WO2013106824A1 - Agents ciblant les récepteurs des éphrines - Google Patents

Agents ciblant les récepteurs des éphrines Download PDF

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WO2013106824A1
WO2013106824A1 PCT/US2013/021434 US2013021434W WO2013106824A1 WO 2013106824 A1 WO2013106824 A1 WO 2013106824A1 US 2013021434 W US2013021434 W US 2013021434W WO 2013106824 A1 WO2013106824 A1 WO 2013106824A1
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imaging
agent
peptide
ephb4
hauns
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Chiyi Xiong
Qizhen Cao
Anil Sood
Chun Li
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • 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
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1241—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins
    • A61K51/1244—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins microparticles or nanoparticles, e.g. polymeric nanoparticles
    • A61K51/1251—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins microparticles or nanoparticles, e.g. polymeric nanoparticles micro- or nanospheres, micro- or nanobeads, micro- or nanocapsules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
    • G01R33/48—NMR imaging systems
    • G01R33/54—Signal processing systems, e.g. using pulse sequences ; Generation or control of pulse sequences; Operator console
    • G01R33/56—Image enhancement or correction, e.g. subtraction or averaging techniques, e.g. improvement of signal-to-noise ratio and resolution
    • G01R33/5601—Image enhancement or correction, e.g. subtraction or averaging techniques, e.g. improvement of signal-to-noise ratio and resolution involving use of a contrast agent for contrast manipulation, e.g. a paramagnetic, super-paramagnetic, ferromagnetic or hyperpolarised contrast agent

Definitions

  • the present invention relates generally to the field of molecular imaging and nanomedicine. More particularly, it concerns cancer targeting agents and methods for targeted imaging and therapy of cancer cells.
  • radiolabeled peptides have been developed as nuclear imaging agents for tumor detection and noninvasive assessment of receptor expression in solid tumors.
  • small radiolabeled somatostatin peptidyl analogs with a molecular weight of -1.5 kDa have been successfully utilized in the clinic for localizing neuroendocrine tumors expressing somatostatin receptors (Bakker et ah, 1991 ; Hammond et ah, 1993).
  • Cyclic Arg-Gly-Asp (RGD) peptide that strongly binds to integrin ⁇ 3 receptors is currently under clinical investigation (Haubner et ah, 2009).
  • peptide-based imaging agents including melanocyte-stimulating hormone (MSH) analog (Chen et ah, 2000), substance P (Hargreaves, 2002), calcitonin (Blower et ah, 1998), atrial natriuretic peptide (ANP) (Liu et ah, 2010), bombesin/gastrin-releasing peptide (GRP), cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1), and neuropeptide-Y (NPY), have also been identified and characterized for tumor receptor imaging (Schottelius and Wester, 2009).
  • MSH melanocyte-stimulating hormone
  • peptides that can be used for the noninvasive detection of prostate, ovarian, melanoma, and colon cancer remain elusive (Schottelius and Wester, 2009).
  • Many sold tumors, including prostate, ovarian, melanoma, and colon cancer overexpress EphB4 receptor, a member of the Ephrin receptor tyrosine kinase family.
  • Noninvasive imaging of EphB4 using peptide-based imaging agents could potentially increase early detection rates, monitor response to therapy directed again EphB4, and improve patient outcomes.
  • specific targeting of therapeutic agents to EphB4 expressing cancers could greatly improve the efficacy and specificity of cancer therapy.
  • an Ephrin receptor targeting agent comprising at least a first peptide comprising the amino acid sequence TNYLFSPNGPIA (SEQ ID NO: 2) conjugated to a therapeutic agent or an imaging agent.
  • the first peptide is defined as a peptide that binds specifically or preferentially to Eph receptor EphB4.
  • the peptide can comprise a sequence TNYLFSPNGPIARAW (SEQ ID NO: 1) or a sequence identical to SEQ ID NO: l, but comprising 1 or 2 amino acid substitutions, insertions or deletions.
