediate may include, for instance, gas-filled microvesicles (or precursors thereof) including a suitable reactive moiety (e.g. maleimide or streptavidin), which can be reacted with a corresponding complementary reactive (e.g. thiol or biotin, respectively) linked to a targeting ligand.
  • a suitable reactive moiety e.g. maleimide or streptavidin
  • intermediate of a Mm-Sp-TL compound includes any Mm-Sp intermediate which can be converted into a Mm-Sp-TL compound, including any Mm-Sp intermediate containing a suitable reactive moiety as illustrated here above.
  • the phrase also includes any Sp-TL intermediate which can be converted into a Mm-Sp-TL compound, including any Sp-TL intermediate containing a suitable reactive moiety.
  • therapeutic agent includes within its meaning any substance, composition or particle which may be used in any therapeutic application, such as in methods for the treatment of a disease in a patient, as well as any substance which is capable of exerting or responsible to exert a biological effect in vitro and/or in vivo.
  • Therapeutic agents thus include any compound or material capable of being used in the treatment (including diagnosis, prevention, alleviation, pain relief or cure) of any pathological status in a patient (including malady, affliction, disease lesion or injury).
  • therapeutic agents are drugs, pharmaceuticals, bioactive agents, cytotoxic agents, chemotherapy agents, radiotherapeutic agents, proteins, natural or synthetic peptides, including oligopeptides and polypeptides, vitamins, steroids and genetic material, including nucleosides, nucleotides, oligonucleotides, polynucleotides and plasmids.
  • Adoa 8-amino-3,6-dioxaoctanoic acid
  • ACN Acetonitrile
  • API-ES Atmospheric pressure ionization electrospray
  • BOP (benzotriazol-l-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate
  • CHCI 3 Chloroform
  • DCC Dicyclohexylcarbodiimide
  • Dga Diglycolyl, 3-oxapentan-l,5-di-oyl
  • DIC N,N'-diisopropylcarbodiimide
  • DIEA N,N-Diisopropylethylamine
  • DMSO Dimethyl sulfoxide
  • DPPE l,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine, commonly also identified as dipalmitoylphosphatidylethanolamine;
  • DPPG l,2-Dipalmitoyl-sn-glycero-3-[phospho-rac-(l-glycerol)] sodium salt, commonly also identified as dipalmitoylphosphatidylglycerol;
  • DPPS 1,2-Dipalmitoyl-sn-glycero-phospho-L-serine, commonly also identified as dipalmitoylphosphatidylserine
  • DSPA 1,2-Distearoyl-sn-glycero-phosphate sodium salt, commonly also identified as distearoylphosphatidic acid
  • DSPE l,2-Distearoyl-sn-glycero-3-phosphoethanolamine, commonly also identified as distearoylphosphatidylethanolamine
  • DSPE-PEG1000 Distearoylphosphatidylethanolamine-N-methoxy(PEGlOOO); DSPS: l,2-Distearoyl-sn-glycero-3-(phospho-L-serine), commonly also identified as distearoylphosphatidylserine;
  • EDAC 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide HCI;
  • ELSD Evaporative Light Scattering Detector
  • Fmoc 9-Fluorenylmethyloxycarbonyl
  • Glut Glutaryl, pentan-l,5-di-oyl
  • HATU N- ⁇ (Dimethylamino)-lH-l,2,3-triazolo(4,5-b)pyridine-l-ylmethylene]-N- methylenemethanaminium hexafluorophosphate-N-oxide;
  • HBTU O-Benzotriazol-l-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • HOAc Acetic acid
  • HOAt l-Hydroxy-7-azabenzotriazole
  • HOBt N-Hydroxybenzotriazole
  • HPLC High performance liquid chromatography
  • ivDde (4,4-Dimethyl-2,6-dioxocyclohex-l-ylidene)-3-methylbutyl
  • MALDI Matrix assisted laser desorption ionization MeOH : Methanol
  • Neg. ion Negative ion
  • NMM N-Methylmorpholine
  • NMP N-Methylpyrrolidone
  • Pd[P(Ph) 3 ] 4 Tetrakis(triphenylphosphine) palladium (O);
  • PEG Polyethylene glycol (if followed by a number, e.g. PEG2000, this identifies the approximate mean molecular weight of the polydispersed PEG polymer, i.e. about 2000 daltons in the example;
  • Reagent B (TFA:H 2 O:phenol:triisopropylsilane, 88: 5: 5:2, v/v/wt/v); SATA: S-Acetylthioacetyl; SPPS: Solid phase peptide synthesis; Su : Succinimidyl
  • the moiety Mm of the Mm-Sp-TL compound can advantageously be selected among those amphiphilic components which are generally employed in the preparation of gas-filled microvesicles, said components being suitably functionalized in order to be linked into the Mm-Sp-TL compound.
  • said moiety is an envelope-forming moiety.
  • amphiphilic components include, for instance, phospholipids; lysophospholipids; fatty acids, such as palmitic acid, stearic acid, arachidonic acid or oleic acid; lipids, phospholipids or lysophospholipids with ether linkages replacing one or more of the ester linkages of the fatty ester moieties (as illustrated e.g. by Abdelmageed, O.; Duclos, R., Jr.; Griffin, R.; Siminovitch, D.; Ruocco, M.; Makriyannis, A. Chem. Phys.
  • Lipids 1989 50, 163-169 lipids, phospholipids or lysophospholipids with a NH group replacing the alkyl oxygen of one or both of the fatty ester linkages of the fatty ester moieties, i.e. amide analogs of phospholipids (as illustrated e.g. Byun, H-S and Bittman, R. J. Org. Chem.
  • ester-linked fatty acids include dicetyl phosphate; stearylamine; sterol esters of aliphatic acids; sterol esters of sugar acids; esters of sugars with aliphatic acids; saponins; glycerol or glycerol esters including glycerol tripalmitate, glycerol distearate, glycerol tristearate, glycerol dimyristate, glycerol trimyristate, glycerol dilaurate, glycerol trilaurate, glycerol dipalmitate; long chain alcohols including n-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, or n-octadecyl alcohol; and mixtures or combinations thereof.
  • the amphiphilic compound is a phospholipid.
  • suitable phospholipids include esters of glycerol with one or preferably two (equal or different) residues of fatty acids and with phosphoric acid, wherein the phosphoric acid residue is in turn bound to a hydrophilic group, such as choline (phosphatidylcholines - PC), serine (phosphatidylserines - PS), glycerol (phosphatidylglycerols - PG), ethanolamine
  • Phospholipids with only one residue of fatty acid are generally referred to in the art as the "lyso" forms of the phospholipid, i.e. lysophospholipids.
  • Fatty acid residues present in the phospholipids are in general long chain aliphatic acids, typically containing from 12 to 24 carbon atoms, preferably from 14 to 22; the aliphatic chain may contain one or more unsaturations or is preferably completely saturated.
  • Suitable fatty acids included in the phospholipids are, for instance, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid.
  • saturated fatty acids such as myristic acid, palmitic acid, stearic acid and arachidic acid are employed.
  • phospholipids are phosphatidic acids, i.e. the diesters of glycerol- phosphoric acid with fatty acids; sphingolipids such as sphingomyelins, i.e. those phosphatidylcholine analogs where the residue of glycerol diester with fatty acids is replaced by a ceramide chain; cardiolipins, i.e. the esters of 1,3-diphosphatidylglycerol with a fatty acid; glycolipids such as gangliosides GMl (or GM2) or cerebrosides; glucolipids; sulfatides and glycosphingolipids.
  • phosphatidic acids i.e. the diesters of glycerol- phosphoric acid with fatty acids
  • sphingolipids such as sphingomyelins, i.e. those phosphatidylcholine analogs where the residue of glycerol diester with
  • phospholipids includes either naturally occurring, semisynthetic or synthetically prepared products that can be employed either singularly or as mixtures.
  • naturally occurring phospholipids are natural lecithins (phosphatidylcholine
  • PC soybean or egg yolk lecithins
  • Examples of semisynthetic phospholipids are the partially or fully hydrogenated derivatives of the naturally occurring lecithins.
  • Preferred phospholipids are di-esters of fatty acids (preferably Ci 2 -Ci 8 ) of phosphatidylcholine, ethylphosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine or of sphingomyelin, phosphatidylethanolamine being particularly preferred.
  • Preferred phosphatidylethanolamines are dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoyl phosphatidyl-ethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), diarachidoylphosphatidylethanolamine (DAPE) and dilinoleoylphosphatidylethanolamine (DLPE).
  • DMPE dimyristoyl-phosphatidylethanolamine
  • DPPE dipalmitoylphosphatidylethanolamine
  • DSPE distearoyl phosphatidyl-ethanolamine
  • DOPE dioleoylphosphatidylethanolamine
  • DAPE diarachidoylphosphatidylethanolamine
  • DLPE dilinoleoylphosphatidylethanolamine
  • Examples of synthetic phospholipids are ether phospholipids such as 1,2-di-O- hexadecyl-sn-glycero-3-phosphoethanolamine (see the above cited Abdelmageed, O.; Duclos, R., Jr.; Griffin, R.; Siminovitch, D.; Ruocco, M.; Makriyannis, A. Chem. Phys. Lipids 1989 50, 163-169) wherein the carbonyl groups of the fatty ester linkages are replaced by methylene groups, and amide phospholipids (see the above cited Byun, H-S and Bittman, R. J. Org. Chem. 1996, 61, 8706-8708) wherein the fatty ester linkages are replaced with fatty amide linkages, specifically for both of the fatty ester linkages the ester alkyl oxygen is replaced with a NH group.
  • ether phospholipids such as 1,2-di-O- hexadecyl
  • the above compounds either contain or are functionalized with suitable reactive groups capable of reacting with corresponding complementary reactive groups, in order to stably link the Mm moiety in the Mm-Sp-TL compound.
  • Said reactive groups include, for instance, primary amino groups, secondary amino groups, carboxylic groups, hydroxyl groups, alkyl halides and alkyl-aryl halides.
  • the reactive group can be inserted according to conventional techniques.
  • primary or secondary amino groups can be inserted to create, for example, derivatives of distearoylphosphoethanolamine, distearoyl N-alkyl-phosphoethanolamine, distearylbromophosphoethyl, distearoylphosphoethanol, distearoylphosphoacetyl, dipalmitoylphosphoethanolamine, dipalmitoyl N-alkyl- phosphoethanolamine, dipalmitoylbromophosphoethyl, dipalmitoylphosphoethanol or dipalmitoylphosphoacetyl.
  • the amphiphilic molecule associated with the microvesicle's envelope already includes a reactive group.
  • a reactive group such as a carboxylic group in the form of an activated carboxylic acid ester, for example, to link the Mm moiety into the Mm-Sp-TL compound.
  • the lipid molecule is bound to the spacer "Sp", directly or through a suitable linker (as discussed in detail in the following of the specification), preferably by means of a covalent bond obtained by reacting the reactive group with a suitable corresponding reactive group.
  • suitable reactive moieties on the linker or spacer may be isothiocyanate groups (to form a thiourea bond), carboxylic groups or reactive esters (to form an amide bond), or aldehyde groups (for the formation of an imine bond to be reduced to an alkylamine bond);
  • suitable complementary reactive moieties include haloacetyl derivatives (to form a thioether bond), maleimides (to form a thioether bond) or 2-thiopyridylalkyl disulfides (meaning disulfides wherein one of the sulphur atoms is bound to a 2-pyridyl
  • the spacer "Sp” of the Mm-Sp- TL compound is a moiety comprising a group of formula (I) as above defined.
  • Suitable binding groups are preferably provided at the respective ends of the spacer Sp, for covalently binding with corresponding complementary binding groups in the Mm-Sp-TL compound.
  • said binding group is the amino or carboxy group of the proline unit
  • the spacer comprises a plurality of proline units of formula (I) where R is hydrogen.
  • said proline units are from 5 to 25 and, more preferably, there are nine proline units.
  • the above proline-based spacer can be modified to increase its hydrophilicity. This can be obtained by either using proline rings bearing suitable hydrophilic residues, or by combining the proline-based spacer with other more hydrophilic components; the combination of sequences of modified proline rings with other hydrophilic components is also envisaged.
  • Mm-Sp-TL comprises a combination of proline units of formula (I) comprising at least one modified proline unit where the R residue is selected among a group of formula -NH-R 2 , -
  • any combination of a proline unit with any of the above substituted proline units can be prepared, e.g. depending on the desired degree of hydrophilicity to be conferred to the spacer. For instance, sequences where a proline and a substituted proline unit are alternated can be prepared. Alternatively, sequences where each third unit is a proline unit can be prepared, the other units of the sequence being any of the above substituted proline units.
  • sequences where each third unit is a proline unit can be prepared, the other units of the sequence being any of the above substituted proline units.
  • the desired sequence of proline units of formula (I) can be obtained according to conventional peptide synthesis protocols.
  • the sequence comprises only unmodified proline units of formula (I)
  • synthesis can be accomplished beginning with Fmoc-Pro-2- chlortrityl resin or Fmoc-Pro-TGT resin using an Applied Biosystems Inc. model 433A automated peptide synthesizer which employs the "Fastmoc" synthesis protocol supplied by the manufacturer.
  • the protocol consists of deprotection of the Fmoc group of the resin- bound amino acid, in this case Fmoc-Pro, for 10-29 min, using 20% piperidine in NMP or
  • the smallest scale reaction (0.1 mmol) employs 10 equivalents of activator and incoming amino acid
  • the intermediate (0.25 mmol) scale employs 4 equivalents of activator and incoming amino acid
  • the 1.0 mmol scale reaction employs 3 equivalents of activator and incoming amino acid; a 2-fold excess of DIEA vs the incoming amino acid and activator is generally employed.
  • the larger scale reaction is conducted for the longest reaction time.
  • the Fmoc-polyproline C-terminal acid is removed from the 2-chlorotrityl resin using 5% TFA in dichloromethane (45 min), or in the case of the TGT resin, 5-50% TFA in DCM for a similar period.
  • the volatiles can be removed by evaporation and the crude Fmoc-polyproline C-terminal acid can be triturated with ether to provide a solid from which the ether layer, containing impurities, is decanted.
  • the trituration-washing procedure can be repeated several times to insure optimal removal of ether soluble impurities.
  • Purification of the Fmoc-polyproline acid can be effected by dissolution in ACN-H 2 O followed by reversed phase HPLC employing a linear gradient of ACN (0.1% TFA) into H 2 O (0.1% TFA).
  • the product-containing fractions can be detected by monitoring the eluate using an ultraviolet detector and/or an ELS (evaporative light scattering) detector. Pure fractions can be combined and the desired product isolated by lyophilization.
  • the 4-hydroxyl group of trans-4- hydroxyproline and the 4-amino group of trans-4-aminoproline may require protection.
  • the hydroxyl group may be protected with acid-cleavable protecting groups such as as the tert- butyl ether or other acid sensitive or oxidatively cleavable protecting groups such as the 2,4-dimethoxybenzyl ether, the 3,4-dimethoxybenzyl ether or the 2,4,6-trimethoxybenzyl ether.
  • acid-cleavable protecting groups such as as the tert- butyl ether or other acid sensitive or oxidatively cleavable protecting groups such as the 2,4-dimethoxybenzyl ether, the 3,4-dimethoxybenzyl ether or the 2,4,6-trimethoxybenzyl ether.
  • Other groups that can be employed are the allyl ether (Hu Y-J, Dominique R, Das SK, Roy R. Can. J.
  • the protecting group can be chosen such that it is less sensitive to acid than the resin-peptide linkage so that the protecting group is retained when the assembled spacer is removed from the resin; for example the use of tert-butyl to protect the hydroxyl group of a trans-4-hydroxyprolyl moiety will withstand the acid conditions (1-5% TFA in DCM) required for cleavage of the protected Fmoc-polyproline spacer (which contains a O-tert-butyl protected trans-4-(hydroxy)-prolyl moiety) from the resin.
  • trans-4-aminoproline residues of the spacer Sp can be protected with the Boc protecting group, with an aloe (allyloxycarbonyl) protecting group or with the Dde or ivDde protecting group all of which can withstand the above described conditions for removal of the spacer from the resin.
  • trans-4-hydroxy or trans- 4-aminoproline derivatives bearing ether or acyl groups meaning that the 4-hydroxy is etherified or the 4-amino group is acylated, for example
  • any hydroxy or amino substituents on the ether or acyl groups can also be protected with the protecting groups illustrated for trans-4-amino and trans-4-hydroxyproline, where required.
  • Carboxyl groups of such solubilizing groups can be protected as the tert-butyl ester, or as the (4[N-(l-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutylamino]- benzyl ester (Dmab ester) while amino groups can be protected with the Boc, Dde, ivDde, or the aloe groups as the needs of the synthesis and the requirements for other protecting groups for the overall synthesis of the final Mm-Sp-TL compound dictate.
