US20090131636A1 - Targeting vector-phospholipid conjugates - Google Patents

Targeting vector-phospholipid conjugates Download PDF

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US20090131636A1
US20090131636A1 US12/257,552 US25755208A US2009131636A1 US 20090131636 A1 US20090131636 A1 US 20090131636A1 US 25755208 A US25755208 A US 25755208A US 2009131636 A1 US2009131636 A1 US 2009131636A1
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peptide
phospholipid
tfa
gly
disulfide
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US12/257,552
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Philippe Bussat
Samir Cherkaoui
Hong (Helen) Fan
Bernard Lamy
Palaniappa Nanjappan
Radhakrishna Pillai
Sibylle Pochon
Bo Song
Rolf E. Swenson
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Bracco Suisse SA
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Bracco International BV
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Priority claimed from PCT/US2003/006731 external-priority patent/WO2003074005A2/en
Priority claimed from US10/661,156 external-priority patent/US20050100963A1/en
Application filed by Bracco International BV filed Critical Bracco International BV
Priority to US12/257,552 priority Critical patent/US20090131636A1/en
Assigned to BRACCO INTERNATIONAL B.V. reassignment BRACCO INTERNATIONAL B.V. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: POCHON, SIBYLLE, BUSSAT, PHILIPPE, CHERKAOUI, SAMIR, LAMY, BERNARD, SWENSON, ROLF E., FAN, HONG HELEN, NANJAPPAN, PALANIAPPA, PILLAI, RADHAKRISHNA, SONG, BO
Publication of US20090131636A1 publication Critical patent/US20090131636A1/en
Assigned to BRACCO SUISSE SA reassignment BRACCO SUISSE SA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BRACCO INTERNATIONAL B.V.
Priority to US14/190,860 priority patent/US9295737B2/en
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    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544—Phospholipids
    • 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
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • 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
    • 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
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to targeting vector-phospholipid conjugates and particularly targeting peptide-phospholipid conjugates, which are useful in therapeutic and diagnostic compositions and methods of preparation of the same.
  • the invention includes targeted ultrasound contrast agents, and particularly targeted microbubbles which include such targeting vector-phospholipid conjugates.
  • Angiogenesis the formation of new blood vessels, occurs not only during embryonic development and normal tissue growth and repair, but is also involved in the female reproductive cycle, establishment and maintenance of pregnancy, and repair of wounds and fractures.
  • angiogenesis that occurs in the normal individual, angiogenic events are involved in a number of pathological processes, notably tumor growth and metastasis, and other conditions in which blood vessel proliferation is increased, such as diabetic retinopathy, psoriasis and arthropathies.
  • angiogenesis is important in the transition of a tumor from hyperplastic to neoplastic growth. Consequently, inhibition of angiogenesis has become an active cancer therapy research field.
  • VEGF vascular endothelial growth factor
  • VEGF also referred to as VEGF-A
  • VEGF-A is synthesized as five different splice isoforms of 121, 145, 165, 189, and 206 amino acids.
  • VEGF 121 and VEGF 165 are the main forms produced, particularly in tumors (see Cohen et al. 1999, supra).
  • VEGF 121 lacks a basic domain encoded by exons 6 and 7 of the VEGF gene and does not bind to heparin or extracellular matrix, unlike VEGF 165 .
  • Each of the references cited in this paragraph is incorporated by reference in its entirety.
  • VEGF family members act primarily by binding to receptor tyrosine kinases.
  • receptor tyrosine kinases are glycoproteins having an extracellular domain capable of binding one or more specific growth factors, a transmembrane domain (usually an alpha helix), a juxtamembrane domain (where the receptor may be regulated, e.g., by phosphorylation), a tyrosine kinase domain (the catalytic component of the receptor), and a carboxy-terminal tail, which in many receptors is involved in recognition and binding of the substrates for the tyrosine kinase.
  • VEGFR-1 Flt-1
  • VEGFR-2 VEGFR-2
  • Flk-1 VEGFR-3
  • Flt4 Flt-1 and KDR (also known as VEGFR-2 or Flk-1, which are used interchangeably herein) have been identified as the primary high affinity VEGF receptors. While Flt-1 has higher affinity for VEGF, KDR displays more abundant endothelial cell expression (Bikfalvi et al., J. Cell. Physiol., 149: 50-59 (1991)). Moreover, KDR is thought to dominate the angiogenic response and is therefore of greater therapeutic and diagnostic interest (see Cohen et al.
  • KDR kinase domain region
  • the glycosylated form of KDR migrates on an SDS-PAGE gel with an apparent molecular weight of about 205 kDa.
  • KDR contains seven immunoglobulin-like domains in its extracellular domain, of which the first three are the most important in VEGF binding (Cohen et al. 1999, supra).
  • VEGF itself is a homodimer capable of binding to two KDR molecules simultaneously. The result is that two KDR molecules become dimerized upon binding and autophosphorylate, becoming much more active. The increased kinase activity in turn initiates a signaling pathway that mediates the KDR-specific biological effects of VEGF.
  • VEGF binding activity of KDR in vivo critical to angiogenesis, but the ability to detect KDR upregulation on endothelial cells or to detect VEGF/KDR binding complexes would be extremely beneficial in detecting or monitoring angiogenesis.
  • gas filled ultrasound contrast agents are exceptionally efficient ultrasound reflectors for echography.
  • Such ultrasound contrast agents include, for example, gas-filled microvesicles such as gas-filled microbubbles and gas filled microballoons.
  • Gas filled microbubbles are particularly preferred ultrasound contrast agents.
  • the term of “microbubble” specifically designates a gaseous bubble surrounded or stabilized by phospholipids. For instance injecting into the bloodstream of living bodies suspensions of air- or gas-filled microbubbles in a carrier liquid will strongly reinforce ultrasonic echography imaging, thus aiding in the visualization of internal anatomical structures. Imaging of vessels and internal organs can strongly help in medical diagnosis, for instance for the detection of neoplastic, cardiovascular and other diseases.
  • targeting vector compositions which exhibit high binding affinity for a desired target (such as, for example, KDR or the VEGF/KDR complex).
  • a desired target such as, for example, KDR or the VEGF/KDR complex.
  • targeting vector-phospholipid conjugates and particularly targeting peptide-phospholipid conjugates may be used to prepare targeted, gas filled ultrasound contrast agents.
  • methods for large scale production of highly purified forms of such targeting vector-phospholipid conjugates Such compositions and methods would allow for production of compositions for use in diagnostic or therapeutic applications such as, for example, precise targeting of reporter moieties, tumoricidal agents or angiogenesis inhibitors to the target site.
  • the present invention provides targeting vector-phospholipid conjugates and particularly targeting peptide-phospholipid conjugates which are useful in the preparation of gas filled ultrasound contrast agents.
  • the targeting peptide-phospholipid conjugates include targeting peptides which exhibit high KDR binding affinity and thus are useful components of contrast agents for imaging of angiogenesis processes.
  • the present invention also provides monomeric and dimeric peptide phospholipid conjugates (also referred to herein as lipopeptides) which are useful in preparing gas filled ultrasound contrast agents, and particularly in preparing ultrasound contrast agents which target KDR and may be used for imaging of angiogenesis processes.
  • monomeric and dimeric peptide phospholipid conjugates also referred to herein as lipopeptides
  • the present invention also provides methods and processes for the large scale production of highly pure monomeric and dimeric peptide phospholipid conjugates, particularly monomeric and dimeric peptide phospholipids conjugates having high KDR binding affinity.
  • the present invention also provides methods and processes for the large scale production of highly pure dimeric peptide phospholipid conjugates having minimal levels of trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • the present invention also provides methods for synthesizing monomeric peptides in high purity and the construction of peptide phospholipid conjugates from multiple peptide sub-units.
  • the present invention also provides monomeric peptides which bind KDR or the VEGF/KDR complex with high affinity, as well as methods of synthesizing and using such monomeric peptides.
  • the present invention also provides targeted ultrasound contrast agents prepared from such targeting vector-phospholipid conjugates.
  • targeted ultrasound contrast agents are useful for imaging target-bearing tissue.
  • the targeted ultrasound contrast agents are targeted microbubbles and the targeting vector-phospholipid conjugates include targeting peptides which exhibit high KDR binding affinity and thus are useful components of contrast agents for imaging KDR-bearing tissue and particularly for imaging of tumors and angiogenesis processes. Methods of preparing and using such targeted ultrasound contrast agents are also provided.
  • FIG. 1 illustrates a method for the production of a monomeric peptide phospholipid conjugate (1) from a linear peptide monomer (2).
  • FIG. 2 illustrates a monomeric peptide phospholipid conjugate (1) including a peptide with high binding affinity for KDR.
  • FIG. 3 illustrates a method for the production of a precursor dimer peptide (16) from peptide monomers.
  • FIG. 4 illustrates a method for the conjugation of the precursor dimer peptide shown in FIG. 1 to DSPE-PEG2000-NH 2 to form a dimeric peptide phospholipid conjugate (11) containing peptides which bind with high affinity to KDR.
  • FIG. 5 illustrates a dimeric peptide-phospholipid conjugate (11) containing peptides which bind with high affinity to KDR.
  • FIG. 6 illustrates a method for the production of dimer peptide-phospholipid conjugates (such as (21)) having minimal levels of TFA.
  • FIG. 7 illustrates another method for the production of dimer peptide-phospholipid conjugates (such as (21)) having minimal levels of TFA.
  • FIG. 8 illustrates another method for the production of dimer peptide-phospholipid conjugates having minimal levels of TFA.
  • FIG. 9 illustrates another representative monomeric peptide (32) having a high binding affinity for KDR.
  • FIG. 10 illustrates another monomeric peptide-phospholipid conjugate (31) which includes the monomeric peptide shown in FIG. 9 .
  • FIGS. 11A-C show images obtained by using the dimer peptide-phospholipid conjugate (11) (shown in FIG. 52 ) in a contrast agent at: 1) baseline ( FIG. 11A ); 2) after 25 minutes ( FIG. 11B ); and 3) after subtraction of the baseline and free circulating bubbles ( FIG. 11C ).
  • FIGS. 12A-C show images obtained by using the monomeric phospholipid peptide conjugate (1) (shown in FIG. 2 ) in a contrast agent at baseline ( FIG. 12A ); after 25 minutes ( FIG. 12B ); and after subtraction of the baseline and free circulating bubbles ( FIG. 12C ).
  • Applicants have unexpectedly discovered peptide phospholipid conjugates, which are useful in producing targeted ultrasound contrast agents and which have exceptional KDR binding efficiency.
  • Two of these compounds are monomeric peptide phospholipid conjugates which include a linear peptide monomer which binds with high affinity to KDR while the other is a dimeric peptide phospholipid conjugate which includes two distinct monomer subunits, each binding to KDR.
  • highly efficient methods for large scale production of purified forms of these conjugates and precursor materials have been discovered. Such methods include the production of dimeric peptide phospholipid conjugates having minimal levels of TFA.
  • the phospholipid may be selected from the group consisting of: phosphatidylethanolamines and modified phosphatidylethanolamines Particularly preferred phospholipids include phosphatidylethanolamines modified by linking a hydrophilic polymer thereto.
  • modified phosphatidylethanolamines are phosphatidylethanolamines (PE) modified with polyethylenglycol (PEG), in brief “PE-PEGs”, i.e. phosphatidylethanolamines where the hydrophilic ethanolamine moiety is linked to a PEG molecule of variable molecular weight (e.g.
  • a salt form of the phospholipid may be used, such as, for example, the trimethyl ammonium salt, the tetramethylammonium salt, the triethylammonium salt, sodium salt, etc.
  • gas filled ultrasound contrast agents such as, for example, gas filled microbubbles to form contrast agents that provide excellent imaging of target-bearing tissue.
  • gas filled ultrasound contrast agents such as, for example, gas filled microbubbles
  • targeting vector-phospholipid conjugates which include targeting peptides which bind with high affinity to KDR are incorporated into targeted microbubbles. As shown herein, such targeted microbubbles selectively localize at KDR-bearing tissue, permitting imaging of such tissue, and, in particular imaging of tumors and angiogenic processes, including those processes associated with neoplastic development.
  • Table 1 provides a description for the identification labels shown in FIGS. 1 , 2 , 9 and 10 .
  • This conjugate is also referred to as Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH 2 (SEQ ID NO. 1) and Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Lys(DSPE-PEG2000-NH-Glut)-NH 2 .
  • FIG. 10 provides the structure of another monomeric peptide phospholipid conjugate (31), N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-glycyl- ⁇ N6-[1,2-d]stearoyl-sn-glycero-3-phosphoethanolamin
  • This conjugate is also referred to as Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH 2 (SEQ ID NO. 4) and Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Lys(DSPE-PEG2000-NH-Glut)-NH 2 . As shown in FIG.
  • the conjugate comprises a 28 amino acid linear peptide monomer (32), N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide, which is also referred to as Ac-AQDWYYDEILSMADQLRHAFLSGGG
  • ultrasound contrast agents such as gas filled microbubbles formulated with the monomeric peptide phospholipid conjugates (1) and (31) displayed high KDR binding which was confirmed using echographic examination of VX2 tumors in rabbits.
  • the linear peptide monomer (2) or (32) should be prepared in bulk. Then conjugation of the purified linear peptide monomer (2) or (32) to the phospholipid, such as, for example, a pegylated phospholipid in salt form, e.g., DSPE-PEG2000-NH 2 phospholipid ammonium salt (4) via the linker disuccinimidyl glutarate (DSG), may be used to provide monomeric peptide phospholipid conjugates (1) or (31).
  • a pegylated phospholipid in salt form e.g., DSPE-PEG2000-NH 2 phospholipid ammonium salt (4)
  • DSG linker disuccinimidyl glutarate
  • methods according to the present invention provide at least the following advantages: increased yield of peptide synthesis; reduced extent of racemization; avoidance of previously observed piperidine amide formation during synthesis, efficient purification of peptide monomers (2) and (32), development of a procedure for conjugation of peptide monomers (2) and (32) on larger scale; and development of purification protocols that would allow the ready separation of the monomeric peptide phospholipid conjugates (1) and (31) from the starting DSPE-PEG2000-NH 2 phospholipid ammonium salt (4).
  • Monomeric peptide phospholipid conjugates may be prepared as described below. It should be appreciated that the numerical values referred to in this representative description of the synthesis of monomeric peptide phospholipid conjugates are representative.
  • Linear peptide monomers may be prepared by SPPS.
  • the sequence of the linear peptide monomers may be constructed as a C-terminal carboxamide on Pal-Peg-PS-resin (substitution level: 0.2 mmol/g).
  • Peptide synthesis may be accomplished using Fmoc chemistry on a SONATA®/Pilot Peptide Synthesizer. Problems previously observed with this process have been racemization, incomplete couplings and piperidine amide formation, each of which contribute to suboptimal yield and purity. A dramatic decrease in the formation of the piperidine amide may be attained by the use of 25% piperidine in DMF containing HOBt (0.1M) as the reagent for Fmoc removal.
  • Racemization may be considerably reduced by using DIC/HOBt as the activator for most couplings; a 3 h coupling time using a four-fold excess of pre-activated Fmoc-amino acid with an intervening wash with anhydrous DMF (6 ⁇ ).
  • N′-Fmoc amino acids may be dissolved just before their coupling turn and pre-activated with DIC/HOBt in DMF for 4 min and transferred to the reaction vessel. This may be accomplished on the Sonata instrument by loading the solid Fmoc-amino acids into the amino acid vessels of the instrument and then programming the instrument to add DMF, HOBt/DMF and DIC/DMF sequentially with bubbling of the solution.
  • Additional optimization may be accomplished by reducing the number of couplings by using Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, in lieu of serial coupling of Fmoc-Gly-OH.
  • Activation of -Gly-Gly-OH segments may lead to cyclization of the activated acid function with the distal amide function to produce an inactive diketopiperazine; this may reduce coupling yields in a time dependant manner.
  • the Fmoc may be removed from the N-terminus.
  • the peptide and the free amino group may be acetylated.
  • the peptide sequence may be cleaved from the resin and deprotected using “Reagent B” (TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) for 4 h.
  • Reagent B TAA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v
  • the crude peptide may be isolated as a solid by evaporation of the volatiles, trituration of the residue with diethyl ether and washing of the solid thus obtained using the same solvent.
  • the peptide may be precipitated from the reaction mixture by addition of diethyl ether to the reaction mixture, collecting the solid thus formed and washing with the same solvent.
  • Linear peptide monomers may be purified as described below. Again, the numerical references are representative. Crude linear peptide monomers (0.5 g) may be dissolved in CH 3 CN (40 mL/g) and this solution may be diluted to a final volume of 100 mL with water. The solution may then be filtered. The filtered solution may be loaded onto the preparative HPLC column (Waters, XTerra® Prep MS C18, 10 ⁇ , 300 ⁇ , 50 ⁇ 250 mm) equilibrated with 10% CH 3 CN in water (0.1% TFA).
  • the composition of the eluent may then be ramped to 20% CH 3 CN-water (0.1% TFA) over 1 min, and a linear gradient may be initiated at a rate of 0.6%/min of CH 3 CN (0.1% TFA) into water (0.1% TFA) and run for 50 min. Eluted fractions may be checked for purity on an analytical reversed phase C18 column (Waters XTerra MS-C18, 10 ⁇ , 120 ⁇ , 4.6 ⁇ 50 mm) and fractions containing the product in >95% purity may be combined and freeze-dried. For each purification of 0.5 g of crude peptide 0.12 g (24%) of linear peptide monomer may be consistently isolated and will provide the peptide in the same yield and purity.
  • Synthesis of monomeric peptide phospholipid conjugates may be performed as described below. The numerical references are again representative.
  • the last step in the synthesis may be the conjugation of the phospholipid, such as, for example, a pegylated phospholipid such as DSPE-PEG2000-NH 2 phospholipid ammonium salt to a linear peptide monomer.
  • the PEG2000 moiety of DSPE-PEG2000-NH 2 phospholipid ammonium salt (4) is nominally comprised of 45 ethylene glycol units. It should be understood, however, that this material is a distribution of PEG containing species whose centroid is the nominal compound containing 45 ethylenoxy units.
  • the conjugation of a linear peptide monomer with DSPE-PEG2000-NH 2 phospholipid ammonium salt may be accomplished by preparation of the glutaric acid monoamide mono NHS ester of a linear peptide monomer and reaction of this with the free amino group of the phospholipid ammonium salt.
  • a linear peptide monomer may be reacted with DSG (4 eq.) in DMF in the presence of DIEA (5 eq.) for 30 min.
  • the reaction mixture may be diluted with ethyl acetate, which may result in precipitation of the peptide glutaric acid monoamide mono-NHS ester.
  • the supernatant containing un-reacted DSG may be decanted and the intermediate peptide mono-NHS ester may be washed several times with ethyl acetate to remove traces of DSG.
  • Mass spectral data confirms the formation of the peptide mono-NHS ester as a clean product.
  • the solid mono-NHS ester may be dissolved in DMF and reacted with DSPE-PEG2000-NH 2 phospholipid ammonium salt (0.9 eq.) in the presence of DIEA (4 eq.) for 24 h.
  • linear peptide monomer glutaric acid monoamide mono-NHS ester may be used in excess to maximize the consumption of the phospholipid ammonium salt because free phospholipid ammonium salt may complicate the isolation of monomeric peptide phospholipid conjugates in highly pure form.
  • the reaction mixture may be diluted with a 1:1 mixture of water (0.1% TFA) and CH 3 CN—CH 3 OH (1:1, v/v) (0.1% TFA) (100 mL), applied to a reversed phase C2 column (Kromasil® Prep C2, 10 ⁇ , 300 ⁇ , 50 ⁇ 250 mm, flow rate 100 mL/min) and the column may be eluted with a 3:1 mixture of water (0.1% TFA) and CH 3 CN—CH 3 OH (1:1, v/v) (0.1% TFA) to remove hydrophilic impurities.
  • the product may be eluted using a gradient of CH 3 CN—CH 3 OH (1:1) (0.1% TFA) into water (0.1% TFA) (see Experimental Section for details).
  • the collected fractions may be analyzed by reversed phase HPLC using an ELS detector which allows the detection of the desired product and the often difficult-to-separate DSPE-PEG2000-NH 2 phospholipid which has very little UV absorbance. This indicates the clear separation of the monomeric peptide phospholipid conjugates and DSPE-PEG2000-NH 2 phospholipid.
  • the pure product-containing fractions may be collected, concentrated on a rotary evaporator (to reduce the content of methanol) and freeze-dried to provide monomeric peptide phospholipid conjugates as a colorless solid.
  • a rotary evaporator to reduce the content of methanol
  • several runs may be conducted employing 0.5 g to 1.0 g of linear peptide monomer.
  • the target monomeric peptide phospholipid conjugates may be were isolated in high yield and purity (e.g., 57-60% yield and >99% purity).
  • Table 2 provides a description for the identification labels shown in FIGS. 3 , 4 and 5 .
  • the dimeric peptide phospholipid conjugate (11) Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-1-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-L
  • Ultrasound contrast agents e.g. gas filled microbubbles
  • the dimeric peptide phospholipid conjugate (11) displayed high KDR binding which was confirmed using echographic examination of VX2 tumors in rabbits.
  • the monomers used for this purpose optimally should be prepared in bulk. Then the monomers may be tethered to each other using di-succinimidyl glutarate as a linker to form the precursor dimer peptide (16), Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-L-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl-L-valy
  • methods according to the present invention provide at least the following advantages: increased yield of automated chain elongation of the peptide sequences; reduced extent of racemization encountered during synthesis; avoidance of previously observed piperidine amide formation during synthesis of peptide monomer (13); cyclization of linear di-cysteine containing peptide precursors of (12) and (13) using procedures amenable to multigram scale yet allowing efficient and practical sample handling; efficient purification of monomer peptides (12) and (13); maximized yield and purity of precursor dimer peptide (16); development of a procedure for conjugation of the precursor dimer peptide (16) on larger scale; and development of purification protocols that would allow the ready separation of the target dimeric peptide phospholipid conjugate (11) from phospholipid ammonium salt (18).
  • the dimeric peptide phospholipid conjugate (11) may be prepared by automated synthesis of the peptide monomers (12), Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH 2 cyclic (6-13) disulfide, and (13), Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH 2 cyclic (2-12) disulfide, their efficient coupling using disuccinimidyl glutarate (DSG) to give an ivDde-protected dimer, its deprotect
  • monomer peptides may be synthesized on a 10 mmol scale without complication and after HPLC purification may be obtained in about 20% yield and >95% purity.
  • Such methods allow dimer formation reactions and the subsequent conjugation to the phospholipid component providing formation of dimeric peptide phospholipid conjugate (11) to be carried out on a gram scale.
  • the precursor dimer peptide (16) may be obtained from the monomer peptides routinely in about 32% yield and >95% purity.
  • the dimeric peptide phospholipid conjugate (11) may be produced from the precursor dimer peptide (16) in 57-60% yield and >99% purity.
  • Dimeric peptide phospholipid conjugates may be prepared as described below. It should be appreciated that the numerical values referred to in this representative description of the synthesis of dimeric peptide phospholipid conjugates are representative.
  • a representative method for the solid phase synthesis and disulfide cyclization of a peptide monomer (12) Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH 2 cyclic (6-13) disulfide, and a peptide monomer (13), Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH 2 cyclic (2-12) disulfide.
  • the peptides may be constructed as their C-terminal carboxamides on Pal-Peg-PS-resin (substitution level: 0.2 mmol/g). Chain elongation may be accomplished using Fmoc chemistry employing optimized deprotection and coupling protocols on a SONATA®/Pilot Peptide Synthesizer on a 10 mmol synthesis scale.
  • the optimized synthesis of the peptides by automated SPSS may be developed by study of peptide impurities and the effect of changes of particular elements of the protocols on the overall yield and purity of the peptides obtained.
  • N- ⁇ umlaut over ( ⁇ ) ⁇ Fmoc amino acids may be dissolved just before their coupling turn and pre-activated with DIC/HOBt in DMF for 4 min and transferred to the reaction vessel. This may be accomplished on the Sonata instrument by loading the solid Fmoc-amino acids into the amino acid vessels of the instrument and then programming the instrument to add DMF, HOBt/DMF and DIC/DMF sequentially with bubbling of the solution after each addition.
  • the pseudo-orthogonally protected derivative, Fmoc-Lys(ivDde)-OH may be used to enable the selective unmasking of the ⁇ -amine of the C-terminal lysine of the monomer and dimer peptides and their subsequent functionalization, which also may be optimized.
  • the ivDde group on the ⁇ -amine of the C-terminal lysine of each of the peptide monomers may be removed using 10% hydrazine in DMF.
  • Fmoc-Adoa for monomer (13) or Lys(ivDde) for monomer (12) may be appended to the exposed lysine ⁇ -amino group using 4 equivalents of the Fmoc amino acid and 4 equivalents each of DIC and HOBt in DMF for 10 h.
  • the peptide sequence may be cleaved from the resin and deprotected using “Reagent B” (TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) for 4 h. After the cleavage reaction was complete the peptide may be precipitated, washed with diethyl ether and dried.
  • the following procedures for cyclization of the linear di-cysteine containing peptides may be used to provide optimal scale-up of monomer peptides.
  • the aerial oxidation of linear di-cysteine peptides may be carried out at a concentration of approximately 0.5-5 mg/mL (for the disclosed peptide monomers ⁇ 0.18-1.8 mM in peptide, ⁇ 0.36-3.6 mM in cysteine thiol).
  • DMSO-assisted cyclization of di-cysteine peptides allows the cyclization of ⁇ 10 g of the linear peptides in good yields in as little as ⁇ 50 mL of solution.
  • the crude linear di-cysteine peptides may be cyclized in 95% DMSO-H 2 O (5 mL/g) at pH 8.5 at ambient temperature.
  • the progress of the cyclization may be routinely followed by mass spectroscopy and HPLC.
  • cyclization may be essentially complete in ⁇ 36 h, the reaction mixture may be generally stirred for up to 48 h.
  • the cyclic disulfide peptides may be precipitated from the reaction mixture by dilution with CH 3 CN and the resulting off-white crude solid peptides may be collected by filtration. This is a convenient method for removing DMSO from the crude cyclic peptide.
  • C* refers to a cysteine residue that contributes to a disulfide bond.
  • Attempts to dissolve 0.5 g of the crude peptide in up to 300 mL of 30% CH 3 CN in water (0.1% TFA) have been unsuccessful. Therefore, as an alternative, the crude peptide, (0.5 g) may be dissolved in DMSO (5 mL/g) and this solution may be diluted to a final volume of 100 mL with 20% CH 3 CN-water. The solution may be filtered.
  • the filtered solution may be loaded onto the preparative HPLC column (Waters, XTerra® Prep MS C18, 10 ⁇ , 300 ⁇ , 50 ⁇ 250 mm) equilibrated with 10% CH 3 CN (0.1% TFA) in water (0.1% TFA), and the column may be eluted with 10% CH 3 CN (0.1% TFA) in water (0.1% TFA) to wash DMSO from the column.
  • the composition of the eluent then may be ramped to 35% CH 3 CN-water (01% TFA) over 1 min, and a linear gradient may be initiated at a rate of 0.5%/min of CH 3 CN (0.1% TFA) into water (0.1% TFA) and run for 50 min.
  • Eluted fractions may be checked for purity on an analytical reversed phase C18 column (Waters XTerra MS-C18, 10 ⁇ , 120 ⁇ , 4.6 ⁇ 50 mm) and fractions containing the product in >95% purity may be combined and freeze-dried.
  • an analytical reversed phase C18 column Waters XTerra MS-C18, 10 ⁇ , 120 ⁇ , 4.6 ⁇ 50 mm
  • fractions containing the product in >95% purity may be combined and freeze-dried.
  • the peptide monomer (13), Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH 2 may be purified and isolated as described for peptide monomer (12) except that the subject peptide may be dissolved in 20% CH 3 CN (0.1% TFA) in 0.1% aqueous TFA (0.5 g peptide/100 mL) instead of a DMSO-containing diluent.
  • the resulting solution of crude peptide may be loaded onto the preparative HPLC column (Waters, XTerra® Prep MS C18, 10 ⁇ , 300 ⁇ , 50 ⁇ 250 mm, flow rate 100 mL/min) equilibrated with 10% CH 3 CN in water (0.1% TFA).
  • the column may be eluted with 10% CH 3 CN (0.1% TFA)/water (0.1% TFA) at 100 mL/min for 5 min. Then the composition of the eluent may be ramped to 30% CH 3 CN (0.1% TFA)/water (0.1% TFA) over 1 min and a linear gradient rate of 0.5%/min of CH 3 CN (0.1% TFA) into water (0.1% TFA) may be initiated, and maintained until the desired peptide is completely eluted from the column.
  • Product-containing fractions may be analyzed on a Waters XTerra analytical reversed phase C-18 column (10 ⁇ , 120 ⁇ ) and fractions containing the product in >95% purity may be pooled and freeze-dried to afford the cyclic disulfide peptide monomer (13) (0.12 g, 24% yield) in >95% purity.
  • the 10 g of crude peptide monomer may be purified serially in this manner.
  • Described below is a representative method for preparing the precursor dimer peptide (16), Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH 2 cyclic (2-12) disulfide]-NH 2 cyclic (6-13) disulfide.
  • the preparation of the precursor dimer peptide may be accomplished by the tethering of the monomer peptides in a two step procedure.
  • Ac-AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDde)]-NH 2 (12) may be reacted with disuccinimidyl glutarate (DSG, 5 eq.) in DMF in the presence of DIEA (5 eq.) for 30 min.
  • the reaction mixture may be diluted with ethyl acetate, which results in precipitation of the glutaric acid monoamide mono-NHS ester of the peptide.
  • the supernatant, containing unreacted DSG, may be decanted and the mono-NHS ester may be washed several times with ethyl acetate to remove traces of DSG.
  • Mass spectral data confirms the formation of the mono-NHS ester as a clean product. This may be redissolved in DMF and reacted with monomer peptide Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH 2 (13) in the presence of DIEA (5 eq). HPLC and MS results indicate the formation of the ivDde-bearing dimer, as a single major product. The ivDde protecting group on the ⁇ -amine of Lys of the dimer may be removed by stirring the reaction mixture with hydrazine (10%) in DMF for 20 min.
  • the solution then may be acidified with TFA and diluted with 10% CH 3 CN (0.1% TFA)-water (0.1% TFA), applied to a preparative reversed phase C18 HPLC column and purified by a gradient elution of acetonitrile (0.1% TFA) into 0.1% aqueous TFA.
  • the reaction may be conducted employing from 0.5 g to as much as 1 g of each of the monomer peptides. In every case the required precursor dimer peptide may be isolated in ⁇ 32% yield and >95% purity confirming the reproducibility and scalability of the procedures.
  • the final step in the synthesis may be the conjugation of DSPE-PEG2000-NH 2 phospholipid ammonium salt (18) to the precursor dimer peptide.
  • the PEG2000 moiety of DSPE-PEG2000-NH 2 is nominally comprised of 45 ethylene glycol units. It should be understood, however, that this material is a distribution of PEG containing species whose centroid is the nominal compound containing 45 ethylenoxy units.
  • Conjugation of the DSPE-PEG2000-NH 2 to the precursor dimer peptide may be accomplished by preparation of a glutaric acid monoamide mono NHS ester of the precursor dimer and reaction of this with the free amino group of the phospholipid ammonium salt.
  • the ivDde bearing precursor dimer peptide (16) may be reacted with DSG (4 eq.) in DMF in the presence of DIEA (5 eq.) for 30 min.
  • the solution may be diluted with ethyl acetate to precipitate the glutaric acid monoamide mono-NHS ester of the dimer (17), as a solid. The supernatant may be decanted to remove the un-reacted DSG.
  • the solid glutaric acid monoamide mono-NHS ester of the dimer peptide (17) may then be washed several times with ethyl acetate to remove traces of DSG. Mass spectral results confirm the formation of the glutaric acid monoamide mono-NHS ester of the peptide dimer as a clean product.
  • the dimer glutaric acid monoamide mono-NHS ester (17) may be dissolved in DMF-CH 2 Cl 2 (8:2) and reacted with DSPE-PEG2000-NH 2 phospholipid ammonium salt (0.9 eq.) in the presence of DIEA (4 eq.) for 24 h.
  • the NHS ester (17) may be used in excess to maximize the consumption of the phospholipid ammonium salt because any free phospholipid may complicate the purification and isolation of the final product.
  • the reaction mixture may be diluted with water (0.1% TFA)-CH 3 CN—CH 3 OH (1:1) (0.1% TFA) (100 mL), applied to a reversed phase C4 column (Kromasil® Prep C4, 10 ⁇ , 300 ⁇ , 50 ⁇ 250 mm, flow rate 100 mL/min) and the column may be eluted with water (0.1% TFA)-CH 3 CN—CH 3 OH (1:1) (0.1% TFA) solvent mixture to remove hydrophilic impurities. Then the product may be eluted using a gradient of CH 3 CN—CH 3 OH (1:1) (0.1% TFA) into water (0.1% TFA).
  • the collected fractions may be analyzed by reversed phase HPLC using an ELS detector which allows the detection of the desired product and the often difficult to separate DSPE-PEG2000-NH 2 phospholipid ammonium salt which has no strong UV chromophore. This indicates the clear separation of dimeric peptide phospholipid conjugate and DSPE-PEG2000-NH 2 phospholipid ammonium salt.
  • the pure product-containing fractions may be collected, concentrated on a rotary evaporator (to reduce the content of methanol) and freeze-dried to provide the dimer peptide phospholipid conjugate as a colorless solid.
  • dimer peptide phospholipid conjugate In order to prepare the required quantity of the dimer peptide phospholipid conjugate, several runs may be conducted employing 0.5 g to 1.0 g of the precursor dimer peptide. In all cases the target dimer peptide phospholipid conjugate may be isolated in 57-60% yield and in >99% purity.
  • the bulk quantity of dimer peptide phospholipid conjugate, obtained from the serial runs described above may be obtained by dissolution of the product from the individual runs in t-butanol-acetonitrile-water (1:1:3) followed by lyophilization.
  • the procedure of Ellman for quantitative estimation of free thiol may be applied to the bulk sample of the dimeric peptide phospholipid conjugate; free thiol, if present will be below the limit of detection.
  • Amino acid composition analysis gives results within the acceptable limits, supporting the assigned structure of the peptide derivative.
  • MALDI-TOF mass spectral analysis also supports the presumed structure of the dimeric peptid
  • the present invention also provides methods for producing dimeric peptide-phospholipid conjugates having very low levels of TFA. While certain methods provide for the synthesis and purification of such conjugates on a gram scale, formation of a lyso-version of the conjugates has been observed upon storage of lyophilized material at 5° C. or upon storage of aqueous solutions of the conjugates. It is believed that the lyso-compound is formed by TFA-promoted acid hydrolysis of one of the phospholipid fatty acid esters in dimer peptide-phospholipid conjugates.