  • the first peptide is a cyclic peptide, such as a peptide comprising a lactam bridge (e.g., a cyclic peptide comprising the structure shown in FIG. 25).
  • a targeting agent of the embodiments further comprises a second peptide targeting agent.
  • the second peptide can bind to the same target as the first peptide or to a second target.
  • the second peptide binds to an EphA2 receptor.
  • a targeting agent is provided that binds to EphA2 and EphB4 receptors.
  • the second peptide comprises an amino acid sequence of SEQ ID NO: 4.
  • a first and second peptide of the embodiments are comprised in a fusion protein.
  • the first and second peptides are connected by a linker. Examples of linkers include, without limitation, linker peptides, carbohydrate polymers and fatty acids. In some cases, the linker is a polyethylene glycol (PEG) moiety.
  • the first peptide of a targeting agent is conjugated to a radioisotope, a nanoparticle, a toxin, a chemotherapeutic agent, a fluorescent dye or a combination thereof.
  • the conjugate may be through covalent linkage or a non-covalent association, such as via chelator moiety (e.g., 1,4,7, 10-tetraazadodecane-N, N',N",N"'-tetraacetic acid (DOTA)).
  • the first peptide is conjugated to an imaging agent, such as a SPECT imaging agent, PET imaging agent an MRI contrast agent, or a fluorescent dye.
  • the first peptide is conjugated to a radioisotope, such as a gamma emitter, a positron emitter or a beta-emitter.
  • a radioisotope such as a gamma emitter, a positron emitter or a beta-emitter.
  • the radioisotope is conjugated to the first peptide through a chelating moiety (e.g., DOTA).
  • the radioisotope is conjugated to the first peptide as part of a molecule that comprises the radioisotope.
  • radioisotopes for use in accordance with the invention include, without limitation, astatine-21 1, chromium-51, cobalt-57, cobalt-58, copper-60, copper-61, copper-62, copper-64, copper-66, copper-67, Eu-152, gallium-67, gallium-68, indium-I l l, iron-59, lutetium-177m, rhenium-186, rhenium-188, selenium-75, strontium-89, technicium- 99m, thorium-227, and/or yttrium-90.
  • a first peptide of the embodiments is conjugated to a therapeutic agent, such as chemotherapeutic agent, a radioisotope, or a therapeutic nanoparticle.
  • a therapeutic agent such as chemotherapeutic agent, a radioisotope, or a therapeutic nanoparticle.
  • the chemotherapeutic agent is an anthracycline antibiotic, such as doxorubicin (Dox).
  • therapeutic nanoparticles include, without limitation, nanoparticles that may be used to apply a photothermal ablation therapy, such as a hollow gold nanosphere (HAuNS).
  • a nanoparticle of the embodiments is further loaded with or conjugated to a chemotherapeutic agent, such as Dox.
  • a method of imaging a subject comprising: (a) administering an effective amount of a targeting agent of the embodiments to the subject (e.g., a EphB4 and/or EphA2 targeting agent); and (b) imaging the subject to detect the presence of the targeting agent.
  • a targeting agent of the embodiments e.g., a EphB4 and/or EphA2 targeting agent
  • imaging the subject can comprise positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), or photoacoustic imaging.
  • SPECT single photon emission computed tomography
  • MRI magnetic resonance imaging
  • a method of treating a subject comprising administering an effective amount of a targeting agent of the embodiments to the subject.
  • a method of the embodiment can further comprise administering one or more secondary therapy to the patient.
  • the secondary therapy can be a surgical therapy, chemotherapy, radiation therapy or hormonal therapy.
  • the secondary therapy can facilitate the activity the targeting agent.
  • a targeting agent that comprises a nanoparticle such as a HAuNS
  • a photothermal ablation therapy can be applied to heat the tissues surrounding the targeting agent.
  • a nanoparticle can be loaded with a chemotherapy agent (e.g., Dox) such that heating of the nanoparticle activates local release of the chemotherapy agent.