  • Groups such as guanidino and carboxamide can also be present and in protected form can be present as the Pmc, nitro or tosyl derivatives (for guanidino groups) and the trityl or 9-xanthyl derivatives (for the amido nitrogen, which may also be employed in unprotected form).
  • Preparation of polyproline spacers based on 4-substituted prolines can be accomplished as follows.
  • trans-4-aminoprolines employed in the spacer Sp for example, trans-4-(Boc-amino)
  • N ⁇ -Fmoc-proline can be reacted with a resin such as 2-chlorotrityl resin (Barlos K, Chatzi O, Gatos D, et.al. Int. J. Peptide and Protein Res. 1991, 37, 513-520) to give trans-4-(Boc-amino), N ⁇ -Fmoc-prolyl-2-chlorotrityl resin.
  • the resulting resin can be treated with 20% piperidine in DMF to effect removal of the Fmoc group from the cyclic nitrogen.
  • the resulting resin-bound trans-4-Boc-aminoproline can be reacted with trans-4- (Boc-amino)-N ⁇ -Fmoc-proline in the presence of a coupling reagent combination such as HBTU/DIEA, HATU/DIEA, PyBOP/DIEA or DIC/HOBt to form trans-4-(Boc-amino)-N ⁇ -Fmoc- prolyl-trans-4-(Boc-amino)-proline-2-chlorotrityl resin.
  • a coupling reagent combination such as HBTU/DIEA, HATU/DIEA, PyBOP/DIEA or DIC/HOBt
  • trans-4-(Boc-amino)-N ⁇ - Fmoc-prolyl units can be appended to the growing peptide chain by peptide coupling with intervening Fmoc removal.
  • the resin-bound, fully protected poly trans-4-(Boc-amino)-proline, still bearing its N-terminal Fmoc group can be cleaved from the resin using reagents that cleave the side chain-protected peptide from the 2-chlorotrityl resin without cleavage of the side chain protecting groups.
  • Such reagent combinations that accomplish this cleavage that are known in the art are: 1% TFA in dichloromethane, acetic acid/trifluoroethanol/dichloromethane (1/2/7 ', v/v/v) or hexafluoroisopropanol/dichloromethane (3/7, v/v), to obtain the fully side-chain protected poly-(trans-4-Boc-aminoproline) bearing an unprotected carboxyl group at its C-terminus.
  • the obtained product can then be activated and reacted with suitable Mm and/or TL moieties, as explained herein.
  • trans-4-hydroxyproline containing spacers the same chemistry as described above for the protected trans-4-amino proline containing spacers can be carried out, except that the allyloxycarbonyl or allyl protecting groups for the trans-4-hydroxy group are used instead of those employed for the amino analog. Those protecting groups can be removed in a highly efficient manner by using [CpRu(IV)( ⁇ -C 3 H 5 )(2- quinolinecarboxylato)]PF 6 , MeOH, 30 0 C for 0.5 h, as described above.
  • a spacer Sp comprising a proline unit of formula (I) as above defined (preferably where R is hydrogen) can be combined with an additional more hydrophilic spacer moiety.
  • Said additional hydrophilic spacer moiety can be, for instance, a hydrophilic synthetic polymer or a polypeptide comprising a sequence of suitable hydrophilic amino acids.
  • Said additional spacer moiety can be inserted between the spacer Sp and the Mm moiety, between the spacer Sp and the moiety TL or inserted between the spacer Sp and the moieties Mm and T, respectively.
  • hydrophilic synthetic polymers include for instance, polyethylenglycol, polyvinylpyrrolidone, polyacrylic acid, polyhydroxymethyl acrylate, polypeptides comprising synthetic amino acids such as L-arginine, D-arginine, L-lysine, D- lysine, L-ornithine, D-ornithine, L-aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid, glycine, L-serine and D-serine, either as homopolymers or as polypeptides which incorporate any combination of these amino acids in the polymer as well as copolymers of PEG and any of the amino acids described above.
  • polyethylenglycol PEG
  • the synthetic polymer may include from 2 to about 500 monomer units, preferably from about 12 to about 250 and even more preferably from about 20 to about 130 monomer units.
  • a polymeric spacer, such as PEG may help to reduce the antigenicity and increase the blood half time of the gas-filled microvesicles, as they are not readily opsonised and consequently less readily recognized by the reticuloendothelial system (also referred to as the macrophage monocytic phagocytic system - MMPS), present in the Kuppfer cells of the liver and spleen, for example.
  • a potential problem with a targeting vector attached to the end of a polymeric spacer is that a flexible polymeric spacer is more sterically hindered and thus could wrap itself around or otherwise hinder the targeting peptide's access to the receptor or other macromolecular target. Such hindrance may slow down the kinetics of the crucial binding interactions that result in adhesion of the targeting peptide to the macromolecular target, extending the time required to obtain adhesion of the targeting peptide-bearing microvesicles to the target, and, in the worst case, precluding adhesion of the microvesicles to the target.
  • the targeting peptide is advantageously separated from the potentially hindering polymer spacer and thus optimally presented to the biomolecular, macromolecular or cellular target.
  • the hydrophilic synthetic polymer Sp' can be linked to the other components of the Mm-Sp-TL compound either directly through a covalent bond or through suitable reactive linking groups.
  • suitable linking groups can be schematized by a moiety of general formula (II): -L 1 -.. 2 -.. 3 - (II)
  • L 2 represents a group selected from (Ci-Cio)alkyl; (Ci-Cio)alkylidene; (Ci-Cio)aminoalkyl; (Ci-Cio)oxyalkyl; a (C 3 -C 8 ) alicyclic compound; arylenes, such as o-, m- or p-phenylene; 5-membered heteroarylenes, such as 2,5-furylene, 2,3-furylene, 2,4- furylene, 3,4-furylene, the analogous pyrrole or thiophene derivatives, or their saturated analogs tetrahydrofurylene, pyrrolindinylene or tetrahydrothienylene; 6-membered heteroarylenes such as 2,3-pyridylene, 2,4-pyridylene, 2,5-pyridylene, 2,6-pyridylene, 3,4- pyridylene, 3,5-pyridylene 3,
  • L 3 represents a covalent bond or a group selected from amino, (Ci-Cio)alkylamino, oxy, (Ci-Cio)alkyloxy, (Ci-Cio)oxyalkyl, -S-S-(CH 2 )i-io, -CO-, -CS-, -0-C0-, -HN-CO-, -CH 2 -CO-, -CO-O-, -CO-NH-, -CS-NH-, -HN-CO-NH-, -CH 2 -CO-NH-, -HN-CO-O-, -CO-NH-, NH-CS-NH-.
  • linking groups can also be used for linking the hydrophilic polymer to a proline spacer when this latter is linked to a TL moiety, in particular a targeting peptide.
  • the above linking groups will be terminated with a suitable reactive group "Z", compatible with the free linking atom on the proline residue, which is in turn determined by the way the proline spacer is linked to the moiety TL.
  • amide linkages can be formed by reaction of a carboxylic acid and an amine with an activating agent such as DIC in the presence of a coupling additive such as HOBt or NHS to provide the two components linked by an amide linkage.
  • an activating agent such as DIC
  • a coupling additive such as HOBt or NHS
  • Other coupling agents that can be employed for formation of amide linkages are uronium coupling agents such as HBTU and HATU or the phosphonium-based coupling agents such as BOP and PyBOP in the presence of a base such as DIEA (See e.g. Principles and Practice of Solid-Phase Synthesis Fields GB, Tian Z and Barany G, In : Synthetic Peptides - A Users Guide, G. A.
  • Carbamate linkages may be formed by reaction of an alcohol with 4-nitrophenylchlorocarbonate (which provides the mixed carbonic acid ester of 4-nitrophenol and the alcohol) followed by reaction of the product with an amine (Jeffrey SC, Andreyka JB, Bernhardt SX et. al. Bioconj. Chem. 2006, 17, 831-840).
  • carbamates can be formed by reaction of bis-p-nitrophenylcarbonate with an amine to provide a carbamate which can then be reacted with an alcohol (Garbaccio RM, Fraley ME, Tasber ES et. al. Bioorg. Med. Chem. Lett. 2006, 16, 1780-1783).
  • Urea linkages can be prepared by reaction of an amine with phosgene or a phosgene equivalent such as diphosgene or triphosgene (Muller PRM, Specialty Chem. Mag. 1994, 14, 357-360) to provide the isocyanate which upon reaction of a second amine provides the urea linkage.
  • Urea linkages may also be formed by reaction of an amine with an excess of dicarbonylimidazole to provide a mixed alkylamino-imidazolidinyl urea, which upon reaction with a second amine provides the desired mixed urea linkage.
  • ureas may be prepared by sequential reaction of two different amines with bis-p-nitrophenylcarbonate; the first reaction gives an intermediate p-nitrophenyl carbamate which is then reacted with the second amine to give the mixed urea (For a review of urea synthesis and green chemistry approaches to urea synthesis see: Bigi F, Maggi R and Sartori G Green Chemistry, 2000, 2, 140-148 and references therein).
  • Thioureas can be prepared by reaction of thiocarbonyl chloride or an equivalent such as l-(methyldithiocarbonyl)imidazole with an amine to afford an intermediate isothiocyanate or a monosubstitution product, which upon reaction with a second amine provides the desired thiourea (See: Salameh BA, Sundin A, Leffler H et. al. Bioorg. Med. Chem. 2006, 14, 1215-1220; and Ivanov NA, Vlasova RV, Goncharova VA et al.
  • Diamide linkages can be prepared by reaction of an amine with a large excess of a activated diester such as disuccinimidyl glutarate; this provides a glutaric acid monoamide, mono-NHS ester.
  • the glutaric acid monoamide mono- NHS ester can be reacted with a second amine component to give the desired construct containing the diamide linkage.
  • an amine can be reacted with a cyclic anhydride such as glutaric anhydride to provide a glutaric acid monoamide monoacid which then can be activated using reagents such as DIC/HOBt (as described above) and reacted with a second amine to give the desired construct (See for example: Arbogast C, Bussat P, Dransfield DT et. al. PCT Int. Appl. 2003, 278 pp. and Chen Q, Sowa DA, Cai J, et. al. Synth. Commun. 2003, 33, 2401-2421).
  • a cyclic anhydride such as glutaric anhydride
  • DIC/HOBt as described above
  • Imine linkages can be prepared by reaction of an amine reaction partner with an aldehyde derivative bearing the other partner compound to be linked, optionally in the presence of a dehydrating agent for example magnesium sulfate (Kawabata T, Kimura Y, Ito Y et. al. Tetrahedron 1988, 44, 2149-2165).
  • a dehydrating agent for example magnesium sulfate
  • the linkage of the reacting partners as an amine linkage can be accomplished using methods of reductive amination wherein the imine first formed by reaction of an amine with an aldehyde is then reduced using a reducing agent such as sodium cyanoborohydride under mildly acidic conditions (pH 4.5) or in the presence of neat acetic acid (Kreevoy MM, Borch RF and Hutchins JEC U.S. Patent 1975, 8 pp. US 3873621 19750325. Hart DJ and Leroy V. Tetrahedron 1995, 51, 5757-70. Wrobel JE and Ganem B Tetrahedron Lett. 1981, 22, 3447-3450).
  • a reducing agent such as sodium cyanoborohydride under mildly acidic conditions (pH 4.5) or in the presence of neat acetic acid (Kreevoy MM, Borch RF and Hutchins JEC U.S. Patent 1975, 8 pp. US 3873621 19750325. Hart DJ and Leroy V.
  • the disulfide linkage can be formed by treatment of the symmetrical disulfide of one of the reactants to be linked with a suitable reducing agent such as tris-2- carboxyethyl-phoshine or tributyl phosphine under inert atmosphere in aqueous solution or sodium dithionite in aqueous solution, or Cleland's Reagent (dithiothreitol) in nonaqueous solvents to give the free thiol (Bernardi D, Battaglia E and Kirsch G Bioorg. Med. Chem. Lett.
  • a suitable reducing agent such as tris-2- carboxyethyl-phoshine or tributyl phosphine under inert atmosphere in aqueous solution or sodium dithionite in aqueous solution, or Cleland's Reagent (dithiothreitol) in nonaqueous solvents to give the free thiol (Bernard
  • a polypeptide comprising a sequence of suitable hydrophilic amino acids can be combined with a spacer Sp.
  • the hydrophilic polypeptide can be a sequence comprising from 3 to 25 aminoacids selected from Arginine (Arg), Aspartic acid (Asp), Glutamic acid (GIu), Serine (Ser), Glycine (GIy) and mixtures and derivatives thereof.
  • suitable hydrophilic polypeptides are, for instance: -Lys-Arg-Lys-Arg-,
  • Suitable derivatives of the above hydrophilic amino acids are those where additional hydrophilic functionality is appended to these amino acids, particularly by either acylation (acylation of the Lysine ⁇ -amino group), etherification (etherification of the serine hydroxyl group) or amidation (amidation of the side-chain carboxyl group of Aspartic acid and Glutamic acid) of the side chain.
  • acylation acylation of the Lysine ⁇ -amino group
  • etherification etherification of the serine hydroxyl group
  • amidation amidation of the side-chain carboxyl group of Aspartic acid and Glutamic acid
  • modified derivatives of Lys, Ser, Asp, or GIu can be employed in the same manner in combination with each other, or with the unmodified amino acids as well, with no limitation on the combinations.
  • solubilizing moieties comprising such modified amino acids are shown below:
  • the above exemplary embodiments do not limit in any way the possible combinations of the amino acids Lys, Arg, Asp, GIu, Ser, GIy or the modified derivatives such as those shown above, that can be used to prepare the additional polypeptide spacer.
  • Such combinations can be employed to adjust the charge and size of the spacer in order to optimize solubility, e.g. for incorporation of the Mm-Sp-TL component into the microvesicles in a manner to facilitate presentation of the TL to a naturally occurring or artificially constructed relevant biomolecular target whether in vitro or in vivo.
  • the polypeptide spacer as defined above can be prepared by Fmoc peptide synthesis using the appropriate side-chain protected amino acids, as normally employed for Fmoc peptide synthesis (See e.g. "Principles and Practice of Solid-Phase Synthesis Fields GB", Tian Z. and Barany G., in : “Synthetic Peptides - A Users Guide", G. A. Grant Ed. W. H. Freeman, New York, 1992. pp 83-103, 119-124 and 133-135). Similarly, if amino acid derivatives are employed (e.g.
  • the ether or acyl groups can also be protected with protecting groups known in the art for protection of amino acids bearing hydroxyl groups, amino groups or carboxyl groups, as is done for the protection of side chains in Fmoc-peptide synthesis chemistry (see e.g. : "Basic Principles", White P. D. and Chan W. C, in “Fmoc Solid Phase Synthesis - A Practical Approach", P. D. White and W. C. Chan eds. Oxford University Press, New York, 2000, pp 20- 29).
  • Carboxyl groups of such amino acids can be protected as the tert-butyl ester or the 4- [N-(l-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutylamino]-benzyl ester (see e.g. Chan W. C. et. al., J. Chem. Soc. Chem Commun. 1995, 2209), while amino groups can be protected with Dde (Bycroft B. W. et. al., 1993, J. Chem. Soc. Chem Commun., 778-9), ivDde (Chabra SR, et. al. Tetrahedron Lett.
  • orthogonal sets of protecting groups that would allow selective manipulation of the peptide or the side-chain protected peptide in the presence of the spacer, or of the spacer moiety in the presence of the peptide or the side-chain protected peptide, on or off resin, as described above for the trans-4-amino or trans-4- hydroxy proline moieties that can be employed as hydrophilic spacers, is anticipated by the specification above.
  • the compounds obtained according to any the above synthesis methods, comprising a protected spacer moiety Sp optionally combined with any of the above additional spacers, is then partially deprotected for the subsequent preparation of the Mm-Sp-TL compounds.
  • the spacer Sp does not need however to be synthesized as a separate compound.
  • any of the above illustrated methods of synthesis can alternatively be performed by introducing the Mm or TL moiety (suitably functionalized) at any step of the synthesis of the spacer, so to directly obtain an intermediate compound Mm-Sp or Sp-TL.
  • a first portion of the spacer may be synthesized by linking it to an Mm moiety; then, said first portion, suitably functionalized with a reactive moiety, is reacted with a second portion of the spacer (and optionally a third or further one, if necessary), to obtain the intermediate Mm-Sp compound.
  • an Sp-TL intermediate compound can be synthesized by reacting a first portion of the spacer linked to the TL moiety with a second portion of the spacer.
  • a first portion of the spacer, linked to the Mm moiety and suitably functionalized with a reactive moiety can be reacted with a second portion of the spacer linked to a moiety TL (optionally through a third or further portion of the spacer), and containing a corresponding reactive moiety, to obtain the Mm-Sp-TL compound.
  • the spacer can be directly bound to the moiety Mm, on one side, and to the moiety TL, on the other side.
  • said binding can be effected covalently via an amide bond, to link a carboxy (or amino) group of the spacer with and amino (or carboxy, respectively) group of the Mm or TL moiety.