  • Table 3 provides a description for the identification labels shown in FIGS. 6 , 7 and 8 .
  • monomer peptide components of heterodimer peptide (27), namely TFA salts compounds (22) and (25), are subjected to ion exchange chromatography on the macroporous sulfonic acid cation exchange resin AG MP-50 using a step gradient of ammonium acetate to convert them to their acetate salts. Then the two peptide monomer acetates (23) and (26) may be tethered through a glutaryl linker to form the dimer (27) as an acetate salt.
  • compounds (22), (25) and (27) all bear side-chain carboxylic acid and amino groups.
  • AG MP-50 a macroporous cation-exchange resin, may be used to allow full penetration of the resin by the peptides and to exploit the immobilization of the peptides via their basic (amino and guanidine) groups.
  • TFA salts of the peptides may be adsorbed to an AG MP-50 column (sulfonic acid form) and the column may be washed with water and then eluted with a step gradient of NH 4 OAc in 0 or 30% CH 3 CN/H 2 O, depending on the solubility of the peptides.
  • the peptides may be eluted at about 600 mM NH 4 OAc and the acetate form of the peptides then may be obtained in pure form. Both IC fluorine analysis and CE TFA counter-ion analysis consistently show very low TFA content of the peptides.
  • Preferred methods also include redissolution/relyophilization of the final peptides several times to remove residual NH 4 OAc. Otherwise, residual traces of NH 4 OAc present in the peptides may give rise to free ammonia in the presence of DIEA. This may result in the formation of unwanted peptide-Glut-amide as a major product in subsequent preparation of (27) from the monomers (23) and (26) or final phospholipid-peptide conjugate (21) from the acetate salt of (27).
  • another embodiment provides the conversion of the TFA salt of dimer (27) to its analogous acetate salt by ion exchange chromatography on the macroporous sulfonic acid cation exchange resin AG MP-50.
  • This dimer acetate then may be conjugated with DSPE-PEG2000-NH 2 followed by purification of the crude material by C-3 preparative column using CH 3 CN/H 2 O/NH 4 OAc to give the final compound (21) as an acetate salt.
  • FIG. 8 another embodiment provides methods for providing dimeric conjugates having minimal amounts of TFA utilizing the size differential between the phospholipid-peptide conjugate (21) and TFA ions.
  • 21 ⁇ nTFA adduct may be eluted down a size exclusion column in the presence of ammonium bicarbonate buffer.
  • the crude 21 ⁇ nTFA initially may be freed of the lyso-compound by preparative HPLC on a Zorbax C-3 column using a linear gradient of acetonitrile into water. Both phases may be buffered with 10 mM ammonium acetate. This provides separation of the lyso-compound as indicated by analytical HPLC.
  • the material may be applied to a Sephadex G-25 column and eluted with aqueous ammonium bicarbonate solution.
  • the eluate may be monitored by HPLC.
  • Product-containing fractions may be pooled and lyophilized to afford the desired material (21) essentially free of TFA and with high recovery rates. Additional detail is provided below in the Examples section.
  • Both the monomeric and dimeric peptide phospholipid conjugates described herein may be incorporated into ultrasound contrast agents such as, for example, gas filled microvesicles.
  • gas filled microvesicles include, for example, gas filled microbubbles, gas filled microballoons, gas filled microcapsules, etc.
  • the peptide phospholipid conjugates may be incorporated into ultrasound contrast agents comprising gas filled microbubbles.
  • Methods of preparation of gas filled microbubbles from phospholipids and phospholipid conjugates are known to those skilled in the art.
  • microbubbles according to the present invention can be prepared by methods described in any one of the following patents: EP 554213, WO 04/069284, U.S. Pat. No.
  • Suitable phospholipids include esters of glycerol with one or two molecules of fatty acids (the same or different) and phosphoric acid, wherein the phosphoric acid residue is in turn bonded to a hydrophilic group, such as choline, serine, inositol, glycerol, ethanolamine, and the like groups.
  • Fatty acids present in the phospholipids are in general long chain aliphatic acids, typically containing from 12 to 24 carbon atoms, preferably from 14 to 22, that may be saturated or may contain one or more unsaturations.
  • Suitable fatty acids are lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid.
  • Mono esters of phospholipids are known in the art as the “lyso” forms of the phospholipid.
  • phospholipids are phosphatidic acids, i.e., the diesters of glycerol-phosphoric acid with fatty acids, 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, gangliosides, cerebrosides, etc.
  • phosphatidic acids i.e., the diesters of glycerol-phosphoric acid with fatty acids
  • 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
  • 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) derivatives) such as, typically, soya bean or egg yolk lecithins.
  • PC phosphatidylcholine
  • semisynthetic phospholipids are the partially or fully hydrogenated derivatives of the naturally occurring lecithins.
  • Examples of synthetic phospholipids are e.g., dilauryloyl-phosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine (“DMPC”), dipalmitoyl-phosphatidylcholine (“DPPC”), diarachidoylphosphatidylcholine (“DAPC”), distearoyl-phosphatidylcholine (“DSPC”), 1-myristoyl-2-palmitoylphosphatidylcholine (“MPPC”), 1-palmitoyl-2-myristoylphosphatidylcholine (“PMPC”), 1-palmitoyl-2-stearoylphosphatidylcholine (“PSPC”), 1-stearoyl-2-palmitoyl-phosphatidylcholine (“SPPC”), dioleoylphosphatidylycholine (“DOPC”), 1,2 Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DS
  • Suitable phospholipids further include phospholipids modified by linking a hydrophilic polymer thereto.
  • modified phospholipids are phosphatidylethanolamines (PE) modified with polyethylenglycol (PEG), in brief “PE-PEGs”, 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, DSPE-PEG, DMPE-PEG or DAPE-PEG (where DAPE is 1,2-diarachidoyl-sn-glycero-3-phosphoethanolamine).
  • PE phosphatidylethanolamines
  • PEG polyethylenglycol
  • PE-PEGs i.e. phosphatidylethanolamines where the hydrophilic ethanolamine moiety is linked to a PEG molecule of variable molecular weight (e.g
  • compositions also may contain other amphiphilic compounds including, for instance, fatty acids, such as palmitic acid, stearic acid, arachidonic acid or oleic acid; sterols, such as cholesterol, or esters of sterols with fatty acids or with sugar acids; glycerol or glycerol esters including glycerol tripalmitate, glycerol distearate, glycerol tristearate, glycerol dimyristate, glycerol trimyristate, glycerol dilaurate, glycerol trilaurate, glycerol dipalmitate; tertiary or quaternary alkyl-ammonium salts, such as 1,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), and mixtures or combinations thereof.
  • fatty acids such as palmitic
  • the formulation comprises at least one component bearing an overall net charge, such as, for instance, phosphatidic acid, PE-PEG, palmitic acid, stearic acid, Ethyl-DSPC or DSTAP, preferably in a molar amount of less than about 50%.
  • Particularly preferred formulations may include mixtures of two or more of the following components: DSPC, DPPG, DPPA, DSPE-PEG1000, DSPE-PEG2000, palmitic acid and stearic acid.
  • phospholipids and formulations are set forth in the examples Any of the gases disclosed herein or known to the skilled artisan may be employed; however, inert gases, such as SF 6 or perfluorocarbons like CF 4 , C 3 F 8 and C 4 F 10 , are preferred, optionally in admixture with other gases such as air, nitrogen, oxygen or carbon dioxide
  • the preferred microbubble suspensions of the present invention may be prepared from phospholipids using known processes such as a freeze-drying or spray-drying solutions of the crude phospholipids in a suitable solvent or using the processes set forth in EP 554213; WO 04/069284; U.S. Pat. No. 5,413,774; U.S. Pat. No. 5,578,292; EP 744962; EP 682530; U.S. Pat. No. 5,556,610; U.S. Pat. No. 5,846,518; U.S. Pat. No. 6,183,725; EP 474833; U.S. Pat. No. 5,271,928; U.S. Pat. No. 5,380,519; U.S. Pat. No.
  • a microemulsion can be prepared which contains the phospholipids (e.g. DSPC and/or DSPA) in admixture with a lyoprotecting agent (such as, for instance, carbohydrates, sugar alcohols, polyglycols and mixtures thereof, as indicated in detail hereinafter) and optionally other amphiphilic materials (such as stearic acid), dispersed in an emulsion of water and of a water immiscible organic solvent.
  • a lyoprotecting agent such as, for instance, carbohydrates, sugar alcohols, polyglycols and mixtures thereof, as indicated in detail hereinafter
  • amphiphilic materials such as stearic acid
  • Preferred organic solvents are those having solubility in water of 1.0 g/l or lower, preferably lower than about 0.01 g/l, and include, for instance, pentane, hexane, heptane, octane, nonane, decane, 1-pentene, 2-pentene, 1-octene, cyclopentane, cyclohexane, cyclooctane, 1-methyl-cyclohexane, benzene, toluene, ethylbenzene, 1,2-dimethylbenzene, 1,3-dimethylbenzene, di-butyl ether and di-isopropylketone, chloroform, carbon tetrachloride, 2-chloro-1-(difluoromethoxy)-1,1,2-trifluoroethane (enflurane), 2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane (is
  • the peptide-phospholipid conjugate of the invention can be admixed together with the phospholipid forming the microvesicle's envelope, in the microemulsion.
  • an aqueous suspension of the peptide-phospholipid conjugate and of a PE-PEG e.g. DSPE-PEG2000
  • a PE-PEG e.g. DSPE-PEG2000
  • said mixing is effected under heating, e.g. form about 40° C. to 80° C.
  • the freeze dried or spray dried phospholipid powders Prior to formation of the suspension of microbubbles by dispersion in an aqueous carrier, the freeze dried or spray dried phospholipid powders are contacted with air or another gas. When contacted with the aqueous carrier the powdered phospholipids whose structure has been disrupted will form lamellarized or laminarized segments that will stabilize the microbubbles of the gas dispersed therein.
  • This method permits production of suspensions of microbubbles that are stable even when stored for prolonged periods and are obtained by simple dissolution of the dried laminarized phospholipids (which have been stored under a desired gas) without shaking or any violent agitation.
  • microbubbles can be prepared by suspending a gas into an aqueous solution at high agitation speed, as disclosed e.g. in WO 97/29783.
  • a further process for preparing microbubbles is disclosed in WO 2004/069284, herein incorporated by reference, which comprises preparing an emulsion of an organic solvent in an aqueous medium in the presence of a phospholipid and subsequently lyophilizing said emulsion, after optional washing and/or filtration steps.
  • the formulation for the preparation of the gas-filled microbubbles may advantageously further comprise 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 polyglycol such as polyethylene glycol (e.g. PEG-4000).
  • 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 cyclo
  • any of these ultrasound compositions should also be, as far as possible, isotonic with blood.
  • small amounts of isotonic agents may be added to any of above ultrasound contrast agent suspensions.
  • the isotonic agents are physiological solutions commonly used in medicine and they comprise aqueous saline solution (0.9% NaCl), 2.6% glycerol solution, 5% dextrose solution, etc.
  • the ultrasound compositions may include standard pharmaceutically acceptable additives, including, for example, emulsifying agents, viscosity modifiers, cryoprotectants, lyoprotectants, bulking agents etc.
  • gas includes any substances (including mixtures) substantially in gaseous form at the normal human body temperature.
  • the gas may thus include, for example, air, nitrogen, oxygen, CO 2 , argon, xenon or krypton, fluorinated gases (including for example, perfluorocarbons, SF 6 , SeF 6 ) a low molecular weight hydrocarbon (e.g., containing from 1 to 7 carbon atoms), for example, an alkane such as methane, ethane, a propane, a butane or a pentane, a cycloalkane such as cyclopropane, cyclobutane or cyclopentene, an alkene such as ethylene, propene, propadiene or a butene, or an alkyne such as acetylene or propyne and/or mixtures thereof.
  • fluorinated gases including for example, perfluorocarbons, SF 6 , SeF 6
  • Fluorinated gases include materials that contain at least one fluorine atom such as SF 6 , freons (organic compounds containing one or more carbon atoms and fluorine, i.e., CF 4 , C 2 F 6 , C 3 F 8 , C 4 F 8 , C 4 F 10 , CBrF 3 , CCl 2 F 2 , C 2 ClF 5 , and CBrClF 2 ) and perfluorocarbons.
  • perfluorocarbon refers to compounds containing only carbon and fluorine atoms and includes, in particular, saturated, unsaturated, and cyclic perfluorocarbons.
  • the saturated perfluorocarbons which are usually preferred, have the formula C n F n +2, where n is from 1 to 12, preferably from 2 to 10, most preferably from 3 to 8 and even more preferably from 3 to 6.
  • Suitable 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 , C 6 F 2 , C 7 F 14 , C 8 F 18 , and C 9 F 20 .
  • the gas or gas mixture comprises SF 6 or a perfluorocarbon selected from the group consisting of C 3 F 8 , C 4 F 8 , C 4 F 10 , C 5 F 2 , C 6 F 12 , C 7 F 14 , C 8 F 18 , with C 4 F 10 being particularly preferred.
  • C 4 F 10 being particularly preferred.
  • the gas comprises C 4 F 10 or SF 6 , optionally in admixture with air, nitrogen, oxygen or carbon dioxide.
  • a precursor to a gaseous substance e.g., a material that is capable of being converted to a gas in vivo, often referred to as a “gas precursor”.
  • gas precursor e.g., a material that is capable of being converted to a gas in vivo
  • the gas precursor may be pH-activated, photo-activated, temperature activated, etc.
  • certain perfluorocarbons may be used as temperature activated gas precursors. These perfluorocarbons, such as perfluoropentane, 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 phase shift and are converted to a gas within the human body.
  • the gas can comprise a mixture of gases.
  • the following combinations are particularly preferred gas mixtures: a mixture of gases (A) and (B) in which, at least one of the gases (B), present in an amount of between 0.5-41% by vol., has a molecular weight greater than 80 daltons and is a fluorinated gas and (A) is selected from the group consisting of air, oxygen, nitrogen, carbon dioxide and mixtures thereof, the balance of the mixture being gas A.
  • the lyophilized product may be stored and transported without need of temperature control of its environment and in particular it may be supplied to hospitals and physicians for on site formulation into a ready-to-use administrable suspension without requiring such users to have special storage facilities.
  • it can be supplied in the form of a two-component kit, which can include two separate containers or a dual-chamber container.
  • the container is a conventional septum-sealed vial, wherein the vial containing the lyophilized residue of step b) is sealed with a septum through which the carrier liquid may be injected using an optionally prefilled 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 a dual-chamber syringe and once the lyophilizate has been reconstituted and then suitably mixed or gently shaken, the container can be used directly for injecting the contrast agent.
  • the size of the gas microbubbles is substantially independent of the amount of agitation energy applied to the reconstituted dried product. Accordingly, no more than gentle hand shaking is generally required to give reproducible products with consistent microbubble size.
  • aqueous phase can be interposed between the water-insoluble gas and the environment, to increase shelf life of the product.
  • a material necessary for forming the contrast agent is not already present in the container (e.g. a targeting ligand to be linked to the phospholipid during reconstitution)
  • it can be packaged with the other components of the kit, preferably in a form or container adapted to facilitate ready combination with the other components of the kit.
  • the stopper may be a compound or multicomponent formulation based on an elastomer, such as poly(isobutylene) or butyl rubber.
  • the contrast agents formed by phospholipid stabilized microbubbles can be administered, for example, in doses such that the amount of phospholipid injected is in the range 0.1 to 200 ⁇ g/kg body weight, preferably from about 0.1 to 30 ⁇ g/kg.
  • Ultrasound imaging techniques that can be used in accordance with the present invention include known techniques, such as color Doppler, power Doppler, Doppler amplitude, stimulated acoustic imaging, and two- or three-dimensional imaging techniques. Imaging may be done in harmonic (resonant frequency) or fundamental modes, with the second harmonic preferred.
  • the ultrasound contrast agents of the present invention may further be used in a variety of therapeutic imaging methods.
  • 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 agent 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 an ultrasound burst 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 localized release of a suitable therapeutic agent.
  • said therapeutic imaging may include the delivery of a therapeutic agent 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).
  • 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.
  • Solvents for reactions, chromatographic purification and HPLC analyses were E. Merck Omni grade solvents from VWR Corporation (West Chester, Pa.). N-Methylpyrrolidinone (NMP) and N,N-dimethylformamide (DMF) were obtained from Pharmco Products Inc. (Brookfield, Conn.), and were peptide synthesis grade or low water/amine-free Biotech grade quality. Piperidine (sequencing grade, redistilled 99+%) and trifluoroacetic acid (TFA) (spectrophotometric grade or sequencing grade) were obtained from Sigma-Aldrich Corporation (Milwaukee, Wis.) or from the Fluka Chemical Division of Sigma-Alrich Corporation.
  • N,N′-Diisopropylcarbodiimide DIC
  • DIEA N,N-diisopropylethylamine
  • TIS triisopropylsilane
  • Fmoc-protected amino acids, pseudoproline dipeptides, Fmoc-Asp(O-tBu)-Ser( ⁇ Me,Me pro)-OH and Fmoc-Gly-Thr( ⁇ Me,Me pro)-OH and N-hydroxybenzotriazole (HOBt) were obtained from Novabiochem (San Diego, Calif.).
  • Fmoc-8-amino-3,6-dioxaoctanoic acid was obtained from NeoMPS Corp (San Diego, Calif.) or Suven Life Sciences (Hyderabad, India).
  • Disuccinimidyl glutarate (DSG) and 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[amino (polyethyleneglycol)2000] ammonium salt, [DSPE-PEG2000-NH 2 ] were obtained from Pierce Chemical Co. (Rockford, Ill.) and Avanti® Polar Lipids (Alabaster, Ala.), respectively.
  • Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH were prepared in-house from the corresponding triglycine or diglycine by the reaction with Fmoc-OSu.
  • An AG MP-50 ion-exchange resin was obtained from Bio-Rad (Hercules, Calif.).
  • Analytical HPLC data were generally obtained using a Shimadzu LC-10AT VP dual pump gradient system employing a Waters XTerra MS-C18 4.6 ⁇ 50 mm column, (particle size: 5 ⁇ ; 120 ⁇ pore size) and gradient or isocratic elution systems using water (0.1% TFA) as eluent A and CH 3 CN (0.1% TFA) or CH 3 CN—CH 3 OH (1:1, v/v) (0.1% TFA) as eluent B. Detection of compounds was accomplished using UV at 220 and 254 nm.
  • the purity of the phospholipid-PEG-peptide derivatives was determined on a YMC C-4 (5 ⁇ M, 300 ⁇ , 4.6 ⁇ 250 mm) column or on a Zorbax 300 SB-C3 (3.5 ⁇ M; 300 ⁇ , 3 ⁇ 150 mm) column using a SEDEX 55 Light Scattering Detector (LSD) and with a UV detector.
  • LSD Light Scattering Detector
  • Preparative HPLC was conducted on a Shimadzu LC-8A dual pump gradient system equipped with a SPD-10AV UV detector fitted with a preparative flow cell. Generally the solution containing the crude peptide was loaded onto a reversed phase C18, C4 or C3 column, depending on the compound characteristics, using a third pump attached to the preparative Shimadzu LC-8A dual pump gradient system. After the solution of the crude product mixture was applied to the preparative HPLC column the reaction solvents and solvents employed as diluents, 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 eluent B into eluent A. Product-containing fractions were combined based on their purity as determined by analytical HPLC and mass spectral analysis. The combined fractions were freeze-dried to provide the desired product.
  • diluents such as DMF or DMSO
  • non-limiting Examples provide additional detail on efficient processes used for obtaining large quantities of highly purified forms of the monomeric and dimeric peptide phospholipid conjugates. These non-limiting Examples also describe the preparation of representative targeted microbubbles which include these monomeric and dimeric peptide phospholipid conjugates. These Examples also describe the use of such targeted microbubbles in static binding tests on KDR-transfected cells and dynamic binding tests on rh VEGF-R2/Fc chimeric protein. The Examples further describe the evaluation of ultrasound contrast agents containing KDR binding lipopeptides in a rabbit VX2 tumor model.
  • Examples 1-2 below refer to the monomeric peptide phospholipid conjugate shown in FIG. 2 .
  • a process for synthesizing this compound is shown in FIG. 1 .
  • these Examples refer more specifically to the process for synthesizing the compound shown in FIG. 2
  • a similar process may be used to prepare the monomeric peptide phospholipid conjugate shown in FIG. 10 and the linear peptide monomer (32) shown in FIG. 9 as well as other monomer peptide-phospholipid conjugates.
  • co-pending U.S. application Ser. No. 10/661,156 filed Sep. 11, 2003, sets forth methods for the preparation of the peptide monomers and is incorporated by reference herein in its entirety.
  • the linear peptide monomer (2) was synthesized by an established automated protocol on a SONATA®/Pilot Peptide Synthesizer using Fmoc-Pal-Peg-PS resin (0.2 mmol/g), Fmoc-protected amino acids and DIC-mediated HOBt ester activation in DMF.
  • the peptide sequence was synthesized in stepwise fashion by SPPS methods on the Fmoc-Pal-Peg-PS resin, typically on a 10 mmol scale.
  • the amino acid couplings were carried out with a 4-fold excess each of amino acid and the DIC-HOBt reagent pair in DMF.
  • the software of the instrument was employed to deliver successively the chosen volume of DMF (for dilution of the amino acid) and HOBt (4 eq.) in DMF and DIC (4 eq.) in DMF and mixing by nitrogen bubbling was initiated and conducted for 4 min. This served to pre-activate the amino acid and to insure complete dissolution of all components of the mixture.
  • the software mediated the transfer of the solution of the activated Fmoc-amino acid to the reaction vessel containing the resin. After transfer was complete the vessel was agitated for 3 h with recurrent nitrogen bubbling.
  • the resin was washed thoroughly with DMF (5 mL/g, 6 ⁇ ) and the cleavage of the Fmoc-group was performed with 25% piperidine in DMF (5 mL/g) containing HOBt (0.1 M) (2 ⁇ 10 min).
  • the resin was thoroughly washed with DMF (5 mL/g, 6 ⁇ ) to assure complete removal of piperidine from the resin in preparation for the ensuing amino acid coupling.
  • the pre-activation in the amino acid bottle was not conducted in order to minimize the formation of diketopiperazine during the activation time as discussed in the text. Therefore, in these two cases, the solutions of amino acid, HOBt and DIC were added to the reaction vessel sequentially and the coupling process was conducted with ‘in situ’ activation.
  • the Fmoc group of the N-terminal amino acid was removed in the standard manner followed by the standard wash with DMF (vide supra).
  • the N-terminal amino acid was then capped by treatment with freshly prepared acetylation mixture (0.5M acetic anhydride, 0.125M DIEA and 0.015M HOBt in DMF/6 mL/g of resin), 2 ⁇ 20 min.
  • the resin was treated with the cleavage cocktail, ‘Reagent B’ (TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) (10 mL/g of resin) for 4 h.
  • a ⁇ 0.5 g portion of the crude linear peptide monomer (2) was dissolved in a minimum amount of CH 3 CN ( ⁇ 20 mL).
  • the volume of the solution was adjusted to ⁇ 100 mL with water and employing a third pump the solution was loaded onto a reversed phase C18 preparative column (Waters, XTerra® Prep MS C18, 10 ⁇ , 300 ⁇ , 50 ⁇ 250 mm, flow rate 100 mL/min) which had been pre-equilibrated with 10% CH 3 CN in water (0.1% TFA).
  • the column was not eluted with the equilibrating eluent during application of the sample solution.
  • the monomeric peptide phospholipid conjugate (1) Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH 2 (SEQ ID NO. 1), was prepared by conjugation of (3), the glutaric acid monoamide mono-NHS ester of peptide monomer (2), with DSPE-PEG2000-NH 2 phospholipid ammonium salt (4).
  • the reaction mixture was diluted to ⁇ 50 mL with anhydrous ethyl acetate; this resulted in precipitation of the intermediate mono-NHS ester (3), the glutaric acid monoamide mono-NHS ester of peptide monomer (2).
  • the solution was centrifuged to bring down mono-NHS ester (3) as a colorless solid.
  • the supernatant containing excess DSG was decanted from the compacted solid mono-NHS ester (3) which was again dispersed in ethyl acetate, centrifuged and washed twice more to remove the remaining traces of DSG.
  • the solid intermediate mono-NHS ester (3) thus obtained was dissolved in anhydrous DMF (10.0 mL); diisopropylethylamine (0.10 g, 0.78 mmol) was added; and the mixture was stirred.
  • DSPE-PEG2000-NH 2 phospholipid ammonium salt (4) (0.38 g, 0.14 mmol, 0.9 eq.) was suspended in dry dichloromethane (2 mL) in a separate flask and trifluoroacetic acid (2 drops) was added to protonate the phosphodiester oxygen facilitating solubilization of phospholipid ammonium salt in dichloromethane.
  • the clear solution was then evaporated on a rotary evaporator to remove the volatiles and dried further under vacuum.
  • the solid phospholipid ammonium salt (4) was dissolved in DMF (5 mL) and transferred to the stirred solution of mono-NHS ester (3) and the resulting mixture was stirred for 24 h at ambient temperature.
  • the reaction mixture was diluted to 100 mL with a 1:1 mixture of CH 3 OH and CH 3 CN-water (1:1, v/v) and the insolubles were filtered.
  • composition of the eluent was ramped to 70% CH 3 OH—CH 3 CN (1:1, 0.1% TFA) over 9 min and a linear gradient of 0.75%/min of CH 3 OH—CH 3 CN (1:1, 0.1% TFA) into water (0.1% TFA) was initiated and run for 40 min.
  • Fractions (15 mL) were collected using UV (220 nm) as an indicator of product elution. Fractions were checked for purity on an analytical HPLC system (column: YMC C-4, 5 ⁇ , 300 ⁇ , 4.6 ⁇ 250 mm) using UV at 220 nm and an evaporative light scattering detector (ELSD).
  • ELSD evaporative light scattering detector
  • the latter detector was employed to detect DSPE-PEG2000-NH 2 phospholipid ammonium salt (4) which has very little UV absorbance at 220 nm.
  • Product-containing fractions of >98% purity, and devoid of DSPE-PEG2000-NH 2 phospholipid ammonium salt (4) were combined and concentrated on a rotary evaporator to reduce the content of CH 3 OH.
  • the concentrated solution was then diluted with 10% CH 3 CN in water until a faint flocculent precipitate formed.
  • the resulting solution was freeze-dried to provide monomeric peptide phospholipid conjugate (1) as a colorless solid.
  • the second portion of crude monomeric peptide phospholipid conjugate (1) was purified as described above.
  • the combined yield of the target monomeric peptide phospholipid conjugate (1) was 0.40 g (47% yield).
  • Examples 3-5 below refer to the dimeric peptide phospholipid conjugate shown in FIG. 5 .
  • Representative methods of synthesizing the dimeric conjugate are shown in FIGS. 3 , 4 , 6 , 7 and 8 .
  • the linear peptides were synthesized by an established automated protocol on a SONATA®/Pilot Peptide Synthesizer using Fmoc-Pal-Peg-PS resin (0.2 mmol/g), Fmoc-protected amino acids and DCI-mediated HOBt ester activation in DMF.
  • the peptide sequence on the Fmoc-Pal-Peg-PS resin was synthesized in stepwise fashion by SPPS methods typically on a 10 mmol scale.
  • the amino acid coupling was carried out with a 4-fold excess each of amino acid and DIC-HOBt reagent in DMF.
  • the chosen volume of DMF and HOBt (4 eq.) in DMF and DIC (4 eq.) in DMF were delivered successively and after each delivery mixing by nitrogen bubbling was conducted. After the last reagent was delivered mixing by nitrogen bubbling was initiated and conducted for 4 min. This served to preactivate the amino acid and to insure complete dissolution of all components of the mixture.
  • the solution of the activated Fmoc-amino acid was transferred to the reaction vessel containing the resin. After transfer was complete the vessel was agitated for 3 h with recurrent nitrogen bubbling. After the 3 h coupling time, the resin was washed thoroughly with DMF (5 mL/g, 6 ⁇ ) and the cleavage of the Fmoc-group was performed with 25% piperidine in DMF (5 mL/g) containing HOBt (0.1M) (2 ⁇ 10 min). The resin was thoroughly washed with DMF (5 mL/g, 6 ⁇ ) to assure complete removal of piperidine from the resin in preparation for the ensuing amino acid coupling.
  • N-terminal amino acid was then capped by treatment with freshly prepared acetylation mixture (0.5M acetic anhydride, 0.125M DIEA and 0.015M HOBt in DMF—6 mL/g of resin), 2 ⁇ 20 min.
  • acetylation mixture 0.5M acetic anhydride, 0.125M DIEA and 0.015M HOBt in DMF—6 mL/g of resin
  • Functionalization of the ⁇ -amino group of C-terminal Lysine moieties of the monomer peptides was accomplished by first removing the ivDde group of the ⁇ -amino group with freshly prepared 10% hydrazine in DMF (5 mL/g of resin—2 ⁇ 10 min). For appending of Fmoc-Adoa or Fmoc-Lys(ivDde) the coupling time was increased to 10 h.
  • Cyclic disulfide peptides were prepared from the corresponding linear di-cysteine peptides by DMSO-assisted oxidation using DMSO/water (95/5, v/v).
  • the crude linear peptide was dissolved in the solvent mixture (5 mL/g) in a wide mouth beaker, and the pH of the solution was adjusted to 8.5 by the addition of solid N-methyl-D-glucamine in portions.
  • the resulting mixture was stirred for 36 h at ambient temperature.
  • the solution was then diluted with acetonitrile (50 mL/g) and the mixture was stirred for 2 min.
  • the solid cyclic disulfide peptide was collected by filtration, washed with diethyl ether and dried.
  • Peptide Monomer (12) Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH 2 ; Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys[Lys(ivDde)]-NH 2 cyclic (6-13) disulfide
  • a ⁇ 0.5 g portion of the crude cyclic disulfide peptide monomer (12) was dissolved in a minimum amount of DMSO ( ⁇ 3 mL).
  • the volume of the solution was adjusted to ⁇ 100 mL with 20% CH 3 CN-water and employing a third pump, the solution was loaded onto a reversed phase C18 preparative column (Waters, XTerra® Prep MS C18, 10 ⁇ , 300 ⁇ , 50 ⁇ 250 mm, flow rate 100 mL/min), which had been pre-equilibrated with 10% CH 3 CN in water (0.1% TFA).
  • a reversed phase C18 preparative column Waters, XTerra® Prep MS C18, 10 ⁇ , 300 ⁇ , 50 ⁇ 250 mm, flow rate 100 mL/min
  • Peptide Monomer (13) Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH 2 ; Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH 2 cyclic (2-12) disulfide
  • Preparation and Purification of Precursor Dimer Peptide (16) Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH 2 cyclic (2-12) disulfide]-NH 2 cyclic (6-13) disulfide; Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys[Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(-Adoa-Adoa-Glut-
  • disuccinimidyl glutarate (DSG, 0.28 g, 0.86 mmol) was dissolved in stirred anhydrous dimethylformamide (2.0 mL) and diisopropylethylamine (0.11 g, 0.85 mmol) was added in one portion. Then solid peptide monomer (12) Ac-AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDde)]-NH 2 (0.50 g, 0.17 mmol) was added in portions to the stirred solution of DSG over a period of two min.
  • the solid mono-NHS ester (14) was dissolved in stirred anhydrous dimethylformamide (2.0 mL) and diisopropylethylamine (0.11 g, 0.85 mmol) was added. Then solid peptide monomer (13), Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH 2 , (0.50 g, 0.19 mmol, 1.12 eq.) was added in portions to the stirred solution over a three min. period and the resulting mixture was stirred for 18 h.
  • reaction was monitored by mass spectrometry; after the complete consumption of the peptide monomer glutaric acid monoamide mono-NHS ester (14) was confirmed, neat hydrazine (0.1 mL) was added to remove the ivDde protecting group of the ivDde-bearing dimer (15) and the mixture was stirred for 20 min at room temperature.
  • the solution was then acidified by dropwise addition of TFA and the mixture was diluted to 100 mL with 10% CH 3 CN (0.1% TFA) in water (0.1% TFA).
  • the solution was filtered to remove particulates and half of the clarified solution was loaded onto a reversed phase C18 preparative column (Waters, XTerra® Prep MS C18, 10 ⁇ , 50 ⁇ 250 mm, flow rate 100 mL/min) pre-equilibrated with 10% CH 3 CN in water (0.1% TFA).
  • a reversed phase C18 preparative column Waters, XTerra® Prep MS C18, 10 ⁇ , 50 ⁇ 250 mm, flow rate 100 mL/min
  • the fractions were analyzed on a reversed phased C18 column (Waters MS C18, 4.6 mm i.d. ⁇ 50 mm, 5 ⁇ particle, 120 ⁇ pore) and the product-containing fractions of >95% purity were pooled and freeze-dried to provide precursor dimer peptide (16) as a colorless, fluffy solid.
  • the remaining crude precursor dimer peptide (16) was purified in the same manner. From 0.5 g each of monomer peptides (12) and (13), 320 mg (overall yield 33%) of the desired dimer (16) was obtained (>95% purity).
  • the KDR-binding dimer (11) may be prepared by conjugation of precursor dimer peptide (16), Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH 2 cyclic (2-12) disulfide]-NH 2 cyclic (6-13) disulfide, with DSPE-PEG2000-NH 2 phospholipid ammonium salt (18) as shown in FIG. 4 .
  • Solid precursor dimer peptide (16) (0.5 g, 0.092 mmol) was added portionwise to a solution of disuccinimidyl glutarate (DSG, 0.15 g, 0.46 mmol), and diisopropylethylamine (0.06 g, 0.47 mmol) in anhydrous DMF (3.0 mL) with stirring over a period of 3 min. Then the solution was stirred at ambient temperature for 30 min. The reaction mixture was diluted to 50 mL with anhydrous ethyl acetate; this resulted in precipitation of the dimer glutaric acid monoamide mono-NHS ester (17), the glutaric acid monoamide mono-NHS ester of the precursor dimer peptide (16).
  • the solution was centrifuged to pellet 6 (m/z, neg. ion, 1887.3 (M-3H)/3, 1415.1 (M-4H)/4, 1131.9 (M-5H)/5) as a colorless solid.
  • the supernatant ethyl acetate layer containing excess DSG was decanted from the compacted solid dimer glutaric acid monoamide mono-NHS ester (17) which was again resuspended in ethyl acetate, centrifuged and washed twice more to remove the remaining traces of DSG.
  • the solid intermediate glutaric acid monoamide mono-NHS ester dimer derivative (17) thus obtained was dissolved in anhydrous DMF/CH 2 Cl 2 (8:2, v/v) (3.0 mL); diisopropylethylamine (0.06 g, 0.47 mmol) was added and the solution was stirred.