  • a chemotherapy agent e.g., Dox
  • a method for dual imaging and therapy of a subject comprising administering an effective amount of a targeting agent of the embodiments to a subject (i.e., wherein the targeting agent comprises a therapeutic and imaging agent); and imaging the subject.
  • a subject refers to an animal, such a human, canine or feline subject.
  • the subject has or is suspected of a cancer, such as an EphB4- or EphA2-overexpressing cancer.
  • an imaging probe for noninvasive imaging of EphB4 receptors comprising a heterodimer peptide, wherein said peptide has motifs which target both EphA2 and EphB4.
  • the heterodimer bispecific polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 or 2 and 4 (e.g., the amino acid sequence of SEQ ID NO: 1 and 4).
  • the heterodimer, bispecific polypeptide comprises an EphA2-targeting motif and an EphB4- targeting motif separated by a linker, such as PEG linker.
  • the heterodimer polypeptide comprises from amino- to carboxy-terminus the amino acid sequence of SEQ ID NO: 1 ; a PEG linker; and the amino acid sequence of SEQ ID NO: 4.
  • a method of monitoring a cancer patient comprising the steps of (i) injecting the patient with a heterodimer peptide of the embodiments; (ii) imaging the patient using a PET/CT scan imaging device wherein the device produces a PET image; (iii) acquiring the PET image of a tumor in the patient; (iv) determining tumor-to-muscle ratio in the patient; and (v) administering a cancer treatment to the patient based on the tumor-to-muscle ratio.
  • a method is provided of identifying EphB4 receptor expression in a subject in need thereof comprising administering to the subject a therapeutic amount of the heterodimer peptide of the embodiments, and determining uptake or binding of the peptide in said subject.
  • embodiments of the present invention contemplate the use of targeting nanoparticles in combination with physiological and physical approaches, such as tumor priming, vascular disruption, degradation of the extracellular matrix, and vessel normalization.
  • a method of reducing renal uptake of a radiolabeled agent in a subject comprising administering the radio-labeled agent in conjunction with metformin.
  • the metformin can be administered before, after or essentially simultaneously with the radio-labeled agent.
  • the radio-labeled agent is a radio-labeled therapeutic or imaging agent (e.g., an Eph Receptor targeting agent of the embodiments).
  • a pharmaceutical composition comprising a radio-labeled therapeutic or imaging agent and metformin formulated together in a pharmaceutically acceptable carrier.
  • Such a composition may be used for instance to reduce renal uptake of a radio-labeled agent in subject (e.g., a subject being treated or imaged with the agent).
  • FIG. 1 Structure of 64 Cu-DOTA-TNYL-RAW and nat Cu-DOTA -TNYL-RAW peptides.
  • FIG. 2 SPR sensorgrams of nat Cu-DOTA-TNYL-RAW and a scrambled peptide on sensor chips coated with EphB4.
  • the peptides were injected as ten two-fold concentration series from 1.6 nM to 800 nM and were analyzed in duplicate binding cycles. Data sets (shown in black) are overlaid with curves fit to a 1 : 1 mass transfer interaction model (gray lines).
  • the vertical axes in response units represent binding of each peptide to immobilized EphB4.
  • FIG. 3 Uptake of ⁇ Cu-DOTA -TNYL-RAW in EphB4-expressing PC-3M and CT26 cells and EphB4-negative A549 cells. The cell-to-medium uptake ratio is expressed as [cprn ⁇ g protein in pellet]/[cpm ⁇ g medium]. ⁇ Cu-DOTA-TNYL-RAW exhibited increased uptake over time in PC-3M and CT26 cells. This uptake was blocked by the parent TNYL-RAW peptide.
  • FIG. 3A CT26 cells.
  • FIG. 3B PC-3M cells.
  • FIG. 3C A549 cells.