  • the spacer can be covalently bound to the moiety M, on one side, and to a suitable reactive moiety (e.g. a moiety of an affinity binding pair, such as streptavidin), for non-covalently binding it with a corresponding complementary reactive moiety (e.g. biotin) linked to a moiety TL.
  • a suitable reactive moiety e.g. a moiety of an affinity binding pair, such as streptavidin
  • the spacer is bound to the moiety TL (or to a moiety of an affinity binding pair) or to the moiety Mm, through one or more linker groups.
  • said linker bound to the spacer Sp comprises one or more amino acids or a moiety of formula:
  • the linker preferably further comprises suitable binding moieties at its respective ends, for covalently binding with corresponding complementary binding moieties on the envelope-forming moiety, on one side, and with corresponding complementary binding moieties on the moiety T, on the other side.
  • reactive moieties include amino moieties (-NH-), carboxy moieties (- CO-).
  • binding moiety is an amino or a carboxy moiety.
  • linker groups are the following:
  • said linker is formed by two, equal or different, moieties defined by the above formula (III).
  • Particularly preferred examples of combined linker moieties are:
  • said linker is first bound to the targeting moiety (e.g. a peptide) and then to the spacer.
  • the targeting ligand is a peptide
  • the synthesis of the peptide comprises the incorporation of the spacer as a final portion of the peptide sequence.
  • the TL moiety of the Mm-Sp-TL compound is preferably a targeting ligand.
  • Materials or substances which may serve as targeting ligands include, for example, proteins, including antibodies, antibody fragments, receptor molecules, receptor binding molecules, glycoproteins and lectins; peptides, including oligopeptides and polypeptides; peptidomimetics; saccharides, including mono and polysaccharides; vitamins; steroids, steroid analogs, hormones, cofactors, bioactive agents including substituted small molecules (and genetic material, including nucleosides, nucleotides and polynucleotides and mimetics thereof, such as peptide nucleic acids (see e.g.
  • Suitable specific targets to which a targeted microvesicle of the invention can be directed are, for instance, fibrin and the GPIIbIIIa receptor on activated platelets. Fibrin and platelets are generally present in "thrombi", i.e. coagula which may form in the blood stream and cause a vascular obstruction. In addition, fibrin may also be associated with various tumoral processes. Preferred binding peptides specific for fibrin- targeting are disclosed, for instance, in co-pending provisional patent application U. S. S. N. 60/869,472 here incorporated by reference. Further preferred binding peptides specific for fibrin-targeting are also disclosed in International patent applications WO 01/09188 and WO 02/055544, which are also herein incorporated by reference.
  • KDR kinase insert domain receptor
  • VEGF vascular endothelial growth factor
  • binding peptides suitable for targeting KDR or VEGF/KDR complex are disclosed, for instance WO 03/74005, WO 03/084574, and WO 06/031885, as well as in the co-pending International Patent Application PCT/US2006/061793, all herein incorporated by reference.
  • tumor specific ligands are, for instance, transferrin, folic acid, arginine-glycine-aspartic acid sequence (RGD), NRG sequence (for targeting aminopeptidase expressed on newly formed vessels) or GA3 peptide sequence (target Tie2 receptor involved in tumor angiogenesis), Tuftsin-like sequences (targeting NRP-I receptor as described by von Wronski et al. J. Biol. Chem..2006; 281 : 5702-5710)
  • RGD arginine-glycine-aspartic acid sequence
  • NRG sequence for targeting aminopeptidase expressed on newly formed vessels
  • GA3 peptide sequence target Tie2 receptor involved in tumor angiogenesis
  • Tuftsin-like sequences targeting NRP-I receptor as described by von Wronski et al. J. Biol. Chem..2006; 281 : 5702-5710
  • the targeting ligand is a peptide comprising from about 10 to about 40 amino acids, more preferably from about 15 to about 30 amino acids.
  • Particularly preferred are those targeting peptides disclosed in International patent applications WO 01/91805, WO 02/055544, WO 03/74005, WO 03/084574, and WO 06/031885, as well as in co- pending International Patent Application PCT/US2006/061793, and in co-pending provisional patent application U. S. S. N. 60/869,472, including those listed in the following table 1 : Table 1 : Targeting peptides
  • the above peptides may be used with or without the -GGGK sequence at the C terminus. Furthermore, new peptide sequences illustrated in the following table 2 may also be advantageously employed.
  • peptides SeqOO ⁇ and Seq009 have been found particularly useful as binding peptides suitable for targeting fibrin, while peptides SeqOlO and SeqO l l have been found particularly useful as binding peptides suitable for targeting KDR or VEGF/KDR complex.
  • the targeting ligand is preferably bound to the spacer, directly or through a suitable linker, through a covalent bond obtained by reacting a reactive moiety on the targeting ligand with a respective reactive moiety on the spacer or linker, as previously illustrated.
  • the targeting ligand can be bound to the spacer through a non-covalent bond (e.g. biotin-streptavidin interaction).
  • said moiety TL is a therapeutic agent as above defined.
  • the Mm-Sp-TL compound can be prepared either as a separate compound to be used in the methods for preparing gas-filled microvesicles as described hereinafter, or can be formed as a final compound during the preparation of the gas-filled microvesicles.
  • an intermediate (Mm-Sp) containing a suitable reactive group can be employed for the preparation of gas-filled microvesicles, which is then reacted with the desired moiety "TL", containing a complementary reactive group, at any stage of the preparation processes illustrated hereinafter.
  • the moieties Mm, Sp and TL of the Mm-Sp-TL can be linked to each other according to conventional bond forming reactions techniques, involving the reaction of suitable complementary reactive moieties.
  • a moiety of the Mm-Sp-TL compound includes a reactive amino group
  • it can be reacted with any of the other moieties containing a suitable corresponding reactive moiety, such as an isothiocyanate group (to form a thiourea bond), a reactive ester (to form an amide bond), or an aldehyde group (to form an imine bond, which may be reduced to an alkylamine bond).
  • a suitable corresponding reactive moiety such as an isothiocyanate group (to form a thiourea bond), a reactive ester (to form an amide bond), or an aldehyde group (to form an imine bond, which may be reduced to an alkylamine bond).
  • suitable complementary reactive moieties on the other moieties may include haloacetyl derivatives, maleimides (to form a thioether bond) or a mixed disulfide comprising a sulphide in the form of a 2-pyridylthio group which upon reaction with a thiol derived from the thiol-bearing moiety results in the formation of a stable disulfide bond and the assembly of Mm-Sp-TL.
  • suitable reactive moieties on the other moieties can be amines and hydrazides (to form amide or N-acyl, N'-alkylhydrazide functions).
  • amines and hydrazides to form amide or N-acyl, N'-alkylhydrazide functions.
  • an Mm-Sp-TL compound of the invention can be synthesized by first reacting a spacer (e.g. prepared as described above, comprising a protective group and a free carboxylic group) with a suitable phospholipid (preferably a phosphatidylethanolamine PE, e.g. DPPE), to obtain a compound "PE-Sp-PG" where the phospholipid PE is linked to the spacer Sp having a protecting group PG (e.g. benzyl).
  • a spacer e.g. prepared as described above, comprising a protective group and a free carboxylic group
  • a suitable phospholipid preferably a phosphatidylethanolamine PE, e.g. DPPE
  • the reaction is preferably performed in the presence of suitable activating agents, such as, for instance carbodiimides such as DIC, DCC or EDAC, in the presence of additives such as NHS, HOBt, HOAt, or by the use of uronium coupling agents such as HBTU, HATU, or phosphonium coupling agents such as BOP or PyBOP, in the presence of a base such as DIEA, TEA, NMM, 2,6-lutidine (2,6-dimethylpyridine), sym-collidine (2,4,6- trimethylpyridine), proton sponge (1,8-bis-dimethylaminonaphthalene) or mixtures thereof, in a suitable solvent, such as, for instance, NMP, DMF, DMA, DMSO or DCM or mixtures thereof.
  • suitable activating agents such as, for instance carbodiimides such as DIC, DCC or EDAC
  • additives such as NHS, HOBt, HOAt
  • uronium coupling agents
  • the residue is washed with water, filtered and dried under vacuum and the PE-S-PG compound is preferably recrystallized in ACN.
  • the compound is then subjected to hydrogenation (e.g. Pd-C 10% in THF) to give, upon filtration and solvent removal, the deprotected compound PE-S-COOH.
  • Compound PE-S-COOH preferably in the presence of DIC and HOBT or DIC and NHS in DMF/DCM
  • TL preferably a targeting peptide
  • a peptide TL and a polyproline spacer Sp may be assembled first and the resulting intermediate appended to a Mm moiety, e.g. a phospholipid.
  • a peptide is assembled on a resin such as 2- chlorotrityl resin using Fmoc chemistry.
  • the N-terminal amino acid can bear an acid cleavable protecting group such as the Boc protecting group.
  • a C-terminal N ⁇ -ivDde lysine residue, or alternatively a C-terminal N ⁇ -aloc-lysine residue is in place.
  • the ivDde protecting group can be removed with 2 - 10% hydrazine in DMF for 10-30 min.
  • the aloe group can be removed with NMM :HOAc:DMF (1 :2: 10), Pd(PPh 3 ) 4 (1.5 equiv), for 30 min (see e.g. Albericio F, et. al. In : Peptides 1992, Schneider CH and Eberle AN, Eds. ESCOM Science Publishers B. V., Leiden pp 191-19).
  • the resulting unmasked amino group can be reacted with a Fmoc-poly(trans-4-Boc-amino)-L-proline derivative such as Fmoc-[(trans-4-Boc- amino)-L-proline] 9 -OH in the presence of a coupling agent such as DIC/HOBT or HBTU/DIEA to provide the resin-bound peptide coupled to the C-terminal acid group of Fmoc-[(trans-4- Boc-amino)-L-proline] 9 via the N ⁇ of the C-terminal lysine of the peptide.
  • the Fmoc group at the N-terminus of the spacer can be removed using 20% piperidine in DMF.
  • the [(trans-4-Boc-amino)-L-proline] 9 spacer-functionalized peptide can be cleaved from the resin using using 1% TFA in dichloromethane, acetic acid / trifluoroethanol / dichloromethane (1/2/7, v/v/v) or hexafluoroisopropanol/dichloromethane (3/7, v/v) and purified by precipitation by a suitable solvent such as diethyl ether or ethyl acetate for example, or by silica gel chromatography or HPLC (either reversed or normal phase) or a combination of these techniques as necessary.
  • a suitable solvent such as diethyl ether or ethyl acetate for example, or by silica gel chromatography or HPLC (either reversed or normal phase) or a combination of these techniques as necessary.
  • a phospholipid derivative such as DSPE- glutaric acid monoamide
  • activating agents such as DIC/NHS, DIC/HOBt, HBTU/DIEA or PyBOP/DIEA
  • the resulting phospholipid glutaric acid monoamide active ester can then be treated with the purified side chain-protected peptide TL in dichloromethane, chloroform or a mixture of dichloromethane or chloroform in DMF to provide the fully side-chain protected phospholipid-[(trans-4-Boc- amino)-L-proline] 9 -peptide TL compound.
  • the side chain protecting groups on the [(trans- 4-Boc-amino)-L-proline] 9 portion of the molecule and the targeting vector can then be removed using cleavage reagents well known in the art such as Reagent B to provide the deprotected phospholipid-[(trans-4- amino)-L-proline] 9 -peptide TL compound which can be purified by reverse phase HPLC.
  • the assembly of the compound Mm-Sp-Peptide TL can also be conducted on the resin as a solid phase synthesis.
  • the peptide TL can be assembled on a resin such as NovaSyn TGR resin and at the C-terminus of the peptide an N ⁇ -ivDde-Lysine moiety is in place.
  • the ivDde group can be removed using 2-10% hydrazine in DMF.
  • a Fmoc- polyproline spacer can be coupled to the Lysine-N ⁇ -amino group using coupling agents such as HATU, HBTU or PyBOP in DMF in the presence of DIEA.
  • the polyproline spacer can be constructed by serial coupling of Fmoc-Pro or any combination of Fmoc-Pro and Fmoc-proline derivatives bearing substituents on the proline ring.
  • Fmoc group of the terminating residue of the spacer is removed from the N-terminus of the polyproline spacer.
  • This amino group can be coupled to an Mm moiety such as DSPE-glutaric acid monoamide mono NHS ester to provide a resin-bound protected Mm-Sp-peptide TL, which then can be cleaved from the resin and side-chain deprotected using reagent B.
  • the product can then be purified by preparative HPLC to give the required compound in pure form.
  • the chain elongation of the peptide TL can be conducted using Fmoc- amino acids and coupling agents such as HBTU, HATU or PyBOP and DIEA in DMF. Then the N-terminal amino group of the resin-bound peptide TL is exposed by Fmoc removal using piperidine in DMF. The chain elongation of the polyproline spacer is conducted using Fmoc- proline derivatives and coupling reagents such as HBTU, HATU or PyBOP in the presence of DIEA in DMF.
  • the N-terminal Fmoc of the polyproline spacer is removed using piperidine in DMF. Then an active ester of an Mm moiety such as DSPE-glutaric acid monoamide mono- NHS ester is employed to append the Mm moiety to the resin-bound Sp-peptide TL. The compound is cleaved from the resin and side-chain deprotected followed by purification by preparative HPLC.
  • an active ester of an Mm moiety such as DSPE-glutaric acid monoamide mono- NHS ester
  • polyproline side chain groups can be protected as the allyloxycarbonyl or allyl ether (for the hydroxyl group) and the allyloxycarbonyl group (for the amino group).
  • allyloxycarbonyl or allyl ether for the hydroxyl group
  • allyloxycarbonyl group for the amino group
  • a fully side-chain protected poly-(trans-4-Boc-aminoproline), or poly- (trans-4-Boc-hydroxyproline), bearing an unprotected carboxyl group at its C-terminus can be activated with an activating agent such as HBTU/DIEA, HATU/DIEA, PyBOP/DIEA, DIC/HOBt, or DIC/HOAt and reacted with a corresponding reactive moiety, e.g. an amino moiety on a Mm moiety, such as phosphatidylethanolamine.
  • an activating agent such as HBTU/DIEA, HATU/DIEA, PyBOP/DIEA, DIC/HOBt, or DIC/HOAt
  • a corresponding reactive moiety e.g. an amino moiety on a Mm moiety, such as phosphatidylethanolamine.
  • the resulting intermediate can be treated with 20% piperidine in DMF to remove the Fmoc group of the proline moiety distal to the just-appended Mm moiety.
  • the free amino group can then be coupled to a protected, partially protected or unprotected targeting peptide ("peptide TL" in the following) which has been treated with disuccinimidylglutarate to effect monoacylation of a free amino group of the peptide TL to provide the glutaric acid monoamide mono-NHS ester of the peptide TL.
  • the peptide TL glutaric acid monoamide mono-NHS ester can be employed to acylate the free N-terminal amino group of the Mm- polyproline intermediate.
  • the resulting product can be treated with trifluoroacetic acid or mixtures of trifluoroacetic acid containing additives (cleavage cocktails) such as Reagent B (trifluoroacetic acid/water/phenol/triisopropylsilane, 88/5/5/2, v/v/v) (Synthesis and cleavage and subsequent coupling of protected peptides is discussed in: Barlos K and Gatos D Convergent peptide synthesis in Fmoc Solid Phase Peptide Synthesis, Chan WC and White PD Eds. Oxford University Press, New York, 2000, pp 215-228).
  • the resulting Mm- polyproline-peptide compound can then be purified by preparative HPLC.
  • a free amino group on a protected peptide TL, on a partially protected peptide TL or on an unprotected peptide TL can be reacted with a Mm-Sp intermediate (whose free amino group has been reacted with DSG to provide the glutaric acid monoamide mono NHS ester of the Mm-Sp intermediate) to provide the compound Mm-Sp-peptide TL where either or both the Sp and the peptide TL may be at various stages of protection or fully deprotected.
  • Aloe groups are removed using NMM : HOAc:DMF (1 :2: 10), Pd(PPh 3 ) 4 (1.5 equiv) for 30 min while the O-allyloxycarbonyl group or the O-allyl groups are removed using a ruthenium catalyst [[CpRu(IV)( ⁇ -C 3 H 5 )(2-quinolinecarboxylato)]PF 6 , MeOH, 30 0 C, for 0.5 hours (see e.g.Tanaka, S. and Saburi, H. J. Organomet. Chem. 2007, 692, 295-298). These reagents do not affect Fmoc, Boc or ivDde protecting groups used in peptide chemistry.
  • the moiety TL may be associated with the spacer through a non-covalent bond via physical and/or electrostatic interaction.
  • a functional moiety of an affinity binding pair can be covalently linked to the spacer moiety (directly or through a linker), while the complementary moiety of the binding pair will be linked to the moiety TL.
  • an avidin (or streptavidin) moiety having high affinity for biotin
  • the complementary biotin moiety can be incorporated into a suitable moiety T, e.g. a peptide or an antibody.