  • DSPE-PEG2000-NH 2 phospholipid ammonium salt (18) (0.235 g, 0.084 mmol, 0.9 eq.) was suspended in dry dichloromethane (2 mL) in a separate flask and TFA (2 drops) was added to protonate the phosphodiester oxygen, facilitating solubilization of phospholipid ammonium salt (18) in dichloromethane.
  • TFA 2 drops
  • the solid phospholipid ammonium salt (18) was dissolved in DMF (2 mL) and transferred to the stirred solution of glutaric acid monoamide mono-NHS ester dimer derivative (17) and the resulting mixture was stirred for 24 h at ambient temperature.
  • the reaction mixture was diluted with a solution of 50% CH 3 OH, 25% CH 3 CN and 25% water (1:1) to 100 mL and the insolubles were filtered.
  • composition of the eluent was then ramped to 70% CH 3 OH—CH 3 CN (1:1, 0.1% TFA)-water (0.1% TFA) over 1 min and a linear gradient of 0.75%/min of CH 3 OH—CH 3 CN (1:1, 0.1% TFA) into water (0.1% TFA) was initiated.
  • the elution was continued after reaching 100% B in order to achieve complete elution of the product from the column.
  • Fractions (15 mL) were collected using UV (220 nm) as an indicator of product elution and after the main product was eluted fraction collection was continued for several minutes in order to insure elution of trace amounts of starting phospholipid ammonium salt (18).
  • the resulting solution was freeze-dried to afford the dimeric peptide phospholipid conjugate (11) as a colorless solid.
  • the second portion of crude dimeric peptide phospholipid conjugate (11) was purified as described above.
  • the combined yield of the target dimeric peptide phospholipid conjugate (11) was 0.39 g (57% yield).
  • the samples of the dimeric peptide phospholipid conjugate (11) made from different sample purification runs were pooled together, dissolved in tert-butanol-acetonitrile-water mixture and re-lyophilized to provide the dimeric peptide phospholipid conjugate (11) as a colorless, fluffy solid which was further dried under vacuum.
  • Examples 6-8 below refer to the preparation of the dimer peptide-phospholipid conjugate shown in FIG. 5 , wherein the dimeric conjugate contains very low levels of TFA.
  • FIGS. 6-8 illustrate the methods described in the Examples below.
  • a gradient of NH 4 OAc in 20% of CH 3 CN/H 2 O was applied at 200 mM, 400 mM, 600 mM and 800 mM, 250 mL each.
  • the compound came out at 600 mM NH 4 OAc.
  • the fractions were analyzed by HPLC and the ones containing the compound were combined and lyophilized several times until the weight of the material was constant. 176 mg of the pure material (23) was obtained as a white fluffy solid. The yield was 83.8%.
  • dimer peptide (27) acetate salt similar to the procedure for compound (23), an AG MP-50 column (100 mL wet volume) was washed with 30% CH 3 CN/H 2 O until the conductivity was below 1 ⁇ s/cm.
  • Compound (27) as the TFA salt 120 mg in 80 mL of 30% of CH 3 CN/H 2 O was loaded onto the column and the column was washed with the same eluent until the conductivity was stable at 1 ⁇ s/cm.
  • a step gradient of NH 4 OAc 30% of CH 3 CN/H 2 O into 30% of CH 3 CN/H 2 O was run as for compound (23) and the compound was eluted at ca 600 mM NH 4 OAc.
  • the combined fractions were lyophilized and then relyophilized several times until the material displayed a constant weight to provide 104 mg of the pure material (27) as an acetate salt.
  • the yield was 86.7%.
  • the column was washed with the same eluent until the DMF plug was eluted.
  • the eluent composition was increased to 25% B over 2 min. and then ramped to 65% B over 40 min.
  • the fractions were analyzed on an analytical reverse phase C-18 column (Waters MS C-18, 4.6 ⁇ 50 mm, 5 ⁇ m particle, 100 ⁇ pore, flow rate 3 mL/min) and the product-containing fractions of >95% purity were pooled and freeze-dried to afford 25 mg of the dimer peptide (27) as its acetate salt as a fluffy white solid. The yield was 21.8%.
  • the crude reaction mixture was purified by preparative HPLC equipped using a new Zorbax 300SB-C3 column (21.2 ⁇ 150 mm, 5 ⁇ particle) which had never been exposed to TFA.
  • the HPLC system was pre-washed by CH 3 CN/H 2 O/NH 4 OAc extensively to remove traces of TFA.
  • the reaction mixture was loaded onto the column which was pre-equilibrated with 20% B (A: 10 mM NH 4 OAc in H 2 O; B: 10 mM NH 4 OAc in CH 3 CN/H 2 O, 9/1 v/v) at a flow rate of 30 mL/min.
  • the column was eluted at 30 mL/min with the same eluent until the plug of DMF was eluted.
  • the eluent composition was then increased to 40% B over 3 min and then ramped to 90% B over 50 min.
  • the collected fractions were analyzed on an analytical reverse phase C-3 column (Zorbax 300SB-C3, 3 ⁇ 150 mm, 3.5 ⁇ m particle, 300 ⁇ pore, flow rate: 0.5 mL/min), where detection was accomplished using UV at 220 nm and an evaporative light scattering detector (ELSD).
  • the fractions containing the pure product were pooled and lyophilized. A 6.5 mg portion of the final product (21) acetate salt was obtained. The yield was 33.0%.
  • the crude compound was loaded at a concentration of 30% eluent B.
  • Materials used and conditions include: Conditions: Column: Waters Zorbax 300SB C-3; 21.2 mm i.d. ⁇ 150 mm; 3.5 ⁇ m particle; Eluents: Eluent A: H 2 O(HPLC Grade with 10 mM NH 4 OAc); Eluent B: CH 3 CN/H 2 O, 9/1 (final NH 4 OAc concentration: 10 mM).
  • composition of the eluent was then changed to 45% B over 2 min, then the column was eluted with a linear gradient of 45-100% B over 40 min; Flow rate: 30 mL/min; Detection: UV at 220 nm.
  • the crude compound (100 mg) was dissolved in 25 mL of a solution of 30% B.
  • the preparative HPLC system was equilibrated at 30% B.
  • the compound was loaded on to the Zorbax C-3 column.
  • the mobile phase composition was ramped to 45% B over 2 min.
  • a linear gradient from 45-100% B over 40 min was used for the elution of (21).
  • the product eluted between 26.5-33 min.
  • a Sephadex G-25 column (100 g resin, bead size 20-80 ⁇ m, total gel volume 500 mL, column height: 27 cm) was equilibrated with 4 L of 50 mM ammonium bicarbonate. Then (21) (70 mg) was dissolved in 30 mL (final volume) of 60 mM ammonium bicarbonate in 10% aqueous acetonitrile. The solution was filtered and then loaded on to the Sephadex G-25 column. The column was eluted with 50 mM ammonium bicarbonate buffer with collection of 10 mL fractions. The collected fractions were monitored by analytical HPLC (UV detection at 220 nm). The results are provided in Table 4 below.
  • Fractions 20-28 were pooled and lyophilized.
  • the lyophilized material obtained was re-dissolved in a small volume of water and the solution was frozen and lyophilized to remove residual amounts of ammonium bicarbonate.
  • the final weight of the desired material was 58 mg.
  • the recovery was 83%.
  • Table 5 below provides definitions for the abbreviations used and the sources of materials referred to in Examples 9-12.
  • Example 9A was repeated using a mixture of DSPC/DPPG/ and the monomeric peptide phospholipid conjugate (31) shown in FIG. 10 (molar ratio 49.5/49.5/1, corresponding to 182.8, 172.3 and 28.2 mg of the three components, respectively)
  • micellar suspension An aqueous suspension of DSPE-PEG1000 (0.43 mg-0.24 ⁇ mole) and the monomeric peptide phospholipid conjugate (31) shown in FIG. 10 (3.0 mg-0.5 ⁇ mole) was prepared in 500 ⁇ L of distilled water at 60° C. to obtain a micellar suspension.
  • DPPE 15.8 mg-22.8 ⁇ moles
  • DPPG 4.2 mg-5.7 ⁇ moles
  • the dispersion was then cooled to room temperature.
  • Perfluoroheptane 1.6 mL was emulsified in the aqueous phase using a high speed homogenizer (Polytron PT3000, probe diameter of 3 cm) for 1 minute at 10500 rpm to obtain an emulsion.
  • micellar suspension was added to the emulsion and the resulting mixture was heated at 60° C. for 1 hour under stirring. After cooling to room temperature (1 hour), the obtained emulsion was divided in 4 mL fractions in 50 mL round bottom flasks. The emulsion was frozen at ⁇ 45° C. for 5 minutes and freeze-dried at 0.2 mBar for 24 hours (Freeze-Drier Christ Beta 1-8K).
  • the lyophilisate was exposed to an atmosphere containing C4F10/nitrogen (50/50 by volume). The lyophilized product was then dispersed in a volume of water twice the initial one by gentle hand shaking.
  • micellar suspension An aqueous suspension of DSPE-PEG1000 (0.5 mg-0.27 ⁇ mole) and dimeric peptidephospholipid conjugate (11) shown in FIG. 5 (5.3 mg-0.63 ⁇ mole) was prepared in 500 L of distilled water at 60° C. to obtain a micellar suspension.
  • DPPE 15.8 mg-22.8 ⁇ moles
  • DPPG 4.2 mg-5.7 ⁇ moles
  • a solution of PEG4000 10% in distilled water (20 mL) at 70° C. for 20 minutes.
  • the dispersion was then cooled to room temperature.
  • Perfluoroheptane 1.6 mL was emulsified in the aqueous phase using a high speed homogenizer (Polytron PT3000, probe diameter of 3 cm) for 1 minute at 10000 rpm to obtain an emulsion.
  • micellar suspension was added to the emulsion and the resulting mixture was heated at 80° C. for 1 hour under stirring. After cooling to room temperature (1 hour), the obtained emulsion was washed once by centrifugation (200 g/10 min—Sigma centrifuge 3K10) to eliminate the excess of phospholipid. The separated pellet (containing emulsified microdroplets of solvent) was recovered and re-suspended with the initial volume of a 10% PEG4000 aqueous solution.
  • the obtained emulsion was sampled into DIN8R vials (1 mL/vial). Then vials were cooled at ⁇ 50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at ⁇ 25° C. and 0.2 mBar for 12 hours with a final drying step at 30° C. and 0.1 mBar for 7 hours. Vials were exposed to an atmosphere containing C4F10/nitrogen (35/65 by volume) and sealed. The lyophilized product was redispersed in a volume of water twice the initial one by gentle hand shaking.
  • DIN8R vials (1 mL/vial). Then vials were cooled at ⁇ 50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at ⁇ 25° C. and 0.2 mBar for 12 hours with a final drying step at 30° C. and 0.1 mBar for 7 hours. Vials were exposed to an atmosphere containing C4F10/nitrogen (35/65
  • micellar suspension An aqueous suspension of DSPE-PEG1000 (2.5 mg-1.4 ⁇ mole) and dimeric peptide conjugate (11) shown in FIG. 5 (7.0 mg-0.84 ⁇ mole) was prepared in 1 mL of distilled water at 60° C. to obtain a micellar suspension.
  • DSPC (16.3 mg-20.6 ⁇ moles) and DSPA (3.7 mg-5.15 ⁇ moles) were dissolved in cyclooctane (1.6 mL) at 80° C.
  • This organic phase was added to a PEG4000 10% solution in water (20 mL) using a high speed homogenizer (Polytron T3000, probe diameter of 3 cm) for 1 minute at 8000 rpm, to obtain an emulsion.
  • micellar suspension was mixed with the emulsion and the resulting mixture was heated at 80° C. for 1 hour under agitation. After cooling to room temperature (1 hour), the obtained emulsion was washed once by centrifugation (1500 g/10 min—Sigma centrifuge 3K10) to eliminate the excess of the phospholipid. The separated supernatant (containing emulsified microdroplets of solvent) was recovered and re-suspended in twice the initial volume of a 10% PEG 4000 aqueous solution.
  • the obtained suspension was sampled into DIN8R vials (1 mL/vial). Then vials were cooled to ⁇ 50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at ⁇ 25° C. and 0.2 mbar for 12 hours, with a final drying step at 30° C. and 0.1 mbar for 7 hours. Vials were exposed to an atmosphere containing C 4 F 10 /Nitrogen (35/65 by volume) and sealed. The lyophilized product was then dispersed in a volume of water twice the initial one by gentle hand shaking.
  • DIN8R vials (1 mL/vial). Then vials were cooled to ⁇ 50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at ⁇ 25° C. and 0.2 mbar for 12 hours, with a final drying step at 30° C. and 0.1 mbar for 7 hours. Vials were exposed to an atmosphere containing C 4 F 10 /Nitrog
  • Example 11A was repeated, but using 0.7 mg of DSPE-PEG2000 (0.26 ⁇ moles) and 1.6 mg of monomeric peptide-phospholipid conjugate (1) shown in FIG. 2 (0.26 ⁇ mole) to prepare the micellar suspension.
  • DSPC (16.3 mg-20.6 ⁇ moles), DSPA (3.7 mg-5.15 ⁇ moles) and monomeric peptide phospholipid conjugate (1) shown in FIG. 1 (1.6 mg-0.26 ⁇ mole) were dissolved in cyclooctane (1.6 mL) at 80° C.
  • This 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.
  • the resulting emulsion was heated at 80° C. for 1 hour under stirring. After cooling to room temperature (1 hour), the obtained emulsion was diluted with 20 ml of a PEG4000 10% aqueous solution.
  • the emulsion was sampled into DIN8R vials (1 mL/vial). Then vials were cooled at ⁇ 50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at ⁇ 25° C. and 0.2 mBar for 12 hours with a final drying step at 30° C. and 0.1 mBar for 7 hours. Vials were exposed to an atmosphere containing C4F10/nitrogen (35/65 by volume) and sealed. The lyophilized product was redispersed in a volume of water twice the initial one by gentle hand shaking.
  • DIN8R vials (1 mL/vial). Then vials were cooled at ⁇ 50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at ⁇ 25° C. and 0.2 mBar for 12 hours with a final drying step at 30° C. and 0.1 mBar for 7 hours. Vials were exposed to an atmosphere containing C4F10/nitrogen (35/65 by
  • micellar suspension An aqueous suspension of DSPE-PEG2000 (2.5 mg-0.9 ⁇ moles) and the dimeric phospholipid conjugate (11) shown in FIG. 5 (2.5 mg-0.3 ⁇ moles) was prepared in 660 ⁇ L of distilled water at 60° C. to obtain the micellar suspension.
  • DSPC 18.2 mg-23.1 ⁇ moles
  • stearate 1.8 mg-5.8 ⁇ moles
  • This organic phase was added to a 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, to obtain an emulsion.
  • micellar solution was mixed with the emulsion and the resulting mixture was heated at 80° C. for 1 hour under agitation. After cooling to room temperature (1 hour), the obtained emulsion was washed once by centrifugation (1500 g/10 min—Sigma centrifuge 3K10) to eliminate the excess of phospholipids. The separated supernatant (containing emulsified microdroplets of solvent) was recovered and re-suspended with twice the initial volume of a 10% PEG 4000 aqueous solution.
  • the obtained suspension was sampled into DIN8R vials (1 mL/vial). Then vials were cooled to ⁇ 50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at ⁇ 25° C. and 0.2 mbar for 12 hours, with a final drying step at 30° C. and 0.1 mbar for 7 hours. Vials were exposed to an atmosphere containing C 4 F 10 /Nitrogen (35/65 by volume) and sealed. The lyophilized product was dispersed in a volume of water twice the initial one by gentle hand shaking.
  • Example 12A was repeated by replacing the dimeric peptide phospholipid conjugate (11) shown in FIG. 5 with the same relative molar amount of the monomeric peptide phospholipid conjugate (1) shown in FIG. 2 .
  • Example 11C was repeated with DSPC (18.2 mg-23.1 ⁇ moles), sodium stearate (1.8 mg-5.8 ⁇ moles) and the dimeric peptide phospholipid conjugate (11) shown in FIG. 5 (2.2 mg-0.26 ⁇ mole).
  • the agitation speed for emulsification was fixed to 9000 rpm.
  • the obtained emulsion was washed once by centrifugation (1500 g/10 min—Sigma centrifuge 3K10) to eliminate the excess of the phospholipid.
  • the separated supernatant (containing emulsified microdroplets of solvent) was recovered and re-suspended in twice the initial volume of a 10% PEG 4000 aqueous solution.
  • Cells were grown on poly-D-lysine-coated Thermanox® circular coverslips in 24-well plate. Transfection was done as recommended in the lipofectamine 2000 protocol (Invitrogen, cat# 11668-019) using 1 ⁇ g of DNA (pc-DNA6-fKDR)/per coverslip (1.3 cm2) in 0.1 mL. Transfection was done in serum-free media, the transfection reagent mix was removed from cells after 2 hours and replaced with regular serum-containing medium. Some of the cell-coated coverslips were mock-transfected (with no DNA). The next day, expression of the KDR receptor was assessed by immunocytochemistry and the binding assay was performed.
  • the transfected cells were incubated with KDR-targeted microbubbles resuspended in 50% human plasma in PBS.
  • a small plastic cap was filled with a suspension containing a 1.3 ⁇ 10 8 bubbles and the cap was covered with an inverted Thermanox® coverslip so as to put the transfected cells in contact with the targeted microbubbles.
  • the coverslip was lifted with tweezers, rinsed three times in PBS and examined under a microscope to assess binding of the targeted microbubbles.
  • the same peptide may show different binding activities when included (as a lipopeptide) in different phospholipid formulations forming the stabilizing envelope of the microbubble.
  • Microbubbles containing KDR binding lipopeptides of the invention bind specifically to KDR-expressing cells while they did not bind appreciably to mock transfected cells.
  • a surface of dimensions 14 ⁇ 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.
  • the number of microbubbles 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 microbubbles per minute).
  • the slope represents the bubble binding rate on the target substrate. For instance, a slope value of 8 indicates that an average of eighty (80) microbubbles was bound on the coated coverslip in ten minutes. A higher slope indicates a better capacity of bubbles to bind to the target under flow conditions.
  • the same peptide may show different binding activities when included (as a peptide-phospholipid conjugate or lipopeptide) in different phospholipid formulations forming the stabilizing envelope of the microbubble.
  • VX2 rabbit carcinoma was serially implanted in the dorsal muscle of New Zealand rabbits (Charles River Laboratories, France) weighting 2.5/3 kg.
  • Tumor was surgically removed, placed into McCoy's culture medium containing 10% fetal calf serum, antibiotics, 1.5 mM Glutamax I and cut into small pieces that were rinsed to remove blood and debris. Then tumor pieces (3 to 5 cm 3 ) were placed in a 50 ml Falcon tube containing 5 mL of complete medium. The tumor tissue was ground (Polytron) until no more solid pieces were visible. The murky fluid was centrifuged for 5 minutes at 300 g and the supernatant discarded. Seven mL of fresh medium was added per 5 mL of pellet.
  • VX2 tumor homogenate was injected intramuscularly. Fifteen days after implantation of VX2 tumors, animals were anesthetized again with the same mixture, plus subcutaneous injection of 50% Urethane (2 mL/kg, s.c.) (Sigma) for imaging experiments.
  • VX2 tumor imaging was performed using an ultrasound imaging system ATL HDI 5000 apparatus equipped with a L7-4 linear probe.
  • the linear probe was fixed on the skin directly over the implanted tumors.
  • Example 16 After bubble injection (0.1 ⁇ L/kg of gas) using the preparations of either Example 16 or Example 17, insonation was stopped allowing bubbles to accumulate for 25 minutes. Then, insonation was reactivated at high acoustic power (MI 0.9) destroying all the bubbles present in the tumor. The amount of free circulating bubbles was then assessed by recording the signal obtained after 20 sec accumulation without insonation. Video frames from VX2 tumor imaging experiments were captured with video-capture and analysed with Image-Pro Plus 2.0 software. The image representing free circulating bubbles was subtracted from the image obtained at 25 min, to provide an image representing bound bubbles. Referring to FIG. 11 (which shows the results with the preparation of Example 16) and FIG. 12 (which shows the results with the preparation of Example 17), FIGS.
  • FIGS. 11A and 12A show an image before bubble injection (baseline); FIGS. 11B and 12B show retention of bubble contrast in the tumor 25 minutes post injection; and FIGS. 11C and 12C show the result obtained after subtraction of the baseline and free circulating bubbles and represent bound microbubbles containing KDR lipopeptides according to the present invention.
  • Examples 15-17 and FIGS. 11 and 12 confirm that ultrasound contrast agents bearing such KDR binding moieties localize to KDR expressing (and thus angiogenic) tissue in animal models.
  • Example 12A was repeated by replacing DSPE-PEG2000 with DSPE-PEG1000 (2.7 mg, 1.54 ⁇ mol) and using 2.5 mg (0.31 ⁇ mol) of dimeric peptide phospholipid conjugate (11) shown in FIG. 5 .
  • Example 16 was repeated by replacing the dimeric peptide phospholipid conjugate with the same molar amount of monomeric phospholipid conjugate (1) shown in FIG. 2 .

Abstract

Peptide vectors having high KDR binding affinity and processes for making such vectors are provided. The peptide vectors may be conjugated to phospholipids and included in ultrasound contrast agent compositions. Such ultrasound contrast agents are particularly useful in therapeutic and diagnostic methods, such as in imaging KDR-containing tissue and in the evaluation and treatment of angiogenic processes associated with neoplastic conditions. The present invention also provides processes for the large scale production of highly pure dimeric and monomeric peptide phospholipid conjugates as well as precursor materials used to form the conjugates. The present invention further provides processes for the large scale production of highly pure peptide phospholipid conjugates which contain very low levels of TFA.

Description

    RELATED APPLICATIONS
  • This application is a divisional of U.S. application Ser. No. 11/608,395, filed Dec. 8, 2006 which claims priority to and benefit of U.S. Provisional Application No. 60/833,342, filed Jul. 25, 2006 and U.S. Provisional Application No. 60/749,240, filed Dec. 9, 2005, and is a continuation-in-part of U.S. application Ser. No. 10/661,156, filed Sep. 11, 2003, which is a continuation-in-part of U.S. application Ser. No. 10/382,082, filed Mar. 3, 2003, and a continuation in-part of International Application No. PCT/US03/06731, filed Mar. 3, 2003, both of which claim priority to and benefit of U.S. Provisional Application No. 60/440,411, filed Jan. 15, 2003; and U.S. Provisional Application No. 60/360,851, filed Mar. 1, 2002, the contents of each which are hereby incorporated herein by reference.
    • FIELD OF THE INVENTION
  • The present invention relates to targeting vector-phospholipid conjugates and particularly targeting peptide-phospholipid conjugates, which are useful in therapeutic and diagnostic compositions and methods of preparation of the same. The invention includes targeted ultrasound contrast agents, and particularly targeted microbubbles which include such targeting vector-phospholipid conjugates.
    • BACKGROUND OF THE INVENTION
  • Angiogenesis, the formation of new blood vessels, occurs not only during embryonic development and normal tissue growth and repair, but is also involved in the female reproductive cycle, establishment and maintenance of pregnancy, and repair of wounds and fractures. In addition to angiogenesis that occurs in the normal individual, angiogenic events are involved in a number of pathological processes, notably tumor growth and metastasis, and other conditions in which blood vessel proliferation is increased, such as diabetic retinopathy, psoriasis and arthropathies. In addition, angiogenesis is important in the transition of a tumor from hyperplastic to neoplastic growth. Consequently, inhibition of angiogenesis has become an active cancer therapy research field.
  • Tumor-induced angiogenesis is thought to depend on the production of pro-angiogenic growth factors by the tumor cells, which overcome other forces that tend to keep existing vessels quiescent and stable. The best characterized of these pro-angiogenic agents or growth factors is vascular endothelial growth factor (VEGF) (Cohen et al., FASEB J., 13: 9-22 (1999)). VEGF is produced naturally by a variety of cell types in response to hypoxia and some other stimuli. Many tumors also produce large amounts of VEGF, and/or induce nearby stromal cells to make VEGF (Fukumura et al., Cell, 94: 715-725 (1998)). VEGF, also referred to as VEGF-A, is synthesized as five different splice isoforms of 121, 145, 165, 189, and 206 amino acids. VEGF121 and VEGF165 are the main forms produced, particularly in tumors (see Cohen et al. 1999, supra). VEGF121 lacks a basic domain encoded by exons 6 and 7 of the VEGF gene and does not bind to heparin or extracellular matrix, unlike VEGF165. Each of the references cited in this paragraph is incorporated by reference in its entirety.
  • VEGF family members act primarily by binding to receptor tyrosine kinases. In general, receptor tyrosine kinases are glycoproteins having an extracellular domain capable of binding one or more specific growth factors, a transmembrane domain (usually an alpha helix), a juxtamembrane domain (where the receptor may be regulated, e.g., by phosphorylation), a tyrosine kinase domain (the catalytic component of the receptor), and a carboxy-terminal tail, which in many receptors is involved in recognition and binding of the substrates for the tyrosine kinase. There are three endothelial cell-specific receptor tyrosine kinases known to bind VEGF: VEGFR-1 (Flt-1), VEGFR-2 (KDR or Flk-1), and VEGFR-3 (Flt4). Flt-1 and KDR (also known as VEGFR-2 or Flk-1, which are used interchangeably herein) have been identified as the primary high affinity VEGF receptors. While Flt-1 has higher affinity for VEGF, KDR displays more abundant endothelial cell expression (Bikfalvi et al., J. Cell. Physiol., 149: 50-59 (1991)). Moreover, KDR is thought to dominate the angiogenic response and is therefore of greater therapeutic and diagnostic interest (see Cohen et al. 1999, supra). Expression of KDR is highly upregulated in angiogenic vessels, especially in tumors that induce a strong angiogenic response (Veikkola et al., Cancer Res., 60: 203-212 (2000)). The critical role of KDR in angiogenesis is highlighted by the complete lack of vascular development in homozygous KDR knockout mouse embryos (Folkman et al., Cancer Medicine, 5th Edition (B. C. Decker Inc.; Ontario, Canada, 2000) pp. 132-152).
  • KDR (kinase domain region) is made up of 1336 amino acids in its mature form. The glycosylated form of KDR migrates on an SDS-PAGE gel with an apparent molecular weight of about 205 kDa. KDR contains seven immunoglobulin-like domains in its extracellular domain, of which the first three are the most important in VEGF binding (Cohen et al. 1999, supra). VEGF itself is a homodimer capable of binding to two KDR molecules simultaneously. The result is that two KDR molecules become dimerized upon binding and autophosphorylate, becoming much more active. The increased kinase activity in turn initiates a signaling pathway that mediates the KDR-specific biological effects of VEGF.
  • Thus, not only is the VEGF binding activity of KDR in vivo critical to angiogenesis, but the ability to detect KDR upregulation on endothelial cells or to detect VEGF/KDR binding complexes would be extremely beneficial in detecting or monitoring angiogenesis.
  • It is well known that gas filled ultrasound contrast agents are exceptionally efficient ultrasound reflectors for echography. Such ultrasound contrast agents include, for example, gas-filled microvesicles such as gas-filled microbubbles and gas filled microballoons. Gas filled microbubbles are particularly preferred ultrasound contrast agents. (In this disclosure the term of “microbubble” specifically designates a gaseous bubble surrounded or stabilized by phospholipids). For instance injecting into the bloodstream of living bodies suspensions of air- or gas-filled microbubbles in a carrier liquid will strongly reinforce ultrasonic echography imaging, thus aiding in the visualization of internal anatomical structures. Imaging of vessels and internal organs can strongly help in medical diagnosis, for instance for the detection of neoplastic, cardiovascular and other diseases.
  • For both diagnostic and therapeutic purposes it would be particularly beneficial to incorporate into gas filled ultrasound contrast agents, targeting vector compositions which exhibit high binding affinity for a desired target (such as, for example, KDR or the VEGF/KDR complex). For example, targeting vector-phospholipid conjugates and particularly targeting peptide-phospholipid conjugates may be used to prepare targeted, gas filled ultrasound contrast agents. In addition, it would be particularly beneficial to have methods for large scale production of highly purified forms of such targeting vector-phospholipid conjugates. Such compositions and methods would allow for production of compositions for use in diagnostic or therapeutic applications such as, for example, precise targeting of reporter moieties, tumoricidal agents or angiogenesis inhibitors to the target site.
    • SUMMARY OF THE INVENTION
  • The present invention provides targeting vector-phospholipid conjugates and particularly targeting peptide-phospholipid conjugates which are useful in the preparation of gas filled ultrasound contrast agents. In a preferred embodiment the targeting peptide-phospholipid conjugates include targeting peptides which exhibit high KDR binding affinity and thus are useful components of contrast agents for imaging of angiogenesis processes.
  • The present invention also provides monomeric and dimeric peptide phospholipid conjugates (also referred to herein as lipopeptides) which are useful in preparing gas filled ultrasound contrast agents, and particularly in preparing ultrasound contrast agents which target KDR and may be used for imaging of angiogenesis processes.
  • The present invention also provides methods and processes for the large scale production of highly pure monomeric and dimeric peptide phospholipid conjugates, particularly monomeric and dimeric peptide phospholipids conjugates having high KDR binding affinity.
  • The present invention also provides methods and processes for the large scale production of highly pure dimeric peptide phospholipid conjugates having minimal levels of trifluoroacetic acid (TFA).
  • The present invention also provides methods for synthesizing monomeric peptides in high purity and the construction of peptide phospholipid conjugates from multiple peptide sub-units.
  • The present invention also provides monomeric peptides which bind KDR or the VEGF/KDR complex with high affinity, as well as methods of synthesizing and using such monomeric peptides.
  • The present invention also provides targeted ultrasound contrast agents prepared from such targeting vector-phospholipid conjugates. Such targeted ultrasound contrast agents are useful for imaging target-bearing tissue. In a preferred embodiment, the targeted ultrasound contrast agents are targeted microbubbles and the targeting vector-phospholipid conjugates include targeting peptides which exhibit high KDR binding affinity and thus are useful components of contrast agents for imaging KDR-bearing tissue and particularly for imaging of tumors and angiogenesis processes. Methods of preparing and using such targeted ultrasound contrast agents are also provided.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 illustrates a method for the production of a monomeric peptide phospholipid conjugate (1) from a linear peptide monomer (2).
  • FIG. 2 illustrates a monomeric peptide phospholipid conjugate (1) including a peptide with high binding affinity for KDR.
  • FIG. 3 illustrates a method for the production of a precursor dimer peptide (16) from peptide monomers.
  • FIG. 4 illustrates a method for the conjugation of the precursor dimer peptide shown in FIG. 1 to DSPE-PEG2000-NH2 to form a dimeric peptide phospholipid conjugate (11) containing peptides which bind with high affinity to KDR.
  • FIG. 5 illustrates a dimeric peptide-phospholipid conjugate (11) containing peptides which bind with high affinity to KDR.
  • FIG. 6 illustrates a method for the production of dimer peptide-phospholipid conjugates (such as (21)) having minimal levels of TFA.
  • FIG. 7 illustrates another method for the production of dimer peptide-phospholipid conjugates (such as (21)) having minimal levels of TFA.
  • FIG. 8 illustrates another method for the production of dimer peptide-phospholipid conjugates having minimal levels of TFA.
  • FIG. 9 illustrates another representative monomeric peptide (32) having a high binding affinity for KDR.
  • FIG. 10 illustrates another monomeric peptide-phospholipid conjugate (31) which includes the monomeric peptide shown in FIG. 9.
  • FIGS. 11A-C show images obtained by using the dimer peptide-phospholipid conjugate (11) (shown in FIG. 52) in a contrast agent at: 1) baseline (FIG. 11A); 2) after 25 minutes (FIG. 11B); and 3) after subtraction of the baseline and free circulating bubbles (FIG. 11C).
  • FIGS. 12A-C show images obtained by using the monomeric phospholipid peptide conjugate (1) (shown in FIG. 2) in a contrast agent at baseline (FIG. 12A); after 25 minutes (FIG. 12B); and after subtraction of the baseline and free circulating bubbles (FIG. 12C).
    •  DETAILED DESCRIPTION
  • Applicants have unexpectedly discovered peptide phospholipid conjugates, which are useful in producing targeted ultrasound contrast agents and which have exceptional KDR binding efficiency. Two of these compounds are monomeric peptide phospholipid conjugates which include a linear peptide monomer which binds with high affinity to KDR while the other is a dimeric peptide phospholipid conjugate which includes two distinct monomer subunits, each binding to KDR. In addition, highly efficient methods for large scale production of purified forms of these conjugates and precursor materials have been discovered. Such methods include the production of dimeric peptide phospholipid conjugates having minimal levels of TFA.
  • The phospholipid may be selected from the group consisting of: phosphatidylethanolamines and modified phosphatidylethanolamines Particularly preferred phospholipids include phosphatidylethanolamines modified by linking a hydrophilic polymer thereto. Examples of modified phosphatidylethanolamines are phosphatidylethanolamines (PE) modified with polyethylenglycol (PEG), in brief “PE-PEGs”, 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, DSPE-PEG, DMPE-PEG or DAPE-PEG. DSPE-PEG2000, DSPE-PEG3400, DPPE-PEG2000 and DPPE-PEG3400 are preferred, with DSPE-PEG2000 particularly preferred. Note that a salt form of the phospholipid may be used, such as, for example, the trimethyl ammonium salt, the tetramethylammonium salt, the triethylammonium salt, sodium salt, etc.
  • These compounds may be incorporated into gas filled ultrasound contrast agents, such as, for example, gas filled microbubbles to form contrast agents that provide excellent imaging of target-bearing tissue. In a preferred embodiment, targeting vector-phospholipid conjugates which include targeting peptides which bind with high affinity to KDR are incorporated into targeted microbubbles. As shown herein, such targeted microbubbles selectively localize at KDR-bearing tissue, permitting imaging of such tissue, and, in particular imaging of tumors and angiogenic processes, including those processes associated with neoplastic development.
    • Monomer Conjugates
  • Generally
  • Table 1 provides a description for the identification labels shown in FIGS. 1, 2, 9 and 10.
  • TABLE 1
    1 Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-
    NH2 (SEQ ID NO. 1)
    2 Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH2 (SEQ ID NO. 2)
    3 mono-NHS ester of glutaryl-peptide monomer (2)
    Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(NHS-Glut)-NH2
    (SEQ ID NO. 3)
    4 DSPE-PEG2000-NH2 phospholipid
    1,2-distearoyl-sn-glycero-3-phosphoethanolaminocarbonyloxy-
    (PEG2000)-amine
    31 Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH2
    (SEQ ID NO. 4)
    32 Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK-NH2 (SEQ ID NO. 5)
  • As shown if FIGS. 1 and 2 the monomeric peptide phospholipid conjugate (1) N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-L-serinyl-L-methionyl-L-alanyl-L-aspartyl-L-glutaminyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L1-phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl-{N6-[1,2-d]stearoyl-sn-glycero-3-phosphoethanolaminocarbonyloxy-(PEG2000)-aminoglutaryl]}-L-lysinamide, is a phospholipid conjugate. This conjugate is also referred to as Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH2 (SEQ ID NO. 1) and Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys(DSPE-PEG2000-NH-Glut)-NH2. It comprises a 29 amino acid linear peptide monomer (2) N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-L-serinyl-L-methionyl-L-alanyl-L-aspartyl-L-glutaminyl-L-leucyl-L1-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl-L-lysinamide, also referred to as Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH2 (SEQ ID NO. 2) and Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH2. This novel peptide monomer binds with high affinity to KDR. It should be understood that analogs and derivatives of the monomeric peptide phospholipid conjugate (1) and the linear peptide monomer (2) are intended to be included within the scope of the present invention.