  • FIG. 5 Representative ⁇ /CT images showing blocking of ⁇ Cu-DOTA- TNYL-RAW uptake in CT26 tumors at 4 h and in PC-3M tumors at 24 h after radiotracer injection.
  • 64 Cu-DOTA-TNYL-RAW was co-injected with cold TNYL-RAW (50 ⁇ g/mouse).
  • FIG. 7A The reaction scheme for the synthesis of nat Cu/ 64 Cu-DOTA- TNYL-RAW.
  • FIG. 8 SPR sensorgrams of TNYL-RAW and DOTA-TNYL-RAW peptides on sensor chips coated with EphB4.
  • the peptides were injected as ten two-fold concentration series from 1.6 nM to 800 nM and were analyzed in duplicate binding cycles. Data sets (shown in black) are overlaid with curves fit to a 1 : 1 mass transfer interaction model (gray lines).
  • the vertical axes in response units represent binding of each peptide to immobilized EphB4.
  • FIG. 9 Reaction scheme for the synthesis of SH-PEG-c(TNYL-RAW) and its conjugation to HAuNS.
  • FIG. 10 Cellular uptake of T-DOX@HAuNS.
  • FIG. 10A Representative photomicrographs of Hey cells after incubation with T-DOX@HAuNS for 2 h. The scattering signal from the HAuNS was visualized using a dark-field condenser, and fluorescence was from DOX. Cell nuclei were counterstained with DAPI. Bar, 20 ⁇ .
  • FIG. 10B Quantitative cellular uptake of nanoparticles in Hey and A549 cells after 3 h incubation with m In-labeled T-DOX@HAuNS and T-DOX@HAuNS plus free c(TNYL-RAW) (blocking). **P ⁇ 0.005;*P ⁇ 0.05.
  • FIG. 11 T-DOX@HAuNS pharmacokinetics, biodistribution, and tumor uptake.
  • FIG. 1 IB Biodistribution of U 1 ln-labeled T-DOX@HAuNS, T-DOX@HAuNS with blocking, and DOX@HAuNS in nude mice at 24 h after injection.
  • FIG. 11C Biodistribution of U 1 ln-labeled T-DOX@HAuNS, T-DOX@HAuNS with blocking, and DOX@HAuNS in nude mice at 24 h after injection.
  • FIG. 11C Biodistribution of U 1 ln-labeled T-DOX@HAuNS, T-DOX
  • FIG. 12 NIR-induced temperature change in tumors injected with HAuNS.
  • FIG. 13 Antitumor activity of various treatments against Hey tumors.
  • FIG. 13B Average tumor weights (left panel) and photographs (right panel) of tumors from different treatment groups. Tumors were removed on day 22 for all groups except the saline-plus-laser group, in which tumors were removed on day 9.
  • FIG. 13C Representative photomicrographs of hematoxylin and eosin-stained slides from scar tissue from a mouse treated with T-DOX@HAuNS-plus-laser or tumors from mice treated with saline or DOX@HAuNS-plus-laser on day 22 after treatment.
  • FIG. 14 Physical properties of HAuNS.
  • FIG. 14A Absorption spectrum of HAuNS.
  • FIG. 14B TEM image of HAuNS.
  • FIG. 14C High-resolution TEM of a single HAuNS.
  • FIG. 15A SPR sensorgrams of c(TNYL-RAW) peptide on sensor chips coated with EphB4. Peptides were injected as ten two-fold concentration series from 1.6 nM to 800 nM and were analyzed in duplicate binding cycles. Data sets (shown in black) are overlaid with curves fit to a 1 : 1 mass transfer interaction model (gray lines). The vertical axes in response units represent binding of each peptide to immobilized EphB4.
  • FIG. 15B Comparison of peptide stability in mouse plasma. 64 Cu- DOTA-TNYL-RAW or 64 Cu-DOTA-c(TNYL-RAW) was incubated in mouse plasma at 37°C.