  • the biotin-labelled moiety TL will thus be non-covalently associated with the (strept)avidin- labelled Mm-Sp intermediate by means of the avidin-biotin coupling system.
  • both the Mm-Sp intermediate and the moiety TL can be provided with a biotin moiety and subsequently coupled to each other by means of avidin (which is a bifunctional component capable of bridging the two biotin moieties).
  • avidin which is a bifunctional component capable of bridging the two biotin moieties.
  • biotin/avidin coupling are also disclosed in the above cited US 6,139,819.
  • Alternative binding pairs include any of the moieties previously exemplified.
  • Gas- filled microvesicles The compound of formula Mm-Sp-TL is preferably included in a formulation of gas-filled microvesicles.
  • said compound is included in formulations of gas-filled microvesicles comprising a stabilizing envelope of an amphiphilic compound, hereinafter referred to as gas-filled microbubbles.
  • Amphiphilic components suitable for forming a stabilizing envelope of microbubbles comprise, for instance, phospholipids; lysophospholipids; fatty acids, such as palmitic acid, stearic acid, arachidonic acid or oleic acid; lipids bearing polymers, such as chitin, hyaluronic acid, polyvinylpyrrolidone or polyethylene glycol (PEG), also referred as "pegylated lipids"; lipids bearing sulfonated mono- di-, oligo- or polysaccharides; cholesterol, cholesterol sulfate or cholesterol hemisuccinate; tocopherol hemisuccinate; lipids with ether or ester-linked fatty acids; polymerized lipids; diacetyl phosphate; dicetyl phosphate; ceramides; polyoxyethylene fatty acid esters (such as polyoxyethylene fatty acid stearates), polyoxyethylene fatty alcohols, polyoxyethylene fatty alcohol
  • the above listed exemplary compounds may be employed as the main compound for forming the microbubble's envelope or as simple additives, thus being present only in minor amounts.
  • At least one of the compounds forming the microbubbles' envelope is a phospholipid, optionally in admixture with any of the other above-cited materials.
  • phospholipid is intended to encompass any amphiphilic phospholipid compound, the molecules of which are capable of forming a stabilizing film of material (typically in the form of a mono-molecular layer) at the gas-water boundary interface in the final microbubbles suspension. Accordingly, these materials are also referred to in the art as "film-forming phospholipids".
  • Amphiphilic phospholipid compounds typically contain at least one phosphate group and at least one, preferably two, lipophilic long-chain hydrocarbon groups. Examples of suitable phospholipids include esters of glycerol with one or preferably two
  • esters of phospholipids with only one residue of fatty acid are generally referred to in the art as the "lyso" forms of the phospholipid or "lysophospholipids”.
  • Fatty acids residues present in the phospholipids are in general long chain aliphatic acids, typically containing from 12 to 24 carbon atoms, preferably from 14 to 22; the aliphatic chain may contain one or more unsaturations or is preferably completely saturated.
  • suitable fatty acids included in the phospholipids are, for instance, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid.
  • saturated fatty acids such as myristic acid, palmitic acid, stearic acid and arachidic acid are employed.
  • phospholipids are phosphatidic acids, i.e. the diesters of glycerol- phosphoric acid with fatty acids; sphingolipids such as sphingomyelins, i.e. those phosphatidylcholine analogs where the residue of glycerol diester with fatty acids is replaced by a ceramide chain; cardiolipins, i.e. the esters of 1,3-diphosphatidylglycerol with a fatty acid; glycolipids such as gangliosides GMl (or GM2) or cerebrosides; glucolipids; sulfatides and glycosphingolipids.
  • the term phospholipids include either naturally occurring, semisynthetic or synthetically prepared products that can be employed either singularly or as mixtures.
  • phospholipids examples include natural lecithins (phosphatidylcholine (PC) derivatives) such as, typically, soya bean or egg yolk lecithins.
  • PC phosphatidylcholine
  • Examples of semisynthetic phospholipids are the partially or fully hydrogenated derivatives of the naturally occurring lecithins.
  • Preferred phospholipids are fatty acid diesters of phosphatidylcholine, ethylphosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol or of sphingomyelin.
  • phospholipids are, for instance, dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), diarachidoyl-phosphatidylcholine (DAPC), distearoyl-phosphatidylcholine (DSPC), dioleoyl- phosphatidylcholine (DOPC), 1,2 Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC), dipentadecanoyl- phosphatidylcholine (DPDPC), l-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC), l-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC), l-palmitoyl-2-stearoyl- phosphatidylcholine (PSPC),
  • Suitable phospholipids further include phospholipids modified by linking a hydrophilic polymer, such as polyethyleneglycol (PEG) or polypropyleneglycol (PPG), thereto.
  • modified phospholipids are phosphatidylethanolamines (PE) modified with PEG ("PE- PEGs"in brief) i.e. phosphatidylethanolamines where the hydrophilic ethanolamine moiety is linked to a PEG molecule of variable molecular weight (e.g. from 300 to 5000 daltons), such as DPPE-PEG (or DSPE-PEG, DMPE-PEG or DAPE-PEG).
  • DPPE-PEG2000 refers to DPPE having attached thereto a PEG polymer having a mean average molecular weight of about 2000.
  • phospholipids are DAPC, DSPC, DPPA, DSPA, DMPS, DPPS, DSPS and Ethyl-DSPC. Most preferred are DPPS or DSPC. Mixtures of phospholipids can also be used, such as, for instance, mixtures of DSPE,
  • DPPE, DPPC, DSPC and/or DAPC with DSPS, DPPS, DSPA, DPPA, DSPG, DPPG, Ethyl-DSPC and/or Ethyl-DPPC.
  • the phospholipid is the main component of the stabilizing envelope of microbubbles, amounting to at least 50% (w/w) of the total amount of components forming the envelope of the gas-filled microbubbles.
  • substantially the totality of the envelope i.e. at least 90% and up to 100% by weight
  • the phospholipids can conveniently be used in admixture with any of the above listed amphiphilic compounds.
  • substances such as cholesterol, ergosterol, phytosterol, sitosterol, lanosterol, tocopherol, propyl gallate or ascorbyl palmitate, fatty acids such as myristic acid, palmitic acid, stearic acid, arachidic acid and derivatives thereof or butylated hydroxytoluene and/or other non-phospholipid compounds can optionally be added to one or more of the foregoing phospholipids in proportions ranging from zero to 50% by weight, preferably up to 25%. Particularly preferred is palmitic acid.
  • the envelope of microbubbles forming a composition of the invention includes a compound bearing an overall (positive or negative) net charge.
  • Said compound can be a charged amphiphilic material, preferably a lipid or a phospholipid.
  • Examples of phospholipids bearing an overall negative charge are derivatives, in particular fatty acid di-ester derivatives, of phosphatidylserine, such as DMPS, DPPS, DSPS; of phosphatidic acid, such as DMPA, DPPA, DSPA; of phosphatidylglycerol such as DMPG, DPPG and DSPG or of phosphatidylinositol, such as DMPI, DPPI or DPPI.
  • modified phospholipids in particular PEG-modified phosphatidylethanolamines, such as DPPE-PEG or DSPE-PEG, can be used as negatively charged molecules.
  • lyso- form of the above cited phospholipids such as lysophosphatidylserine derivatives (e.g. lyso-DMPS, -DPPS or - DSPS), lysophosphatidic acid derivatives (e.g. lyso-DMPA, -DPPA or -DSPA) and lysophosphatidylglycerol derivatives (e.g. lyso-DMPG, -DPPG or -DSPG), can advantageously be used as negatively charged compounds.
  • lysophosphatidylserine derivatives e.g. lyso-DMPS, -DPPS or - DSPS
  • lysophosphatidic acid derivatives e.g. lyso-DMPA, -DPPA or -DSPA
  • lysophosphatidylglycerol derivatives e.g. lyso-DMPG, -DPPG or -DSPG
  • negatively charged compounds are bile acid salts such as cholic acid salts, deoxycholic acid salts or glycocholic acid salts; and (Ci 2 -C 24 ), preferably (Ci 4 -C 22 ) fatty acid salts such as, for instance, palmitic acid salts, stearic acid salts, l,2-dipalmitoyl-sn-3-succinylglycerol salts or l,3-dipalmitoyl-2-succinylglycerol salts.
  • bile acid salts such as cholic acid salts, deoxycholic acid salts or glycocholic acid salts
  • (Ci 2 -C 24 ) preferably (Ci 4 -C 22 ) fatty acid salts such as, for instance, palmitic acid salts, stearic acid salts, l,2-dipalmitoyl-sn-3-succinylglycerol salts or l,3-dipalmitoyl
  • the negatively charged compound is selected among DPPA, DPPS, DSPG, DSPE-PEG2000, DSPE-PEG5000 or mixtures thereof.
  • the negatively charged component is typically associated with a corresponding positive counter-ion, which can be mono- (e.g. an alkali metal or ammonium), di- (e.g. an alkaline earth metal) or tri-valent (e.g. aluminium).
  • the counter-ion is selected among alkali metal cations, such as Li + , Na + , or K + , more preferably Na + .
  • Examples of phospholipids bearing an overall positive charge are derivatives of ethylphosphatidylcholine, in particular di-esters of ethylphosphatidylcholine with fatty acids, such as l,2-distearoyl-sn-glycero-3-ethylphosphocholine (Ethyl-DSPC or DSEPC), 1,2- dipalmitoyl-sn-glycero-3-ethylphosphocholine (Ethyl-DPPC or DPEPC).
  • the negative counterion is preferably a halide ion, in particular chloride or bromide ion.
  • Examples of positively charged compounds that can be incorporated into the envelope of microbubbles are mono-, di- tri-, or tetra-alkylammonium salts with a halide counter ion (e.g. chloride or bromide) comprising at least one (Ci O -C 2O ), preferably (Ci 4 -Ci 8 ), alkyl chain, such as, for instance mono- or di-stearylammonium chloride, mono or di-hexadecylammonium chloride, dimethyldioctadecylammonium bromide (DDAB) or hexadecyltrimethylammonium bromide (CTAB).
  • a halide counter ion e.g. chloride or bromide
  • alkyl chain such as, for instance mono- or di-stearylammonium chloride, mono or di-hexadecylammonium chloride, dimethyldioctadecylammoni
  • positively charged compounds that can be incorporated into the envelope of microbubbles are tertiary or quaternary ammonium salts with a halide counter ion (e.g. chloride or bromide) comprising one or preferably two (Ci O -C 2O ), preferably (Ci 4 -Ci 8 ), acyl chains linked to the N-atom through a (C 3 -C 6 ) alkylene bridge, such as, for instance, l,2-distearoyl-3-trimethylammonium-propane (DSTAP), l,2-dipalmitoyl-3- trimethylammonium-propane (DPTAP), l,2-oleoyl-3-trimethylammonium-propane (DOTAP) or l,2-distearoyl-3-dimethylammonium-propane (DSDAP).
  • a halide counter ion e.g. chloride or bromide
  • DSEPC, DPEPC and/or DSTAP are preferably employed as positively charged compounds in the microbubble envelope.
  • the positively charged component is typically associated with a corresponding negative counter-ion, which can be mono- (e.g. halide), di- (e.g. sulphate) or tri-valent (e.g. phosphate).
  • a corresponding negative counter-ion can be mono- (e.g. halide), di- (e.g. sulphate) or tri-valent (e.g. phosphate).
  • the counter-ion is selected from among the halide ions, such as F " (fluorine), Cl “ (chlorine) or Br " (bromine).
  • the amount of charged lipid or phospholipid may vary from about 95 mol % to about 1 mol %, with respect to the total amount of lipid and phospholipid, preferably from 80 mol % to 20 mol %.
  • Preferred mixtures of neutral phospholipids and charged lipids or phospholipids are, for instance, DPPG/DSPC, DSTAP/DAPC, DPPS/DSPC, DPPS/DAPC, DPPE/DPPG, DSPA/DAPC, DSPA/DSPC and DSPG/DSPC.
  • excipients or additives may be present either in the dry formulation of the microbubbles or may be added together with the aqueous carrier used for the reconstitution thereof, without necessarily being involved (or only partially involved) in the formation of the stabilizing envelope of the microbubble.
  • excipients or additives include pH regulators, osmolality adjusters, viscosity enhancers, emulsifiers, bulking agents, etc. and may be used in conventional amounts.
  • viscosity enhancers or stabilizers are compounds selected from linear and cross-linked poly- and oligo-saccharides, sugars and hydrophilic polymers such as polyethylene glycol.
  • a lyophilization additive such as an agent with cryoprotective and/or lyoprotective effect and/or a bulking agent, for example an amino-acid such as glycine; a carbohydrate, e.g. a sugar such as sucrose, mannitol, maltose, trehalose, glucose, lactose or a cyclodextrin, or a polysaccharide such as dextran; or a polyoxyalkyleneglycol such as polyethylene glycol.
  • a lyophilization additive such as an agent with cryoprotective and/or lyoprotective effect and/or a bulking agent, for example an amino-acid such as glycine; a carbohydrate, e.g. a sugar such as sucrose, mannitol, maltose, trehalose, glucose, lactose or a cyclodextrin, or a polysaccharide such as dextran; or a polyoxyal
  • microbubbles of a composition according to the invention can be produced according to any known method in the art.
  • the manufacturing method involves the preparation of a dried powdered material comprising an amphiphilic material as indicated above, preferably by lyophilization (freeze drying) of an aqueous or organic suspension comprising said material.
  • film-forming amphiphilic compounds can be first converted into a lamellar form by any method employed for formation of liposomes.
  • an aqueous solution comprising the film forming lipids and optionally other additives (e.g. viscosity enhancers, non-film forming surfactants, electrolytes etc.) can be submitted to high-speed mechanical homogenisation or to sonication under acoustic or ultrasonic frequencies, and then freeze dried to form a free flowing powder which is then stored in the presence of a gas.
  • Optional washing steps as disclosed for instance in US 5,597,549, can be performed before freeze drying.
  • a film forming compound and a hydrophilic stabiliser e.g. polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, glycolic acid, malic acid or maltol
  • a hydrophilic stabiliser e.g. polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, glycolic acid, malic acid or maltol
  • an organic solvent e.g. tertiary butanol, 2-methyl-2-butanol or C 2 CI 4 F 2
  • a phospholipid selected among those cited above and including at least one of the above-identified charged phospholipids
  • a lyoprotecting agent such as those previously listed, in particular carbohydrates, sugar alcohols, polyglycols, polyoxyalkylene glycols and mixtures thereof
  • a water immiscible organic solvent e.g. branched or linear alkanes, alkenes, cyclo-alkanes, aromatic hydrocarbons, alkyl ethers, ketones, halogenated hydrocarbons, perfluorinated hydrocarbons or mixtures thereof
  • the emulsion can be obtained by submitting the aqueous medium and the solvent in the presence of at least one phospholipid to any appropriate emulsion-generating technique known in the art, such as, for instance, sonication, shaking, high pressure homogenization, micromixing, membrane emulsification, high speed stirring or high shear mixing.
  • a rotor-stator homogenizer can be employed, such as Polytron ® PT3000.
  • the agitation speed of the rotor-stator homogenizer can be selected depending from the components of the emulsion, the volume of the emulsion, the relative volume of organic solvent, the diameter of the vessel containing the emulsion and the desired final diameter of the microdroplets of solvent in the emulsion.
  • a micromixing technique can be employed for emulsifying the mixture, e.g. by introducing the organic solvent into the mixer through a first inlet (at a flow rate of e.g. 0.05-5 mL/min), and the aqueous phase a second inlet (e.g. at a flow rate of 2-100 mL/min).
  • the organic solvent can be introduced gradually during the emulsification step or at once before starting the emulsification step.
  • the aqueous medium can be gradually added to the water immiscible solvent during the emulsification step or at once before starting the emulsification step.
  • the phospholipid is dispersed in the aqueous medium before this latter is admixed with the organic solvent.
  • the phospholipid can be dispersed in the organic solvent or it may be separately added the aqueous-organic mixture before or during the emulsification step.
  • the so obtained microemulsion which contains microdroplets of solvent surrounded and stabilized by the phospholipid material (and optionally by other amphiphilic film-forming compounds and/or additives), is then lyophilized according to conventional techniques to obtain a lyophilized material, which is stored (e.g.
  • a further process for preparing gas-filled microbubbles comprises generating a gas microbubble dispersion by submitting an aqueous medium comprising a phospholipid (and optionally other amphiphilic film-forming compounds and/or additives) to a controlled high agitation energy (e.g. by means of a rotor stator mixer) in the presence of a desired gas and subjecting the obtained dispersion to lyophilisation to yield a dried reconstitutable product.
  • a controlled high agitation energy e.g. by means of a rotor stator mixer
  • An example of this process is given, for instance, in WO97/29782, here enclosed by reference.
  • Spray drying techniques can also be used to obtain a dried powder, reconstitutable upon contact with physiological aqueous carrier to obtain gas-filled microbubbles.