  • FIG. 10 provides the structure of another monomeric peptide phospholipid conjugate (31), N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-{N6-[1,2-d]stearoyl-sn-glycero-3-phosphoethanolaminocarbonyloxy-(PEG2000)-aminoglutaryl]}-L-lysine-amide, a phospholipid conjugate. This conjugate is also referred to as Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH2 (SEQ ID NO. 4) and Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys(DSPE-PEG2000-NH-Glut)-NH2. As shown in FIG. 9, the conjugate comprises a 28 amino acid linear peptide monomer (32), N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide, which is also referred to as Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK-NH2 (SEQ ID NO. 5) and Ac-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH2. As shown in co-pending application, U.S. application Ser. No. 10/661,156, filed Sep. 11, 2003, this peptide monomer binds with high affinity to KDR. It should be understood that analogs and derivatives of the monomeric peptide phospholipid conjugate and the linear peptide monomer are intended to be included within the scope of the present invention.
  • As shown in the Examples, ultrasound contrast agents such as gas filled microbubbles formulated with the monomeric peptide phospholipid conjugates (1) and (31) displayed high KDR binding which was confirmed using echographic examination of VX2 tumors in rabbits.
  • Ideally, to facilitate production of the monomeric peptide phospholipid conjugate (1) or (31), the linear peptide monomer (2) or (32) should be prepared in bulk. Then conjugation of the purified linear peptide monomer (2) or (32) to the phospholipid, such as, for example, a pegylated phospholipid in salt form, e.g., DSPE-PEG2000-NH2 phospholipid ammonium salt (4) via the linker disuccinimidyl glutarate (DSG), may be used to provide monomeric peptide phospholipid conjugates (1) or (31).
  • Methods of Preparation of Monomer Peptide-Phospholipid Conjugates
  • In preparing monomeric peptide phospholipid conjugates (1) and (31), methods according to the present invention provide at least the following advantages: increased yield of peptide synthesis; reduced extent of racemization; avoidance of previously observed piperidine amide formation during synthesis, efficient purification of peptide monomers (2) and (32), development of a procedure for conjugation of peptide monomers (2) and (32) on larger scale; and development of purification protocols that would allow the ready separation of the monomeric peptide phospholipid conjugates (1) and (31) from the starting DSPE-PEG2000-NH2 phospholipid ammonium salt (4).
  • Monomeric peptide phospholipid conjugates may be prepared as described below. It should be appreciated that the numerical values referred to in this representative description of the synthesis of monomeric peptide phospholipid conjugates are representative.
  • Linear peptide monomers may be prepared by SPPS. The sequence of the linear peptide monomers may be constructed as a C-terminal carboxamide on Pal-Peg-PS-resin (substitution level: 0.2 mmol/g). Peptide synthesis may be accomplished using Fmoc chemistry on a SONATA®/Pilot Peptide Synthesizer. Problems previously observed with this process have been racemization, incomplete couplings and piperidine amide formation, each of which contribute to suboptimal yield and purity. A dramatic decrease in the formation of the piperidine amide may be attained by the use of 25% piperidine in DMF containing HOBt (0.1M) as the reagent for Fmoc removal. Racemization may be considerably reduced by using DIC/HOBt as the activator for most couplings; a 3 h coupling time using a four-fold excess of pre-activated Fmoc-amino acid with an intervening wash with anhydrous DMF (6×). N′-Fmoc amino acids may be dissolved just before their coupling turn and pre-activated with DIC/HOBt in DMF for 4 min and transferred to the reaction vessel. This may be accomplished on the Sonata instrument by loading the solid Fmoc-amino acids into the amino acid vessels of the instrument and then programming the instrument to add DMF, HOBt/DMF and DIC/DMF sequentially with bubbling of the solution.
  • To optimize the yield, the problem of aggregation of the resin during the synthesis of longer peptides, which can be devastating even when optimal coupling reagents are employed, may be addressed. To reduce aggregation during peptide assembly the strategy of using pseudoproline dipeptides to incorporate X-Thr or X-Ser as dipeptides instead of sequential couplings of X and Thr or X and Ser, may be employed. For linear peptide monomers sequential couplings of Leu11-Ser12 and Leu22-Ser23 may be replaced by the single coupling of the pseudoproline dipeptide, Fmoc-Leu-Ser(ψMe,Mepro)-OH. Additional optimization may be accomplished by reducing the number of couplings by using Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, in lieu of serial coupling of Fmoc-Gly-OH. Activation of -Gly-Gly-OH segments may lead to cyclization of the activated acid function with the distal amide function to produce an inactive diketopiperazine; this may reduce coupling yields in a time dependant manner. This problem may be avoided by addition of Fmoc-Glyn-OH (n=2, 3) to the reaction vessel and sequential addition of HOBt and DIC; the activated Fmoc-Glyn-OH may be intercepted by the resin-bound amino group before appreciable cyclization to the diketopiperazine takes place. With these improvements, the synthesis of linear peptide monomers may be completed on the Sonata Peptide Synthesizer on a 10 mmol synthesis scale.
  • After chain elongation, the Fmoc may be removed from the N-terminus. The peptide and the free amino group may be acetylated. Then the peptide sequence may be cleaved from the resin and deprotected using “Reagent B” (TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) for 4 h. After the cleavage reaction the crude peptide may be isolated as a solid by evaporation of the volatiles, trituration of the residue with diethyl ether and washing of the solid thus obtained using the same solvent. In another variation the peptide may be precipitated from the reaction mixture by addition of diethyl ether to the reaction mixture, collecting the solid thus formed and washing with the same solvent.
  • Linear peptide monomers may be purified as described below. Again, the numerical references are representative. Crude linear peptide monomers (0.5 g) may be dissolved in CH3CN (40 mL/g) and this solution may be diluted to a final volume of 100 mL with water. The solution may then be filtered. The filtered solution may be loaded onto the preparative HPLC column (Waters, XTerra® Prep MS C18, 10μ, 300 Å, 50×250 mm) equilibrated with 10% CH3CN in water (0.1% TFA). After loading, the composition of the eluent may then be ramped to 20% CH3CN-water (0.1% TFA) over 1 min, and a linear gradient may be initiated at a rate of 0.6%/min of CH3CN (0.1% TFA) into water (0.1% TFA) and run for 50 min. Eluted fractions may be checked for purity on an analytical reversed phase C18 column (Waters XTerra MS-C18, 10μ, 120 Å, 4.6×50 mm) and fractions containing the product in >95% purity may be combined and freeze-dried. For each purification of 0.5 g of crude peptide 0.12 g (24%) of linear peptide monomer may be consistently isolated and will provide the peptide in the same yield and purity.
  • Synthesis of monomeric peptide phospholipid conjugates may be performed as described below. The numerical references are again representative. The last step in the synthesis may be the conjugation of the phospholipid, such as, for example, a pegylated phospholipid such as DSPE-PEG2000-NH2 phospholipid ammonium salt to a linear peptide monomer. The PEG2000 moiety of DSPE-PEG2000-NH2 phospholipid ammonium salt (4) is nominally comprised of 45 ethylene glycol units. It should be understood, however, that this material is a distribution of PEG containing species whose centroid is the nominal compound containing 45 ethylenoxy units. The conjugation of a linear peptide monomer with DSPE-PEG2000-NH2 phospholipid ammonium salt may be accomplished by preparation of the glutaric acid monoamide mono NHS ester of a linear peptide monomer and reaction of this with the free amino group of the phospholipid ammonium salt. Thus a linear peptide monomer may be reacted with DSG (4 eq.) in DMF in the presence of DIEA (5 eq.) for 30 min. The reaction mixture may be diluted with ethyl acetate, which may result in precipitation of the peptide glutaric acid monoamide mono-NHS ester. The supernatant containing un-reacted DSG may be decanted and the intermediate peptide mono-NHS ester may be washed several times with ethyl acetate to remove traces of DSG. Mass spectral data confirms the formation of the peptide mono-NHS ester as a clean product. The solid mono-NHS ester may be dissolved in DMF and reacted with DSPE-PEG2000-NH2 phospholipid ammonium salt (0.9 eq.) in the presence of DIEA (4 eq.) for 24 h. The linear peptide monomer glutaric acid monoamide mono-NHS ester may be used in excess to maximize the consumption of the phospholipid ammonium salt because free phospholipid ammonium salt may complicate the isolation of monomeric peptide phospholipid conjugates in highly pure form.
  • The reaction mixture may be diluted with a 1:1 mixture of water (0.1% TFA) and CH3CN—CH3OH (1:1, v/v) (0.1% TFA) (100 mL), applied to a reversed phase C2 column (Kromasil® Prep C2, 10μ, 300 Å, 50×250 mm, flow rate 100 mL/min) and the column may be eluted with a 3:1 mixture of water (0.1% TFA) and CH3CN—CH3OH (1:1, v/v) (0.1% TFA) to remove hydrophilic impurities. Then the product may be eluted using a gradient of CH3CN—CH3OH (1:1) (0.1% TFA) into water (0.1% TFA) (see Experimental Section for details). The collected fractions may be analyzed by reversed phase HPLC using an ELS detector which allows the detection of the desired product and the often difficult-to-separate DSPE-PEG2000-NH2 phospholipid which has very little UV absorbance. This indicates the clear separation of the monomeric peptide phospholipid conjugates and DSPE-PEG2000-NH2 phospholipid. The pure product-containing fractions may be collected, concentrated on a rotary evaporator (to reduce the content of methanol) and freeze-dried to provide monomeric peptide phospholipid conjugates as a colorless solid. In order to prepare the required quantity of the monomeric peptide phospholipid conjugates, several runs may be conducted employing 0.5 g to 1.0 g of linear peptide monomer. In all cases the target monomeric peptide phospholipid conjugates may be were isolated in high yield and purity (e.g., 57-60% yield and >99% purity).
    • Dimer Conjugate
  • Generally
  • Table 2 provides a description for the identification labels shown in FIGS. 3,4 and 5.
  • TABLE 2
    11 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWLDSWGGEVCFRYDP-GGGK[-
    Adoa-Adoa-Glut-K(DSPE-PLG2000-NH-Glut)]-NH2 cyclic (2-12)
    disulfide}-NH2 cyclic (6-13) disulfide
    12 Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH2 cyclic (6-13)
    disulfide
    13 Ac-VCWEDSWGGLVCFRYDPGGGK(Adoa-Adoa)-NH2 cyclic (2-12)
    disulfide
    14 mono-NHS ester of glutaryl-peptide 12
    Ac-AGPTWCEDDWYYCWLFGTGGGK[NHS-Glut-K(ivDde)]-NH2 cyclic
    (6-13) disulfide
    15 ivDde-bearing dimer
    Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK[-Adoa-
    Adoa-Glut-K(ivDde)]-NH2 cyclic (2-12) disulfide}-
    NH2 cyclic (6-13) disulfide
    16 Ac-AGPTWCLDDWYYCWLFGTGGGK[Ac-VCWLDSWGGLVCFRYDPGGGK(-
    Adoa-Adoa-Glut-K)-NH2 cyclic (2-12) disulfide]-NH2
    cyclic (6-13) disulfide
    17 Mono-NHS ester of glutaryl-peptide 16
    Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK[-Adoa-
    Adoa-Glut-K(NHS-Glut)]-NH2 cyclic (2-12) disulfide}-
    NH2 cyclic (6-13) disulfide
    18 DSPE-PLG2000-NH2 phospholipid
  • As shown in those figures the dimeric peptide phospholipid conjugate (11) Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-1-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(distearylphosphoethanolaminocarbonoxy-PEG2000-amino-8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl) amide cyclic (2-12) disulfide]-amide cyclic (6-13) disulfide, consists of two monomeric peptide chains which bind KDR: a 21 amino acid cyclic disulfide peptide monomer (13) Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl)amide cyclic (2-12) disulfide, and a 22 amino acid cyclic disulfide peptide monomer (12) Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-L-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysinamide cyclic 6-13 disulfide tethered by a glutaryl linker. It should be understood that analogs and derivatives of the dimeric peptide phospholipid conjugate (11) and the cyclic disulfide peptide monomers (12) and (13) are intended to be included within the scope of the present invention.
  • Ultrasound contrast agents (e.g. gas filled microbubbles) formulated with the dimeric peptide phospholipid conjugate (11) displayed high KDR binding which was confirmed using echographic examination of VX2 tumors in rabbits.
  • Methods of Preparation of Dimer-Phospholipid Conjugates
  • To accomplish synthesis of the dimeric peptide phospholipid conjugate (11), the monomers used for this purpose optimally should be prepared in bulk. Then the monomers may be tethered to each other using di-succinimidyl glutarate as a linker to form the precursor dimer peptide (16), Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-L-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl) amide cyclic (2-12) disulfide]-amide cyclic (6-13) disulfide. Then conjugation of the purified precursor dimer peptide (16) to a DSPE-PEG2000-NH2 phospholipid ammonium salt (18) again via disuccinimidyl glutarate may be used in order to provide the target dimeric peptide phospholipid conjugate (11).
  • In preparing dimeric peptide phospholipid conjugate (11), methods according to the present invention provide at least the following advantages: increased yield of automated chain elongation of the peptide sequences; reduced extent of racemization encountered during synthesis; avoidance of previously observed piperidine amide formation during synthesis of peptide monomer (13); cyclization of linear di-cysteine containing peptide precursors of (12) and (13) using procedures amenable to multigram scale yet allowing efficient and practical sample handling; efficient purification of monomer peptides (12) and (13); maximized yield and purity of precursor dimer peptide (16); development of a procedure for conjugation of the precursor dimer peptide (16) on larger scale; and development of purification protocols that would allow the ready separation of the target dimeric peptide phospholipid conjugate (11) from phospholipid ammonium salt (18).
  • The dimeric peptide phospholipid conjugate (11) may be prepared by automated synthesis of the peptide monomers (12), Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH2 cyclic (6-13) disulfide, and (13), Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH2 cyclic (2-12) disulfide, their efficient coupling using disuccinimidyl glutarate (DSG) to give an ivDde-protected dimer, its deprotection and subsequent coupling to DSPE-PEG2000-NH2, also via a glutaryl linkage. Using procedures according to the present invention, monomer peptides may be synthesized on a 10 mmol scale without complication and after HPLC purification may be obtained in about 20% yield and >95% purity. Such methods allow dimer formation reactions and the subsequent conjugation to the phospholipid component providing formation of dimeric peptide phospholipid conjugate (11) to be carried out on a gram scale. The precursor dimer peptide (16) may be obtained from the monomer peptides routinely in about 32% yield and >95% purity. The dimeric peptide phospholipid conjugate (11) may be produced from the precursor dimer peptide (16) in 57-60% yield and >99% purity.
  • Dimeric peptide phospholipid conjugates may be prepared as described below. It should be appreciated that the numerical values referred to in this representative description of the synthesis of dimeric peptide phospholipid conjugates are representative.
  • Described below is a representative method for the solid phase synthesis and disulfide cyclization of a peptide monomer (12) Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH2 cyclic (6-13) disulfide, and a peptide monomer (13), Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH2 cyclic (2-12) disulfide.
  • The peptides may be constructed as their C-terminal carboxamides on Pal-Peg-PS-resin (substitution level: 0.2 mmol/g). Chain elongation may be accomplished using Fmoc chemistry employing optimized deprotection and coupling protocols on a SONATA®/Pilot Peptide Synthesizer on a 10 mmol synthesis scale. The optimized synthesis of the peptides by automated SPSS may be developed by study of peptide impurities and the effect of changes of particular elements of the protocols on the overall yield and purity of the peptides obtained.
  • Analysis of the impurities obtained from nonoptimized syntheses of the monomer peptides indicates that the major problems are racemization, incomplete couplings and piperidine amide formation (presumably via an intermediate aspartimide or glutarimide intermediate), each of which contributes to suboptimal yield and purity. A dramatic decrease in formation of the piperidine amide may be attained by the use of 25% piperidine in DMF containing HOBt (0.1 M) as the reagent for finoc removal. Racemization may be considerably reduced by using DIC/HOBt as the activator for most couplings; and a 3 h coupling time using a four-fold excess of pre-activated Fmoc-amino acid with an intervening wash with anhydrous DMF (6×). N-{umlaut over (α)}Fmoc amino acids may be dissolved just before their coupling turn and pre-activated with DIC/HOBt in DMF for 4 min and transferred to the reaction vessel. This may be accomplished on the Sonata instrument by loading the solid Fmoc-amino acids into the amino acid vessels of the instrument and then programming the instrument to add DMF, HOBt/DMF and DIC/DMF sequentially with bubbling of the solution after each addition.
  • To optimize the yield, the problem of aggregation of the resin during the synthesis of longer peptides, which can be devastating even when optimal coupling reagents are employed, may be addressed. To reduce aggregation during peptide assembly the strategy of using pseudoproline dipeptides to incorporate X-Thr or X-Ser (X refers to the n-1 amino acid of the sequence) as dipeptides instead of sequential couplings of X and Thr or X and Ser, may be employed. Thus, for the monomer (12), Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH2 cyclic (6-13) disulfide, sequential coupling of suitably protected Thr and Gly (shown in bold above) may be replaced by the single coupling of the pseudoproline dipeptide, Fmoc-Gly-Thr(ψMe,Mepro)-OH. Similarly, in the synthesis of the monomer (13), Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH2 cyclic (2-12) disulfide, the pseudoproline dipeptide, Fmoc-Asp(OtBu)-Ser(ψMe,Mepro)-OH may be employed to replace the sequential coupling of suitably protected Ser and Asp (shown in bold font above). Further optimization may be accomplished by reducing the number of couplings by using Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, in lieu of serial coupling of Fmoc-Gly-OH. Activation of -Gly-Gly-OH segments can lead to cyclization of the activated acid function with the distal amide function to produce an inactive diketopiperazine; this may reduce coupling yields in a time dependant manner. This problem may be avoided by addition of Fmoc-Glyn-OH (n=2, 3) to the reaction vessel and sequential addition of HOBt and DIC; the activated Fmoc-Glyn-OH may be intercepted by the resin-bound amino group before appreciable cyclization to the diketopiperazine takes place. After chain elongation is completed the N-terminal Fmoc protecting group may be removed from each of the peptides and the free amino group may be acetylated.
  • The pseudo-orthogonally protected derivative, Fmoc-Lys(ivDde)-OH may be used to enable the selective unmasking of the ε-amine of the C-terminal lysine of the monomer and dimer peptides and their subsequent functionalization, which also may be optimized. The ivDde group on the ε-amine of the C-terminal lysine of each of the peptide monomers may be removed using 10% hydrazine in DMF. Then Fmoc-Adoa, for monomer (13) or Lys(ivDde) for monomer (12) may be appended to the exposed lysine ε-amino group using 4 equivalents of the Fmoc amino acid and 4 equivalents each of DIC and HOBt in DMF for 10 h. After completion of the synthesis, the peptide sequence may be cleaved from the resin and deprotected using “Reagent B” (TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) for 4 h. After the cleavage reaction was complete the peptide may be precipitated, washed with diethyl ether and dried.
  • The following procedures for cyclization of the linear di-cysteine containing peptides may be used to provide optimal scale-up of monomer peptides. Generally the aerial oxidation of linear di-cysteine peptides may be carried out at a concentration of approximately 0.5-5 mg/mL (for the disclosed peptide monomers ˜0.18-1.8 mM in peptide, ˜0.36-3.6 mM in cysteine thiol). In order to work at significantly higher concentrations DMSO-assisted cyclization of di-cysteine peptides allows the cyclization of ˜10 g of the linear peptides in good yields in as little as ˜50 mL of solution. Therefore the crude linear di-cysteine peptides may be cyclized in 95% DMSO-H2O (5 mL/g) at pH 8.5 at ambient temperature. The progress of the cyclization may be routinely followed by mass spectroscopy and HPLC. Although cyclization may be essentially complete in ˜36 h, the reaction mixture may be generally stirred for up to 48 h. The cyclic disulfide peptides may be precipitated from the reaction mixture by dilution with CH3CN and the resulting off-white crude solid peptides may be collected by filtration. This is a convenient method for removing DMSO from the crude cyclic peptide.
  • Purification and isolation of monomer peptide (12), Ac-AGPTWC*EDDWYYC*WLFGTGGGK [K(ivDde)]-NH2 may be accomplished as described below. Note that as used herein the designation “C*” refers to a cysteine residue that contributes to a disulfide bond. Attempts to dissolve 0.5 g of the crude peptide in up to 300 mL of 30% CH3CN in water (0.1% TFA) have been unsuccessful. Therefore, as an alternative, the crude peptide, (0.5 g) may be dissolved in DMSO (5 mL/g) and this solution may be diluted to a final volume of 100 mL with 20% CH3CN-water. The solution may be filtered. The filtered solution may be loaded onto the preparative HPLC column (Waters, XTerra® Prep MS C18, 10μ, 300 Å, 50×250 mm) equilibrated with 10% CH3CN (0.1% TFA) in water (0.1% TFA), and the column may be eluted with 10% CH3CN (0.1% TFA) in water (0.1% TFA) to wash DMSO from the column. The composition of the eluent then may be ramped to 35% CH3CN-water (01% TFA) over 1 min, and a linear gradient may be initiated at a rate of 0.5%/min of CH3CN (0.1% TFA) into water (0.1% TFA) and run for 50 min. Eluted fractions may be checked for purity on an analytical reversed phase C18 column (Waters XTerra MS-C18, 10μ, 120 Å, 4.6×50 mm) and fractions containing the product in >95% purity may be combined and freeze-dried. For each purification of 0.5 g of crude peptide 0.1 g (20%) for (12), Ac-AGPTWC*EDDWYYC*WLFGTGGGK [K(ivDde)]-NH2 may be isolated. Repeat purifications have been found to provide the peptide consistently in the same yield and purity.
  • The peptide monomer (13), Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2 may be purified and isolated as described for peptide monomer (12) except that the subject peptide may be dissolved in 20% CH3CN (0.1% TFA) in 0.1% aqueous TFA (0.5 g peptide/100 mL) instead of a DMSO-containing diluent. The resulting solution of crude peptide may be loaded onto the preparative HPLC column (Waters, XTerra® Prep MS C18, 10μ, 300 Å, 50×250 mm, flow rate 100 mL/min) equilibrated with 10% CH3CN in water (0.1% TFA). The column may be eluted with 10% CH3CN (0.1% TFA)/water (0.1% TFA) at 100 mL/min for 5 min. Then the composition of the eluent may be ramped to 30% CH3CN (0.1% TFA)/water (0.1% TFA) over 1 min and a linear gradient rate of 0.5%/min of CH3CN (0.1% TFA) into water (0.1% TFA) may be initiated, and maintained until the desired peptide is completely eluted from the column. Product-containing fractions may be analyzed on a Waters XTerra analytical reversed phase C-18 column (10μ, 120 Å) and fractions containing the product in >95% purity may be pooled and freeze-dried to afford the cyclic disulfide peptide monomer (13) (0.12 g, 24% yield) in >95% purity. The 10 g of crude peptide monomer may be purified serially in this manner.
  • Described below is a representative method for preparing the precursor dimer peptide (16), Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH2 cyclic (2-12) disulfide]-NH2 cyclic (6-13) disulfide. The preparation of the precursor dimer peptide may be accomplished by the tethering of the monomer peptides in a two step procedure. First, Ac-AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDde)]-NH2 (12) may be reacted with disuccinimidyl glutarate (DSG, 5 eq.) in DMF in the presence of DIEA (5 eq.) for 30 min. The reaction mixture may be diluted with ethyl acetate, which results in precipitation of the glutaric acid monoamide mono-NHS ester of the peptide. The supernatant, containing unreacted DSG, may be decanted and the mono-NHS ester may be washed several times with ethyl acetate to remove traces of DSG. Mass spectral data confirms the formation of the mono-NHS ester as a clean product. This may be redissolved in DMF and reacted with monomer peptide Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2 (13) in the presence of DIEA (5 eq). HPLC and MS results indicate the formation of the ivDde-bearing dimer, as a single major product. The ivDde protecting group on the ε-amine of Lys of the dimer may be removed by stirring the reaction mixture with hydrazine (10%) in DMF for 20 min. The solution then may be acidified with TFA and diluted with 10% CH3CN (0.1% TFA)-water (0.1% TFA), applied to a preparative reversed phase C18 HPLC column and purified by a gradient elution of acetonitrile (0.1% TFA) into 0.1% aqueous TFA. In order to provide the needed quantity of the precursor dimer peptide, the reaction may be conducted employing from 0.5 g to as much as 1 g of each of the monomer peptides. In every case the required precursor dimer peptide may be isolated in ˜32% yield and >95% purity confirming the reproducibility and scalability of the procedures.
  • The final step in the synthesis may be the conjugation of DSPE-PEG2000-NH2 phospholipid ammonium salt (18) to the precursor dimer peptide. As mentioned previously, the PEG2000 moiety of DSPE-PEG2000-NH2 is nominally comprised of 45 ethylene glycol units. It should be understood, however, that this material is a distribution of PEG containing species whose centroid is the nominal compound containing 45 ethylenoxy units.
  • Conjugation of the DSPE-PEG2000-NH2 to the precursor dimer peptide may be accomplished by preparation of a glutaric acid monoamide mono NHS ester of the precursor dimer and reaction of this with the free amino group of the phospholipid ammonium salt. Thus the ivDde bearing precursor dimer peptide (16) may be reacted with DSG (4 eq.) in DMF in the presence of DIEA (5 eq.) for 30 min. As in the preparation of the precursor dimer peptide the solution may be diluted with ethyl acetate to precipitate the glutaric acid monoamide mono-NHS ester of the dimer (17), as a solid. The supernatant may be decanted to remove the un-reacted DSG. The solid glutaric acid monoamide mono-NHS ester of the dimer peptide (17) may then be washed several times with ethyl acetate to remove traces of DSG. Mass spectral results confirm the formation of the glutaric acid monoamide mono-NHS ester of the peptide dimer as a clean product.
  • The dimer glutaric acid monoamide mono-NHS ester (17) may be dissolved in DMF-CH2Cl2 (8:2) and reacted with DSPE-PEG2000-NH2 phospholipid ammonium salt (0.9 eq.) in the presence of DIEA (4 eq.) for 24 h. The NHS ester (17) may be used in excess to maximize the consumption of the phospholipid ammonium salt because any free phospholipid may complicate the purification and isolation of the final product. The reaction mixture may be diluted with water (0.1% TFA)-CH3CN—CH3OH (1:1) (0.1% TFA) (100 mL), applied to a reversed phase C4 column (Kromasil® Prep C4, 10μ, 300 Å, 50×250 mm, flow rate 100 mL/min) and the column may be eluted with water (0.1% TFA)-CH3CN—CH3OH (1:1) (0.1% TFA) solvent mixture to remove hydrophilic impurities. Then the product may be eluted using a gradient of CH3CN—CH3OH (1:1) (0.1% TFA) into water (0.1% TFA). The collected fractions may be analyzed by reversed phase HPLC using an ELS detector which allows the detection of the desired product and the often difficult to separate DSPE-PEG2000-NH2 phospholipid ammonium salt which has no strong UV chromophore. This indicates the clear separation of dimeric peptide phospholipid conjugate and DSPE-PEG2000-NH2 phospholipid ammonium salt. The pure product-containing fractions may be collected, concentrated on a rotary evaporator (to reduce the content of methanol) and freeze-dried to provide the dimer peptide phospholipid conjugate as a colorless solid.
  • In order to prepare the required quantity of the dimer peptide phospholipid conjugate, several runs may be conducted employing 0.5 g to 1.0 g of the precursor dimer peptide. In all cases the target dimer peptide phospholipid conjugate may be isolated in 57-60% yield and in >99% purity. The bulk quantity of dimer peptide phospholipid conjugate, obtained from the serial runs described above may be obtained by dissolution of the product from the individual runs in t-butanol-acetonitrile-water (1:1:3) followed by lyophilization. The procedure of Ellman for quantitative estimation of free thiol may be applied to the bulk sample of the dimeric peptide phospholipid conjugate; free thiol, if present will be below the limit of detection. Amino acid composition analysis gives results within the acceptable limits, supporting the assigned structure of the peptide derivative. MALDI-TOF mass spectral analysis also supports the presumed structure of the dimeric peptide phospholipid conjugate.
  • Methods of Preparation of Dimer-Phospholipid Conjugates Having Low or Negligible Levels of TFA
  • The present invention also provides methods for producing dimeric peptide-phospholipid conjugates having very low levels of TFA. While certain methods provide for the synthesis and purification of such conjugates on a gram scale, formation of a lyso-version of the conjugates has been observed upon storage of lyophilized material at 5° C. or upon storage of aqueous solutions of the conjugates. It is believed that the lyso-compound is formed by TFA-promoted acid hydrolysis of one of the phospholipid fatty acid esters in dimer peptide-phospholipid conjugates.
  • To obtain the phospholipid peptide as a stable material bearing a pharmaceutically acceptable counterion, highly efficient methods for obtaining dimer peptide-phospholipid conjugates were discovered which convert the TFA salts of the dimer peptide-phospholipid conjugate, or any suitable precursor(s), to analogous pharmaceutical acetate salt(s). Representative embodiments of these methods are provided below.
  • Table 3 provides a description for the identification labels shown in FIGS. 6, 7 and 8.
  • TABLE 3
    21 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-
    VCWEDSWGGEVCFRYDPGGGK[-Adoa-Adoa-Glut-K(DSPE-PLG2000-NH-
    Glut)]-NH2 cyclic (2-12) disulfide}-NH2 cyclic (6-13) disulfide
    22 Ac-AGPTWCLDDWYYCWLFGTGGGK[K(ivDde)]-NH2 cyclic (2-12) disulfide
    •nTFA
    23 Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH2 cyclic (2-12)
    disulfide•xHOAc
    24 mono-NHS ester of glutaryl-peptide 23
    Ac-AGPTWCEDDWYYCWLFGTGGGK[NHS-Glut-K(ivDde)]-NH2 cyclic (2-
    12) disulfide
    25 Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH2 cyclic (2-12) disulfide
    •yTFA
    26 Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH2 cyclic (2-12) disulfide
    •zHOAc
    27 Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-
    VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)-NH2 cyclic (2-12)
    disulfide]-NH2 cyclic (6-13) disulfide•X HOAc
    28 Mono-NHS ester of glutaryl-peptide 27
    Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-
    VCWEDSWGGEVCFRYDPGGGK [-Adoa-Adoa-Glut-K(NHS-Glut)]-NH2
    cyclic (2-12) disulfide}-NH2 cyclic (6-13) disulfide
    29 DSPL-PLG2000-NH2
    Where m, n, x, y, z are variable depending on lyophilization conditions.
  • Referring now to FIGS. 6 and 7, in certain embodiments monomer peptide components of heterodimer peptide (27), namely TFA salts compounds (22) and (25), are subjected to ion exchange chromatography on the macroporous sulfonic acid cation exchange resin AG MP-50 using a step gradient of ammonium acetate to convert them to their acetate salts. Then the two peptide monomer acetates (23) and (26) may be tethered through a glutaryl linker to form the dimer (27) as an acetate salt. Purification of the crude dimer acetate salt of (27), by C-18 preparative HPLC using a linear gradient method employing CH3CN/H2O each containing 10 mM NH4OAc provides the pure dimer acetate (27). Conjugation of this dimer to DSPE-PEG2000-NH2 (29) and final purification of the crude mixture by C-3 preparative HPLC using CH3CN/H2O/NH4OAc provides compound (21) as the acetate salt.
  • More specifically, compounds (22), (25) and (27) all bear side-chain carboxylic acid and amino groups. AG MP-50, a macroporous cation-exchange resin, may be used to allow full penetration of the resin by the peptides and to exploit the immobilization of the peptides via their basic (amino and guanidine) groups. TFA salts of the peptides may be adsorbed to an AG MP-50 column (sulfonic acid form) and the column may be washed with water and then eluted with a step gradient of NH4OAc in 0 or 30% CH3CN/H2O, depending on the solubility of the peptides. The peptides may be eluted at about 600 mM NH4OAc and the acetate form of the peptides then may be obtained in pure form. Both IC fluorine analysis and CE TFA counter-ion analysis consistently show very low TFA content of the peptides.
  • Preferred methods also include redissolution/relyophilization of the final peptides several times to remove residual NH4OAc. Otherwise, residual traces of NH4OAc present in the peptides may give rise to free ammonia in the presence of DIEA. This may result in the formation of unwanted peptide-Glut-amide as a major product in subsequent preparation of (27) from the monomers (23) and (26) or final phospholipid-peptide conjugate (21) from the acetate salt of (27).
  • Referring now to FIG. 7, another embodiment provides the conversion of the TFA salt of dimer (27) to its analogous acetate salt by ion exchange chromatography on the macroporous sulfonic acid cation exchange resin AG MP-50. This dimer acetate then may be conjugated with DSPE-PEG2000-NH2 followed by purification of the crude material by C-3 preparative column using CH3CN/H2O/NH4OAc to give the final compound (21) as an acetate salt.
  • While the methods described above and in FIGS. 6 and 7 provide excellent results, the second approach has the advantage of requiring fewer steps. Additional details are provided below in the Examples section.
  • Turning to FIG. 8, another embodiment provides methods for providing dimeric conjugates having minimal amounts of TFA utilizing the size differential between the phospholipid-peptide conjugate (21) and TFA ions. In this embodiment 21nTFA adduct may be eluted down a size exclusion column in the presence of ammonium bicarbonate buffer. The crude 21nTFA initially may be freed of the lyso-compound by preparative HPLC on a Zorbax C-3 column using a linear gradient of acetonitrile into water. Both phases may be buffered with 10 mM ammonium acetate. This provides separation of the lyso-compound as indicated by analytical HPLC.
  • To further reduce the amount of TFA, the material may be applied to a Sephadex G-25 column and eluted with aqueous ammonium bicarbonate solution. The eluate may be monitored by HPLC. Product-containing fractions may be pooled and lyophilized to afford the desired material (21) essentially free of TFA and with high recovery rates. Additional detail is provided below in the Examples section.