  • a 100 ⁇ , of plasma was removed from the incubation solution at 0, 2, 4, 8, 12, and 24 h time points and subjected to solid phase extraction on a CI 8 cartridge SPE column (Waters, Milford, MA). Then 20 ⁇ of the extract was analyzed by reversed phase-high-performance liquid chromatography (RP-HPLC) on an Agilent 1100 system (C-18, Vydac, 4.6 x 250 mm, 10 ⁇ ) equipped with a radiodetector. The system was eluted with a linear gradient of 10%- 90% acetonitrile in a 0.1% aqueous trifluoroacetic acid solution over 35 min at a flow rate of 1.0 mL/min.
  • RP-HPLC reversed phase-high-performance liquid chromatography
  • FIG. 16 Western blot analysis of EphB4 expression in MDA-MB-231, A549, A2780, and Hey cell lines.
  • FIG. 17 Cell viability as a function of equivalent DOX concentration.
  • A549 (FIG. 17A) and A2780 (FIG. 17B) cells were treated with T-DOX@HAuNS, DOX@HAuNS, or free DOX. The viability of cells was determined using MTT assay. Circles are T-DOX@HAuNS; Squares are DOX@HAuNA; Triangles are Free DOX.
  • FIG. 18 Percentage of body weight change after various treatments. All data are presented as mean ⁇ standard deviation. Diamonds are Saline + Laser; Squares are HAuNS + Laser; Triangles are DOX@HAuNS + Laser; X is T-DOX@HAuNS + Laser.
  • FIG. 19A Structure of heterodimer peptides with motifs targeting both EphA2 and EphB4 receptors.
  • FIG. 19B Characterization of heterodimer peptides by ESI-MS.
  • FIG. 19C Characterization of heterodimer peptides by HPLC.
  • FIG. 20 Surface competition assays.
  • FIG. 20A Surface competition assays were performed with YSA-TNYL-RAW dimer and ephrinB2 with the surface coated with EphB4 using an increasing concentration of heterodimer and a constant concentration of ephrinB2, the natural ligand of EphB4.
  • FIG. 20B Surface competition assays were also performed with YSA-TNYL-RAW dimer and EphA2 with the surface coated with ephrinAl, the natural ligand of EphA2, using an increasing concentration of heterodimer and a constant concentration of EphA2. The highest concentration corresponds to the bottom line on the sensorgrams while the lowest corresponds to the top line.
  • FIG. 21 Cell binding and inhibition in ovarian cancer Hey cells.
  • FIG. 21 A Cell binding of 64CU-DOTA-YSA-TNYL-RAW dimer over time.
  • FIG. 2 IB Cell binding at a single time point with various blocking agents. The bars, from left to right, are as follows: No blocking, Block w/ Dimer, Block w/ TNYL-RAW, Block w/ YSA.
  • FIG. 22 Biodistribution of 64 Cu-labeled YSA-TNYL-RAW bispecific heterodimer targeting both EphA2 and EphB4 receptors in ovarian cancer xenografts.
  • FIG. 23 Structure of the dual-labeled EphB4-binding peptide DOTA-TNYL-
  • FIG. 24 Immunohistochemistry of tumor sections confirming the binding of ⁇ Cu-DOTA-TNYL-CyS.S to tumor cells and tumor microvessels.
  • U251 and U87 tumors were stained and imaged for CD 31 and EphB4.
  • FIG. 25 Structure of EphB4-targeting cyclic peptide c(TNYL-RAW).
  • FIG. 26 Surface plasmon resonance curves for binding of c(TNYL-RAW) to
  • FIG. 27 Stability of peptides in mouse serum as assayed by LC-MS. Cyclic peptide c(TNYL-RAW) was more stable in mouse serum than its corresponding linear peptide. Top line is c(TNYL-RAW); bottom line is TNYL-RAW.