  • the dried or lyophilized product obtained with any of the above techniques will generally be in the form of a powder or a cake, and can be stored (e.g. in a vial) in contact with the desired gas.
  • the product is readily reconstitutable in a suitable physiologically acceptable aqueous liquid carrier, which is typically injectable, to form the gas-filled microbubbles, upon gentle agitation of the suspension.
  • suitable physiologically acceptable liquid carriers are sterile water, aqueous solutions such as saline (which may advantageously be balanced so that the final product for injection is not hypotonic), or solutions of one or more tonicity adjusting substances such as salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials (eg. glucose, sucrose, sorbitol, mannitol, glycerol, polyethylene glycols, propylene glycols and the like).
  • Mean dimensions and size distribution of the final reconstituted microbubbles can in general be controlled by suitable modification of the parameters of the preparation process. Different values of mean size and size distribution of a final preparation can be obtained, for instance, by selecting different envelope-stabilizing phospholipids and/or (when required by the process) by selecting different organic solvents and/or different volumes thereof (relative to the volume of aqueous phase). In addition, for the specific preparation processes disclosed in WO2004/069284 or WO97/29782, a variation of the mixing speed generally results in a variation of the mean dimensions of the final microbubble preparation (typically, the higher the mixing speeds, the smaller the obtained microbubbles).
  • the Mm-Sp-TL compound can be inserted as a component of the stabilizing envelope of the gas-filled microbubble according to conventional techniques, in any of the of the above illustrated preparation methods.
  • the Mm-Sp-TL compound can be admixed with the film-forming components of the microbubble at the initial step of the preparation process, so to be incorporated into the stabilizing envelope upon reconstitution of the freeze-dried material obtained according to any of the above preparation methods.
  • the compound can be admixed as a suitably functionalized intermediate (i.e. including any of the previously illustrated reactive moieties, e.g. Mm-Sp-maleimide) to the initial formulation, to produce a freeze-dried material containing said intermediate; the moiety T, containing a suitable complementary reactive moiety (e.g. thiol), can then be linked to the respective reactive moiety of the Mm-Sp compound already incorporated in the envelope of the reconstituted microbubbles.
  • a suitably functionalized intermediate i.e. including any of the previously illustrated reactive moieties, e.g. Mm-Sp-maleimide
  • T containing a suitable complementary reactive moiety (e.g. thiol)
  • the Mm-Sp-TL compound can also be admixed with the components of the initial mixture, undergoing to the emulsion and lyophilisation steps.
  • the Mm-Sp-TL compound can be separately prepared as a micellar suspension and subsequently added to the already formed emulsion (containing the other film-forming components), preferably under heating.
  • a functionalized Mm-Sp intermediate can alternatively be used, which can then be reacted at any step of the process (e.g. in the emulsion phase or upon reconstitution of the lyophilized compound) with a complementary reactive moiety on the "TL" component.
  • a functionalized Mm-Sp compound is added as a micellar suspension to the formed emulsion, under agitation.
  • a compound comprising the moiety TL is then added to the obtained emulsion.
  • the functionalized Mm- Sp compound in the emulsion is covalently reacted with a compound comprising a moiety of an affinity binding pair (e.g. streptavidin), to obtain an Mm-Sp compound comprising said binding moiety.
  • the emulsion is then lyophilized and subsequently reconstituted, in the presence of a gas, with an aqueous carrier, to form a suspension of gas-filled microbubbles comprising said Mm-Sp compound bound to said binding moiety.
  • These microbubbles can then be associated with a compound comprising a TL moiety and the complementary moiety of said affinity binding pair.
  • maleimide-derivatized microbubbles by incorporating 5% (w/w) of a maleimide derivative of a Mm-Sp compound in the phospholipid formulation.
  • said maleimide derivative can be added to the emulsion as a micellar suspension.
  • a solution of a mercaptoacetylated targeting peptide or of a mercaptoacetylated streptavidin (10 mg/mL in DMF) which has been incubated in deacetylation solution (50 mM sodium phosphate, 25 mM EDTA, 0.5 M hydroxylamine.HCI, pH 7.5) is added to the maleimide-activated microbubble suspension.
  • deacetylation solution 50 mM sodium phosphate, 25 mM EDTA, 0.5 M hydroxylamine.HCI, pH 7.5
  • microcapsules are those having a stabilizing envelope comprising a polymer, preferably a biodegradable polymer, or a biodegradable water-insoluble lipid (such as tripalmitine) optionally in admixture with a biodegradable polymer.
  • suitable microcapsules and of the preparation thereof are disclosed, for instance in US 5,711,933 and US 6,333,021, herein incorporated by reference in their entirety.
  • Microcapsules having a proteinaceous envelope, i.e. made of natural proteins (albumin, haemoglobin) such as those described in US-A-4,276,885 or EP-A-O 324 938 (here incorporated by reference), can also be employed.
  • Microvesicles according to the invention may contain any combination of Mm-Sp-TL compounds as above defined, e.g. compounds bearing different targeting ligands or therapeutic agent or combinations of different compounds bearing a targeting ligand and a therapeutic agent, respectively.
  • the gas may comprise, for example, air; nitrogen; oxygen; carbon dioxide; hydrogen; nitrous oxide; a noble or inert gas such as helium, argon, xenon or krypton; a radioactive gas such as Xe 133 or Kr 81 ; a hyperpolarized noble gas such as hyperpolarized helium, hyperpolarized xenon or hyperpolarized neon; a low molecular weight hydrocarbon (e.g.
  • halogenated gases preferably fluorinated gases, such as or halogenated, fluorinated or prefluorinated low molecular weight hydrocarbons (e.g. containing up to 7 carbon atoms); or a mixture of any of the foregoing.
  • a halogenated hydrocarbon preferably at least some, more preferably all, of the halogen atoms in said compound are fluorine atoms.
  • Fluorinated gases are preferred, in particular perfluorinated gases, especially in the field of ultrasound imaging.
  • Fluorinated gases include materials which contain at least one fluorine atom such as, for instance fluorinated hydrocarbons (organic compounds containing one or more carbon atoms and fluorine); sulfur hexafluoride; fluorinated, preferably perfluorinated, ketones such as perfluoroacetone; and fluorinated, preferably perfluorinated, ethers such as perfluorodiethyl ether.
  • Preferred compounds are perfluorinated gases, such as SF 6 or perfluorocarbons (perfluorinated hydrocarbons), i.e. hydrocarbons where all the hydrogen atoms are replaced by fluorine atoms, which are known to form particularly stable microbubble suspensions, as disclosed, for instance, in EP 0554 213, which is herein incorporated by reference.
  • perfluorocarbon includes saturated, unsaturated, and cyclic perfluorocarbons.
  • biocompatible, physiologically acceptable perfluorocarbons are : perfluoroalkanes, such as perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes (e.g. perfluoro-n-butane, optionally in admixture with other isomers such as perfluoro-isobutane), perfluoropentanes, perfluorohexanes or perfluoroheptanes; perfluoroalkenes, such as perfluoropropene, perfluorobutenes (e.g.
  • perfluorobut-2ene or perfluorobutadiene
  • perfluoroalkynes e.g. perfluorobut-2-yne
  • perfluorocycloalkanes e.g. perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutanes, perfluorotrimethylcyclobutanes, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentanes, perfluorocyclohexane, perfluoromethylcyclohexane and perfluorocycloheptane).
  • Preferred saturated perfluorocarbons include, for example, CF 4 , C 2 F 6 / C 3 F 8 , C 4 F 8 , C 4 F 10 , C 5 F 12 and C 6 F 12 . It may also be advantageous to use a mixture of any of the above gases in any ratio.
  • the mixture may comprise a conventional gas, such as nitrogen, air or carbon dioxide and a gas forming a stable microbubble suspension, such as sulfur hexafluoride or a perfluorocarbon as indicated above. Examples of suitable gas mixtures can be found, for instance, in WO 94/09829, which is herein incorporated by reference.
  • the amount of gas (B) can represent from about 0.5% to about 95% v/v of the total mixture, preferably from about 5% to 80%.
  • Particularly preferred gases are SF 6 , C 3 F 8 , C 4 Fi 0 or mixtures thereof, optionally in admixture with air, oxygen, nitrogen, carbon dioxide or mixtures thereof.
  • a precursor to a gaseous substance i.e. a material that is capable of being converted to a gas in vivo.
  • the gaseous precursor and the gas derived therefrom are physiologically acceptable.
  • the gaseous precursor may be pH-activated, photo-activated, temperature activated, etc.
  • certain perfluorocarbons may be used as temperature activated gaseous precursors. These perfluorocarbons, such as perfluoropentane or perfluorohexane, have a liquid/gas phase transition temperature above room temperature (or the temperature at which the agents are produced and/or stored) but below body temperature; thus, they undergo a liquid/gas phase transition and are converted to a gas within the human body.
  • the microvesicles will preferably contain a hyperpolarized noble gas such as hyperpolarized neon, hyperpolarized helium, hyperpolarized xenon, or mixtures thereof, optionally in admixture with air, CO2, oxygen, nitrogen, helium, xenon, or any of the halogenated hydrocarbons as defined above.
  • a hyperpolarized noble gas such as hyperpolarized neon, hyperpolarized helium, hyperpolarized xenon, or mixtures thereof, optionally in admixture with air, CO2, oxygen, nitrogen, helium, xenon, or any of the halogenated hydrocarbons as defined above.
  • the microvesicle will preferably contain radioactive gases such as Xe 133 or Kr 81 or mixtures thereof, optionally in admixture with air, CO 2 , oxygen, nitrogen, helium, kripton or any of the halogenated hydrocarbons as defined above.
  • radioactive gases such as Xe 133 or Kr 81 or mixtures thereof, optionally in admixture with air, CO 2 , oxygen, nitrogen, helium, kripton or any of the halogenated hydrocarbons as defined above.
  • the formulations of gas-filled microvesicles according to the invention may further comprise a therapeutic agent as above defined.
  • the therapeutic agent may be associated the microvesicle's structure by aggregation, incorporation or binding thereto, e.g. by any of the covalent or non-covalent interactions previously illustrated.
  • a contrast agent according to the invention is preferably stored in dried powdered form and as such can advantageously be packaged in a two component diagnostic and/or therapeutic kit.
  • the kit preferably comprises a first container, containing the lyophilized composition in contact with a selected microvesicle-forming gas and a second container, containing a physiologically acceptable aqueous carrier.
  • suitable carriers are water, typically sterile, pyrogen free water (to prevent as much as possible contamination in the intermediate lyophilized product), aqueous solutions such as saline (which may advantageously be balanced so that the final product for injection is not hypotonic), or aqueous solutions of one or more tonicity adjusting substances such as salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials (eg. glucose, sucrose, sorbitol, mannitol, glycerol, polyethylene glycols, propylene glycols and the like).
  • Said two component kit can include two separate containers or a dual-chamber container.
  • the container is preferably a conventional septum-sealed vial, wherein the vial containing the lyophilized residue is sealed with a septum through which the carrier liquid may be injected using an optionally pre-filled syringe.
  • the syringe used as the container of the second component is also used then for injecting the contrast agent.
  • the dual-chamber container is preferably a dual-chamber syringe and once the lyophilisate has been reconstituted and then suitably mixed or gently shaken, the container can be used directly for injecting the contrast agent.
  • the contrast agents of the present invention may be used in a variety of diagnostic and/or therapeutic imaging methods, including in particular ultrasound and magnetic resonance imaging.
  • a patient is administered an effective amount of the contrast agent (e.g. by injection) and the body part or tissue to be imaged or treated is subjected to ultrasound scanning to image or treat said body part or tissue.
  • Diagnostic imaging includes any contrast enhanced imaging of a body part or tissue, as well as any other diagnostic technique or method such as, for instance, quantification diagnostic techniques (including e.g. blood pressure, flow and/or perfusion assessment).
  • Therapeutic imaging includes within its meaning any method for the treatment of a disease in a patient which comprises the use of a contrast imaging agent (e.g. for the delivery of a therapeutic compound to a selected receptor or tissue), and which is capable of exerting or is responsible to exert a biological effect in vitro and/or in vivo.
  • Therapeutic imaging may advantageously be associated with the controlled localized destruction of the gas-filled microvesicles, e.g. by means of ultrasound waves at high acoustic pressure (typically higher than the one generally employed in non-destructive diagnostic imaging methods).
  • This controlled destruction may be used, for instance, for the treatment of blood clots (a technique also known as sonothrombolysis), optionally in combination with the release of a suitable therapeutic compound associated with the contrast agent.
  • said therapeutic imaging may include the delivery of a therapeutic compound into cells, as a result of a transient membrane permeabilization at the cellular level induced by the localized burst of the microvesicles.
  • This technique can be used, for instance, for an effective delivery of genetic material into the cells; optionally, a drug can be locally delivered in combination with genetic material, thus allowing a combined pharmaceutical/genetic therapy of the patient (e.g. in case of tumor treatment).
  • a variety of imaging techniques may be employed in ultrasound applications, for example including fundamental and harmonic B-mode imaging, pulse or phase inversion imaging and fundamental and harmonic Doppler imaging; if desired three-dimensional imaging techniques may be used.
  • diagnostic techniques entailing the destruction of gas-filled microvesicles (e.g. by means of ultrasound waves at high acoustical pressure) are also contemplated, for instance in methods for assessing blood perfusion.
  • Microvesicles according to the invention can typically be administered in a concentration of from about 0.01 to about 1.0 ⁇ l of gas per kg of patient, depending e.g. on their respective composition, the tissue or organ to be imaged and/or the chosen imaging technique. This general concentration range can of course vary depending from specific imaging applications, e.g. when signals can be observed at very low doses such as in color Doppler or power pulse inversion.
  • Possible other diagnostic imaging applications include scintigraphy, light imaging, and X-ray imaging, including X-ray phase contrast imaging.
  • Fmoc-Pal-Peg-PS resin was obtained from Applied Biosystems (Foster City, CA). 2-CI-Trt resin, Fmoc-Pro-NovaSyn-TGT resin, Fmoc-, Boc- and other side-chain protected amino acids, the pseudoproline dipeptides Fmoc-Gly-Thr( ⁇ Me ' Me pro)-OH and Fmoc-Asp(O-tBu)- Ser( ⁇ Me ' Me pro)-OH, HBTU (O-Benzotriazol-l-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate), HATU (N- ⁇ (Dimethylamino)- IH- 1,2, 3-triazolo(4,5-b) pyridine- 1- ylmethylene]-N-methylenemethanaminium hexafluorophosphate-N-oxide) and N- hydroxybenzotriazole (HOBt) were
  • Fmoc-8-amino-3,6- dioxaoctanoic acid was obtained from NeoMPS Corp (San Diego, CA) or Suven Life Sciences (Hyderabad, India).
  • DSPE-PEG2000-NH 2 ammonium salt
  • DPPE l,2dipalmitoyl-sn-glycero-3-phosphoethanolamine
  • DSG (disuccinimidylglutarate) was obtained from Pierce Chemical Co. (Rockford, IL). Fmoc-(Pro) 5 -OH was obtained from Bachem Bioscience (King of Prussia, PA) or prepared as described in the experimental procedures. Methylpyrrolidinone (NMP) and N 7 N- dimethylformamide (DMF) were purchased from Pharmco Products Inc. (Brookfield, CT), and were peptide synthesis grade or low H 2 O/amine-free Biotech grade quality. Other solvents for reactions, chromatographic purification and HPLC analyses were E. Merck Omni grade solvents from VWR Corporation (West Chester, PA). Other reagents were procured from Aldrich Chemical Co. (Milwaukee, WI) and VWR Scientific Products (Bridgeport, NJ).
  • Analytical HPLC data for peptides, spacer and linker and related synthetic intermediates were generally obtained using a Shimadzu LC-IOAT VP dual pump gradient system employing Waters XTerra MS-C18 4.6 x 50 mm column, (particle size: 5 ⁇ , pore size 12 ⁇ A) and gradient or isocratic elution systems using H 2 O (0.1% TFA) and ACN (0.1% TFA) as eluent A and eluent B respectively. Detection of compounds was accomplished using UV at 220, 230 and 254 nm.
  • Preparative HPLC purification was conducted on a Shimadzu LC-8A dual pump gradient system equipped with a Shimadzu SPD-10AV UV detector. Generally the solution containing the crude peptide was loaded onto a reversed phase C18 or C4 column, depending on the compound characteristics, using a third pump attached to the preparative Shimadzu LC-8A dual pump gradient system. After the compound was applied to the preparative HPLC column the reaction solvents, such as DMF or DMSO, were eluted from the column at low organic phase composition; then the desired product was eluted using a gradient elution of the stronger eluent into the weaker eluent.