  • Both the monomeric and dimeric peptide phospholipid conjugates described herein may be incorporated into ultrasound contrast agents such as, for example, gas filled microvesicles. Such gas filled microvesicles include, for example, gas filled microbubbles, gas filled microballoons, gas filled microcapsules, etc. In a preferred embodiment, the peptide phospholipid conjugates may be incorporated into ultrasound contrast agents comprising gas filled microbubbles. Methods of preparation of gas filled microbubbles from phospholipids and phospholipid conjugates are known to those skilled in the art. For example, microbubbles according to the present invention can be prepared by methods described in any one of the following patents: EP 554213, WO 04/069284, U.S. Pat. No. 5,413,774, U.S. Pat. No. 5,578,292, EP 744962, EP 682530, U.S. Pat. No. 5,556,610, U.S. Pat. No. 5,846,518, U.S. Pat. No. 6,183,725, EP 474833, U.S. Pat. No. 5,271,928, U.S. Pat. No. 5,380,519, U.S. Pat. No. 5,531,980, U.S. Pat. No. 5,567,414, U.S. Pat. No. 5,658,551, U.S. Pat. No. 5,643,553, U.S. Pat. No. 5,911,972, U.S. Pat. No. 6,110,443, U.S. Pat. No. 6,136,293, EP 619743, U.S. Pat. No. 5,445,813, U.S. Pat. No. 5,597,549, U.S. Pat. No. 5,686,060, U.S. Pat. No. 6,187,288, and U.S. Pat. No. 5,908,610, which are incorporated by reference herein in their entirety. The methods disclosed in WO 04/069284 are particularly preferred.
  • Suitable phospholipids include esters of glycerol with one or two molecules of fatty acids (the same or different) and phosphoric acid, wherein the phosphoric acid residue is in turn bonded to a hydrophilic group, such as choline, serine, inositol, glycerol, ethanolamine, and the like groups. Fatty acids present in the phospholipids are in general long chain aliphatic acids, typically containing from 12 to 24 carbon atoms, preferably from 14 to 22, that may be saturated or may contain one or more unsaturations. Examples of suitable fatty acids are lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid. Mono esters of phospholipids are known in the art as the “lyso” forms of the phospholipid.
  • Further examples of phospholipids are phosphatidic acids, i.e., the diesters of glycerol-phosphoric acid with fatty acids, 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, gangliosides, cerebrosides, etc.
  • As used herein, the term phospholipids includes either naturally occurring, semisynthetic or synthetically prepared products that can be employed either singularly or as mixtures.
  • Examples of naturally occurring phospholipids are natural lecithins (phosphatidylcholine (PC) derivatives) such as, typically, soya bean or egg yolk lecithins. Examples of semisynthetic phospholipids are the partially or fully hydrogenated derivatives of the naturally occurring lecithins.
  • Examples of synthetic phospholipids are e.g., dilauryloyl-phosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine (“DMPC”), dipalmitoyl-phosphatidylcholine (“DPPC”), diarachidoylphosphatidylcholine (“DAPC”), distearoyl-phosphatidylcholine (“DSPC”), 1-myristoyl-2-palmitoylphosphatidylcholine (“MPPC”), 1-palmitoyl-2-myristoylphosphatidylcholine (“PMPC”), 1-palmitoyl-2-stearoylphosphatidylcholine (“PSPC”), 1-stearoyl-2-palmitoyl-phosphatidylcholine (“SPPC”), dioleoylphosphatidylycholine (“DOPC”), 1,2 Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC), dilauryloyl-phosphatidylglycerol (“DLPG”) and its alkali metal salts, diarachidoylphosphatidylglycerol (“DAPG”) and its alkali metal salts, dimyristoylphosphatidylglycerol (“DMPG”) and its alkali metal salts, dipalmitoyl-phosphatidylglycerol (“DPPG”) and its alkali metal salts, distearolyphosphatidylglycerol (“DSPG”) and its alkali metal salts, dioleoylphosphatidylglycerol (“DOPG”) and its alkali metal salts, dimyristoyl phosphatidic acid (“DMPA”) and its alkali metal salts, dipalmitoyl phosphatidic acid (“DPPA”) and its alkali metal salts, distearoyl phosphatidic acid (“DSPA”), diarachidoyl phosphatidic acid (“DAPA”) and its alkali metal salts, dimyristoyl phosphatidyl-ethanolamin-e (“DMPE”), dipalmitoyl phosphatidylethanolamine (“DPPE”), distearoyl phosphatidyl-ethanolamine (“DSPE”), dimyristoyl phosphatidylserine (“DMPS”), diarachidoyl phosphatidylserine (“DAPS”), dipalmitoyl phosphatidylserine (“DPPS”), distearoylphosphatidylserine (“DSPS”), dioleoylphosphatidylserine (“DOPS”), dipalmitoyl sphingomyelin (“DPSP”), and distearoyl sphingomyelin (“DSSP”).
  • Suitable phospholipids further include phospholipids modified by linking a hydrophilic polymer thereto. Examples of modified phospholipids are phosphatidylethanolamines (PE) modified with polyethylenglycol (PEG), in brief “PE-PEGs”, 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, DSPE-PEG, DMPE-PEG or DAPE-PEG (where DAPE is 1,2-diarachidoyl-sn-glycero-3-phosphoethanolamine). The compositions also may contain other amphiphilic compounds including, for instance, fatty acids, such as palmitic acid, stearic acid, arachidonic acid or oleic acid; sterols, such as cholesterol, or esters of sterols with fatty acids or with sugar acids; glycerol or glycerol esters including glycerol tripalmitate, glycerol distearate, glycerol tristearate, glycerol dimyristate, glycerol trimyristate, glycerol dilaurate, glycerol trilaurate, glycerol dipalmitate; tertiary or quaternary alkyl-ammonium salts, such as 1,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), and mixtures or combinations thereof.
  • Preferably, the formulation comprises at least one component bearing an overall net charge, such as, for instance, phosphatidic acid, PE-PEG, palmitic acid, stearic acid, Ethyl-DSPC or DSTAP, preferably in a molar amount of less than about 50%. Particularly preferred formulations may include mixtures of two or more of the following components: DSPC, DPPG, DPPA, DSPE-PEG1000, DSPE-PEG2000, palmitic acid and stearic acid. Some preferred phospholipids and formulations are set forth in the examples Any of the gases disclosed herein or known to the skilled artisan may be employed; however, inert gases, such as SF6 or perfluorocarbons like CF4, C3F8 and C4F10, are preferred, optionally in admixture with other gases such as air, nitrogen, oxygen or carbon dioxide
  • The preferred microbubble suspensions of the present invention may be prepared from phospholipids using known processes such as a freeze-drying or spray-drying solutions of the crude phospholipids in a suitable solvent or using the processes set forth in EP 554213; WO 04/069284; U.S. Pat. No. 5,413,774; U.S. Pat. No. 5,578,292; EP 744962; EP 682530; U.S. Pat. No. 5,556,610; U.S. Pat. No. 5,846,518; U.S. Pat. No. 6,183,725; EP 474833; U.S. Pat. No. 5,271,928; U.S. Pat. No. 5,380,519; U.S. Pat. No. 5,531,980; U.S. Pat. No. 5,567,414; U.S. Pat. No. 5,658,551; U.S. Pat. No. 5,643,553; U.S. Pat. No. 5,911,972; U.S. Pat. No. 6,110,443; U.S. Pat. No. 6,136,293; EP 619743; U.S. Pat. No. 5,445,813; U.S. Pat. No. 5,597,549; U.S. Pat. No. 5,686,060; U.S. Pat. No. 6,187,288; and U.S. Pat. No. 5,908,610, which are incorporated by reference herein in their entirety. Preferably, as disclosed in International patent application WO 04/069284, a microemulsion can be prepared which contains the phospholipids (e.g. DSPC and/or DSPA) in admixture with a lyoprotecting agent (such as, for instance, carbohydrates, sugar alcohols, polyglycols and mixtures thereof, as indicated in detail hereinafter) and optionally other amphiphilic materials (such as stearic acid), dispersed in an emulsion of water and of a water immiscible organic solvent. Preferred organic solvents are those having solubility in water of 1.0 g/l or lower, preferably lower than about 0.01 g/l, and include, for instance, pentane, hexane, heptane, octane, nonane, decane, 1-pentene, 2-pentene, 1-octene, cyclopentane, cyclohexane, cyclooctane, 1-methyl-cyclohexane, benzene, toluene, ethylbenzene, 1,2-dimethylbenzene, 1,3-dimethylbenzene, di-butyl ether and di-isopropylketone, chloroform, carbon tetrachloride, 2-chloro-1-(difluoromethoxy)-1,1,2-trifluoroethane (enflurane), 2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane (isoflurane), tetrachloro-1,1-difluoroethane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorononane, perfluorobenzene, perfluorodecalin, methylperfluorobutylether, methylperfluoroisobutylether, ethylperfluorobutylether, ethylperfluoroisobutylether and mixtures thereof. The peptide-phospholipid conjugate of the invention can be admixed together with the phospholipid forming the microvesicle's envelope, in the microemulsion. Preferably, an aqueous suspension of the peptide-phospholipid conjugate and of a PE-PEG (e.g. DSPE-PEG2000) is first prepared, which is then admixed together with an aqueous-organic emulsion comprising the phospholipid and the lyoprotecting agent. Preferably said mixing is effected under heating, e.g. form about 40° C. to 80° C.
  • Prior to formation of the suspension of microbubbles by dispersion in an aqueous carrier, the freeze dried or spray dried phospholipid powders are contacted with air or another gas. When contacted with the aqueous carrier the powdered phospholipids whose structure has been disrupted will form lamellarized or laminarized segments that will stabilize the microbubbles of the gas dispersed therein. This method permits production of suspensions of microbubbles that are stable even when stored for prolonged periods and are obtained by simple dissolution of the dried laminarized phospholipids (which have been stored under a desired gas) without shaking or any violent agitation.
  • Alternatively, microbubbles can be prepared by suspending a gas into an aqueous solution at high agitation speed, as disclosed e.g. in WO 97/29783. A further process for preparing microbubbles is disclosed in WO 2004/069284, herein incorporated by reference, which comprises preparing an emulsion of an organic solvent in an aqueous medium in the presence of a phospholipid and subsequently lyophilizing said emulsion, after optional washing and/or filtration steps. Some preferred preparation methods are disclosed in the examples.
  • The formulation for the preparation of the gas-filled microbubbles may advantageously further comprise 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 polyglycol such as polyethylene glycol (e.g. PEG-4000).
  • Any of these ultrasound compositions should also be, as far as possible, isotonic with blood. Hence, before injection, small amounts of isotonic agents may be added to any of above ultrasound contrast agent suspensions. The isotonic agents are physiological solutions commonly used in medicine and they comprise aqueous saline solution (0.9% NaCl), 2.6% glycerol solution, 5% dextrose solution, etc. Additionally, the ultrasound compositions may include standard pharmaceutically acceptable additives, including, for example, emulsifying agents, viscosity modifiers, cryoprotectants, lyoprotectants, bulking agents etc.
  • Any biocompatible gas may be used in the ultrasound contrast agents of the invention. The term “gas” as used herein includes any substances (including mixtures) substantially in gaseous form at the normal human body temperature. The gas may thus include, for example, air, nitrogen, oxygen, CO2, argon, xenon or krypton, fluorinated gases (including for example, perfluorocarbons, SF6, SeF6) a low molecular weight hydrocarbon (e.g., containing from 1 to 7 carbon atoms), for example, an alkane such as methane, ethane, a propane, a butane or a pentane, a cycloalkane such as cyclopropane, cyclobutane or cyclopentene, an alkene such as ethylene, propene, propadiene or a butene, or an alkyne such as acetylene or propyne and/or mixtures thereof. However, fluorinated gases are preferred. Fluorinated gases include materials that contain at least one fluorine atom such as SF6, freons (organic compounds containing one or more carbon atoms and fluorine, i.e., CF4, C2F6, C3F8, C4F8, C4F10, CBrF3, CCl2F2, C2ClF5, and CBrClF2) and perfluorocarbons. The term perfluorocarbon refers to compounds containing only carbon and fluorine atoms and includes, in particular, saturated, unsaturated, and cyclic perfluorocarbons. The saturated perfluorocarbons, which are usually preferred, have the formula CnFn+2, where n is from 1 to 12, preferably from 2 to 10, most preferably from 3 to 8 and even more preferably from 3 to 6. Suitable perfluorocarbons include, for example, CF4, C2F6, C3F8, C4F8, C4F10, C5F12, C6F2, C7F14, C8F18, and C9F20. Most preferably the gas or gas mixture comprises SF6 or a perfluorocarbon selected from the group consisting of C3F8, C4F8, C4F10, C5F2, C6F12, C7F14, C8F18, with C4F10 being particularly preferred. See also WO 97/29783, WO 98/53857, WO 98/18498, WO 98/18495, WO 98/18496, WO 98/18497, WO 98/18501, WO 98/05364, WO 98/17324. In a preferred embodiment the gas comprises C4F10 or SF6, optionally in admixture with air, nitrogen, oxygen or carbon dioxide.
  • In certain circumstances it may be desirable to include a precursor to a gaseous substance (e.g., a material that is capable of being converted to a gas in vivo, often referred to as a “gas precursor”). Preferably the gas precursor and the gas it produces are physiologically acceptable. The gas precursor may be pH-activated, photo-activated, temperature activated, etc. For example, certain perfluorocarbons may be used as temperature activated gas precursors. These perfluorocarbons, such as perfluoropentane, 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 phase shift and are converted to a gas within the human body.
  • As discussed above, the gas can comprise a mixture of gases. The following combinations are particularly preferred gas mixtures: a mixture of gases (A) and (B) in which, at least one of the gases (B), present in an amount of between 0.5-41% by vol., has a molecular weight greater than 80 daltons and is a fluorinated gas and (A) is selected from the group consisting of air, oxygen, nitrogen, carbon dioxide and mixtures thereof, the balance of the mixture being gas A.
  • Unless it contains a hyperpolarized gas, known to require special storage conditions, the lyophilized product may be stored and transported without need of temperature control of its environment and in particular it may be supplied to hospitals and physicians for on site formulation into a ready-to-use administrable suspension without requiring such users to have special storage facilities. Preferably in such a case it can be supplied in the form of a two-component kit, which can include two separate containers or a dual-chamber container. In the former case preferably the container is a conventional septum-sealed vial, wherein the vial containing the lyophilized residue of step b) is sealed with a septum through which the carrier liquid may be injected using an optionally prefilled syringe. In such a case the syringe used as the container of the second component is also used then for injecting the contrast agent. In the latter case, preferably the dual-chamber container is a dual-chamber syringe and once the lyophilizate has been reconstituted and then suitably mixed or gently shaken, the container can be used directly for injecting the contrast agent. In both cases means for directing or permitting application of sufficient bubble forming energy into the contents of the container are provided. However, as noted above, in the stabilised contrast agents according to the invention the size of the gas microbubbles is substantially independent of the amount of agitation energy applied to the reconstituted dried product. Accordingly, no more than gentle hand shaking is generally required to give reproducible products with consistent microbubble size.
  • It can be appreciated by one of ordinary skilled in the art that other two-chamber reconstitution systems capable of combining the dried powder with the aqueous solution in a sterile manner are also within the scope of the present invention. In such systems, it is particularly advantageous if the aqueous phase can be interposed between the water-insoluble gas and the environment, to increase shelf life of the product. Where a material necessary for forming the contrast agent is not already present in the container (e.g. a targeting ligand to be linked to the phospholipid during reconstitution), it can be packaged with the other components of the kit, preferably in a form or container adapted to facilitate ready combination with the other components of the kit.
  • No specific containers, vial or connection systems are required; the present invention may use conventional containers, vials and adapters. The only requirement is a good seal between the stopper and the container. The quality of the seal, therefore, becomes a matter of primary concern; any degradation of seal integrity could allow undesirable substances to enter the vial. In addition to assuring sterility, vacuum retention is essential for products stoppered at ambient or reduced pressures to assure safe and proper reconstitution. The stopper may be a compound or multicomponent formulation based on an elastomer, such as poly(isobutylene) or butyl rubber.
  • In ultrasound applications the contrast agents formed by phospholipid stabilized microbubbles can be administered, for example, in doses such that the amount of phospholipid injected is in the range 0.1 to 200 μg/kg body weight, preferably from about 0.1 to 30 μg/kg.
  • Ultrasound imaging techniques that can be used in accordance with the present invention include known techniques, such as color Doppler, power Doppler, Doppler amplitude, stimulated acoustic imaging, and two- or three-dimensional imaging techniques. Imaging may be done in harmonic (resonant frequency) or fundamental modes, with the second harmonic preferred.
  • The ultrasound contrast agents of the present invention may further be used in a variety of therapeutic imaging methods. The term 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 agent 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 an ultrasound burst 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 localized release of a suitable therapeutic agent. Alternatively, said therapeutic imaging may include the delivery of a therapeutic agent 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).
  • The term “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). Examples of 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.
  • Materials and Analytical Methods
  • Solvents for reactions, chromatographic purification and HPLC analyses were E. Merck Omni grade solvents from VWR Corporation (West Chester, Pa.). N-Methylpyrrolidinone (NMP) and N,N-dimethylformamide (DMF) were obtained from Pharmco Products Inc. (Brookfield, Conn.), and were peptide synthesis grade or low water/amine-free Biotech grade quality. Piperidine (sequencing grade, redistilled 99+%) and trifluoroacetic acid (TFA) (spectrophotometric grade or sequencing grade) were obtained from Sigma-Aldrich Corporation (Milwaukee, Wis.) or from the Fluka Chemical Division of Sigma-Alrich Corporation. N,N′-Diisopropylcarbodiimide (DIC), phenol (99%), N,N-diisopropylethylamine (DIEA) and triisopropylsilane (TIS) were purchased from Sigma-Aldrich Corporation. Fmoc-protected amino acids, pseudoproline dipeptides, Fmoc-Asp(O-tBu)-Ser(ψMe,Mepro)-OH and Fmoc-Gly-Thr(ψMe,Mepro)-OH and N-hydroxybenzotriazole (HOBt) were obtained from Novabiochem (San Diego, Calif.). Fmoc-8-amino-3,6-dioxaoctanoic acid (Adoa) was obtained from NeoMPS Corp (San Diego, Calif.) or Suven Life Sciences (Hyderabad, India). Disuccinimidyl glutarate (DSG) and 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[amino (polyethyleneglycol)2000] ammonium salt, [DSPE-PEG2000-NH2] were obtained from Pierce Chemical Co. (Rockford, Ill.) and Avanti® Polar Lipids (Alabaster, Ala.), respectively. Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH were prepared in-house from the corresponding triglycine or diglycine by the reaction with Fmoc-OSu. An AG MP-50 ion-exchange resin was obtained from Bio-Rad (Hercules, Calif.).
  • Analytical HPLC data were generally obtained using a Shimadzu LC-10AT VP dual pump gradient system employing a Waters XTerra MS-C18 4.6×50 mm column, (particle size: 5μ; 120 Å pore size) and gradient or isocratic elution systems using water (0.1% TFA) as eluent A and CH3CN (0.1% TFA) or CH3CN—CH3OH (1:1, v/v) (0.1% TFA) as eluent B. Detection of compounds was accomplished using UV at 220 and 254 nm. The purity of the phospholipid-PEG-peptide derivatives was determined on a YMC C-4 (5 μM, 300 Å, 4.6×250 mm) column or on a Zorbax 300 SB-C3 (3.5 μM; 300 Å, 3×150 mm) column using a SEDEX 55 Light Scattering Detector (LSD) and with a UV detector.
  • Preparative HPLC was conducted on a Shimadzu LC-8A dual pump gradient system equipped with a SPD-10AV UV detector fitted with a preparative flow cell. Generally the solution containing the crude peptide was loaded onto a reversed phase C18, C4 or C3 column, depending on the compound characteristics, using a third pump attached to the preparative Shimadzu LC-8A dual pump gradient system. After the solution of the crude product mixture was applied to the preparative HPLC column the reaction solvents and solvents employed as diluents, 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 eluent B into eluent A. Product-containing fractions were combined based on their purity as determined by analytical HPLC and mass spectral analysis. The combined fractions were freeze-dried to provide the desired product.
  • Amino acid composition analyses were performed at the Keck Biotechnology Resource Laboratory at Yale University, New Haven, Conn. Mass spectral data were obtained from MScan Inc. (606 Brandywine Parkway, West Chester Pa. 19380) or obtained in-house on an Agilent LC-MSD 1100 Mass Spectrometer. For the purposes of fraction selection and characterization of the products mass spectral values were usually obtained using API-ES in negative ion mode. Generally the molecular weight of the target peptides was ˜3000; the mass spectra usually exhibited doubly or triply negatively charged ion mass values rather than [M-H]−. These were generally employed for selection of fractions for collection and combination to obtain the pure peptide during HPLC purification. In some cases fractions exhibited dominant peaks attributable to [M-2H]/2+57 or [M-2H]/2+114 in the mass spectrum. These peaks are due to the formation of adducts of one or two molecules of trifluoroacetic acid per molecule of the peptide. After careful collection of fractions by comparing MS results and HPLC purities and freeze-drying process, a small amount of the isolated fluffy solid was dissolved in water (0.5 mg/mL) and treated with a drop of aqueous N-methyl-D-glucamine (˜0.5 M). This solution was analyzed by HPLC and MS for final purity results of the purified peptide. Peptide solutions in the presence of N-methyl-D-glucamine did not exhibit [M-2H]/2+57 or [M-2H]/2+114 mass value peaks in the mass spectrum, instead the expected [M-2H]/2 or [M-3H]/3 peaks were observed.
  • The following non-limiting Examples provide additional detail on efficient processes used for obtaining large quantities of highly purified forms of the monomeric and dimeric peptide phospholipid conjugates. These non-limiting Examples also describe the preparation of representative targeted microbubbles which include these monomeric and dimeric peptide phospholipid conjugates. These Examples also describe the use of such targeted microbubbles in static binding tests on KDR-transfected cells and dynamic binding tests on rh VEGF-R2/Fc chimeric protein. The Examples further describe the evaluation of ultrasound contrast agents containing KDR binding lipopeptides in a rabbit VX2 tumor model.
    • EXAMPLES
  • Examples 1-2 below refer to the monomeric peptide phospholipid conjugate shown in FIG. 2. A process for synthesizing this compound is shown in FIG. 1. Although these Examples refer more specifically to the process for synthesizing the compound shown in FIG. 2, a similar process may used to prepare the monomeric peptide phospholipid conjugate shown in FIG. 10 and the linear peptide monomer (32) shown in FIG. 9 as well as other monomer peptide-phospholipid conjugates. Additionally, co-pending U.S. application Ser. No. 10/661,156, filed Sep. 11, 2003, sets forth methods for the preparation of the peptide monomers and is incorporated by reference herein in its entirety.
    • Example 1 Solid Phase Synthesis (SPPS) and Purification of Linear Peptide Monomer (2) Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH2, (SEQ ID NO. 2) Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH2, N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide
  • The linear peptide monomer (2) was synthesized by an established automated protocol on a SONATA®/Pilot Peptide Synthesizer using Fmoc-Pal-Peg-PS resin (0.2 mmol/g), Fmoc-protected amino acids and DIC-mediated HOBt ester activation in DMF. The peptide sequence was synthesized in stepwise fashion by SPPS methods on the Fmoc-Pal-Peg-PS resin, typically on a 10 mmol scale. The amino acid couplings were carried out with a 4-fold excess each of amino acid and the DIC-HOBt reagent pair in DMF.
  • In a typical coupling of an amino acid, 5 mL of dry DMF per gram of resin was used. The total volume of DMF, calculated on the basis of resin used, was allocated among amino acid, HOBt and DIC for solution preparation. For example, for the synthesis involving 50 g (10 mmol scale) of resin, the calculated volume of 250 mL of DMF was distributed among amino acid (150 mL), HOBt (50 mL) and DIC (50 mL). The amino acid vessel on the Sonata Pilot Peptide Synthesizer was charged with the solid dry amino acid (4-fold excess with respect to the resin). At inception of the coupling step, the software of the instrument was employed to deliver successively the chosen volume of DMF (for dilution of the amino acid) and HOBt (4 eq.) in DMF and DIC (4 eq.) in DMF and mixing by nitrogen bubbling was initiated and conducted for 4 min. This served to pre-activate the amino acid and to insure complete dissolution of all components of the mixture. After activation, the software mediated the transfer of the solution of the activated Fmoc-amino acid to the reaction vessel containing the resin. After transfer was complete the vessel was agitated for 3 h with recurrent nitrogen bubbling. After the 3 h coupling time, the resin was washed thoroughly with DMF (5 mL/g, 6×) and the cleavage of the Fmoc-group was performed with 25% piperidine in DMF (5 mL/g) containing HOBt (0.1 M) (2×10 min). The resin was thoroughly washed with DMF (5 mL/g, 6×) to assure complete removal of piperidine from the resin in preparation for the ensuing amino acid coupling. In the case of Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, the pre-activation in the amino acid bottle was not conducted in order to minimize the formation of diketopiperazine during the activation time as discussed in the text. Therefore, in these two cases, the solutions of amino acid, HOBt and DIC were added to the reaction vessel sequentially and the coupling process was conducted with ‘in situ’ activation.
  • After chain elongation was completed, the Fmoc group of the N-terminal amino acid was removed in the standard manner followed by the standard wash with DMF (vide supra). The N-terminal amino acid was then capped by treatment with freshly prepared acetylation mixture (0.5M acetic anhydride, 0.125M DIEA and 0.015M HOBt in DMF/6 mL/g of resin), 2×20 min. After completion of the peptide synthesis, the resin was treated with the cleavage cocktail, ‘Reagent B’ (TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) (10 mL/g of resin) for 4 h. The volatiles were removed and the paste thus obtained was triturated with ether to provide a solid which was washed with ether (3×) with intervening centrifugation (to compact the suspended solids in order to allow decantation of the supernatant) and then dried under vacuum to provide the required peptide as an off-white solid. A 10 mmol scale synthesis of the linear peptide monomer (2) gave 33.82 g (103% of theory) of the crude peptide. The greater than theoretical yield was most likely due to moisture and residual solvents.
  • Purification of the Linear Peptide Monomer (2) Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH2 (SEQ ID NO. 2); Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH2; N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide
  • A ˜0.5 g portion of the crude linear peptide monomer (2) was dissolved in a minimum amount of CH3CN (˜20 mL). The volume of the solution was adjusted to ˜100 mL with water and employing a third pump the solution was loaded onto a reversed phase C18 preparative column (Waters, XTerra® Prep MS C18, 10μ, 300 Å, 50×250 mm, flow rate 100 mL/min) which had been pre-equilibrated with 10% CH3CN in water (0.1% TFA). The column was not eluted with the equilibrating eluent during application of the sample solution. After the sample solution was applied to the column, the composition of the eluent was ramped to 20% CH3CN-water (0.1% TFA) over 1 min, and a linear gradient at a rate of 0.6%/min of CH3CN (0.1% TFA) into water (0.1% TFA) was initiated and maintained for 50 min. Fractions (15 mL) were manually collected using UV at 220 nm as an indicator of product elution. The collected fractions were analyzed on a Waters XTerra analytical reversed phase C-18 column (5μ particle, 120 Å pore) and product-containing fractions of >95% purity were pooled and freeze-dried to afford the corresponding pure linear peptide monomer (2). Typically the purification of 0.5 g of crude (2) afforded 0.12 g (24% yield) of the desired product (>95% purity).
    • Example 2 Preparation of Monomeric Peptide Phospholipid Conjugate (1) Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH2 (SEQ ID NO. 1); Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-(DSPE-PEG2000-NH-Glut)-NH2; N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-1-serinyl-L-methionyl-L-alanyl-L-aspartyl-L-glutaminyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl-L-lysinamide
  • The monomeric peptide phospholipid conjugate (1), Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH2 (SEQ ID NO. 1), was prepared by conjugation of (3), the glutaric acid monoamide mono-NHS ester of peptide monomer (2), with DSPE-PEG2000-NH2 phospholipid ammonium salt (4).
  • A round-bottomed flask equipped with magnetic stir bar and septum cap was charged sequentially with anhydrous dimethylformamide (7.5 mL), disuccinimidyl glutarate (DSG, 0.25 g, 0.75 mmol) and diisopropylethylamine (0.10 g, 0.78 mmol) with stirring. Solid linear peptide monomer (2) (0.5 g, 0.152 mmol) was added portionwise to the above solution over a period of 2 min; then the solution was stirred for 30 min at ambient temperature. The reaction mixture was diluted to ˜50 mL with anhydrous ethyl acetate; this resulted in precipitation of the intermediate mono-NHS ester (3), the glutaric acid monoamide mono-NHS ester of peptide monomer (2). The solution was centrifuged to bring down mono-NHS ester (3) as a colorless solid. The supernatant containing excess DSG was decanted from the compacted solid mono-NHS ester (3) which was again dispersed in ethyl acetate, centrifuged and washed twice more to remove the remaining traces of DSG. The solid intermediate mono-NHS ester (3) thus obtained was dissolved in anhydrous DMF (10.0 mL); diisopropylethylamine (0.10 g, 0.78 mmol) was added; and the mixture was stirred.
  • Meanwhile, DSPE-PEG2000-NH2 phospholipid ammonium salt (4) (0.38 g, 0.14 mmol, 0.9 eq.) was suspended in dry dichloromethane (2 mL) in a separate flask and trifluoroacetic acid (2 drops) was added to protonate the phosphodiester oxygen facilitating solubilization of phospholipid ammonium salt in dichloromethane. The clear solution was then evaporated on a rotary evaporator to remove the volatiles and dried further under vacuum.
  • The solid phospholipid ammonium salt (4) was dissolved in DMF (5 mL) and transferred to the stirred solution of mono-NHS ester (3) and the resulting mixture was stirred for 24 h at ambient temperature. The reaction mixture was diluted to 100 mL with a 1:1 mixture of CH3OH and CH3CN-water (1:1, v/v) and the insolubles were filtered. Half of the filtered solution was loaded onto a reversed phase C2 preparative column (Kromasil® Prep C2, 10μ, 300 Å, 50×250 mm) which had been pre-equilibrated with 3:1 (v/v) mixture of water (0.1% TFA) and CH3OH—CH3CN (1:1, v/v, 0.1% TFA) 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 ramped to 70% CH3OH—CH3CN (1:1, 0.1% TFA) over 9 min and a linear gradient of 0.75%/min of CH3OH—CH3CN (1:1, 0.1% TFA) into water (0.1% TFA) was initiated and run for 40 min. Fractions (15 mL) were collected using UV (220 nm) as an indicator of product elution. Fractions were checked for purity on an analytical HPLC system (column: YMC C-4, 5μ, 300 Å, 4.6×250 mm) using UV at 220 nm and an evaporative light scattering detector (ELSD). The latter detector (ELSD) was employed to detect DSPE-PEG2000-NH2 phospholipid ammonium salt (4) which has very little UV absorbance at 220 nm. Product-containing fractions of >98% purity, and devoid of DSPE-PEG2000-NH2 phospholipid ammonium salt (4) were combined and concentrated on a rotary evaporator to reduce the content of CH3OH. The concentrated solution was then diluted with 10% CH3CN in water until a faint flocculent precipitate formed. The resulting solution was freeze-dried to provide monomeric peptide phospholipid conjugate (1) as a colorless solid. The second portion of crude monomeric peptide phospholipid conjugate (1) was purified as described above. The combined yield of the target monomeric peptide phospholipid conjugate (1) was 0.40 g (47% yield).
  • Examples 3-5 below refer to the dimeric peptide phospholipid conjugate shown in FIG. 5. Representative methods of synthesizing the dimeric conjugate are shown in FIGS. 3, 4, 6, 7 and 8.
    • Example 3 Solid Phase Synthesis (SPPS), Cyclization and Purification of Monomer Peptides (12) Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH2 and (13) Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2
  • The linear peptides were synthesized by an established automated protocol on a SONATA®/Pilot Peptide Synthesizer using Fmoc-Pal-Peg-PS resin (0.2 mmol/g), Fmoc-protected amino acids and DCI-mediated HOBt ester activation in DMF. The peptide sequence on the Fmoc-Pal-Peg-PS resin was synthesized in stepwise fashion by SPPS methods typically on a 10 mmol scale. The amino acid coupling was carried out with a 4-fold excess each of amino acid and DIC-HOBt reagent in DMF.
  • In a typical coupling of an amino acid in the sequence, 5 mL of dry DMF per gram of resin was used. The total volume of DMF, calculated on the basis of resin used, was allocated among amino acid, HOBt and DIC for solution preparation. For example, for the synthesis involving 50 g of resin, the calculated volume of 250 mL of DMF was distributed among amino acid (150 mL), HOBt (50 mL) and DIC (50 mL). The amino acid vessel on the Sonata® Pilot Peptide Synthesizer was charged with the solid dry amino acid (4-fold excess with respect to the resin). At inception of the coupling step, the chosen volume of DMF and HOBt (4 eq.) in DMF and DIC (4 eq.) in DMF were delivered successively and after each delivery mixing by nitrogen bubbling was conducted. After the last reagent was delivered mixing by nitrogen bubbling was initiated and conducted for 4 min. This served to preactivate the amino acid and to insure complete dissolution of all components of the mixture.
  • After activation, the solution of the activated Fmoc-amino acid was transferred to the reaction vessel containing the resin. After transfer was complete the vessel was agitated for 3 h with recurrent nitrogen bubbling. After the 3 h coupling time, the resin was washed thoroughly with DMF (5 mL/g, 6×) and the cleavage of the Fmoc-group was performed with 25% piperidine in DMF (5 mL/g) containing HOBt (0.1M) (2×10 min). The resin was thoroughly washed with DMF (5 mL/g, 6×) to assure complete removal of piperidine from the resin in preparation for the ensuing amino acid coupling. In the case of Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, the pre-activation in the amino acid bottle was not conducted in order to minimize the formation of diketopiperazine during the activation time as discussed in the text. Therefore, in these two cases, the solution of the amino acid, HOBt and DIC were added to the reaction vessel sequentially and the coupling process was conducted with ‘in situ’ activation. After chain elongation was completed, the fmoc group of the N-terminal amino acid was removed in the standard manner followed by the standard wash with DMF (vide supra). The N-terminal amino acid was then capped by treatment with freshly prepared acetylation mixture (0.5M acetic anhydride, 0.125M DIEA and 0.015M HOBt in DMF—6 mL/g of resin), 2×20 min.
  • Functionalization of the ε-amino group of C-terminal Lysine moieties of the monomer peptides (with Fmoc-Adoa or with Fmoc-Lys(ivDde) as required) was accomplished by first removing the ivDde group of the ε-amino group with freshly prepared 10% hydrazine in DMF (5 mL/g of resin—2×10 min). For appending of Fmoc-Adoa or Fmoc-Lys(ivDde) the coupling time was increased to 10 h. After completion of the peptide synthesis, the resin was treated with the cleavage cocktail, ‘Reagent B’ (TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) (10 mL/g of resin) for 4 h. After evaporation of the volatiles under vacuum, the paste was triturated with ether to provide a solid which was collected by filtration washed with diethyl ether and dried. A 10 mmol scale synthesis of (12), Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH2 gave 30 g (103% of theory) of the crude peptide. In the case of (13) Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2, a 10 mmol scale synthesis gave 28 g (107% of theory) of crude peptide. The greater than theoretical yields are most likely due to moisture and residual solvents.
  • Cyclization of the Linear Di-Cysteine Peptides to Cyclic Disulfide Peptides
  • Cyclic disulfide peptides were prepared from the corresponding linear di-cysteine peptides by DMSO-assisted oxidation using DMSO/water (95/5, v/v). The crude linear peptide was dissolved in the solvent mixture (5 mL/g) in a wide mouth beaker, and the pH of the solution was adjusted to 8.5 by the addition of solid N-methyl-D-glucamine in portions. The resulting mixture was stirred for 36 h at ambient temperature. The solution was then diluted with acetonitrile (50 mL/g) and the mixture was stirred for 2 min. The solid cyclic disulfide peptide was collected by filtration, washed with diethyl ether and dried.
  • Purification of Monomer Peptides
  • Peptide Monomer (12) Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH2; Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys[Lys(ivDde)]-NH2 cyclic (6-13) disulfide
  • A ˜0.5 g portion of the crude cyclic disulfide peptide monomer (12) was dissolved in a minimum amount of DMSO (˜3 mL). The volume of the solution was adjusted to ˜100 mL with 20% CH3CN-water and employing a third pump, the solution was loaded onto a reversed phase C18 preparative column (Waters, XTerra® Prep MS C18, 10μ, 300 Å, 50×250 mm, flow rate 100 mL/min), which had been pre-equilibrated with 10% CH3CN in water (0.1% TFA). During application of the sample solution to the column the flow of the equilibrating eluent from the preparative HPLC system was stopped. After the sample solution was applied to the column, the flow of equilibrating eluent from the gradient HPLC system was reinitiated and the column was eluted with 10% CH3CN-water (0.1% TFA) until the DMSO was eluted. Then the eluent composition was ramped to 35% CH3CN-water (0.1% TFA) over 1 min after which a linear gradient at a rate of 0.5%/min CH3CN (0.1% TFA) into water (0.1% TFA) was initiated and maintained for 50 min. Fractions (15 mL) were manually collected using UV at 220 nm as an indicator of product elution. The collected fractions were analyzed on a Waters XTerra analytical reversed phase C-18 column (5μ particle, 120 Å pore) and product-containing fractions of >95% purity were pooled and freeze-dried to afford the corresponding cyclic disulfide peptide monomer (12). Typically the purification of 0.5 g of crude peptide monomer (12) afforded 0.1 g (20% yield) of the desired product (>95% purity).
  • Peptide Monomer (13) Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2; Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH2 cyclic (2-12) disulfide
  • Following the procedure employed for the HPLC purification of peptide monomer (12), the crude cyclic disulfide peptide monomer (13) Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2 (0.5 g) dissolved in 20% CH3CN-water mixture (100 mL) was loaded onto a reversed phase C18 preparative column (Waters, XTerra® Prep MS C18, 50×250 mm, 101 particle, 300 Å pore, flow rate 100 mL/min), which had been pre-equilibrated with 10% CH3CN (0.1% TFA) in water (0.1% TFA). During application of the sample solution to the column the flow of the equilibrating eluent from the preparative HPLC system was stopped. After the sample solution was applied to the column, the flow of equilibrating eluent from the gradient HPLC system was reinitiated and the column was eluted with 10% CH3CN-water (0.1% TFA) for 5 min. Then the eluent composition was ramped to 30% CH3CN (0.1% TFA)-water (0.1% TFA) over 1 min and a linear gradient elution at a rate of 0.5%/min of CH3CN (0.1% TFA) into water (0.1% TFA) was initiated and maintained for 50 min. Fractions (15 mL) were manually collected using UV at 220 nm as an indicator of product elution. The fractions were analyzed on a Waters XTerra analytical reversed phase C-18 column (4.6 mm i.d.×50 mm, 5μ particle, 120 Å pore) and product-containing fractions of >95% purity were pooled and freeze-dried to afford the corresponding cyclic disulfide peptide monomer (13). Typically the purification of 0.5 g of crude peptide monomer (13) afforded 0.12 g (24% yield) of the desired product (>95% purity).
    • Example 4 Preparation and Purification of Precursor Dimer Peptide (16) Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH2 cyclic (2-12) disulfide]-NH2 cyclic (6-13) disulfide; Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys[Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(-Adoa-Adoa-Glut-Lys)]-NH2 cyclic (2-12) disulfide]-NH2 cyclic (6-13) disulfide
  • As shown in FIG. 3, disuccinimidyl glutarate (DSG, 0.28 g, 0.86 mmol) was dissolved in stirred anhydrous dimethylformamide (2.0 mL) and diisopropylethylamine (0.11 g, 0.85 mmol) was added in one portion. Then solid peptide monomer (12) Ac-AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDde)]-NH2 (0.50 g, 0.17 mmol) was added in portions to the stirred solution of DSG over a period of two min. After stirring for 30 min at room temperature, the solution was diluted with anhydrous ethyl acetate to ˜50 mL, (this served to precipitate intermediate mono-NHS ester (14)). The entire mixture was centrifuged and the supernatant was decanted leaving intermediate mono-NHS ester (14) as a colorless solid. The solid was resuspended with ethyl acetate; the solution containing the suspended solid mono-NHS ester (14) was centrifuged to separate the solid and the supernatant was again decanted. This washing process was repeated twice to remove completely the excess DSG.
  • The solid mono-NHS ester (14) was dissolved in stirred anhydrous dimethylformamide (2.0 mL) and diisopropylethylamine (0.11 g, 0.85 mmol) was added. Then solid peptide monomer (13), Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2, (0.50 g, 0.19 mmol, 1.12 eq.) was added in portions to the stirred solution over a three min. period and the resulting mixture was stirred for 18 h. The reaction was monitored by mass spectrometry; after the complete consumption of the peptide monomer glutaric acid monoamide mono-NHS ester (14) was confirmed, neat hydrazine (0.1 mL) was added to remove the ivDde protecting group of the ivDde-bearing dimer (15) and the mixture was stirred for 20 min at room temperature.
  • The solution was then acidified by dropwise addition of TFA and the mixture was diluted to 100 mL with 10% CH3CN (0.1% TFA) in water (0.1% TFA). The solution was filtered to remove particulates and half of the clarified solution was loaded onto a reversed phase C18 preparative column (Waters, XTerra® Prep MS C18, 10μ, 50×250 mm, flow rate 100 mL/min) pre-equilibrated with 10% CH3CN in water (0.1% TFA). During application of the sample solution to the column the flow of the equilibrating eluent from the preparative HPLC system was stopped. After the sample solution was applied to the column, the flow of equilibrating eluent from the gradient HPLC system was reinitiated and the column was eluted with 10% CH3CN-water (0.1% TFA) in order to flush DMF from the column. After elution of the DMF plug was completed the eluent composition was increased to 20% CH3CN over one min. and the elution was continued with a linear gradient rate of 0.6%/min of CH3CN (0.1% TFA) into water (0.1% TFA). Fractions (15 mL) were collected using UV (220 nm) as an indicator of product elution. The fractions were analyzed on a reversed phased C18 column (Waters MS C18, 4.6 mm i.d.×50 mm, 5μ particle, 120 Å pore) and the product-containing fractions of >95% purity were pooled and freeze-dried to provide precursor dimer peptide (16) as a colorless, fluffy solid. The remaining crude precursor dimer peptide (16) was purified in the same manner. From 0.5 g each of monomer peptides (12) and (13), 320 mg (overall yield 33%) of the desired dimer (16) was obtained (>95% purity).
    • Example 5
  • Preparation of KDR-Binding Dimeric Peptide Phospholipid Conjugate (11) Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-1-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(distearylphosphoethanolaminocarbonoxy-PEG2000-amino-8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl) amide cyclic (2-12) disulfide]-amide cyclic (6-13) disulfide; Ac-AGPTWCEDDWYYCWLFGTGGGK {Ac-VCWEDSWGGEVCFRYDP-GGGK[-Adoa-Adoa-Glut-K(DSPE-PEG2000-NH-Glut)]-NH2 cyclic (2-12) disulfide}-NH2 cyclic (6-13) disulfide; Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys {Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys[-Adoa-Adoa-Glut-Lys(DSPE-PEG2000-NH-Glut)-]-NH2 cyclic (2-12) disulfide}-NH2 cyclic (6-13) Disulfide
  • The KDR-binding dimer (11) may be prepared by conjugation of precursor dimer peptide (16), Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH2 cyclic (2-12) disulfide]-NH2 cyclic (6-13) disulfide, with DSPE-PEG2000-NH2 phospholipid ammonium salt (18) as shown in FIG. 4.
  • Solid precursor dimer peptide (16) (0.5 g, 0.092 mmol) was added portionwise to a solution of disuccinimidyl glutarate (DSG, 0.15 g, 0.46 mmol), and diisopropylethylamine (0.06 g, 0.47 mmol) in anhydrous DMF (3.0 mL) with stirring over a period of 3 min. Then the solution was stirred at ambient temperature for 30 min. The reaction mixture was diluted to 50 mL with anhydrous ethyl acetate; this resulted in precipitation of the dimer glutaric acid monoamide mono-NHS ester (17), the glutaric acid monoamide mono-NHS ester of the precursor dimer peptide (16). The solution was centrifuged to pellet 6 (m/z, neg. ion, 1887.3 (M-3H)/3, 1415.1 (M-4H)/4, 1131.9 (M-5H)/5) as a colorless solid. The supernatant ethyl acetate layer containing excess DSG was decanted from the compacted solid dimer glutaric acid monoamide mono-NHS ester (17) which was again resuspended in ethyl acetate, centrifuged and washed twice more to remove the remaining traces of DSG. The solid intermediate glutaric acid monoamide mono-NHS ester dimer derivative (17) thus obtained was dissolved in anhydrous DMF/CH2Cl2 (8:2, v/v) (3.0 mL); diisopropylethylamine (0.06 g, 0.47 mmol) was added and the solution was stirred.
  • Meanwhile, DSPE-PEG2000-NH2 phospholipid ammonium salt (18) (0.235 g, 0.084 mmol, 0.9 eq.) was suspended in dry dichloromethane (2 mL) in a separate flask and TFA (2 drops) was added to protonate the phosphodiester oxygen, facilitating solubilization of phospholipid ammonium salt (18) in dichloromethane. The clear solution was concentrated to remove the volatiles and dried further under vacuum.
  • The solid phospholipid ammonium salt (18) was dissolved in DMF (2 mL) and transferred to the stirred solution of glutaric acid monoamide mono-NHS ester dimer derivative (17) and the resulting mixture was stirred for 24 h at ambient temperature. The reaction mixture was diluted with a solution of 50% CH3OH, 25% CH3CN and 25% water (1:1) to 100 mL and the insolubles were filtered. Half of the filtered solution was loaded onto a reverse phased C4 preparative column (Kromasil® Prep C4, 10μ, 300 Å, 50×250 mm) which had been pre-equilibrated with 1:1 mixture of CH3OH and CH3CN (1:1, 0.1% TFA) and water (0.1% TFA) at a flow rate of 100 mL/min. During application of the sample solution to the column the flow of the equilibrating eluent from the preparative HPLC system was stopped. After the sample solution was loaded the flow of the equilibrating eluent was reinitiated and the column was washed until the plug of DMF was eluted.
  • The composition of the eluent was then ramped to 70% CH3OH—CH3CN (1:1, 0.1% TFA)-water (0.1% TFA) over 1 min and a linear gradient of 0.75%/min of CH3OH—CH3CN (1:1, 0.1% TFA) into water (0.1% TFA) was initiated. The elution was continued after reaching 100% B in order to achieve complete elution of the product from the column. Fractions (15 mL) were collected using UV (220 nm) as an indicator of product elution and after the main product was eluted fraction collection was continued for several minutes in order to insure elution of trace amounts of starting phospholipid ammonium salt (18). Fractions were checked for purity on an analytical HPLC system (column: YMC C4, 5 μM, 300 Å, 4.6×250 mm) using UV at 220 nm and an evaporative light scattering detector (ELSD). The latter detector is employed to detect DSPE-PEG2000-NH2 which has a weak chromophore at 220 nm. Product-containing fractions of >98% purity, and devoid of DSPE-PEG2000-NH2 phospholipid ammonium salt (8) were combined and concentrated to reduce the content of CH3OH. The solution was then diluted with 10% CH3CN in water until a faint flocculent precipitate formed. The resulting solution was freeze-dried to afford the dimeric peptide phospholipid conjugate (11) as a colorless solid. The second portion of crude dimeric peptide phospholipid conjugate (11) was purified as described above. The combined yield of the target dimeric peptide phospholipid conjugate (11) was 0.39 g (57% yield). The samples of the dimeric peptide phospholipid conjugate (11) made from different sample purification runs were pooled together, dissolved in tert-butanol-acetonitrile-water mixture and re-lyophilized to provide the dimeric peptide phospholipid conjugate (11) as a colorless, fluffy solid which was further dried under vacuum.
  • Examples 6-8 below refer to the preparation of the dimer peptide-phospholipid conjugate shown in FIG. 5, wherein the dimeric conjugate contains very low levels of TFA. FIGS. 6-8 illustrate the methods described in the Examples below.
    • Example 6 Preparation of Dimeric Conjugate Having Low TFA Levels Via the Use of a Glutaryl Linker
  • Preparation of (23), (26) and Dimer Peptide (27) Acetate Salt by Conversion of (22), (25) and Dimer Peptide 27nTFA Salts to Acetates by AG MP-50 Ion-Exchange Resin
  • For compound (23) an AG MP-50 ion-exchange resin (1.5 meq/mL resin bed) was suspended in 20% of CH3CN/H2O. The suspension was packed in a 3×30 cm glass column and the final volume was 150 mL. The column was connected to a pump and a conductivity meter. It was washed with 20% of CH3CN/H2O at 17 mL/min flow rate until the conductivity was below 1 μs/cm. Compound (22) (210 mg) was dissolved in 20% of CH3CN/H2O (80 mL) and the resulting solution was loaded to the column. The column was washed again with the same eluent until its conductivity was below 1 μs/cm. A gradient of NH4OAc in 20% of CH3CN/H2O was applied at 200 mM, 400 mM, 600 mM and 800 mM, 250 mL each. The compound came out at 600 mM NH4OAc. The fractions were analyzed by HPLC and the ones containing the compound were combined and lyophilized several times until the weight of the material was constant. 176 mg of the pure material (23) was obtained as a white fluffy solid. The yield was 83.8%.
  • Additional parameters and results were as follows: HPLC: Ret. Time: 5.6 min; Assay >98% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5μ particle, 120 Å pore; Eluent: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 15% B, linear gradient 15-50% B over 8 min; Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES; Mode: Negative ion; 1441.7 [M-2H]/2, 960.9 [M-3H]/3. CE analysis (counter-ion % wt./wt.): TFA estimated to be 0.3%; acetate 1.1%.
  • For compound (26), following the same procedure for compound (23), 300 mg of the peptide TFA salt (25) in 80 mL of water was loaded at 17 mL/min. to a 150 mL of AG MP-50 column, which was washed with H2O to conductivity of 1 μs/cm. The column was then washed with H2O again after loading, and the same step gradient of aqueous NH4OAc into H2O as employed for the ion exchange of compound (23) was applied. Lyophilization of the combined fractions to a constant weight afforded 200 mg of the acetate (26) as a white fluffy solid. The yield was 66.7%.
  • Additional parameters and results were as follows: HPLC: Ret. Time: 5.6 min; Assay 97.0% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5μ particle, 120 Å pore; Eluent: A: H2O (0.1% TFA), B: CH3CN (01% TFA); Elution: Initial condition: 15% B, linear gradient 15-50% B over 8 min; Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES; Mode: Negative ion; 1336.9 [M-2H]/2, 890.8 [M-3H]/3; CE analysis (counter-ion % wt./wt.): TFA estimated to be 0.4%; acetate 4.2%; IC analysis (F %): 0.26.
  • For the dimer peptide (27) acetate salt, similar to the procedure for compound (23), an AG MP-50 column (100 mL wet volume) was washed with 30% CH3CN/H2O until the conductivity was below 1 μs/cm. Compound (27) as the TFA salt, (120 mg in 80 mL of 30% of CH3CN/H2O) was loaded onto the column and the column was washed with the same eluent until the conductivity was stable at 1 μs/cm. A step gradient of NH4OAc 30% of CH3CN/H2O into 30% of CH3CN/H2O was run as for compound (23) and the compound was eluted at ca 600 mM NH4OAc. The combined fractions were lyophilized and then relyophilized several times until the material displayed a constant weight to provide 104 mg of the pure material (27) as an acetate salt. The yield was 86.7%.
  • Additional parameters and results were as follows: HPLC: Ret. time: 5.2 min; Assay >99% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5μ particle, 120 Å pore; Eluent: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 20% B, linear gradient 20-60% B over 8 min; Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES; Mode: Negative ion; 1816.3 [M-3H]/3, 1362.0 [M-4H]/4, 1089.2 [M-5H]/5; CE analysis (counter-ion % wt./wt.): TFA estimated to be 0.2%; acetate 0.15%.
  • Preparation and Purification of the Dimer Peptide (27) Acetate Salt from Compound (23) and Compound (26)
  • To a solution of disuccinimidyl glutarate (18 mg, 0.055 mmol) in anhydrous DMF (0.1 mL) was added a solution of compound (23) (61 mg, 0.021 mmol) in 0.2 mL of anhydrous DMF dropwise (pH 8, neutralized by DIEA). The clear solution was stirred at RT for 0.5 h. HPLC and MS showed the completion of the reaction. Solvent was removed in vacuo and EtOAc (8 mL) was added to precipitate the intermediate (24). The mixture was centrifuged and decanted to remove excess glutarate. This EtOAc washing was repeated 3 more times and the resulting solid was dried using a stream of dry nitrogen. It was then dissolved in 0.3 mL of anhydrous DMF. Compound (26), (56 mg, 0.021 mmol) was added and the pH of the solution was adjusted to 8 by addition of DIEA. The solution was stirred for 16 h at room temperature after which by HPLC and MS analysis indicated completion of the reaction. A 30 μL aliquot of NH2NH2 was added and the mixture was stirred for 5 min to cleave the ivDde group. The reaction mixture was analyzed by HPLC and MS, which indicated complete removal of the ivDde group.
  • Before purification of the dimer peptide (27) acetate, caution was taken to carefully wash the whole preparative HPLC system including the column with TFA-free eluents, CH3CN/H2O/10 mM NH4OAc. The crude reaction mixture was then applied to a reverse phase C-18 preparative column (Atlantis C-18, 5 μm particle, 100 Å pore, 30×150 mm, flow rate 30 mL/min), pre-equilibrated with 15% B (A: 10 mM NH4OAc in H2O; B: 10 mM NH4OAc in CH3CN/H2O, 9/1, v/v). The column was washed with the same eluent until the DMF plug was eluted. The eluent composition was increased to 25% B over 2 min. and then ramped to 65% B over 40 min. The fractions were analyzed on an analytical reverse phase C-18 column (Waters MS C-18, 4.6×50 mm, 5 μm particle, 100 Å pore, flow rate 3 mL/min) and the product-containing fractions of >95% purity were pooled and freeze-dried to afford 25 mg of the dimer peptide (27) as its acetate salt as a fluffy white solid. The yield was 21.8%.
  • Additional parameters and results were as follows: HPLC: Ret. time: 5.2 min; Assay >99% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5μ particle, 120 Å pore; Eluent: A: H2O (0.1% TFA), B: CH3CN (01% TFA); Elution: Initial condition: 20% B, linear gradient 20-60% B over 8 min; Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES; Mode: Negative ion; [M-3H]/3, 1362.0 [M-4H]/4, 1089.2 [M-5H]/5; CE analysis (counter-ion % wt./wt.): TFA estimated to be less than 0.2%; acetate 1.1%.
    • Example 7 FIG. 7 Preparation of Dimer Peptide-Phospholipid Conjugates Having Low TFA Levels Via Ion Exchange Resin
  • Preparation and Purification of the Phospholipid Peptide Conjugate (21) as its Acetate Salt from Dimer Peptide (27) Acetate Salt
  • To a solution of disuccinimidyl glutarate-DSG (3.7 mg, 11.3 μmol) in anhydrous DMF (0.1 mL) was added a solution of neutralized dimer peptide (27) acetate salt, (15 mg, 2.75 μmol) in anhydrous DMF (0.2 mL), dropwise. The reaction solution was stirred at RT for 0.5 h. HPLC analysis with a Waters Xterra C-18 column and MS showed the completion of the reaction. The solvent was evaporated and EtOAc (8 mL) was added to precipitate the intermediate (28). The vessel containing the precipitated intermediate (28) was centrifuged and the liquid layer was decanted. This procedure was repeated 3 times to remove the excess of DSG. The solid was dried with a stream of dry nitrogen and then dissolved in 0.3 mL of anhydrous DMF. DSPE-PEG2000-NH2 ammonium salt (29) (6.5 mg, 2.33 μmol) was added in solid form and the pH of the mixture was adjusted to (28). The reaction mixture was stirred at RT for 16 h. The mixture was analyzed by MS and HPLC with a Zorbax 300 SB-C3 column and this indicated that the reaction was complete.
  • To minimize the potential contamination of the product with TFA, the crude reaction mixture was purified by preparative HPLC equipped using a new Zorbax 300SB-C3 column (21.2×150 mm, 5μ particle) which had never been exposed to TFA. The HPLC system was pre-washed by CH3CN/H2O/NH4OAc extensively to remove traces of TFA. The reaction mixture was loaded onto the column which was pre-equilibrated with 20% B (A: 10 mM NH4OAc in H2O; B: 10 mM NH4OAc in CH3CN/H2O, 9/1 v/v) at a flow rate of 30 mL/min. The column was eluted at 30 mL/min with the same eluent until the plug of DMF was eluted. The eluent composition was then increased to 40% B over 3 min and then ramped to 90% B over 50 min. The collected fractions were analyzed on an analytical reverse phase C-3 column (Zorbax 300SB-C3, 3×150 mm, 3.5 μm particle, 300 Å pore, flow rate: 0.5 mL/min), where detection was accomplished using UV at 220 nm and an evaporative light scattering detector (ELSD). The fractions containing the pure product were pooled and lyophilized. A 6.5 mg portion of the final product (21) acetate salt was obtained. The yield was 33.0%.
  • Additional parameters and results were as follows: HPLC: Ret. Time: 13.3 min; Assay >99% (area %); Column: Zorbax 300SB-C3, 3×150 mm, 3.5 μm, 300 Å pore; Eluent: A: H2O (0.1% TFA), B: CH3CN/MeOH 1/1 (01% TFA); Elution: Initial condition: 60% B, linear gradient 60-90% B over 3 min; Flow rate: 0.5 mL/min; Detection: UV at 220 nm and ELSD; CE analysis (counter-ion % wt./wt.): % wt. TFA: 0.3%; % wt acetate 0.4%.
    • Example 8 FIG. 8 Preparation of Dimeric Conjugate Having Low TFA Levels Via Sequential Purification Using Zorbax C-3 RP Preparative HPLC and Sephadex G-25 Gel Permeation Chromatography
  • Materials used and conditions for the analytical HPLC system include the following: Column: Zorbax 300SB C-3; 3 mm i.d.×150 mm; 3.5 μm particle; Eluent A: H2O (HPLC Grade with 0.1% TFA by volume); Eluent B: CH3CN (0.1% TFA by volume). Elution: Initial condition: 50% B then a linear gradient of 50-90% B over 3 min, hold at 90% B for 11 min; Flow rate: 0.5 mL/min; Detection: UV at 220 nm. Ret. time: (Compound (21)): 6.77 min, Rt (lyso): 4.06 min.
  • Preparative HPLC Using Preparative Zorbax C-3 Column to Remove the Lyso-Compound from (21)
  • The crude compound was loaded at a concentration of 30% eluent B. Materials used and conditions include: Conditions: Column: Waters Zorbax 300SB C-3; 21.2 mm i.d.×150 mm; 3.5 μm particle; Eluents: Eluent A: H2O(HPLC Grade with 10 mM NH4OAc); Eluent B: CH3CN/H2O, 9/1 (final NH4OAc concentration: 10 mM).
  • The composition of the eluent was then changed to 45% B over 2 min, then the column was eluted with a linear gradient of 45-100% B over 40 min; Flow rate: 30 mL/min; Detection: UV at 220 nm.
  • The crude compound (100 mg) was dissolved in 25 mL of a solution of 30% B. The preparative HPLC system was equilibrated at 30% B. The compound was loaded on to the Zorbax C-3 column. The mobile phase composition was ramped to 45% B over 2 min. A linear gradient from 45-100% B over 40 min was used for the elution of (21). The product eluted between 26.5-33 min.
  • The fractions that contained (21) were combined and lyophilized to give a white fluffy solid. This was dissolved in water-acetonitrile, then lyophilized again. This provided 70 mg product devoid of the lyso-compound. The recovery was about 70%. After chromatography was completed, the system was washed with 95% B for 15 min at a flow rate of 30 mL/min. The column was then washed with CH3CN/H2O (50/50, without TFA or buffer) for 30 min at a flow rate of 15 mL/min. The column was then stored at room temperature for future use. Analytical HPLC confirmed the absence of the lyso-compound in the isolated material. Further analysis confirmed that no lyso-compound formed after 5 days at room temperature. The material still contained significant amounts (4.2 wt %) of TFA.
  • Removal of TFA from (21) by Gel Permeation Chromatography on Sephadex G-25
  • A Sephadex G-25 column (100 g resin, bead size 20-80 μm, total gel volume 500 mL, column height: 27 cm) was equilibrated with 4 L of 50 mM ammonium bicarbonate. Then (21) (70 mg) was dissolved in 30 mL (final volume) of 60 mM ammonium bicarbonate in 10% aqueous acetonitrile. The solution was filtered and then loaded on to the Sephadex G-25 column. The column was eluted with 50 mM ammonium bicarbonate buffer with collection of 10 mL fractions. The collected fractions were monitored by analytical HPLC (UV detection at 220 nm). The results are provided in Table 4 below.
  • TABLE 4
    Compound present
    (by HPLC analysis
    Fraction # Volume (mL) of fraction)
    1 10 No
    3 10 No
    6 10 No
    9 10 No
    12 10 No
    15 10 No
    18 10 No
    19 10 No
    20 10 Yes
    21 10 Yes
    24 10 Yes
    27 10 Yes
    28 10 Yes
    29 10 No
  • Fractions 20-28 were pooled and lyophilized. The lyophilized material obtained was re-dissolved in a small volume of water and the solution was frozen and lyophilized to remove residual amounts of ammonium bicarbonate. The final weight of the desired material was 58 mg. The recovery was 83%.
  • To ascertain the effective removal of TFA, the sample was subjected to CE analysis for TFA and acetate ions. The TFA is clearly present in the starting material (4.2%) according to the previous assay, while it is hardly detected (0.2%) after the gel permeation procedure. No acetate ion was detected.
  • Analytical Data for (21) Obtained by Serial Zorbax C-3 Preparative HPLC and Sephadex G-25 Gel Permeation Chromatography
  • Materials used and conditions for collecting analytical data include: Fluorine analysis (IC by QTI): 751 ppm (0.15% TFA wt/wt); Mass Spectrum: Method: MALDI-TOF; Mode: Positive Ion; Average molecular weight detected was 8461 the typical PEG2000 mass distribution curve was observed. HPLC: System A: Column: Zorbax 300SB C-3; 3 mm i.d.×150 mm; 3.5 μm particle; Eluent A: Water (HPLC Grade with 0.1% TFA by volume); Eluent B: Acetonitrile (0.1% TFA by volume). Initial condition: 50% B; Elution: linear gradient of 50-90% B over 3 min, hold at 90% B for 11 min; Flow rate: 0.5 mL/min; Detection: UV at 220 nm. Ret time: 6.77 min; Area %:99.6%. System B: Column: Zorbax 300SB C-3; 3 mm i.d.×150 mm; 3.5 μm particle; Eluent A: Water (HPLC Grade with 0.1% TFA by volume); Eluent B: Acetonitrile (0.1% TFA by volume). Initial condition: 50% B; Elution: linear gradient of 50-90% B over 3 min then ramp to 100% B over 12 min. Flow rate: 0.5 mL/min; Detection: LSD; Ret: time: 13.98 min. Area %:99.3%.
  • Table 5 below provides definitions for the abbreviations used and the sources of materials referred to in Examples 9-12.
  • TABLE 5
    DSPA.Na (Genzyme) IUPAC: 1,2-Distearoyl-sn-glycero-3-
    phosphosphatidic acid, sodium salt
    DPPG.Na (Genzyme) IUPAC: 1,2-Dipalmitoyl-sn-glycero-3-
    phosphoglycerol, sodium salt
    DPPE (Genzyme) IUPAC: 1,2-Dipalmitoyl-sn-glycero-3-
    phosphoethanolamine
    DSPC Distearoyl-glycero-phosphatidylcholine (Genzyme)
    IUPAC: 1,2-Distearoyl-sn-glycero-3-phosphocholine
    DSPG.Na (Genzyme) IUPAC: 1,2-Distearoyl-sn-glycero-3-
    phosphoglycerol, sodium salt
    DSPE-PEG1000 Distearoyl-glycero-phosphoethanolamine-N-
    methoxy(polyethylene glycol)1000 (Avanti Polar)
    DSPE-PEG2000 Distearoyl-glycero-phosphoethanolamine-N-
    methoxy(polyethylene glycol)2000 (Avanti Polar)
    Stearate* Sodium Stearate (Fluka)
    PEG4000 (polyethylene glycol) MW 4000 (Fluka)
    Mannitol (Fluka)
    *the acid form, i.e., stearic acid, can also be used in any of the microbubble preparations herein.
    • Example 9 Preparation of Targeted Microbubbles with DSPC/DPPG Envelope Example 9A
  • 383 mg of a mixture of DSPC/DPPG/ and the dimeric peptide phospholipid conjugate (11) shown in FIG. 5 (molar ratio 49.75/49.75/0.5, corresponding to 187.1, 176.4 and 19.8 mg of the three components, respectively) and PEG-4000 (22.6 g) were solubilized in 120 g of t-butyl alcohol at 60° C., in a water bath. The solution was filled in 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 C4F10/Nitrogen (50/50) and vials capped and crimped. The lyophilized samples were reconstituted with 5 mL of H2O per vial.
    • Example 9B
  • Example 9A was repeated using a mixture of DSPC/DPPG/ and the monomeric peptide phospholipid conjugate (31) shown in FIG. 10 (molar ratio 49.5/49.5/1, corresponding to 182.8, 172.3 and 28.2 mg of the three components, respectively)
    • Example 10 Preparation of Targeted Microbubbles with DPPE/DPPG Envelope Example 10A
  • An aqueous suspension of DSPE-PEG1000 (0.43 mg-0.24 μmole) and the monomeric peptide phospholipid conjugate (31) shown in FIG. 10 (3.0 mg-0.5 μmole) was prepared in 500 μL of distilled water at 60° C. to obtain a micellar suspension.
  • Separately, DPPE (15.8 mg-22.8 μmoles) and DPPG (4.2 mg-5.7 μmoles) were dispersed in a solution of mannitol 10% in distilled water (20 mL) at 70° C. for 20 minutes. The dispersion was then cooled to room temperature. Perfluoroheptane (1.6 mL) was emulsified in the aqueous phase using a high speed homogenizer (Polytron PT3000, probe diameter of 3 cm) for 1 minute at 10500 rpm to obtain an emulsion.
  • The micellar suspension was added to the emulsion and the resulting mixture was heated at 60° C. for 1 hour under stirring. After cooling to room temperature (1 hour), the obtained emulsion was divided in 4 mL fractions in 50 mL round bottom flasks. The emulsion was frozen at −45° C. for 5 minutes and freeze-dried at 0.2 mBar for 24 hours (Freeze-Drier Christ Beta 1-8K).
  • Before redispersion, the lyophilisate was exposed to an atmosphere containing C4F10/nitrogen (50/50 by volume). The lyophilized product was then dispersed in a volume of water twice the initial one by gentle hand shaking.
    • Example 10B
  • An aqueous suspension of DSPE-PEG1000 (0.5 mg-0.27 μmole) and dimeric peptidephospholipid conjugate (11) shown in FIG. 5 (5.3 mg-0.63 μmole) was prepared in 500 L of distilled water at 60° C. to obtain a micellar suspension.
  • Separately, DPPE (15.8 mg-22.8 μmoles) and DPPG (4.2 mg-5.7 μmoles) were dispersed in a solution of PEG4000 10% in distilled water (20 mL) at 70° C. for 20 minutes. The dispersion was then cooled to room temperature. Perfluoroheptane (1.6 mL) was emulsified in the aqueous phase using a high speed homogenizer (Polytron PT3000, probe diameter of 3 cm) for 1 minute at 10000 rpm to obtain an emulsion.
  • The micellar suspension was added to the emulsion and the resulting mixture was heated at 80° C. for 1 hour under stirring. After cooling to room temperature (1 hour), the obtained emulsion was washed once by centrifugation (200 g/10 min—Sigma centrifuge 3K10) to eliminate the excess of phospholipid. The separated pellet (containing emulsified microdroplets of solvent) was recovered and re-suspended with the initial volume of a 10% PEG4000 aqueous solution.
  • The obtained emulsion was sampled into DIN8R vials (1 mL/vial). Then vials were cooled at −50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at −25° C. and 0.2 mBar for 12 hours with a final drying step at 30° C. and 0.1 mBar for 7 hours. Vials were exposed to an atmosphere containing C4F10/nitrogen (35/65 by volume) and sealed. The lyophilized product was redispersed in a volume of water twice the initial one by gentle hand shaking.
    • Example 11 Preparation of Targeted Microbubbles with DSPC/DSPA Envelope Example 11A
  • An aqueous suspension of DSPE-PEG1000 (2.5 mg-1.4 μmole) and dimeric peptide conjugate (11) shown in FIG. 5 (7.0 mg-0.84 μmole) was prepared in 1 mL of distilled water at 60° C. to obtain a micellar suspension.
  • Separately, DSPC (16.3 mg-20.6 μmoles) and DSPA (3.7 mg-5.15 μmoles) were dissolved in cyclooctane (1.6 mL) at 80° C. This organic phase was added to a PEG4000 10% solution in water (20 mL) using a high speed homogenizer (Polytron T3000, probe diameter of 3 cm) for 1 minute at 8000 rpm, to obtain an emulsion.
  • The micellar suspension was mixed with the emulsion and the resulting mixture was heated at 80° C. for 1 hour under agitation. After cooling to room temperature (1 hour), the obtained emulsion was washed once by centrifugation (1500 g/10 min—Sigma centrifuge 3K10) to eliminate the excess of the phospholipid. The separated supernatant (containing emulsified microdroplets of solvent) was recovered and re-suspended in twice the initial volume of a 10% PEG 4000 aqueous solution.