  • the reaction solvents such as DMF or DMSO
  • Mass spectra for characterization of peptides and disulfide peptides were obtained using API-ES in negative ion mode on an Agilent LC-MSD (1100) mass spectrometer. In cases where the molecular weight of the target peptides was ca 3000, mass spectra usually exhibited values for the doubly or triply negatively charged ions rather than [M-H] " . These values were diagnostic for the desired peptides and were generally employed for selection of fractions for collection and pooling for isolation of the pure peptide during HPLC purification. In some cases mass spectral analysis of collected fractions of exhibited [M-2H]/2 + 57 or [M-2H]/2 + 114 peaks in the mass spectrum.
  • A Water (0.1% TFA), B: Acetonitrile/Methanol (1 : 1, v/v)(0.1% TFA); Elution : Initial condition : 25% B, linear gradient 25-100% over 7 min; Flow rate; 3 mL/min; Detection : UV @ 220 nm.
  • cysteine-containing peptides were prepared on PAL-Peg-PS-resin ® (1.2 g, 0.18-0.20 mmol/g) or NovaSyn-TGR resin® (0.2 mmol/g) using an ABI 433A instrument (Applied Biosystems, Foster City, CA).
  • the peptides were assembled on resin using the FastMocTM protocol usually on a 0.25 mmol scale. After chain elongation was completed, the Fmoc group was removed from the N-terminal amino acid, the resin was washed with NMP (4x).
  • Pseudoproline dipeptides Fmoc-Trp(Boc)-Thr( ⁇ Me ' Me pro)-OH, Fmoc-Asp(OtBu)- Ser( ⁇ Me ' Me pro)-OH and Fmoc-Leu-Ser( ⁇ Me ' Me pro)-OH were employed in lieu of serial coupling of the single amino acids for chain elongation where these dipeptide units occurred.
  • Trp-Thr and Asp-Ser units were located adjacent to each other a peptide sequence, only one of the pseudoproline dipeptides was employed in order to avoid the incomplete couplings which occur when two pseudoproline dipeptides are employed adjacent to each other.
  • the lysine was incorporated as the N ⁇ -ivDde derivative.
  • the ivDde group was removed with 10% hydrazine in DMF, the resin was washed with DMF and the required derivatization of the N ⁇ -nitrogen of the C-terminal lysine of the resin-bound peptide was performed, usually by manual coupling.
  • Fmoc-Pro-NovaSyn ® TGT resin (0.6 mmol/g) or Fmoc-Pro-2-CI- Trt resin (0.66 mol/g) was used to synthesize Fmoc-(Pro) 9 -OH using the above-described FastMocTM protocol on the ABI-433A peptide synthesizer or the Rainin Sonata Pilot synthesizer.
  • the penta-Gly unit was assembled by sequential coupling of Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH (with intervening removal of the Fmoc group).
  • the pseudoproline dipeptide, Fmoc-Leu- Ser( ⁇ Me ' Me pro)-OH was utilized in lieu of sequential couplings of Fmoc-Ser(tBu)-OH and Fmoc- Leu-OH to introduce the Leu-Ser units in the peptide.
  • the resin containing the ivDde-protected amino acid was treated with 10% (v/v) hydrazine in DMF (10 mL/g resin) for 10 min. The solution was drained from the resin. This procedure was repeated once and then followed by washing the resin with DMF (4x).
  • Fmoc-Adoa (2 equiv) and HATU (2 equiv) were dissolved in DMF and DIEA (4 equiv) was added to the mixture. The mixture was stirred for 1 min before transferring the activated acid to the resin. The concentration of reagents was as discussed above for standard peptide couplings. The coupling was continued for 12 h at ambient temperature. The resin was drained of the reactants and washed with DMF (4x). In cases where two Adoa units were appended to the resin, the Fmoc group of the first appended Fmoc-Adoa unit was removed (procedure C), the resin washed with DMF (4x) and followed by coupling of the second Adoa moiety.
  • Reagent B (88: 5: 5:2 - TFA:water:phenol:TIPS - v/v/wt/v), 15 mL/g resin, was added to about 1 g of the resin and the vessel was shaken for 4-6 h at ambient temperature. The resin was filtered and washed twice with TFA (5 mL/g resin). The filtrates were combined, concentrated to give a syrup which upon trituration with 20 mL of Et 2 O/g of resin gave a solid residue which was stirred for 5-15 min and then centrifuged. The supernatant was decanted and the process was repeated three times. The resulting solid was dried under high vacuum or with a stream of dry nitrogen gas.
  • the title compound was purchased from Bachem Bioscience.
  • Fmoc-Pro-2-CI-Trt resin (1 g, 1.52 mmol) was employed for the synthesis of the title peptide on the ABI-433A instrument as described in procedure A. After chain elongation was completed, the peptide was cleaved from the resin using DCM/TFA (95/5, v/v) for 45 min. The volatiles were removed by evaporation and the residue was triturated with Et 2 O (2x).
  • the peptide was made using Fmoc-Pro-NovaSyn-TGT (0.22 mmol/g) following procedure A on a ABI 433A Peptide Synthesizer. After the completion of the synthesis, the peptide was cleaved from the resin by treating with 5% TFA in DCM for 5 min at ambient temperature. The volatiles were removed on a rotary evaporator and the isolated crude peptide was purified as for compound Ib. Fractions containing the product in >98% purity were collected and freeze-dried to provide the title compound (0.15 g/g resin employed for the synthesis, 62% yield/g of resin).
  • Fmoc-Pro-2-CI-Trt resin (1 g, 0.33 mmol) was employed for chain elongation on an ABI peptide synthesizer to incorporate all of the required proline units using the FastMocTM protocol. After the last Fmoc-Pro was appended, the Fmoc- protecting group was removed and the resin was washed and transferred to a 60 mL peptide synthesizer vessel.
  • Chain elongation of the peptide was conducted on Fmoc-Pal-Peg-PS resin on a 0.2 mmol scale (procedure A) to provide Ac-W(N m -Boc)-Q(Trt)-P-C(Trt)-P-W(N m -Boc)-E(OtBu)-S(tBu)- W(N m -Boc)-T(tBu)-F-C(Trt)-W(N m -Boc)-D(OtBu)-P-GGGK(ivDde)-NH-Pal-Peg-PS resin.
  • the peptide was cleaved from the resin using reagent B (procedure G) and the crude solid peptide was subjected to disulfide cyclization (procedure H).
  • the crude mixture was diluted with water to about five-fold its volume and applied to a Waters XTerra C-18 (250 mm x 50 mm i.d.) column and purified using a linear gradient elution of ACN (0.1% TFA) into H 2 O (0.1% TFA).
  • the pure product-containing fractions were pooled, frozen and lyophilized to provide 130 mg (28% yield) of the peptide as a fluffy white solid.
  • Chain elongation of the peptide was performed using Fmoc-Lys(ivDde)-Pal-Peg-PS resin to give Ac-W(N m -Boc)-Q(Trt)-P-C(Trt)-P-W(N m -Boc)-E(OtBu)-S(tBu)-W(N m -Boc)-T(tBu)-F-C(Trt)- W(N m -Boc)-D(OtBu)-P-GGGK(ivDde)-NH-Pal-Peg-PS resin (procedure A).
  • the resulting resin was subjected to cleavage and side-chain deprotection using reagent B (40 ml_) (procedure G) to give the crude linear peptide Ac-WQPCPWESWTFCWDPGGGK(Adoa- AdOa)-NH 2 and disulfide cyclization was conducted for 48h in 10 ml_ of DMSO-H 2 O (9/1, v/v) after adjusting the pH to 8 using 100 mM aqueous N-methylglucamine (procedure H).
  • reaction mixture was diluted to 50 ml_ with water and purified on a Waters XTerra C-18 (250 mm x 50 mm i.d.) column which was then eluted with a linear gradient of ACN (0.1% TFA) into H 2 O (0.1% TFA). Pure product-containing fractions were pooled, frozen and lyophilized to provide 295 mg (21% yield) of the required peptide.
  • the ivDde group was removed (procedure E) by treatment of the resin with 10% hydrazine in DMF (6.5 ml_) for 10 min (2x) to give Ac- W(N m -Boc)-Q(Trt)-P-C(Trt)-P-A-E(OtBu)-S(tBu)-W(N m -Boc)-T(tBu)-F-C(Trt)-W(N m -Boc)- D(OtBu)-P-GGGK-NH-TGR. Then the resin was washed with DMF (4x).
  • the resin was washed with DMF (4 x 5 ml_) and the Fmoc group was removed by treatment with 20% piperidine in DMF (10 ml_, 2 x 10 min) followed by washing (4 x 10 ml_) with DMF (procedure C) to give Ac-W(N m -Boc)-Q(Trt)-P-C(Trt)-P-A-E(OtBu)-S(tBu)- W(N m -Boc)-T(tBu)-F-C(Trt)-W(N m -Boc)-D(OtBu)-P-GGGK(Adoa)-NH-TGR.
  • Fmoc-Adoa was coupled to the resin as described (vide supra) followed by removal of the Fmoc protecting group (vide supra) and washing of the resin to provide Ac-W(N m -Boc)-Q(Trt)-P- C(Trt)-P-A-E(OtBu)-S(tBu)-W(N m -Boc)-T(tBu)-F-C(Trt)-W(N m -Boc)-D(OtBu)-P-GGGK(Adoa- AdOa)-NH-TGR.
  • the ivDde group was removed using 10% hydrazine in DMF (2 x 10 min) and the resulting resin was washed with DMF (3x). Then the exposed N ⁇ 29 -amino group was reacted with Fmoc-Adoa (2.31 g, 6 mmol, 3 equiv), HOBt (810 mg, 3 mmol, 6 equiv) and DIC (757 mg, 6 mmol, 3 equiv) in DMF for 17h at ambient temperature. The reaction was complete as determined by the ninhydrin test for free amino groups.
  • the Fmoc group was removed and the resin was reacted with Fmoc-Adoa (2.31 g, 6 mmol, 3 equiv), HOBt (810 mg, 3 mmol, 6 equiv) and DIC (757 mg, 6 mmol, 3 equiv) for 1Oh at ambient temperature.
  • the resin was washed with DMF (3x) and DCM (3x) and dried under a stream of dry nitrogen.
  • the resulting resin was treated with Reagent B for 15 h, the volatiles were removed and the residue was triturated with Et 2 O and dried to provide the crude peptide as a white solid.
  • the crude peptide was dissolved in 40 ml_ Of ACN-H 2 O (1/3, v/v), filtered and applied to a Waters XTerra C-18 (250 mm x 50 mm i.d.) column.
  • the column was eluted with 25% ACN (0.1% TFA) in H 2 O (0.1% TFA) to remove the solvents from the column.
  • the column was eluted with a linear gradient of 27% ACN (0.1% TFA) into H 2 O (0.1% TFA) over 50 min at 100 mL/min with collection of 15 ml_ fractions. Pure product- containing fractions were combined, frozen and lyophilized to provide 3.1 g (43% yield) of the target peptide as a fluffy white solid.
  • the chain elongation starting with Fmoc-Arg(NO 2 )-2-CI-Trt resin was carried out using N ⁇ -Fmoc-amino acids bearing side-chain protecting groups employed for Boc chemistry using the method of procedure C on a 3.25 mmol scale. Then the Fmoc-Adoa-T(Bn)- K(Cbz)-PP-Arg(NO 2 )-2-CI-Trt resin thus obtained was treated with HOAc/PFE/DCM (1/1/8, v/v/v) (50 ml_) for 2 h at ambient temperature with agitation of the vessel.
  • Adoa-T(Bn)-K(Cbz)-PP-R(NO 2 )-OBn Adoa-T(Bn)-K(Cbz)-PP-R(NO 2 )-OBn which was carried directly to the next step.
  • the peptide Adoa-T(Bn)-K(Cbz)-PP-R(NO 2 )-OBn was dissolved in ACN (25 mL) and di- tert-butyldicarbonate (2.3 g, 10.54 mmol, 4.34 equiv) was added to the stirred solution of the peptide in ACN and the reaction mixture was stirred 15 h at ambient temperature.
  • Boc-Adoa-T(Bn)-K(Cbz)-PP-R(NO 2 )-OBn (g, 1.1 mmol) was treated with a solution of 20% TFA in DCM for 1 h. The volatiles were removed and the residue was triturated with Et 2 O to provide the TFA salt of the deprotected peptide in quantitative yield. This was neutralized by partition between DCM and saturated aqueous Na 2 CO 3 solution in a separatory funnel followed by drying (Na 2 SO 4 ) the organic phase. Removal of the volatiles gave Adoa-T(Bn)-K(Cbz)-PP- R(NO 2 )-OBn (0.122 g, 1.1 mmol) in quantitative yield.
  • the volume of the solution was adjusted to ⁇ 100 ml_ with 20% ACN-water and employing a third pump, the solution was loaded onto a reversed phase C18 preparative column (Waters, XTerra ® Prep MS C18, 10 ⁇ , 3O ⁇ A, 50 x 250 mm, flow rate 100 mL/min), which had been pre- equilibrated with 10% ACN in water (0.1% TFA).
  • a reversed phase C18 preparative column Waters, XTerra ® Prep MS C18, 10 ⁇ , 3O ⁇ A, 50 x 250 mm, flow rate 100 mL/min
  • the title peptide sequence was prepared starting with Fmoc-Pal PEG resin on a Rainin SonataTM instrument. The peptide was cleaved from resin using Reagent B (procedure G). The volatiles were removed and the crude peptide was precipitated using Et 2 O and the resulting solid was collected and dried under vacuum.
  • Disuccinimidyl glutarate (DSG, 0.28 g, 0.86 mmol, 5.06 equiv) was dissolved in stirred anhydrous dimethylformamide (2.0 mL) and diisopropylethylamine (0.11 g, 0.85 mmol, 5.0 equiv) was added in one portion.
  • Seq005(ivDde) peptide (0.50 g, 0.17 mmol) was added in portions to the stirred solution of DSG over a period of 2 min. After stirring for 30 min at ambient temperature, the solution was diluted with anhydrous ethyl acetate to about 50 mL, to precipitate intermediate NHS ester.
  • Seq006(Adoa-Adoa) peptide (0.50 g, 0.19 mmol, 1.12 eq.) was added in portions to the stirred solution over a 3 min period and the resulting mixture was stirred for 18 h.
  • the reaction was monitored by mass spectrometry; after the complete consumption of the intermediate glutaric acid monoamide mono-NHS ester was confirmed, neat hydrazine (0.1 ml_, 0.102 g, 3.19 mmol, 18.8 equiv) was added to remove the ivDde protecting group of the dimeric peptide [m/z, neg.
  • the resin was washed with DMF (5 x 20 ml_) and then treated with Nl-(tert-butoxycarbonyl)-l,3-diamino-4,7,10-trioxatridecane (0.513 g, 1.6 mmol, 4 eq), HATU (0.608 g, 1.6 mmol, 4 eq) and DIEA (0.413 g, 0.558 mL, 3.2 mmol, 8 eq) in DMF (20 ml_) for 15h.
  • the resin was washed with DMF (5 x 20 mL).
  • the peptide was cleaved from the resin using reagent B (procedure G) and the crude solid peptide was subjected to disulfide cyclization (procedure H).
  • the crude mixture was diluted with water to about five-fold its volume and applied to a Waters XTerra C-18 (250 mm x 50 mm i.d.) column and purified using a linear gradient elution of ACN (0.1% TFA) into H 2 O (0.1% TFA) as described in the procedure titled .
  • the pure product-containing fractions were pooled, frozen and lyophilized to provide 175 mg (16.2 % yield) of the peptide as a fluffy white solid.
  • the resin was washed with DMF (5 x 20 ml_) and then treated with Nl- (tert-butoxycarbonyl)-l,3-diamino-4,7, 10-trioxatridecane (0.385 g, 1.2 mmol, 3 eq), HATU (0.456 g, 1.2 mmol, 3 eq) and DIEA (0.310 g, 0.419 ml_, 2.4 mmol, 6 eq) in DMF (15 ml_) for 15h.
  • the resin was washed with DMF (5 x 20 ml_).
  • the peptide was cleaved from the resin using reagent B (procedure G) and the crude solid peptide was subjected to disulfide cyclization (procedure H).
  • the crude mixture was diluted with water to about five-fold its volume and applied to a Waters XTerra C-18 (250 mm x 50 mm i.d.) column and purified using a linear gradient elution of ACN (0.1% TFA) into H 2 O (0.1% TFA) as described in the procedure titled .
  • the pure product-containing fractions were pooled, frozen and lyophilized to provide 227.7 mg (21.3 % yield) of the peptide as a fluffy white solid.
  • the peptide was prepared on a 0.4 mmol scale using the procedure for Seq009(Ttda-Dga). Preparative HPLC purification and isolation by lyophilization provided the peptide as a white fluffy solid (164 mg, 11.31% yield).
  • the peptide was prepared on a 0.4 mmol scale using the methods described for preparation of SeqOlO. HPLC purification provided the product as a white fluffy solid (80 mg, 5.44% yield).
  • the reaction mixture was diluted with MeOH-ACN (1/1, v/v) to 20 mL and loaded onto a preparative reverse phase C-4 column (YMC, Prep C-4, lO ⁇ M, 100A, 30 x 250 mm, flow rate 30 mL/min) pre-equilibrated with a 1/1 mixture of MeOH and ACN (1/1, v/v, 0.1%TFA) (eluent B) and H 2 O (0.1%TFA) (eluent A).