  • The obtained suspension was sampled into DIN8R vials (1 mL/vial). Then vials were cooled to −50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at −25° C. and 0.2 mbar for 12 hours, with a final drying step at 30° C. and 0.1 mbar for 7 hours. Vials were exposed to an atmosphere containing C4F10/Nitrogen (35/65 by volume) and sealed. The lyophilized product was then dispersed in a volume of water twice the initial one by gentle hand shaking.
    • Example 11B
  • Example 11A was repeated, but using 0.7 mg of DSPE-PEG2000 (0.26 μmoles) and 1.6 mg of monomeric peptide-phospholipid conjugate (1) shown in FIG. 2 (0.26 μmole) to prepare the micellar suspension.
    • Example 11C
  • DSPC (16.3 mg-20.6 μmoles), DSPA (3.7 mg-5.15 μmoles) and monomeric peptide phospholipid conjugate (1) shown in FIG. 1 (1.6 mg-0.26 μmole) were dissolved in cyclooctane (1.6 mL) at 80° C. This 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.
  • The resulting emulsion was heated at 80° C. for 1 hour under stirring. After cooling to room temperature (1 hour), the obtained emulsion was diluted with 20 ml of a PEG4000 10% aqueous solution.
  • The emulsion was sampled into DIN8R vials (1 mL/vial). Then vials were cooled at −50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at −25° C. and 0.2 mBar for 12 hours with a final drying step at 30° C. and 0.1 mBar for 7 hours. Vials were exposed to an atmosphere containing C4F10/nitrogen (35/65 by volume) and sealed. The lyophilized product was redispersed in a volume of water twice the initial one by gentle hand shaking.
    • Example 12 Preparation of Targeted Microbubbles with DSPC/Stearate Envelope Example 12A
  • An aqueous suspension of DSPE-PEG2000 (2.5 mg-0.9 μmoles) and the dimeric phospholipid conjugate (11) shown in FIG. 5 (2.5 mg-0.3 μmoles) was prepared in 660 μL of distilled water at 60° C. to obtain the micellar suspension.
  • Separately, DSPC (18.2 mg-23.1 μmoles) and stearate (1.8 mg-5.8 μmoles) were dissolved in cyclooctane (1.6 mL) at 80° C. This organic phase was added to a 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, to obtain an emulsion.
  • The micellar solution was mixed with the emulsion and the resulting mixture was heated at 80° C. for 1 hour under agitation. After cooling to room temperature (1 hour), the obtained emulsion was washed once by centrifugation (1500 g/10 min—Sigma centrifuge 3K10) to eliminate the excess of phospholipids. The separated supernatant (containing emulsified microdroplets of solvent) was recovered and re-suspended with twice the initial volume of a 10% PEG 4000 aqueous solution.
  • The obtained suspension was sampled into DIN8R vials (1 mL/vial). Then vials were cooled to −50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at −25° C. and 0.2 mbar for 12 hours, with a final drying step at 30° C. and 0.1 mbar for 7 hours. Vials were exposed to an atmosphere containing C4F10/Nitrogen (35/65 by volume) and sealed. The lyophilized product was dispersed in a volume of water twice the initial one by gentle hand shaking.
    • Example 12B
  • Example 12A was repeated by replacing the dimeric peptide phospholipid conjugate (11) shown in FIG. 5 with the same relative molar amount of the monomeric peptide phospholipid conjugate (1) shown in FIG. 2.
    • Example 12C
  • Example 11C was repeated with DSPC (18.2 mg-23.1 μmoles), sodium stearate (1.8 mg-5.8 μmoles) and the dimeric peptide phospholipid conjugate (11) shown in FIG. 5 (2.2 mg-0.26 μmole). The agitation speed for emulsification was fixed to 9000 rpm. After cooling to room temperature (1 hour), the obtained emulsion was washed once by centrifugation (1500 g/10 min—Sigma centrifuge 3K10) to eliminate the excess of the phospholipid. The separated supernatant (containing emulsified microdroplets of solvent) was recovered and re-suspended in twice the initial volume of a 10% PEG 4000 aqueous solution.
    • Example 13 Static Binding Test on KDR-Transfected Cells
  • Plasmid Production and Purification
  • Full-length KDR was cloned into the pcDNA6 vector and the plasmid was amplified in competent DH5α E. coli. Plasmid amplification and purification was performed using E. coli JM 109 and a kit from Quiagen.
  • Transfection of 293H Cells on Thermanox® Coverslips
  • Cells were grown on poly-D-lysine-coated Thermanox® circular coverslips in 24-well plate. Transfection was done as recommended in the lipofectamine 2000 protocol (Invitrogen, cat# 11668-019) using 1 μg of DNA (pc-DNA6-fKDR)/per coverslip (1.3 cm2) in 0.1 mL. Transfection was done in serum-free media, the transfection reagent mix was removed from cells after 2 hours and replaced with regular serum-containing medium. Some of the cell-coated coverslips were mock-transfected (with no DNA). The next day, expression of the KDR receptor was assessed by immunocytochemistry and the binding assay was performed.
  • Bubble Binding Assay
  • The transfected cells were incubated with KDR-targeted microbubbles resuspended in 50% human plasma in PBS. For the incubation with the transfected cells a small plastic cap was filled with a suspension containing a 1.3×108 bubbles and the cap was covered with an inverted Thermanox® coverslip so as to put the transfected cells in contact with the targeted microbubbles. After 30 min of incubation at RT, the coverslip was lifted with tweezers, rinsed three times in PBS and examined under a microscope to assess binding of the targeted microbubbles.
  • Determination of the % of Surface Covered by Microbubbles
  • Images were acquired with a digital camera DC300F (Leica) and the percent of surface covered by bound microbubbles in the imaged area was determined using the software QWin version 3.1 (Leica Microsystem AG, Basel, Switzerland). Pictures were taken of each Thermanox®coverslip. For each preparation of Examples 9 and 10, the binding assay was repeated a minimum of two times thus obtaining an average value of the surface covered. In the following Tables 6 and 7, the binding activity of the microbubbles prepared according to Examples 9 and 10 above are recorded.
  • As indicated by the Tables, the same peptide may show different binding activities when included (as a lipopeptide) in different phospholipid formulations forming the stabilizing envelope of the microbubble. Microbubbles containing KDR binding lipopeptides of the invention bind specifically to KDR-expressing cells while they did not bind appreciably to mock transfected cells.
    • Example 14 Dynamic Binding test on rh VEGF-R2/Fc Chimeric Protein
  • Preparation of Fc-VEGF-R2-Coated Coverslips
  • Glass coverslips (40 mm in diameter, Bioptechs Inc., Butler, Pa., USA) were coated with recombinant human VEGF-R2/Fc Chimeric protein (R&D Systems) according the following methodology.
  • A surface of dimensions 14×25 mm was delimited on the glass coverslip using a special marker (Dako Pen) and 400 μL of Fc-VEGF-R2 solution at 4 μg/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 bubbles 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. The coated coverslip was inserted as a plate of the flow chamber. Microbubbles (5×106 bubbles/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, Ill., 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. After 10 minutes, the flow was stopped and pictures were taken randomly at different positions on the coverslip (on areas of about 0.025 mm2) using a 40× objective and a CCD monochrome camera (F-View II, Soft Imaging Systems, Germany) connected to an inverted Olympus IX 50 microscope.
  • The number of microbubbles 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 microbubbles per minute).
  • For each preparation of Examples 11 and 12, the binding assay was repeated four times thus obtaining an average value of the slope.
  • The slope represents the bubble binding rate on the target substrate. For instance, a slope value of 8 indicates that an average of eighty (80) microbubbles was bound on the coated coverslip in ten minutes. A higher slope indicates a better capacity of bubbles to bind to the target under flow conditions.
  • In the following tables 8 and 9, the binding activity of the microbubbles prepared according to Examples 11 and 12 above were illustrated.
  • As inferable from the tables, the same peptide may show different binding activities when included (as a peptide-phospholipid conjugate or lipopeptide) in different phospholipid formulations forming the stabilizing envelope of the microbubble.
  • TABLE 6
    KDR-
    Example KDR Mock Mock
    9A 28.6% 0.4% 28.3%
    9B 28.0% 0.3% 27.7%
  • TABLE 7
    KDR-
    Example KDR Mock Mock
    10A 23.6% 0.2% 23.5%
    10B 28.0% 0.0% 28.0%
  • TABLE 8
    Example Slope
    11A 8.2
    11B 8.1
    11C 5.8
  • TABLE 9
    Example Slope
    12A 9.0
    12B 8.0
    12C 7.8
    • Example 15 In Vivo Evaluation of Ultrasound Contrast Agents Targeted to KDR
  • The ability of ultrasound contrast agents containing KDR binding lipopeptides of the invention to bind to KDR-expressing tissue in vivo was assessed using a known model of angiogenesis: the rabbit VX2 tumor model.
  • A known model of angiogenic tissue was used to examine the ability of the KDR-targeted ultrasound microbubbles to localize to and provide an image of angiogenic tissue. The VX2 rabbit carcinoma was serially implanted in the dorsal muscle of New Zealand rabbits (Charles River Laboratories, France) weighting 2.5/3 kg.
  • Preparation of Tumor Homogenate
  • Tumor was surgically removed, placed into McCoy's culture medium containing 10% fetal calf serum, antibiotics, 1.5 mM Glutamax I and cut into small pieces that were rinsed to remove blood and debris. Then tumor pieces (3 to 5 cm3) were placed in a 50 ml Falcon tube containing 5 mL of complete medium. The tumor tissue was ground (Polytron) until no more solid pieces were visible. The murky fluid was centrifuged for 5 minutes at 300 g and the supernatant discarded. Seven mL of fresh medium was added per 5 mL of pellet.
  • Tumor Implantation
  • Rabbits received first 0.3 mL of Vetranquil (Acepromazine, Sanofi, injected intramuscularly) and were then anesthetized with an intramuscular injection of Ketaminol®5/Xylazine (Veterinaria AG/Sigma) mixture (50/10 mg/mL, 0.7 mL/kg). One hundred microliters of VX2 tumor homogenate was injected intramuscularly. Fifteen days after implantation of VX2 tumors, animals were anesthetized again with the same mixture, plus subcutaneous injection of 50% Urethane (2 mL/kg, s.c.) (Sigma) for imaging experiments.
  • In Vivo Ultrasound Imaging
  • VX2 tumor imaging was performed using an ultrasound imaging system ATL HDI 5000 apparatus equipped with a L7-4 linear probe. B-mode pulse inversion at high acoustic power (MI=0.9) was used to evaluate accumulation of targeted microbubbles on the KDR receptor expressed on the endothelium of neovessels. The linear probe was fixed on the skin directly over the implanted tumors.
  • After bubble injection (0.1 μL/kg of gas) using the preparations of either Example 16 or Example 17, insonation was stopped allowing bubbles to accumulate for 25 minutes. Then, insonation was reactivated at high acoustic power (MI 0.9) destroying all the bubbles present in the tumor. The amount of free circulating bubbles was then assessed by recording the signal obtained after 20 sec accumulation without insonation. Video frames from VX2 tumor imaging experiments were captured with video-capture and analysed with Image-Pro Plus 2.0 software. The image representing free circulating bubbles was subtracted from the image obtained at 25 min, to provide an image representing bound bubbles. Referring to FIG. 11 (which shows the results with the preparation of Example 16) and FIG. 12 (which shows the results with the preparation of Example 17), FIGS. 11A and 12A show an image before bubble injection (baseline); FIGS. 11B and 12B show retention of bubble contrast in the tumor 25 minutes post injection; and FIGS. 11C and 12C show the result obtained after subtraction of the baseline and free circulating bubbles and represent bound microbubbles containing KDR lipopeptides according to the present invention. Examples 15-17 and FIGS. 11 and 12 confirm that ultrasound contrast agents bearing such KDR binding moieties localize to KDR expressing (and thus angiogenic) tissue in animal models.
    • Example 16
  • Example 12A was repeated by replacing DSPE-PEG2000 with DSPE-PEG1000 (2.7 mg, 1.54 μmol) and using 2.5 mg (0.31 μmol) of dimeric peptide phospholipid conjugate (11) shown in FIG. 5.
    • Example 17
  • Example 16 was repeated by replacing the dimeric peptide phospholipid conjugate with the same molar amount of monomeric phospholipid conjugate (1) shown in FIG. 2.

Claims (19)

1-39. (canceled)
40. A method of making a peptide phospholipid conjugate comprising, conjugating a peptide selected form the group consisting of SEQ ID NO. 2, Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH2 cyclic (6-13) disulfide, Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH2 cyclic (2-12) disulfide, SEQ ID NO. 5 and Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)-NH2 cyclic (2-12) disulfide]-NH2 cyclic (6-13) disulfide with a phospholipid.
41. The method of claim 40, wherein the phospholipid is pegylated.
42. The method of claim 41, wherein the pegylated phospholipid is DSPE-PEG2000-NH2.
43. The method of claim 42, wherein the conjugation comprises reacting a mono NHS ester of the peptide with a free amino group of the phospholipid salt.
44. A method of making a peptide phospholipid conjugate having low levels of TFA comprising the steps of:
converting peptide dimer TFA salts to an acetate salt via anion exchange; and
conjugating the peptide dimer to a phospholipid.
45. The method of claim 44, wherein the peptide comprises a peptide dimer.
46. The method of claim 45, wherein the peptide dimer comprises Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)-NH2 cyclic (2-12) disulfide]-NH2 cyclic (6-13) disulfide.
47. The method of claim 44, wherein the anion exchange chromatography comprises a step gradient of ammonium acetate.
48. The method of claim 44, wherein the phospholipid is pegylated.
49. The method of claim 48, wherein the pegylated phospholipid is DSPE-PEG2000-NH2.
50. A method of making a peptide-phospholipid conjugate having low levels of TFA comprising eluting a peptide phospholipid conjugate and TFA ions in a size exclusion column.
51. The method of claim 50, wherein said eluting is in the presence of ammonium bicarbonate.
52. The method of claim 50, wherein the peptide phospholipid conjugate comprises a pegylated phospholipid.
53. The method of claim 52, wherein the pegylated phospholipid is DSPE-PEG2000-NH2.
54. The method of claim 50, wherein the peptide-phospholipid conjugate comprises a peptide dimer.
55. The method of claim 54, wherein the peptide dimer comprises Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)-NH2 cyclic (2-12) disulfide]-NH2 cyclic (6-13) disulfide.
56. The method of claim 50, wherein the peptide-phospholipid conjugate comprises a peptide monomer.
57. The method of claim 56, wherein the monomer is selected from the group consisting of: SEQ ID NO.2, Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH2 cyclic (6-13) disulfide, Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH2 cyclic (2-12) disulfide, and SEQ ID NO. 5.
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US36085102P 2002-03-01 2002-03-01
US44041103P 2003-01-15 2003-01-15
US38208203A 2003-03-03 2003-03-03
PCT/US2003/006731 WO2003074005A2 (en) 2002-03-01 2003-03-03 Kdr and vegf/kdr binding peptides and their use in diagnosis and therapy
US10/661,156 US20050100963A1 (en) 2002-03-01 2003-09-11 KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110160431A1 (en) * 2009-04-06 2011-06-30 Novetide, Ltd. Production of peptides containing poly-gly sequences using fmoc chemistry
US9056138B2 (en) 2002-03-01 2015-06-16 Bracco Suisse Sa Multivalent constructs for therapeutic and diagnostic applications
US9295737B2 (en) 2002-03-01 2016-03-29 Bracco Suisse Sa Targeting vector-phospholipid conjugates
US9408926B2 (en) 2002-03-01 2016-08-09 Bracco Suisse S.A. KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy
US9446155B2 (en) 2002-03-01 2016-09-20 Bracco Suisse Sa KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy
USRE46830E1 (en) 2004-10-19 2018-05-08 Polypeptide Laboratories Holding (Ppl) Ab Method for solid phase peptide synthesis

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7211240B2 (en) * 2002-03-01 2007-05-01 Bracco International B.V. Multivalent constructs for therapeutic and diagnostic applications
ES2396368T3 (en) * 2003-03-03 2013-02-21 Dyax Corporation Peptides that specifically bind to the HGF receptor (CMET) and uses thereof
HUE043680T2 (en) * 2014-12-18 2019-09-30 Bracco Suisse Sa Targeted gas-filled microvesicles formulation

Citations (74)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4093709A (en) * 1975-01-28 1978-06-06 Alza Corporation Drug delivery devices manufactured from poly(orthoesters) and poly(orthocarbonates)
US4391797A (en) * 1977-01-05 1983-07-05 The Children's Hospital Medical Center Systems for the controlled release of macromolecules
US4544500A (en) * 1982-04-14 1985-10-01 Scripps Clinic And Research Foundation Synthetic foot and mouth disease antigen
US4718433A (en) * 1983-01-27 1988-01-12 Feinstein Steven B Contrast agents for ultrasonic imaging
US4844882A (en) * 1987-12-29 1989-07-04 Molecular Biosystems, Inc. Concentrated stabilized microbubble-type ultrasonic imaging agent
US4899755A (en) * 1985-05-08 1990-02-13 The General Hospital Corporation Hepatobiliary NMR contrast agents
US4900540A (en) * 1983-06-20 1990-02-13 Trustees Of The University Of Massachusetts Lipisomes containing gas for ultrasound detection
US5021556A (en) * 1987-07-22 1991-06-04 Neorx Corporation Method of radiolabeling chelating compounds comprising sulfur atoms with metal radionuclides
US5118797A (en) * 1989-08-28 1992-06-02 E. R. Squibb & Sons, Inc. Rhenium tris dioxime complexes
US5123414A (en) * 1989-12-22 1992-06-23 Unger Evan C Liposomes as contrast agents for ultrasonic imaging and methods for preparing the same
US5137928A (en) * 1990-04-26 1992-08-11 Hoechst Aktiengesellschaft Ultrasonic contrast agents, processes for their preparation and the use thereof as diagnostic and therapeutic agents
US5180816A (en) * 1988-08-24 1993-01-19 Centocor One vial method for labeling protein/linker conjugates with technetium-99M
US5183653A (en) * 1990-04-13 1993-02-02 E. R. Squibb & Sons, Inc. Boronic acid adducts of metal dioxime complexes useful in labelling proteins and other amine-containing compounds
US5223409A (en) * 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5230882A (en) * 1989-12-22 1993-07-27 Unger Evan C Liposomes as contrast agents for ultrasonic imaging and methods for preparing the same
US5316921A (en) * 1992-05-18 1994-05-31 Genentech, Inc. Single-chain hepatocyte growth factor variants
US5380519A (en) * 1990-04-02 1995-01-10 Bracco International B.V. Stable microbubbles suspensions injectable into living organisms
US5387409A (en) * 1990-01-18 1995-02-07 Bracco International B.V. Boronic acid adducts of rhenium dioxime and technetium-99m dioxime complexes containing a biochemically active group
US5413774A (en) * 1992-01-23 1995-05-09 Bracco International B.V. Long-lasting aqueous dispersions or suspensions of pressure-resistant gas-filled microvesicles and methods for the preparation thereof
US5445813A (en) * 1992-11-02 1995-08-29 Bracco International B.V. Stable microbubble suspensions as enhancement agents for ultrasound echography
US5529766A (en) * 1991-03-28 1996-06-25 Nycomed Imaging As Contrast agents
US5536490A (en) * 1991-03-28 1996-07-16 Nycomed Imaging As Contrast agents
US5547856A (en) * 1992-05-18 1996-08-20 Genentech, Inc. Hepatocyte growth factor variants
US5556939A (en) * 1994-10-13 1996-09-17 Merck Frosst Canada, Inc. TC or RE radionuclide labelled chelate, hexapeptide complexes useful for diagnostic or therapeutic applications
US5556610A (en) * 1992-01-24 1996-09-17 Bracco Research S.A. Gas mixtures useful as ultrasound contrast media, contrast agents containing the media and method
US5558857A (en) * 1991-06-03 1996-09-24 Nycomed Imaging As Contrast agents
US5558856A (en) * 1992-01-09 1996-09-24 Nycomed Imaging As Microbubble-generating contrast agents for ultrasound and magnetic resonance imaging
US5607661A (en) * 1991-07-05 1997-03-04 Nycomed Imaging As Aggregates of x-ray microparticles for ultrasound imaging
US5608110A (en) * 1993-06-15 1997-03-04 Bracco International B.V. Heteroatom-bearing ligands and metal complexes thereof
US5637289A (en) * 1992-01-09 1997-06-10 Nycomed Imaging As Contrast agents, consisting of galactose particles
US5662885A (en) * 1994-07-22 1997-09-02 Resolution Pharmaceuticals Inc. Peptide derived radionuclide chelators
US5670133A (en) * 1992-02-20 1997-09-23 Rhomed Incorporated Peptides method for radiolabeling them, and method for detecting inflammation
US5707624A (en) * 1994-06-03 1998-01-13 The Regents Of The University Of Michigan Treatment of Kaposi's sarcoma by inhibition of scatter factor
US5711933A (en) * 1990-05-18 1998-01-27 Bracco International B.V. Method of making polymeric gas or air filled microballoons for ultrasonic echography
US5720934A (en) * 1992-04-30 1998-02-24 Diatide, Inc. Technetium-99M labeled peptides for imaging
US5723584A (en) * 1993-07-30 1998-03-03 Affymax Technologies N.V. Biotinylation of proteins
US5759515A (en) * 1989-08-09 1998-06-02 Rhomed Incorporated Polyvalent peptide pharmaceutical applications
US5759996A (en) * 1994-04-13 1998-06-02 La Jolla Cancer Research Center Peptides useful for altering αv β3 -mediated binding
US5770421A (en) * 1993-12-03 1998-06-23 St. Jude Children's Research Hospital Human ALK protein tyrosine kinase
US5798091A (en) * 1993-07-30 1998-08-25 Alliance Pharmaceutical Corp. Stabilized gas emulsion containing phospholipid for ultrasound contrast enhancement
US5871959A (en) * 1989-12-27 1999-02-16 The United States Of America As Represented By The Department Of Health And Human Services Method of producing hepatocycte growth factor/scatter factor and related cell lines
US5876973A (en) * 1992-08-10 1999-03-02 Cambridge Neuroscience, Inc. Method for making antibodies which specifically bind to glial growth factors
US5877289A (en) * 1992-03-05 1999-03-02 The Scripps Research Institute Tissue factor compositions and ligands for the specific coagulation of vasculature
US5879658A (en) * 1991-02-08 1999-03-09 Diatide, Inc. Technetium-99m labeled peptides for thrombus imaging
US5885866A (en) * 1996-10-03 1999-03-23 Mosel Vitelic Inc. Self-registered cylindrical capacitor of high density DRAMs
US5886142A (en) * 1997-05-20 1999-03-23 Thomas Jefferson University Radiolabeled thrombus imaging agents
US6025331A (en) * 1996-02-16 2000-02-15 Children's Medical Center Corporation Pharmaceutical compositions comprising troponin subunits, fragments and analogs thereof and methods of their use to inhibit angiogenesis
US6093382A (en) * 1998-05-16 2000-07-25 Bracco Research Usa Inc. Metal complexes derivatized with folate for use in diagnostic and therapeutic applications
US6110433A (en) * 1994-12-20 2000-08-29 Varta Batterie Aktiengesellschaft Process for the recovery of metals from used nickel/metal/rare earth hydride storage batteries
US6171614B1 (en) * 1996-10-15 2001-01-09 Emory University Synthesis of glycophospholipid and peptide-phospholipid conjugates and uses thereof
US6183725B1 (en) * 1992-12-16 2001-02-06 Bracco Research S.A. Gas mixtures useful as ultrasound contrast media, contrast agents containing the media and method
US6207152B1 (en) * 1995-06-02 2001-03-27 Genentech, Inc. Hepatocyte growth factor receptor antagonists and uses thereof
US6258378B1 (en) * 1998-02-09 2001-07-10 Bracco Research S.A. Delivery of biologically active substance to target sites in the body of patients
US6274166B1 (en) * 1997-05-29 2001-08-14 Ben Gurion University Of The Negev Research And Development Authority Transdermal delivery system
US20020102217A1 (en) * 1996-10-28 2002-08-01 Nycomed Imaging As Diagnostic/therapeutic agents
US20020119149A1 (en) * 1998-05-19 2002-08-29 Jakobsen Bent Karsten Multivalent T cell receptor complexes
US20020136721A1 (en) * 1998-02-17 2002-09-26 Schwall Ralph H. Hepatocyte growth factor receptor antagonists and uses thereof
US6524533B1 (en) * 1998-03-06 2003-02-25 Biosafe Medical Technologies, Inc. Device for collecting and drying a body fluid
US20030125493A1 (en) * 2001-10-26 2003-07-03 Harris Stephen H. (Meth)acrylates of oxyalkylated phenolic resins and their use as adhesion promoters
US20030129223A1 (en) * 2000-10-11 2003-07-10 Targesome, Inc. Targeted multivalent macromolecules
US20040018974A1 (en) * 2002-03-01 2004-01-29 Christophe Arbogast Multivalent constructs for therapeutic and diagnostic applications
US20040037820A1 (en) * 1992-10-09 2004-02-26 Kari Alitalo Flt4 (VEGFR-3) as a target for tumor imaging and anti-tumor therapy
US6710165B2 (en) * 2000-06-01 2004-03-23 Kyunglim Lee IgE-dependent histamine-releasing factor-binding peptides
US6733755B2 (en) * 2000-02-04 2004-05-11 Supratek Pharma, Inc. Ligand for vascular endothelial growth factor receptor
US20050100963A1 (en) * 2002-03-01 2005-05-12 Dyax Corporation KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy
US20050147555A1 (en) * 2002-03-01 2005-07-07 Hong Fan Methods for preparing multivalent constructs for therapeutic and diagnostic applications and methods of preparing the same
US20050214859A1 (en) * 2003-03-03 2005-09-29 Dyax Corp. Peptides that specifically bind HGF receptor (cMet) and uses thereof
US6984373B2 (en) * 2000-12-23 2006-01-10 Dyax Corp. Fibrin binding moieties useful as imaging agents
US20060063699A1 (en) * 1998-03-09 2006-03-23 Larsen Bjarne D Pharmacologically active peptide conjugates having a reduced tendency towards enzymatic hydrolysis
US20060089307A1 (en) * 2002-12-30 2006-04-27 Kulseth Mari A Peptides that bind to the heparin binding domian of vegf and vegfr-2
US7199100B2 (en) * 2000-10-04 2007-04-03 Commissariat A L'energie Atomique Cyclic peptides, method for preparing and use as angiogenesis inhibitors or activator
US20070172428A1 (en) * 2002-03-01 2007-07-26 Bracco International B.V. Multivalent constructs for therapeutic and diagnostic applications
US20080152594A1 (en) * 2002-03-01 2008-06-26 Philippe Bussat Targeting vector-phospholipid conjugates
US7794693B2 (en) * 2002-03-01 2010-09-14 Bracco International B.V. Targeting vector-phospholipid conjugates

Family Cites Families (180)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US585199A (en) * 1897-06-29 John w
US2913451A (en) 1957-04-04 1959-11-17 Mallinckrodt Chemical Works Iodophenylamidoalkanesulfonic acid compounds
US4131648A (en) 1975-01-28 1978-12-26 Alza Corporation Structured orthoester and orthocarbonate drug delivery devices
US4180646A (en) 1975-01-28 1979-12-25 Alza Corporation Novel orthoester polymers and orthocarbonate polymers
DE3313947A1 (en) 1983-04-15 1984-10-18 Schering AG, 1000 Berlin und 4709 Bergkamen MICROPARTICLES AND GAS BUBBLES CONTAINING ULTRASONIC CONTRASTING AGENTS
DE3313946A1 (en) 1983-04-15 1984-10-18 Schering AG, 1000 Berlin und 4709 Bergkamen MICROPARTICLES AND GAS BUBBLES CONTAINING ULTRASONIC CONTRASTING AGENTS
DE3834705A1 (en) 1988-10-07 1990-04-12 Schering Ag ULTRASONIC CONTRASTING AGENTS FROM GAS BUBBLES AND MICROPARTICLES CONTAINING FATTY ACID
MX174467B (en) 1986-01-23 1994-05-17 Squibb & Sons Inc 1,4,7-TRISCARBOXIMETHYL-1,4,7,10-TETRAAZACICLODO DECAN SUBSTITUTE IN 1 AND ANALOG COMPOUNDS
IE61591B1 (en) 1987-12-29 1994-11-16 Molecular Biosystems Inc Concentrated stabilized microbubble-type ultrasonic imaging agent and method of production
US5075099A (en) 1988-05-31 1991-12-24 Neorx Corporation Metal radionuclide chelating compounds for improved chelation kinetics
US5364613A (en) 1989-04-07 1994-11-15 Sieving Paul F Polychelants containing macrocyclic chelant moieties
JPH05503072A (en) 1989-08-28 1993-05-27 ザ・ゼネラル・ホスピタル・コーポレーション Hydroxy-aryl metal chelates for diagnostic NMR imaging
US5469854A (en) 1989-12-22 1995-11-28 Imarx Pharmaceutical Corp. Methods of preparing gas-filled liposomes
US5305757A (en) * 1989-12-22 1994-04-26 Unger Evan C Gas filled liposomes and their use as ultrasonic contrast agents
US5773024A (en) * 1989-12-22 1998-06-30 Imarx Pharmaceutical Corp. Container with multi-phase composition for use in diagnostic and therapeutic applications
US5585112A (en) * 1989-12-22 1996-12-17 Imarx Pharmaceutical Corp. Method of preparing gas and gaseous precursor-filled microspheres
US20020150539A1 (en) 1989-12-22 2002-10-17 Unger Evan C. Ultrasound imaging and treatment
US6146657A (en) * 1989-12-22 2000-11-14 Imarx Pharmaceutical Corp. Gas-filled lipid spheres for use in diagnostic and therapeutic applications
US5578292A (en) 1991-11-20 1996-11-26 Bracco International B.V. Long-lasting aqueous dispersions or suspensions of pressure-resistant gas-filled microvesicles and methods for the preparation thereof
US5367080A (en) 1990-11-08 1994-11-22 Sterling Winthrop Inc. Complexing agents and targeting radioactive immunoreagents useful in therapeutic and diagnostic imaging compositions and methods
US5849261A (en) 1991-02-08 1998-12-15 Diatide, Inc. Radiolabeled vasoactive intestinal peptides for diagnosis and therapy
CA2083401C (en) 1991-02-22 2003-03-25 Bruce I. Terman Identification of a novel human receptor tyrosine kinase gene
OA10149A (en) * 1991-03-29 1996-12-18 Genentech Inc Vascular endothelial cell growth factor antagonists
US20020032313A1 (en) * 1991-03-29 2002-03-14 Genentech, Inc. Vascular endothelial cell growth factor antagonists
US5205290A (en) 1991-04-05 1993-04-27 Unger Evan C Low density microspheres and their use as contrast agents for computed tomography
US5808091A (en) 1991-10-29 1998-09-15 Bracco International B.V. Rhenium and technetium complexes containing a hypoxia localizing moiety
GB9200391D0 (en) 1992-01-09 1992-02-26 Nycomed As Improvements in or relating to contrast agents
US5861301A (en) * 1992-02-20 1999-01-19 American Cayanamid Company Recombinant kinase insert domain containing receptor and gene encoding same
ES2193143T3 (en) 1992-03-05 2003-11-01 Univ Texas USE OF IMMUNOCONJUGADOS FOR THE DIAGNOSIS AND / OR THERAPY OF VASCULARIZA TUMORS.
US5965132A (en) * 1992-03-05 1999-10-12 Board Of Regents, The University Of Texas System Methods and compositions for targeting the vasculature of solid tumors
US5580563A (en) 1992-05-01 1996-12-03 Tam; James P. Multiple antigen peptide system having adjuvant properties, vaccines prepared therefrom and methods of use thereof
US5338725A (en) 1992-06-30 1994-08-16 The Research Foundation Of The State University Of New York Anti-aggregatory agents for platelets
HU225646B1 (en) 1992-10-28 2007-05-29 Genentech Inc Hvegf receptors as vascular endothelial cell growth factor antagonists
US6177401B1 (en) * 1992-11-13 2001-01-23 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften Use of organic compounds for the inhibition of Flk-1 mediated vasculogenesis and angiogenesis
KR100218642B1 (en) 1993-07-02 1999-09-01 스티븐 로손 Method of making microspheres encapsulated from the temperature-denaturated protein
CA2167167A1 (en) 1993-07-19 1995-02-02 Alfred Pollak Hydrazino-type radionuclide chelators having an n3s configuration
US5574140A (en) 1993-09-03 1996-11-12 Resolution Pharmaceutical Inc. Hydrazino-type N2 S2 chelators
GB9402867D0 (en) 1994-02-15 1994-04-06 Nycomed Imaging As Improvements in or relating to contrast agents
CN1267550C (en) * 1994-03-08 2006-08-02 人体基因组科学有限公司 Vascular endothelial growth factor 2
US6040157A (en) * 1994-03-08 2000-03-21 Human Genome Sciences, Inc. Vascular endothelial growth factor 2
JP2891647B2 (en) 1994-03-11 1999-05-17 株式会社日本触媒 Organic sulfide compound and method for producing the same
US5582814A (en) 1994-04-15 1996-12-10 Metasyn, Inc. 1-(p-n-butylbenzyl) DTPA for magnetic resonance imaging
JPH09512265A (en) 1994-04-20 1997-12-09 ニコムド サルター アイエヌシー Contrast agent
ES2114280T3 (en) * 1994-04-25 1998-05-16 Oreal USE OF A NON-IONIC OR ANIONIC GUAR GUM IN A NON-PERMANENT CONFORMATION PROCEDURE OF KERATINIC FIBERS.