  • the column was washed with this eluent until the plug of solvent (DMF) was completed (about 10 min).
  • Fmoc-(Pro) 5 -OH 250 mg g, 0.34 mmol, 1.21 equiv
  • DMF-DCM 8/2, v/v, 2 mL
  • PyBop 130 mg, 0.34 mmol, 1.21 equiv
  • DIEA 130 mg, 176 ⁇ L, 1 mmol, 3.57 equiv
  • DPPE ammonium salt 190 mg, 0.28 mmol, 1 equiv
  • FIG. 3 illustrates the preparation of the title compound.
  • Fmoc-(Pro) 9 -OH from example Ib 500 mg, 0.45 mmol, 1.05 equiv was dissolved in dry DMF-DCM (8/2, v/v, 3 mL) with stirring. PyBop (180 mg, 0.47 mmol, 1.09 equiv) and DIEA (176 ⁇ L, 130 mg, 1 mmol, 2.33 equiv) were added and the mixture was stirred for 5 min. DPPE ammonium salt (300 mg, 0.43 mmol) was added and the mixture was stirred for 15 h at ambient temperature. MeOH-ACN (1/1, v/v, 50 mL) was added to the reaction mixture and the solution was filtered to remove particulates.
  • the solution was loaded onto a preparative reverse phase C-4 column (YMC, Prep C-4, lO ⁇ M, 100A, 30 x 250 mm, flow rate 30 mL/min) pre-equilibrated with a 1/1 mixture of MeOH and ACN (1/1, v/v, 0.1%TFA) (eluent B) and H 2 O (0.1%TFA)(eluent A).
  • YMC Prep C-4, lO ⁇ M, 100A, 30 x 250 mm, flow rate 30 mL/min
  • the column was eluted with 50% B until the solvent plug was eluted.
  • Fmoc-(Pro) 9 -DPPE 250 mg, 0.14 mmol was added to piperidine (20%) in dry DMF-DCM (1/1, v/v, 2 mL) and the mixture was stirred for 20 min at ambient temperature.
  • FIG. 4 illustrates the preparation of the title compound.
  • the peptide-resin of example Id [SuO-Glut-(Pro) 9 -CI-Trt-Resin] was transferred to a round-bottomed flask and a solution of DPPE (0.92 g, 1.32 mmol, 4 equiv) in dry pyridine (20 ml_) and DIEA (0.194 g, 1.5 mmol) was added. The resulting mixture was heated at reflux for 4 h.
  • the resin was filtered while hot and washed with hot pyridine (2 x 20 ml_) and then with DCM-MeOH (1/1, v/v, 3 x 25 mL). The resin was then washed with DCM (3 x 25 mL) and dried under vacuum for 2 h. The peptide was cleaved from the resin with DCM-HOAc-PFE (8/1/1, v/v/v, 50 mL) for 2h and filtered. The resin was washed with the cleavage cocktail (2 x 20 mL); the filtrates were combined and evaporated under reduced pressure to a paste.
  • Protected polyproline intermediate Fmoc-Gly-(Pro) 9 -OH from example Ic (0.30 g, 0.26 mmol) was dissolved under stirring in anhydrous DCM (4 mL). PyBOP (0.15 g, 0.29 mmol) and DIEA (0.10 g, 0.77 mmol) were added and the mixture was stirred for 5 min. 0.65 g (0.23 mmol) of DSPE-CO-O-PEG2000-NH 2 ammonium salt (l,2-Distearoyl-sn-glycero-3- phosphoethanolamine-N-carbonyloxy-PEG2000-NH 2 ammonium salt, Avanti Lipids) were added and stirring was continued for 5 h at ambient temperature.
  • the Fmoc-protecting group of the polyproline intermediate of example Ic was removed by adding piperidine (1 mL) to the reaction mixture (total concentration of piperidine about 20%) and stirring the reaction mixture for 20 min. The volatiles were removed under vacuum and the residue was dissolved in MeOH- ACN (1 : 1, v/v) (50 ml_).
  • the elution condition was ramped to 70% MeOH-ACN (1 : 1, 10 mM NH 4 OAc) in H 2 O (10 mM NH 4 OAc) in 1 min and the column was eluted with a linear gradient of 1%/min increase in the concentration of MeOH-ACN (1 : 1, 10 mM NH 4 OAc) until the composition if the organic eluent reached 100%. Elution was continued at 100% MeOH-ACN (1 : 1, 10 mM NH 4 OAc) until the product was completely eluted from the column.
  • Protected polyproline intermediate Fmoc-Gly-(Pro) 9 -OH from example Ic (0.30 g, 0.26 mmol, 1.13 equiv) was dissolved in anhydrous DCM (5 mL) and stirred. PyBOP (0.15 g, 0.29 mmol, 1.25 equiv) and DIEA (0.10 g, 0.77 mmol, 3.39 equiv) were added and stirred for 5 min. l ⁇ -distearoyl-sn-glycero-S-phospho-ethanolamine (DSPE) (Avanti ® sample) (0.17 g, 0.23 mmol, 1.0 equiv) was added and the mixture was stirred 4 h at ambient temperature.
  • DSPE l ⁇ -distearoyl-sn-glycero-S-phospho-ethanolamine
  • the Fmoc-protecting group of the obtained product [m/z, pos. ion, 1902.0 (M + H)] was removed by adding piperidine (1 mL, 0.86 g, 10 mmol, 43 equiv) to the reaction mixture (total concentration of piperidine about 20%). After stirring the reaction mixture for 20 min, the volatiles were removed under vacuum and the residue was dissolved in MeOH-ACN (1/1, v/v) (50 mL).
  • the elution condition was ramped to 70% MeOH-ACN (1/1, v/v, 10 mM NH 4 OAc)-H 2 O (10 mM NH 4 OAc) in 1 min and the column was eluted with a linear gradient of 1%/min increase in concentration of MeOH-ACN (1/1, v/v, .10 mM NH 4 OAc) until the composition of the organic eluent was 100%. Elution was continued at 100% MeOH-ACN (1/1, v/v, 10 mM NH 4 OAc) until the product was completely eluted from the column.
  • the solid was dispersed in EtOAc and centrifuged, and this washing process was repeated twice for the complete removal of the excess DSG.
  • the solid thus obtained was dissolved in dry DMF-DCM (8/2, v/v, 2 ml_) and DIEA (0.04 g, 0.31 mmol, 7.2 equiv) was added with stirring. Then the required phospholipid [(Pro) 5 - or (PrO) 9 -DPPE] (0.038 mmol, 0.9 equiv) was added and the mixture was stirred for 24 h at room temperature.
  • the reaction mixture was diluted with 50% MeOH and ACN-H 2 O (1/1, v/v) to about 100 ml_, filtered to remove the insolubles and loaded onto a reversed phase C-4 column (YMC, Prep C-4, lO ⁇ , lOOA, 30 x 250 mm, flow rate 30 mL/min) pre-equilibrated with 1/1 mixture of MeOH and ACN (1/1, v/v, 0.1%TFA) (eluent B) and H 2 O (0.1%TFA)(eluent A). The column was washed with this eluent to effect elution of the plug of DMF.
  • YMC Prep C-4, lO ⁇ , lOOA, 30 x 250 mm, flow rate 30 mL/min
  • the reaction mixture was diluted with anhydrous EtOAc to about 50 mL; a white precipitate formed and this was pelleted by centrifugation to give the intermediate peptide-glutaric acid monoamide mono-NHS ester derivative.
  • the solid was washed twice with EtOAc and dried with a stream of dry nitrogen gas.
  • the solid was dissolved in dry DMF-DCM (8/2, v/v, 2 mL), followed by addition of DIEA (54 ⁇ L, 40 mg, 0.31 mmol, 7.2 equiv) and the mixture was stirred briefly. Then, compound MSOl (40 mg, 0.034 mmol, 0.8 equiv) was added and the mixture was stirred for 24 h at ambient temperature.
  • the reaction mixture was diluted with 50% MeOH and ACN-H 2 O (1/1, v/v) to about 100 mL and the resulting solution was loaded onto a reversed phase C4 column (YMC, Prep C 4 , lO ⁇ , 100A, 30 x 250 mm, flow rate 30 mL/min) pre-equilibrated with 1/1 mixture of MeOH and ACN (1/1, v/v 0.1%TFA) and H 2 O (0.1%TFA). Elution of the column and analysis of fractions was carried out as described in the general procedure. The pure product-containing fractions were pooled, frozen and lyophilized to provide 68 mg (64% yield) of the title compound as a colorless solid. Exam ple 20 Preparation of com pound MST02 [from MS01 and Seq001 ( Adoa-Adoa) ]
  • the reaction mixture was diluted with anhydrous EtOAc to about 50 ml_ resulting in formation of a white precipitate which was pelleted by centrifugation.
  • the precipitate (the peptide-glutaric acid monoamide mono-NHS ester) was triturated twice with EtOAc to remove the excess unreacted DSG and dried under a stream of dry nitrogen.
  • the solid thus obtained was dissolved in dry DMF-DCM (8/2, v/v, 2 mL), DIEA (54 ⁇ l_, 40 mg, 0.31 mmol, 8 equiv) was added and the mixture was stirred briefly.
  • the preparation of the title compound or [DPPE-(Pro) 9 -Glut-Seq001] is illustrated in figure 9.
  • the active ester was then precipitated with EtOAc, pelleted and washed with EtOAc to remove unreacted DSG.
  • the activated ester was then dissolved in dry DMF-DCM (8/2, v/v, 2 mL) and DIEA (0.04 g, 0.31 mmol, 8.15 equiv with respect to phospholipid) was added and the mixture was stirred briefly.
  • DIEA 0.31 mmol, 8.15 equiv with respect to phospholipid
  • the reaction mixture was diluted with 50% MeOH and ACN-H 2 O (1/1, v/v) to about 50 mL and the resulting solution was filtered and loaded onto a reverse phase C4 column (YMC, Prep C 4 , lO ⁇ , 100A, 30 x 250 mm, flow rate 30 mL/min) pre-equilibrated with 1/1 mixture Of H 2 O (0.1%TFA) and MeOH-ACN (1/1, v/v, 0.1%TFA) and purified as described for MSTOl . Pure product-containing fractions were pooled, frozen and lyophilized to give 88 mg (64% yield) of the title compound as a white solid.
  • the activated ester was then dissolved in dry DMF-DCM (8/2, v/v, 2 mL) and DIEA (40 mg, 0.31 mmol, 9 equiv) followed by brief stirring.
  • Compound MS02 from example 12 50 mg, 0.034 mmol, 0.88 equiv with respect to the active ester was added and the mixture was stirred for 24 h at ambient temperature.
  • the reaction mixture was diluted, filtered and purified as described for MSTOl to provide 83 mg (62% yield) of the desired product as a white solid.
  • the activated ester was then dissolved in dry DMF (3 mL) and DIEA (50 mg, 0.39 mmol, 4.8 equiv) followed by brief stirring.
  • Compound MS02 (80 mg, 0.05 mmol, 0.61 equiv with respect to the active ester) was added and the mixture was stirred for 18 h at ambient temperature.
  • the reaction mixture was diluted and filtered as described for MSTOl .
  • the resulting solution was applied to a Kromasil C2 reverse phase column (250 mm x 50 mm i.d.) which had been pre-equilibrated with 40% ACN- MeOH (1/1, v/v, 0.1% TFA) (eluent B) H 2 O (0.1% TFA) (eluent A).
  • the resin was washed with DMF (2 x 20 ml_) and DCM (20 ml_), re-suspended in DMF (10 ml_) and treated with Fmoc-Adoa (154 mg, 0.4 mmol), HOBt (54 mg, 0.4 mmol), DIC (51 mg, 62 ⁇ l_, 0.4 mmol) and DIEA (139 ⁇ l_, 0.8 mmol) for 4 h. The reagents were filtered off and the resin was washed with DMF (2 x 20 ml_) and DCM (20 ml_).
  • the Fmoc group was removed by treatment with 20% piperidine in DMF (2x20 ml_) (modified procedure D) and the resin was washed with DMF (2 x 20 ml_) and DCM (20 ml_). Coupling with Fmoc-Adoa and Fmoc removal were repeated.
  • the resin was re-suspended in DMF (7 ml_) and treated with 3,6,9-trioxaundecane-l,ll- dioic acid anhydride solution [prepared by the reaction of the corresponding acid (1.0 g, 0.45 mmol)) and DIC (0.56 g, 0.45 mmol) in methylene chloride (5.0 ml_) over a period of 12 hr, the solution was filtered and used directly] for 16 hr.
  • the reagents were filtered off and the resin was washed with DCM (2x 20 ml_) and DMF (2x 20 ml_), re-suspended in DCM (10 ml_) and treated with a solution of dipalmitoyl phosphatidyl ethanolamine (690 mg, 1.0 mmol), HATU (450 mg, 1.0 mmol) DIEA (400 mg) in DCM (5.0 ml_) and the mixture was allowed to shake for 26 hr (modified procedure B). The reagents were filtered off and the resin was washed with DMF (2 x 20 ml_) and DCM (2x 20 ml_) and dried.
  • the solution was diluted with water to a volume 40 ml_ and purified by reverse phase preparative HPLC (Kromasil ® Prep C 4 , 10 ⁇ , 3O ⁇ A, 20 x 250 mm, flow rate 10 mL/min) using a gradient of 50-100 % water (0.1 % TFA)/acetonitrile: MeOH (1 : 1, 0.1%TFA) over a period of 15 min.
  • the pure product-containing fractions were collected, combined and lyophilized to afford the target compound (28 mg, 10% yield) as a fluffy white solid.
  • the reaction mixture was diluted to about 50 ml_ with anhydrous ethyl acetate; this resulted in precipitation of the glutaric acid monoamide mono-NHS ester of peptide Seq007.
  • the solution was centrifuged to compact the precipitate as a colorless solid.
  • the supernatant ethyl acetate layer containing excess DSG was decanted from the compacted solid which was again dispersed in ethyl acetate, centrifuged and washed twice more to remove the remaining traces of DSG.
  • the solid intermediate ester of peptide Seq007 thus obtained was dissolved in anhydrous DMF/DCM 8:2 v/v (3.0 mL); diisopropylethylamine (0.02 g, 0.15 mmol) was added and the mixture was stirred.
  • the solid compound MS04 (0.06 g, 0.016 mmol) was dissolved in DMF (2 mL) and transferred to the stirred solution of the intermediate ester of peptide Seq007 and the resulting mixture was stirred for 24 h at ambient temperature.
  • the reaction mixture was diluted with a mixture of MeOH-ACN (1/1, v/v) and H 2 O (1/1) to about 100 mL and the insolubles were filtered.
  • the clarified solution was loaded onto a reversed phase C4 preparative column (Kromasil ® Prep C 4 , 10 ⁇ M, lOOA, 50 x 250 mm) which had been pre-equilibrated with 1/1 mixture of MeOH- ACN (1/1, v/v, 10 mM NH 4 OAc) and H 2 O (10 mM NH 4 OAc) at a flow rate of 100 mL/min. Note that the column was not eluted with the equilibrating eluent during loading of the sample. After the sample solution was loaded the column was washed with the equilibration eluent until the plug of DMF was eluted.
  • the composition of the eluent was then ramped to 70% MeOH-ACN (1/1, v/v, 10 mM NH 4 OAc)-H 2 O (10 mM NH 4 OAc) over 1 min and a linear gradient of 0.75%/min of MeOH-ACN (1/1, v/v, 10 mM NH 4 OAc) into H 2 O (10 mM NH 4 OAc) was initiated. After the composition of the eluent reached 100% of the organic eluent the elution was continued for several minutes after the product was completely eluted from the column. Fractions (15 mL) were collected using UV (220 nm) as an indicator of product elution.
  • the solid intermediate ester of peptide Seq007 obtained from the reaction of disuccinimidyl glutarate (DSG, 0.3 g, 0.15 mmol, 9.38 equiv) and dimer peptide Seq007 (0.1 g, 0.018 mmol, 1.12 equiv) in DMF in the presence of diisopropylethylamine (0.02 g, 0.15 mmol, 9.38 equiv), following the procedure described as for MST09, was dissolved in anhydrous DMF/DCM 8:2 v/v (3.0 mL); diisopropylethylamine (0.02 g, 0.15 mmol, 9.38 equiv) was added and the mixture was stirred.
  • the solid compound MS05 (0.06 g, 0.016 mmol) was dissolved in DMF (2 mL) and transferred to the stirred solution of the intermediate ester of peptide Seq007; the resulting mixture was then stirred for 24 h at ambient temperature.
  • the reaction mixture was diluted with MeOH-ACN (1/1, v/v) and H 2 O (1/1) to ⁇ 100 mL and the insolubles were filtered.