US5650135A (en) 1994-07-01 1997-07-22 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
GB9417941D0 (en) 1994-09-06 1994-10-26 Nycomed Imaging As Improvements in or relating to contrast agents
US6818220B1 (en) * 1994-11-14 2004-11-16 Licentia Ltd. Vascular endothelial growth factor C (VEGF-C) protein and gene mutants thereof, and uses thereof
US6130071A (en) * 1997-02-05 2000-10-10 Helsinki University Licensing, Ltd. Vascular endothelial growth factor C (VEGF-C) ΔCys156 protein and gene, and uses thereof
WO1998033917A1 (en) 1994-11-14 1998-08-06 The Ludwig Institute For Cancer Research Vascular endothelial growth factor c (vegf-c) protein and gene, mutants thereof, and uses thereof
US6645933B1 (en) * 1995-08-01 2003-11-11 Helsinki University Licensing Ltd. Oy Receptor ligand VEGF-C
US6403088B1 (en) * 1995-08-01 2002-06-11 Helsinki University Licensing, Ltd. Antibodies reactive with VEGF-C, a ligand for the Flt4 receptor tyrosine kinase (VEGFR-3)
US6221839B1 (en) * 1994-11-14 2001-04-24 Helsinki University Licensing Ltd. Oy FIt4 ligand and methods of use
US6245530B1 (en) * 1995-08-01 2001-06-12 Ludwig Institute For Cancer Research Receptor ligand
US6333021B1 (en) 1994-11-22 2001-12-25 Bracco Research S.A. Microcapsules, method of making and their use
DE4445065A1 (en) 1994-12-07 1996-06-13 Diagnostikforschung Inst Methods for in-vivo diagnostics using NIR radiation
IL116328A (en) 1994-12-16 1999-09-22 Bracco Research Sa Frozen suspension of gas microbubbles in frozen aqueous carrier for use as contrast agent in ultrasonic imaging
EP0800831B9 (en) 1995-01-30 2004-11-10 Daiichi Pure Chemicals Co., Ltd. Diagnostic marker
TW319763B (en) 1995-02-01 1997-11-11 Epix Medical Inc
GB9502065D0 (en) 1995-02-02 1995-03-22 Nycomed Imaging As Contrast media
US6521211B1 (en) * 1995-06-07 2003-02-18 Bristol-Myers Squibb Medical Imaging, Inc. Methods of imaging and treatment with targeted compositions
ATE265863T1 (en) 1995-06-07 2004-05-15 Imarx Pharmaceutical Corp NEW TARGETED AGENTS FOR DIAGNOSTIC AND THERAPEUTIC USE
US6033645A (en) * 1996-06-19 2000-03-07 Unger; Evan C. Methods for diagnostic imaging by regulating the administration rate of a contrast agent
US6231834B1 (en) * 1995-06-07 2001-05-15 Imarx Pharmaceutical Corp. Methods for ultrasound imaging involving the use of a contrast agent and multiple images and processing of same
US6361946B1 (en) * 1997-02-05 2002-03-26 Licentia Ltd Vascular endothelial growth factor C (VEGF-C) protein and gene, mutants thereof, and uses thereof
US7727761B2 (en) * 1995-08-01 2010-06-01 Vegenics Limited Vascular endothelial growth factor C (VEGF-C) protein and gene, mutants thereof, and uses thereof
US6020473A (en) * 1995-08-25 2000-02-01 Genentech, Inc. Nucleic acids encoding variants of vascular endothelial cell growth factor
JPH11514976A (en) * 1995-09-08 1999-12-21 ジェネンテク・インコーポレイテッド VEGF-related proteins
AU1116297A (en) 1995-11-08 1997-05-29 Immunex Corporation Flk-1 binding protein
SK284200B6 (en) 1996-02-19 2004-10-05 Amersham Health As Improvements in or relating to contrast agents
US5942385A (en) * 1996-03-21 1999-08-24 Sugen, Inc. Method for molecular diagnosis of tumor angiogenesis and metastasis
AU726914B2 (en) 1996-04-01 2000-11-23 Epix Pharmaceuticals, Inc. Bioactivated diagnostic imaging contrast agents
CA2262908A1 (en) 1996-08-02 1998-02-12 David Johnson Contrast agents for ultrasound imaging of the liver
RU2204415C2 (en) 1996-10-21 2003-05-20 Амершем Хелт АС Combined preparation for using as contrasting agent and method of image preparing
IL129445A0 (en) 1996-10-28 2000-02-29 Nycomed Imaging As Improvements in or relating to diagnostic/therapeutic agents
WO1998018498A2 (en) 1996-10-28 1998-05-07 Marsden, John, Christopher Improvements in or relating to diagnostic/therapeutic agents
US6261537B1 (en) * 1996-10-28 2001-07-17 Nycomed Imaging As Diagnostic/therapeutic agents having microbubbles coupled to one or more vectors
BR9712683A (en) * 1996-10-28 1999-10-19 Nyomed Imaging A S Diagnostic and / or therapeutically active targetable agent, combined formulation, process for preparing and using it, combined formulation, and processes for generating enhanced images of a human or non-human animal body and for in vitro targeting investigation by an agent.
WO1998018495A2 (en) 1996-10-28 1998-05-07 Marsden, John, Christopher Improvements in or relating to diagnostic/therapeutic agents
GB9708265D0 (en) * 1997-04-24 1997-06-18 Nycomed Imaging As Contrast agents
ES2264159T3 (en) 1996-10-28 2006-12-16 Amersham Health As IMPROVEMENTS IN / OR RELATED TO DIAGNOSTIC / THERAPEUTIC AGENTS.
EP0971747B1 (en) 1996-10-28 2005-12-28 Amersham Health AS Contrast agents
WO1998018497A2 (en) 1996-10-28 1998-05-07 Nycomed Imaging As Contrast agents
EP0946384A1 (en) 1996-12-24 1999-10-06 Itt Automotive Electrical Systems, Inc. Hydraulically powered fan and power steering in a vehicle
JP2001511152A (en) 1997-02-03 2001-08-07 マリンクロッド・インコーポレイテッド Detection and localization of malignant human tumors
US7125714B2 (en) * 1997-02-05 2006-10-24 Licentia Ltd. Progenitor cell materials and methods
US20020010137A1 (en) * 1997-09-18 2002-01-24 Genentech, Inc. Methods and compositions for inhibiting neoplastic cell growth
US5919967A (en) 1997-04-11 1999-07-06 Epix Medical, Inc. Process for synthesizing phosphodiesters
DE19717904A1 (en) 1997-04-23 1998-10-29 Diagnostikforschung Inst Acid-labile and enzymatically cleavable dye constructs for diagnostics with near infrared light and for therapy
US6723694B1 (en) 1997-05-21 2004-04-20 The Children's Medical Center Corp. Short peptides which selectively modulate intracellular signalling
US6245318B1 (en) * 1997-05-27 2001-06-12 Mallinckrodt Inc. Selectively binding ultrasound contrast agents
GB9712525D0 (en) 1997-06-16 1997-08-20 Nycomed Imaging As Method
US6204011B1 (en) * 1997-06-18 2001-03-20 Merck & Co., Inc. Human receptor tyrosine kinase, KDR
WO2000075167A2 (en) 1999-06-09 2000-12-14 Ljl Biosystems, Inc. Phosphorylation assays
AU742438C (en) 1997-10-02 2003-05-22 Lantheus Medical Imaging, Inc. Contrast-enhanced diagnostic imaging method for monitoring interventional therapies
US6777534B1 (en) * 1997-12-09 2004-08-17 Children's Medical Center Corporation Peptide antagonists of vascular endothelial growth factor
AU1810899A (en) 1997-12-09 1999-06-28 Children's Medical Center Corporation Neuropilins and use thereof in methods for diagnosis and prognosis of cancer
US6165458A (en) 1997-12-26 2000-12-26 Pharmaderm Laboratories Ltd. Composition and method for dermal and transdermal administration of a cytokine
US6469140B1 (en) 1998-02-06 2002-10-22 Millennium Pharmaceuticals, Inc. Molecules of the card-related protein family and uses thereof
WO1999040947A2 (en) 1998-02-11 1999-08-19 Resolution Pharmaceuticals Inc. Angiogenesis targeting molecules
JP4469935B2 (en) 1998-03-23 2010-06-02 ジェネンテック， インコーポレイテッド GFRα3 and uses thereof
US6537520B1 (en) * 1998-03-31 2003-03-25 Bristol-Myers Squibb Pharma Company Pharmaceuticals for the imaging of angiogenic disorders
US6548663B1 (en) * 1998-03-31 2003-04-15 Bristol-Myers Squibb Pharma Company Benzodiazepine vitronectin receptor antagonist pharmaceuticals
CN1295578A (en) 1998-03-31 2001-05-16 杜邦药品公司 Pharmaceuticals for imaging of angiogenic disorders
US6524553B2 (en) 1998-03-31 2003-02-25 Bristol-Myers Squibb Pharma Company Quinolone vitronectin receptor antagonist pharmaceuticals
US6548048B1 (en) * 1998-04-28 2003-04-15 Amersham Health As Lipopeptide stabilized microbubbles as diagnostic/therapeutic agents
GB9809084D0 (en) 1998-04-28 1998-06-24 Nycomed Imaging As Improvements in or relating to diagnostic/therapeutic agents
US6362371B1 (en) 1998-06-08 2002-03-26 Advanced Medicine, Inc. β2- adrenergic receptor agonists
US20030236190A1 (en) * 1998-09-02 2003-12-25 Renuka Pillutla Isulin and IGF-1 receptor agonists and antagonists
US7173005B2 (en) * 1998-09-02 2007-02-06 Antyra Inc. Insulin and IGF-1 receptor agonists and antagonists
US6875741B2 (en) 1998-09-02 2005-04-05 Renuka Pillutla Insulin and IGF-1 receptor agonists and antagonists
KR100731820B1 (en) 1998-10-05 2007-06-25 파멕사 에이/에스 Novel methods for therapeutic vaccination
JP2002529421A (en) 1998-11-06 2002-09-10 ビーエーエスエフ アクチェンゲゼルシャフト Methods for inhibiting vascular hyperpermeability
US6312665B1 (en) 1998-12-21 2001-11-06 Generex Pharmaceuticals Incorporated Aerosol formulations for buccal and pulmonary application
JP2002536968A (en) 1999-01-29 2002-11-05 イムクローン システムズ インコーポレイティド Antibodies specific for KDR and uses thereof
ATE420177T1 (en) 1999-04-16 2009-01-15 Genentech Inc VASCULAR ENDOTHELIAL CELL GROWTH FACTOR (VEGF) VARIANTS AND THEIR USES
DE19941092A1 (en) 1999-08-30 2001-03-01 Philips Corp Intellectual Pty Network with an identifier reduction
US6294525B1 (en) 1999-09-01 2001-09-25 Molecumetics Ltd. Reverse-turn mimetics and methods relating thereto
GB9928950D0 (en) 1999-12-07 2000-02-02 Metris Therapeutics Limited Binding protein
WO2001052875A1 (en) 2000-01-18 2001-07-26 Ludwig Institute For Cancer Research Vegf-d/vegf-c/vegf peptidomimetic inhibitor
US7740841B1 (en) 2000-01-28 2010-06-22 Sunnybrook Health Science Center Therapeutic method for reducing angiogenesis
EP1259626B1 (en) 2000-02-25 2007-10-31 Ludwig Institute For Cancer Research Materials and methods involving hybrid vascular endothelial growth factor dnas and proteins and screening methods for modulators
US20020102260A1 (en) * 2000-03-02 2002-08-01 Marc Achen Methods for treating neoplastic disease characterized by vascular endothelial growth factor D expression, for screening for neoplastic disease or metastatic risk, and for maintaining vascularization of tissue
AU4194601A (en) * 2000-03-02 2001-09-12 Ludwig Institute For Cancer Research Methods for treating, screening for, and detecting cancers expressing vascular endothelial growth factor
DK1259248T3 (en) 2000-03-02 2005-04-11 Ludwig Inst Cancer Res Method of treating cancers expressing vascular endothelial growth factor D
US20010031485A1 (en) 2000-03-22 2001-10-18 Sibtech, Inc. Recombinant proteins containing Shiga-like toxin and vascular endothelial growth factor fragments
DE10013993A1 (en) 2000-03-22 2001-10-25 Ruediger Benters Article with activated surface for binding macromolecules, useful for making e.g. sensors or arrays, comprises dendrimer framework, containing reactive groups, on a substrate
AU2001249393A1 (en) 2000-03-22 2001-10-03 Glaxo Group Limited Pharmaceutical comprising an agent that blocks the cell cycle and an antibody
AU2001242190A1 (en) * 2000-03-31 2001-10-15 Aventis Pasteur Limited Immunogenic peptides derived from prostate-specific membrane antigen (psma) and uses thereof
WO2001072829A2 (en) 2000-03-31 2001-10-04 Institut Pasteur Peptides blocking vascular endothelial growth factor (vegf)-mediated angiogenesis, polynucleotides encoding said peptides and methods of use thereof
JP4966470B2 (en) 2000-04-14 2012-07-04 ダニスコ・ユーエス・インコーポレーテッド Selective targeting method
WO2001083693A2 (en) 2000-04-28 2001-11-08 Glaxo Group Limited Compounds having affinity for the vascular endothelial growth factor receptor-2 (vegfr-2) and associated uses
EP1278771A4 (en) 2000-05-03 2004-06-16 Ludwig Inst Cancer Res A method for activating only the vascular endothelial growth factor receptor-3 and uses thereof
CA2410887C (en) 2000-06-02 2012-07-24 Bracco Research Usa Compounds for targeting endothelial cells, compositions containing the same and methods for their use
CN1254234C (en) 2000-06-09 2006-05-03 莱古伦公司 Plasmid DNA (Lipogenes TM) and nucleas-containing location signal/fusogene conjogates drug encapsulating into targeted liposomes complex
WO2001097860A2 (en) 2000-06-21 2001-12-27 Bristol-Myers Squibb Pharma Company Pharmaceuticals for the imaging of angiogenic disorders for use in combination therapy
EP1166798A1 (en) 2000-06-23 2002-01-02 Schering Aktiengesellschaft Combinations and compositions which interfere with VEGF/VEGF and angiopoietin/ Tie receptor function and their use
JP2003535910A (en) * 2000-06-23 2003-12-02 シエーリング アクチエンゲゼルシャフト Combinations and compositions that interfere with VEGF / VEGF and angiopoietin / Tie receptor function and their use (II)
EP1166799A1 (en) 2000-06-28 2002-01-02 Schering Aktiengesellschaft Combinations and compositions which interfere with VEGF/VEGF and angiopoietin/Tie receptor function and their use (II)
AU2001280552A1 (en) 2000-07-13 2002-01-30 The Ohio State University Research Foundation Multimeric biopolymers as structural elements, sensors and actuators in microsystems
US7175844B2 (en) * 2000-07-18 2007-02-13 Joslin Diabetes Center, Inc. Methods of modulating fibrosis
US6895563B2 (en) * 2000-08-15 2005-05-17 Paul K. Dixon Universal laboratory prototyping interface system
CA2418006A1 (en) 2000-08-18 2002-02-28 Dyax Corp. Binding polypeptides for b lymphocyte stimulator protein (blys)
US20020091082A1 (en) * 2000-09-13 2002-07-11 Aiello Lloyd P. Methods of modulating symptoms of hypertension
US20030082103A1 (en) * 2000-10-11 2003-05-01 Targesome, Inc. Targeted therapeutic lipid constructs having cell surface targets
US7611711B2 (en) 2001-01-17 2009-11-03 Vegenics Limited VEGFR-3 inhibitor materials and methods
CN1494552A (en) 2001-01-19 2004-05-05 ·��ά��֢�о�Ժ Flt 4 (VEGFR-3) as target for tumor imaging and anti-tumor therapy
AU2002245629A1 (en) * 2001-03-08 2002-09-24 Targesome, Inc. Stabilized therapeutic and imaging agents
EP1385862A4 (en) 2001-04-13 2005-03-02 Human Genome Sciences Inc Vascular endothelial growth factor 2
WO2002092573A2 (en) 2001-05-16 2002-11-21 Vertex Pharmaceuticals Incorporated Heterocyclic substituted pyrazoles as inhibitors of src and other protein kinases
EP1401818A2 (en) 2001-05-30 2004-03-31 Targesome, Inc. Targeted multivalent macromolecules
WO2003000842A2 (en) 2001-06-04 2003-01-03 Curagen Corporation Novel proteins and nucleic acids encoding same
JP2005505260A (en) 2001-06-22 2005-02-24 インサイト・ゲノミックス・インコーポレイテッド Protein modification and maintenance molecules
WO2003018797A2 (en) 2001-08-22 2003-03-06 Helix Biopharma Corporation Method and device for integrated protein expression, purification and detection
WO2003028643A2 (en) 2001-10-01 2003-04-10 Targesome, Inc. Targeted therapeutic lipid constructs having cell surface targets
EP1456240A4 (en) 2001-10-24 2006-08-09 Antyra Inc Target specific screening and its use for identifying target binders
AU2003228276B8 (en) 2002-03-01 2010-02-18 Bracco Suisse S.A. Multivalent constructs for therapeutic and diagnostic applications
US8623822B2 (en) 2002-03-01 2014-01-07 Bracco Suisse Sa KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy
US20050250700A1 (en) 2002-03-01 2005-11-10 Sato Aaron K KDR and VEGF/KDR binding peptides
ES2398393T3 (en) 2002-03-01 2013-03-15 Dyax Corp. KDR and VEGF / KDR binding peptides and their use in diagnosis and therapy
SE0200968D0 (en) 2002-03-26 2002-03-26 Lars Baltzer Novel polypeptide scaffolds and use thereof
US20040033949A1 (en) * 2002-05-06 2004-02-19 Genentech, Inc. Use of VEGF for treating bone defects
EP2430923A1 (en) 2002-06-05 2012-03-21 Genentech, Inc. Compositions and methods for liver growth and liver protection
EP1576172A2 (en) 2002-06-21 2005-09-21 Dyax Corporation Serum protein-associated target-specific ligands and identification method therefor
US20040212193A1 (en) 2002-10-08 2004-10-28 Johnstone Ian David Connector
NO20026285D0 (en) 2002-12-30 2002-12-30 Amersham Health As New peptides
US7033822B2 (en) * 2003-01-29 2006-04-25 New Energy Solutions, Inc. Self-contained and streamlined methane and/or high purity hydrogen generation system
DE602004029010D1 (en) 2003-02-04 2010-10-21 Bracco Suisse Sa ULTRASONIC CONTRASTING AGENT AND METHOD OF CREATION
WO2004085617A2 (en) 2003-03-21 2004-10-07 The Cleveland Clinic Foundation Timp3 as vegf inhibitor
US20040248805A1 (en) 2003-06-05 2004-12-09 Michael Rosenblum Vascular endothelial growth factor fusion constructs and uses thereof
US7601341B2 (en) * 2003-06-05 2009-10-13 Research Development Foundation Vascular endothelial growth factor fusion constructs used to inhibit osteoclastogenesis
WO2005016963A2 (en) 2003-06-12 2005-02-24 Ludwig Institute For Cancer Research Heparin binding veger-3 ligands
WO2005011722A2 (en) 2003-06-12 2005-02-10 Ludwig Institute For Cancer Research Use of vegf-c or vegf-d in reconstructive surgery
JP2005110508A (en) 2003-10-02 2005-04-28 Nec Soft Ltd Growth factor-like protein derived from snake venom having specific binding property to vascular endothelial growth factor receptor 2
FI20031528A0 (en) 2003-10-17 2003-10-17 Ctt Cancer Targeting Tech Oy A therapeutic liposome composition and a process for its preparation
DE602005021057D1 (en) 2004-01-20 2010-06-17 Toronto E HIGH FREQUENCY ULTRASONIC PRESENTATION WITH CONTRAST
US8067368B2 (en) 2004-01-27 2011-11-29 The Ohio State University Research Foundation Vascular endothelial growth factors and methods of their use
JP4175298B2 (en) * 2004-07-07 2008-11-05 セイコーエプソン株式会社 Color filter, method for manufacturing the same, electro-optical device, and electronic apparatus
WO2006015385A2 (en) 2004-08-06 2006-02-09 Sopherion Therapeutics, Inc. Anti-angiogenic peptides and methods of use thereof
BRPI0619522B8 (en) 2005-12-09 2021-07-27 Bracco Suisse Sa vector-phospholipid objectification conjugates, composition comprising said conjugates, ultrasound contrast agent composition and method for preparing gas-filled microvesicle comprising a phospholipid and manufacturing peptide-phospholipid conjugate having low levels of tfa
DE102008037723A1 (en) 2008-08-14 2010-02-25 Henkel Ag & Co. Kgaa Toilet basket with Spülwasserverteilelement

Patent Citations (100)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4138344A (en) * 1975-01-28 1979-02-06 Alza Corporation Erodible agent releasing device comprising poly(orthoesters) and poly(orthocarbonates)
US4093709A (en) * 1975-01-28 1978-06-06 Alza Corporation Drug delivery devices manufactured from poly(orthoesters) and poly(orthocarbonates)
US4391797A (en) * 1977-01-05 1983-07-05 The Children's Hospital Medical Center Systems for the controlled release of macromolecules
US4544500A (en) * 1982-04-14 1985-10-01 Scripps Clinic And Research Foundation Synthetic foot and mouth disease antigen
US4718433A (en) * 1983-01-27 1988-01-12 Feinstein Steven B Contrast agents for ultrasonic imaging
US4900540A (en) * 1983-06-20 1990-02-13 Trustees Of The University Of Massachusetts Lipisomes containing gas for ultrasound detection
US4899755A (en) * 1985-05-08 1990-02-13 The General Hospital Corporation Hepatobiliary NMR contrast agents
US4774958A (en) * 1985-12-05 1988-10-04 Feinstein Steven B Ultrasonic imaging agent and method of preparation
US5021556A (en) * 1987-07-22 1991-06-04 Neorx Corporation Method of radiolabeling chelating compounds comprising sulfur atoms with metal radionuclides
US4844882A (en) * 1987-12-29 1989-07-04 Molecular Biosystems, Inc. Concentrated stabilized microbubble-type ultrasonic imaging agent
US5180816A (en) * 1988-08-24 1993-01-19 Centocor One vial method for labeling protein/linker conjugates with technetium-99M
US5223409A (en) * 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5759515A (en) * 1989-08-09 1998-06-02 Rhomed Incorporated Polyvalent peptide pharmaceutical applications
US5118797A (en) * 1989-08-28 1992-06-02 E. R. Squibb & Sons, Inc. Rhenium tris dioxime complexes
US5123414A (en) * 1989-12-22 1992-06-23 Unger Evan C Liposomes as contrast agents for ultrasonic imaging and methods for preparing the same
US5230882A (en) * 1989-12-22 1993-07-27 Unger Evan C Liposomes as contrast agents for ultrasonic imaging and methods for preparing the same
US5871959A (en) * 1989-12-27 1999-02-16 The United States Of America As Represented By The Department Of Health And Human Services Method of producing hepatocycte growth factor/scatter factor and related cell lines
US5387409A (en) * 1990-01-18 1995-02-07 Bracco International B.V. Boronic acid adducts of rhenium dioxime and technetium-99m dioxime complexes containing a biochemically active group
US5531980A (en) * 1990-04-02 1996-07-02 Bracco International Bv Stable microbubbles suspensions injectable into living organisms
US6110443A (en) * 1990-04-02 2000-08-29 Bracco International N.V. Dry stable formation to produce microbubble suspension for ultrasound
US5643553A (en) * 1990-04-02 1997-07-01 Bracco International B.V. Stable microbubbles suspensions injectable into living organisms
US5567414A (en) * 1990-04-02 1996-10-22 Bracco International B.V. Stable microbubbles suspensions injectable into living organisms
US5911972A (en) * 1990-04-02 1999-06-15 Bracco International B.V. Stable microbubbles suspensions injectable into living organisms
US5658551A (en) * 1990-04-02 1997-08-19 Bracco International B.V. Stable microbubbles suspensions injectable into living organisms
US5380519A (en) * 1990-04-02 1995-01-10 Bracco International B.V. Stable microbubbles suspensions injectable into living organisms
US5183653A (en) * 1990-04-13 1993-02-02 E. R. Squibb & Sons, Inc. Boronic acid adducts of metal dioxime complexes useful in labelling proteins and other amine-containing compounds
US5137928A (en) * 1990-04-26 1992-08-11 Hoechst Aktiengesellschaft Ultrasonic contrast agents, processes for their preparation and the use thereof as diagnostic and therapeutic agents
US5863520A (en) * 1990-05-18 1999-01-26 Bracco International B.V. Method of echographic imaging using polymeric gas or air filled microballoons
US5711933A (en) * 1990-05-18 1998-01-27 Bracco International B.V. Method of making polymeric gas or air filled microballoons for ultrasonic echography
US6200548B1 (en) * 1990-05-18 2001-03-13 Bracco International B.V. Gas or air filled polymeric microballoons
US6123922A (en) * 1990-05-18 2000-09-26 Bracco International B.V. Resilient deformable microballoons for echographic imaging
US5879658A (en) * 1991-02-08 1999-03-09 Diatide, Inc. Technetium-99m labeled peptides for thrombus imaging
US5536490A (en) * 1991-03-28 1996-07-16 Nycomed Imaging As Contrast agents
US5529766A (en) * 1991-03-28 1996-06-25 Nycomed Imaging As Contrast agents
US5529766C1 (en) * 1991-03-28 2002-06-04 Nycomed Imaging As Contrast agents
US5558857A (en) * 1991-06-03 1996-09-24 Nycomed Imaging As Contrast agents
US5607661A (en) * 1991-07-05 1997-03-04 Nycomed Imaging As Aggregates of x-ray microparticles for ultrasound imaging
US5558856A (en) * 1992-01-09 1996-09-24 Nycomed Imaging As Microbubble-generating contrast agents for ultrasound and magnetic resonance imaging
US5637289A (en) * 1992-01-09 1997-06-10 Nycomed Imaging As Contrast agents, consisting of galactose particles
US5413774A (en) * 1992-01-23 1995-05-09 Bracco International B.V. Long-lasting aqueous dispersions or suspensions of pressure-resistant gas-filled microvesicles and methods for the preparation thereof
US5556610A (en) * 1992-01-24 1996-09-17 Bracco Research S.A. Gas mixtures useful as ultrasound contrast media, contrast agents containing the media and method
US6187288B1 (en) * 1992-02-11 2001-02-13 Bracco International B.V. Stable microbubble suspensions as enhancement agents for ultrasonic echography
US5670133A (en) * 1992-02-20 1997-09-23 Rhomed Incorporated Peptides method for radiolabeling them, and method for detecting inflammation
US5877289A (en) * 1992-03-05 1999-03-02 The Scripps Research Institute Tissue factor compositions and ligands for the specific coagulation of vasculature
US5720934A (en) * 1992-04-30 1998-02-24 Diatide, Inc. Technetium-99M labeled peptides for imaging
US5316921A (en) * 1992-05-18 1994-05-31 Genentech, Inc. Single-chain hepatocyte growth factor variants
US5547856A (en) * 1992-05-18 1996-08-20 Genentech, Inc. Hepatocyte growth factor variants
US5876973A (en) * 1992-08-10 1999-03-02 Cambridge Neuroscience, Inc. Method for making antibodies which specifically bind to glial growth factors
US20040037820A1 (en) * 1992-10-09 2004-02-26 Kari Alitalo Flt4 (VEGFR-3) as a target for tumor imaging and anti-tumor therapy
US5445813A (en) * 1992-11-02 1995-08-29 Bracco International B.V. Stable microbubble suspensions as enhancement agents for ultrasound echography
US5597549A (en) * 1992-11-02 1997-01-28 Bracco International B.V. Stable microbubble suspensions as enhancement agents for ultrasound echography
US5908610A (en) * 1992-11-02 1999-06-01 Bracco International B.V. Stable microbubble suspensions comprising saturated phospholipios for ultrasonic echography
US6183725B1 (en) * 1992-12-16 2001-02-06 Bracco Research S.A. Gas mixtures useful as ultrasound contrast media, contrast agents containing the media and method
US5608110A (en) * 1993-06-15 1997-03-04 Bracco International B.V. Heteroatom-bearing ligands and metal complexes thereof
US5656254A (en) * 1993-06-15 1997-08-12 Bracco International B.V. Polyaza heteroatom-bearing ligands and metal complexes thereof for imaging or radiotherapy
US5627286A (en) * 1993-06-15 1997-05-06 Bracco International B.V. Heteroatom-bearing ligands and metal complexes thereof
US5665329A (en) * 1993-06-15 1997-09-09 Bracco International B.V. Heteroatom-bearing ligands and metal complexes thereof
US5798091A (en) * 1993-07-30 1998-08-25 Alliance Pharmaceutical Corp. Stabilized gas emulsion containing phospholipid for ultrasound contrast enhancement
US5723584A (en) * 1993-07-30 1998-03-03 Affymax Technologies N.V. Biotinylation of proteins
US5770421A (en) * 1993-12-03 1998-06-23 St. Jude Children's Research Hospital Human ALK protein tyrosine kinase
US5773412A (en) * 1994-04-13 1998-06-30 La Jolla Cancer Research Center Use of peptides for altering αV β3 -mediated binding
US5759996A (en) * 1994-04-13 1998-06-02 La Jolla Cancer Research Center Peptides useful for altering αv β3 -mediated binding
US5707624A (en) * 1994-06-03 1998-01-13 The Regents Of The University Of Michigan Treatment of Kaposi's sarcoma by inhibition of scatter factor
US5780006A (en) * 1994-07-22 1998-07-14 Resolution Pharmaceuticals Inc. Peptide derived radionuclide chelators
US5662885A (en) * 1994-07-22 1997-09-02 Resolution Pharmaceuticals Inc. Peptide derived radionuclide chelators
US5556939A (en) * 1994-10-13 1996-09-17 Merck Frosst Canada, Inc. TC or RE radionuclide labelled chelate, hexapeptide complexes useful for diagnostic or therapeutic applications
US6110433A (en) * 1994-12-20 2000-08-29 Varta Batterie Aktiengesellschaft Process for the recovery of metals from used nickel/metal/rare earth hydride storage batteries
US6207152B1 (en) * 1995-06-02 2001-03-27 Genentech, Inc. Hepatocyte growth factor receptor antagonists and uses thereof
US6025331A (en) * 1996-02-16 2000-02-15 Children's Medical Center Corporation Pharmaceutical compositions comprising troponin subunits, fragments and analogs thereof and methods of their use to inhibit angiogenesis
US5885866A (en) * 1996-10-03 1999-03-23 Mosel Vitelic Inc. Self-registered cylindrical capacitor of high density DRAMs
US6171614B1 (en) * 1996-10-15 2001-01-09 Emory University Synthesis of glycophospholipid and peptide-phospholipid conjugates and uses thereof
US6680047B2 (en) * 1996-10-28 2004-01-20 Amersham Health As Diagnostic/therapeutic agents
US20020102217A1 (en) * 1996-10-28 2002-08-01 Nycomed Imaging As Diagnostic/therapeutic agents
US5886142A (en) * 1997-05-20 1999-03-23 Thomas Jefferson University Radiolabeled thrombus imaging agents
US6274166B1 (en) * 1997-05-29 2001-08-14 Ben Gurion University Of The Negev Research And Development Authority Transdermal delivery system
US6258378B1 (en) * 1998-02-09 2001-07-10 Bracco Research S.A. Delivery of biologically active substance to target sites in the body of patients
US20020136721A1 (en) * 1998-02-17 2002-09-26 Schwall Ralph H. Hepatocyte growth factor receptor antagonists and uses thereof
US6524533B1 (en) * 1998-03-06 2003-02-25 Biosafe Medical Technologies, Inc. Device for collecting and drying a body fluid
US20060063699A1 (en) * 1998-03-09 2006-03-23 Larsen Bjarne D Pharmacologically active peptide conjugates having a reduced tendency towards enzymatic hydrolysis
US6093382A (en) * 1998-05-16 2000-07-25 Bracco Research Usa Inc. Metal complexes derivatized with folate for use in diagnostic and therapeutic applications
US20020119149A1 (en) * 1998-05-19 2002-08-29 Jakobsen Bent Karsten Multivalent T cell receptor complexes
US6733755B2 (en) * 2000-02-04 2004-05-11 Supratek Pharma, Inc. Ligand for vascular endothelial growth factor receptor
US6710165B2 (en) * 2000-06-01 2004-03-23 Kyunglim Lee IgE-dependent histamine-releasing factor-binding peptides
US7199100B2 (en) * 2000-10-04 2007-04-03 Commissariat A L'energie Atomique Cyclic peptides, method for preparing and use as angiogenesis inhibitors or activator
US20030129223A1 (en) * 2000-10-11 2003-07-10 Targesome, Inc. Targeted multivalent macromolecules
US6984373B2 (en) * 2000-12-23 2006-01-10 Dyax Corp. Fibrin binding moieties useful as imaging agents
US20030125493A1 (en) * 2001-10-26 2003-07-03 Harris Stephen H. (Meth)acrylates of oxyalkylated phenolic resins and their use as adhesion promoters
US7666979B2 (en) * 2002-03-01 2010-02-23 Bracco International B.V. Methods for preparing multivalent constructs for therapeutic and diagnostic applications and methods of preparing the same
US20050147555A1 (en) * 2002-03-01 2005-07-07 Hong Fan Methods for preparing multivalent constructs for therapeutic and diagnostic applications and methods of preparing the same
US20040018974A1 (en) * 2002-03-01 2004-01-29 Christophe Arbogast Multivalent constructs for therapeutic and diagnostic applications
US20050100963A1 (en) * 2002-03-01 2005-05-12 Dyax Corporation KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy
US7211240B2 (en) * 2002-03-01 2007-05-01 Bracco International B.V. Multivalent constructs for therapeutic and diagnostic applications
US20070172428A1 (en) * 2002-03-01 2007-07-26 Bracco International B.V. Multivalent constructs for therapeutic and diagnostic applications
US7261876B2 (en) * 2002-03-01 2007-08-28 Bracco International Bv Multivalent constructs for therapeutic and diagnostic applications
US20080152594A1 (en) * 2002-03-01 2008-06-26 Philippe Bussat Targeting vector-phospholipid conjugates
US7794693B2 (en) * 2002-03-01 2010-09-14 Bracco International B.V. Targeting vector-phospholipid conjugates
US7910088B2 (en) * 2002-03-01 2011-03-22 Bracco Suisse Sa Multivalent constructs for therapeutic and diagnostic applications
US7985402B2 (en) * 2002-03-01 2011-07-26 Bracco Suisse Sa Targeting vector-phospholipid conjugates
US20060089307A1 (en) * 2002-12-30 2006-04-27 Kulseth Mari A Peptides that bind to the heparin binding domian of vegf and vegfr-2
US20050214859A1 (en) * 2003-03-03 2005-09-29 Dyax Corp. Peptides that specifically bind HGF receptor (cMet) and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Kirpotin et al., "Sterically stabilized Anti-HER2 immunoliposomes: design and targeting to human breast cancer cells in vitro", Biochemistry 36: 66-75 (1997). *
Rodionov, ABRF Electronic Discussion Group (July 28, 2000); Downloaded July 1, 2012. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9056138B2 (en) 2002-03-01 2015-06-16 Bracco Suisse Sa Multivalent constructs for therapeutic and diagnostic applications
US9295737B2 (en) 2002-03-01 2016-03-29 Bracco Suisse Sa Targeting vector-phospholipid conjugates
US9381258B2 (en) 2002-03-01 2016-07-05 Bracco Suisse S.A. Targeting vector-phospholipid conjugates
US9408926B2 (en) 2002-03-01 2016-08-09 Bracco Suisse S.A. KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy
US9446155B2 (en) 2002-03-01 2016-09-20 Bracco Suisse Sa KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy
USRE46830E1 (en) 2004-10-19 2018-05-08 Polypeptide Laboratories Holding (Ppl) Ab Method for solid phase peptide synthesis
US20110160431A1 (en) * 2009-04-06 2011-06-30 Novetide, Ltd. Production of peptides containing poly-gly sequences using fmoc chemistry

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