  • the clarified solution was loaded onto a C4 reversed phase preparative column (Kromasil ® Prep C 4 , 10 ⁇ M, 100A, 50 x 250 mm) which had been pre- equilibrated with 1/1 mixture of MeOH-ACN (1/1, v/v, 10 mM NH 4 OAc) and H 2 O (10 mM NH 4 OAc) at a flow rate of 100 mL/min.
  • the column was eluted as described above and the fractions were checked for purity by analytical HPLC (Column : YMC C-4, 5 ⁇ , 3O ⁇ A, 4.6 x 50 mm). Fractions containing the product in >99% purity were pooled and freeze-dried as described above to provide 0.055 g (33% yield) of the title compound as a colorless solid.
  • the title compound was prepared from SeqOl l (80 mg, 0.022 mmol) and DPPE-(Pro) 9 -Glut- NHS (50 mg, 0.028 mmol) (prepared from MS02 and DSG) and DIEA (31 mg, 0.242 mmol, 0.042 ml_, 11 equiv with respect to the peptide SeqOl l) in DCM (0.25 ml_) and DMF (0.5 ml_) at 40 0 C for 4h as for MST13.
  • Preparative HPLC purification gave the product which was isolated after lyophilization as a white fluffy solid (69 mg, 58.77% yield).
  • the obtained solutions were filled into vials with 0.8 ml_ of solution each.
  • the samples were frozen at -45°C and lyophilized.
  • the air in the headspace was replaced with a mixture of C 4 Fio/Nitrogen (50/50) and vials capped and crimped.
  • the lyophilized samples were reconstituted with 5 ml_ of H 2 O per vial.
  • micellar suspension (1.9 mg - 0.45 ⁇ mole) was prepared in 500 ⁇ l_ of distilled water at 60 0 C to obtain a micellar suspension.
  • DSPC (18mg - 22.75 ⁇ moles) and DSPG, Na salt (2 mg - 2.53 ⁇ moles) were dissolved in cyclooctane (1.6 ml_) at 80 0 C.
  • This organic phase was emulsified in PEG4000 10% solution in water (20 ml_) using a high speed homogenizer (Polytron T3000, probe diameter of 3 cm) for 1 minute at 9000 rpm.
  • micellar suspension was added to the emulsion and the resulting mixture was heated at 80 0 C for 1 hour under stirring. After cooling to room temperature (1 hour), the obtained emulsion was washed once by centrifugation (1500g/10min - Sigma centrifuge 3K10) to eliminate the excess of the phospholipid and the separated supernatant (microdroplets) was recovered and re-suspended in the initial volume of a 10% PEG 4000 aqueous solution.
  • the emulsion was sampled in DIN8R vials (lmL/vial) and then lyophilized (laboratory freeze-dryer Lyobeta-35 TELSTAR) according the following sequence. Freezing : 2h at -50 0 C
  • the lyophilisate was exposed to an atmosphere containing C 4 Fi 0 /air (50/50 by volume). The lyophilized product was then dispersed in a volume of water twice the initial one by gentle hand shaking.
  • DSPC (18 mg, 22.75 ⁇ moles), DSPG, Na salt (2 mg, 2.53 ⁇ moles) and a compound MST prepared as described above (type and amount of compound being as indicated in the following table 7) were dissolved in cyclooctane (1.6 ml_) at 80 0 C.
  • Each organic suspension was emulsified in a PEG4000 10% aqueous phase (20 ml_) using a high speed homogenizer (Polytron PT3000, probe diameter of 3 cm) for 1 minute at 8000 rpm to obtain an emulsion.
  • the resulting emulsions were heated at 80 0 C for 1 hour under stirring. After cooling at room temperature (1 hour), the emulsions were washed once by centrifugation (1500g/10min - Sigma centrifuge 3K10) to eliminate the excess of the phospholipid and the separated supernatant (microdroplets) was recovered and re-suspended in the initial volume of a 10% PEG 4000 aqueous solution.
  • the emulsions were sampled and lyophilized according to example 34.
  • Type and molar amount Type and molar amount am p l e ( ⁇ moles) of DSPE-PEG ( ⁇ moles) of MST compound
  • DSPC (16.3 mg - 20.58 ⁇ moles) and DSPA (3.7 mg - 5.15 ⁇ moles) were dissolved in cyclooctane (1.6 ml_) at 80 0 C.
  • An emulsion was prepared by adding a 10% PEG4000 aqueous solution (20 ml_) to the organic suspension, using a high speed homogenizer (Polytron T3000, probe diameter of 3 cm, 1 minute at 8000rpm).
  • micellar suspension was mixed with a corresponding emulsion prepared as above and the resulting mixtures were heated at 80 0 C for 1 hour under agitation. After cooling to room temperature (1 hour), the emulsions were washed once by centrifugation
  • the emulsions were sampled and lyophilized according to example 34. Before redispersion, the lyophilisate was exposed to an atmosphere containing
  • the resulting emulsions were heated at 80 0 C for 1 hour under stirring. After cooling at room temperature (1 hour), the emulsions were washed once by centrifugation (1500g/10min - Sigma centrifuge 3K10) to eliminate the excess of the phospholipid and the separated supernatants (microdroplets) were recovered and re-suspended in twice the initial volume of a 10% PEG 4000 aqueous solution.
  • the emulsions were sampled and lyophilized according to example 34.
  • the lyophilisates were exposed to an atmosphere containing C 4 Fio/nitrogen (35/65 by volume). The lyophilized products were then dispersed in a volume of water twice the initial one by gentle hand shaking.
  • Example 37 was repeated by using comparative compound MST06 (0.26 ⁇ moles).
  • DSPC 18.2 mg - 23.1 ⁇ moles
  • Na stearate 1.8 mg - 5.8 ⁇ moles
  • An emulsion was prepared by adding a 10% PEG4000 aqueous solution (20 ml_) to the organic suspension, using a high speed homogenizer (Polytron T3000, probe diameter of 3 cm, 1 minute at 9000rpm).
  • micellar suspension was mixed with a corresponding emulsion prepared as above and the resulting mixtures were heated at 80 0 C for 1 hour under agitation. After cooling to room temperature ( 1 hour), the obtained emulsions were washed once by centrifugation (1500g/10min - Sigma centrifuge 3K10) to eliminate the excess of phospholipids. The separated supernatants (containing emulsified microdroplets of solvent) were recovered and re-suspended with twice the initial volume of a 10% PEG4000 aqueous solution.
  • the emulsions were sampled and lyophilized according example 34.
  • the lyophilisates were exposed to an atmosphere containing C 4 Fio/nitrogen (50/50 by volume). The lyophilized products were then dispersed in a volume of water twice the initial one by gentle hand shaking.
  • the organic phase was emulsified in a PEG4000 10% aqueous phase (20 ml_) using a high speed homogenizer (Polytron PT3000, probe diameter of 3 cm) for 1 minute at 8000 rpm to obtain an emulsion.
  • a high speed homogenizer Polytron PT3000, probe diameter of 3 cm
  • the resulting emulsion was heated at 80 0 C for 1 hour under stirring. After cooling to room temperature (1 hour), the obtained emulsion was washed once by centrifugation
  • the emulsion was sampled and lyophilized according example 2a Before redispersion, the lyophilisate was exposed to an atmosphere containing
  • MST as specified in the following table 12 (2.3 mg - 0.245 ⁇ mole) was prepared in 300 ⁇ l of Tris-buffer (20 mM - pH : 7.6) at 60 0 C to obtain a micellar suspension.
  • a solution of PEG4000 10% in distilled water (20 ml_) was prepared and the aqueous suspension of micelles was added to each of the above prepared solutions.
  • DSPC 18.5 mg - 23.4 ⁇ moles
  • palmitic acid 1.5 mg - 5.85 ⁇ moles
  • cyclooctane 1.6 ml_
  • a respective organic suspension was added to each aqueous solutions using a high speed homogenizer (Polytron PT3000 - probe diameter of 3 cm, for 1 minute at 10000 rpm) to obtain an emulsion.
  • the resulting emulsions were heated at 80 0 C for 1 hour under stirring. After cooling at room temperature (1 hour), the emulsions were diluted with PEG4000 10% solution in distilled water (60 ml_ for 20 ml_ of emulsion).
  • the emulsions were sampled and lyophilized according example 34. Before redispersion, the lyophilisates were exposed to an atmosphere containing
  • Microvesicle preparations were resuspended in 50% human plasma in PBS at a concentration of about 5.5 x 10 7 microvesicles per ml_.
  • the wells were filled to the brim with the suspension, the whole plate was sealed with a piece of ParafilmR and a tape.
  • the plate was covered with a lid and all layers were fastened with two pliers. Then the plate was incubated upside down for 30 min. at RT on a horizontal surface. At the end of the incubation, the suspensions were discarded, three washes were performed with 1 ml_ of PBS.
  • Bound gas-filled microvesicles were observed using an inverted microscope and images were acquired, for the determination of the surface covered by targeted gas-filled microvesicles, with a digital camera DC300F (Leica); the percent of surface covered by bound gas-filled microvesicles in the imaged area was determined using the software QWin (Leica Microsystem AG, Basel, Switzerland).
  • Table 13 illustrates the results of the binding affinity (expressed as percent of surface covered with gas-filled microvesicles in the imaged area) of targeted microvesicles prepared according to previous examples to cross-linked fibrin, showing that the presence of a spacer according to the invention does not negatively affect the binding activity of targeting peptides in microvesicles preparations.
  • Glass coverslips (40 mm in diameter, Bioptechs Inc., Butler, PA, USA) were coated with fibrin according the following methodology.
  • the pump flow rate was adjusted to 1 mL/min to obtain the desired shear rate of about 114 s "1 .
  • the flow was stopped and pictures were taken randomly on different positions on the coverslip (on surfaces of about 0.025 mm 2 ) using a 40 x objective and a CCD monochrome camera (F-View II, Soft Imaging Systems, Germany) connected to an inverted Olympus IX 50 microscope.
  • the number of gas-filled microvesicles on each picture was determined, averaged with respect to the total number of pictures and the obtained value was then divided by ten (to obtain the "slope", i.e. the average amount of bound gas-filled microvesicles per minute).
  • the binding assay was repeated four times, thus obtaining an average value of the slope.
  • the slope represents the microvesicle binding rate on the target substrate. For instance, a slope value of 6 indicates that an average of sixty (60) gas-filled microvesicles was bound on the coated coverslip in ten minutes. A higher (or lower) slope indicates a better (or worst) capacity of microvesicles to bind to the target under flow conditions.
  • gas-filled microvesicles with targeting lipopeptides comprising a polyproline spacer according to the invention provide higher binding activity with respect to corresponding microvesicles where the targeting lipopeptide does not contain a polyproline spacer.
  • the transfected cells were incubated with KDR-targeted gas-filled microvesicles resuspended in 50% human plasma in PBS.
  • a small plastic cap was filled with a suspension containing a 1.3xlO 8 microvesicles and the cap was covered with an inverted Thermanox® coverslip so as to put the transfected cells in contact with the targeted gas-filled microvesicles.
  • the coverslip was lifted with tweezers, rinsed three times in PBS and examined under a microscope to assess binding of the targeted gas-filled microvesicles.
  • a surface of dimensions 14 x 25 mm was delimited on the glass coverslip using a special marker (Dako Pen) and 400 ⁇ l_ of Fc-VEGF-R2 solution at 4 ⁇ g/mL in PBS was deposited on this surface. After an overnight incubation at 4°C, the solution was aspirated, replaced by 0.5 ml_ of a solution of BSA 1% w/v in PBS-0.05% Tween 80, pH 7.4 and incubated for 3 hours at RT. Then the coverslip was washed three times with 5 ml_ of PBS- 0.05% Tween 80.
  • Binding assay Binding studies of targeted gas-filled microvesicles were carried out using a parallel- plate flow chamber (FCS2, Bioptech Inc., Butler, PA, USA) with a chamber gasket of 0.25 mm in thickness, with a customized adapter for upside-down chamber inversion
  • FCS2 parallel- plate flow chamber
  • the coated coverslip was inserted as a plate of the flow chamber.
  • Gas-filled microvesicles (5 x 10 6 microvesicles/mL in 50% human plasma in PBS) were drawn through the flow chamber using an adjustable infusion pump (Auto Syringe ® AS50 Infusion Pump, Baxter, Deerfield, IL, USA) with a 60 ml_ syringe (Terumo).
  • the pump flow rate was adjusted to 1 mL/min to obtain the desired shear rate of about 114 s "1 .
  • the flow was stopped and pictures were taken randomly at different positions on the coverslip (on areas of about 0.025 mm 2 ) using a 40 x objective and a CCD monochrome camera (F-View II, Soft Imaging Systems, Germany) connected to an inverted Olympus IX 50 microscope.
  • the number of gas-filled microvesicles on each picture was determined, averaged with respect to the total number of pictures and the obtained value was then divided by ten (to obtain the "slope", i.e. the average amount of bound microvesicles per minute).
  • the binding assay was repeated four times thus obtaining an average value of the slope.
  • the slope represents the microvesicle binding rate on the target substrate. For instance, a slope value of 8 indicates that an average of eighty (80) microveiscles was bound on the coated coverslip in ten minutes. A higher slope indicates a better capacity of microvesicles to bind to the target under flow conditions.
  • PC-3 Human prostate carcinoma cells (PC-3) were grown in DMEM containing 10% FCS and 1% non essential amino acids. Cultures maintained in flask were split twice a week at a ratio of 1/5 and incubated at 37°C in 10%CO 2 .
  • Binding assay was performed as described in Example 45 except that PC-3 cells were used in place of 293H-transfected cells.
  • Table 18 Static PC-3 cells binding assay
  • HBVEC Human umbilical vein endothelial cells
  • Confluent flask were detached by trypsin at RT, washed and resuspended at 1.5 10 5 cells per ml_.
  • Thermanox® circular coverslips were inserted in the wells of 24-well plate, then each well was filled with one ml_ of the cell suspension and the plate was incubated 24 or 48 hours at 37°C, 5% CO2 depending on time required to obtain confluence.
  • Binding assay was performed as described in Example 45 except that PC-3 cells were used in place of 293H-transfected cells.

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  • Radiology & Medical Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract

L'invention porte sur un composé représenté par la formule Mm-Sp-TL, dans laquelle Mm représente une fraction amphiphile capable d'être associée à une enveloppe d'une microvésicule remplie de gaz ; TL représente un ligand de ciblage ou un agent thérapeutique ; et Sp représente un espaceur, portant ladite fraction et ledit ligand de ciblage ou agent thérapeutique, qui comprend une séquence d'une pluralité d'unités proline ou de type proline. Le composé peut être utilisé pour la préparation de microvésicules remplies de gaz comprenant ledit composé, pour une utilisation en imagerie ultrasonore.
PCT/EP2008/067101 2007-12-11 2008-12-09 Composés de ciblage et thérapeutiques avec un espaceur comprenant de la polyproline et microvésicules remplies de gaz comprenant lesdits composés WO2009074569A1 (fr)

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US708807P 2007-12-11 2007-12-11
US61/007,088 2007-12-11
EP08102238.6 2008-03-04
EP08102238 2008-03-04

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US20130156706A1 (en) * 2010-08-09 2013-06-20 Bracco Suisse S.A. Targeted gas-filled microvesicles
CN104666242A (zh) * 2013-11-26 2015-06-03 信达生物制药(苏州)有限公司 一种稳定的抗TNF-α抗体制剂及其用途
CN113546183A (zh) * 2021-07-30 2021-10-26 潍坊医学院附属医院 双靶点纳米靶向肝癌超声造影剂的制备方法
CN114740105A (zh) * 2022-03-17 2022-07-12 重庆医药高等专科学校 一种脯氨酸和n-甲基脯氨酸的液相色谱分离检测方法及其应用

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130156706A1 (en) * 2010-08-09 2013-06-20 Bracco Suisse S.A. Targeted gas-filled microvesicles
US9211348B2 (en) * 2010-08-09 2015-12-15 Bracco Suisse S.A. Targeted gas-filled microvesicles
US9333273B2 (en) 2010-08-09 2016-05-10 Bracco Suisse S.A. Targeting constructs
CN104666242A (zh) * 2013-11-26 2015-06-03 信达生物制药(苏州)有限公司 一种稳定的抗TNF-α抗体制剂及其用途
CN104666242B (zh) * 2013-11-26 2018-01-02 信达生物制药(苏州)有限公司 一种稳定的抗TNF‑α抗体制剂及其用途
CN113546183A (zh) * 2021-07-30 2021-10-26 潍坊医学院附属医院 双靶点纳米靶向肝癌超声造影剂的制备方法
CN114740105A (zh) * 2022-03-17 2022-07-12 重庆医药高等专科学校 一种脯氨酸和n-甲基脯氨酸的液相色谱分离检测方法及其应用
CN114740105B (zh) * 2022-03-17 2023-11-07 重庆医药高等专科学校 一种脯氨酸和n-甲基脯氨酸的液相色谱分离检测方法及其应用

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