  • FIG. 28 FIG. 28A. ⁇ /CT images of nude mice bearing subcutaneous human melanoma A375SM xenografts using 7.4 MBq (200 ⁇ ) of 68 Ga-NOTA-c(TNYL- RAW) peptide. Reduced retention in the liver and the spleen, as well as minimal retention in the lungs and GI tract, were shown by ⁇ /CT imaging.
  • FIG. 28B Uptake of 68 Ga-NOTA- c(TNYL-RAW) in different organs.
  • NOTA radiometal chelator l,4,7-tetraazacyclododecane-N,N',N"-tetraacetic acid
  • Radionuclide- and fluorescent dye-labeled peptides and a method of using the peptides in imaging EphB4 receptors and Ephrin family of receptors with the radionuclide and/or fluorescent dye labeled peptides are likewise provided.
  • the disclosed peptides can be potentially used for early detection of cancer and for monitoring response to treatment directed at EphB4 receptors.
  • the imaging properties in disease models include, but are not limited to, dosimetry and toxicity.
  • EphB4 receptor a member of the Ephrin receptor tyrosine kinase family.
  • Noninvasive imaging of EphB4 could potentially increase early detection rates, monitor response to therapy directed again EphB4, and improve patient outcomes.
  • a series of peptide-based imaging agents are disclosed with high receptor binding affinity for nuclear imaging of EphB4 receptors.
  • the EphB4-binding peptide TNYLF SPNGPIARA W (TNYL-RAW) was conjugated with 1,4,7, 10-tetraazadodecane-N, N',N",N"'-tetraacetic acid (DOTA).
  • DOTA-TNYL-RAW was labeled with ⁇ Cu with high labeling efficiency.
  • 64 Cu-DOTA-TNYL-RAW displayed high binding affinity to EphB4 (Kd- 2 nM), was selectively taken up by CT26 and PC-3M cells, but not by A549 cells.
  • Binding of FITC-TNYL-RAW and ⁇ Cu-DOTA-TNYL-RAW to CT26 and PC-3M cells could be blocked by an excess amount of TNYL-RAW.
  • 64 Cu-DOTA-TNYL- RAW showed significantly higher uptake in PC-3M tumors than in A549 tumor, with percentages of injected dose per gram of tumor (%ID/g) values of 0.84 ⁇ 0.09 and 0.44 ⁇ 0.09 at 24 hr after radiotracer injection, respectively ⁇ /CT imaging clearly revealed deposition of ⁇ Cu-DOTA-TNYL-RAW in CT26 and PC-3M tumors but not in A549 tumors.
  • EphA2 and EphB4 receptors are over-expressed in a variety of solid tumors including cancers in the ovarian, breast, colorectal, brain, and prostate. These receptors are also expressed in angiogenic blood vessels. Therefore, members of the Ephrin receptor family are attractive targets for cancer imaging and therapy (Pasquale, Nat Rev. Cancer, 10: 165, 2010).
  • imaging probes that simultaneously bind to both EphA2 and EphB4 can potentially increase early detection rates and be used to monitor response to therapy directed against EphA2 and EphB4.
  • a dual labeling approach is provided by introducing both a radionucle and a near-infrared dye to EphB4-targeting peptide that allow dual modal imaging of the receptors.
  • imaging probes can provide increased information content and are useful for both diagnostic imaging and guiding surgery intraoperatively.
  • PEG polyethylene glycol
  • YSA-TNYL was then labeled with the positron emitter ⁇ Cu through 1,4,7, 10-tetraazacyclododecane-N, N', N", N"'-tetraacetic acid (DOTA) chelator.
  • DOTA 10-tetraazacyclododecane-N, N', N", N"'-tetraacetic acid
  • SPR Surface Plasmon Resonance
  • YSA-TNYL-DOTA peptide had comparable binding affinity to EphB4 compared with TNYL peptide and higher binding affinity to EphA2 than YSA peptide.
  • YSA-TNYL-DOTA- 64 Cu dimer showed significantly higher tumor uptake value compared with monomeric 64 Cu-DOTA- TNYL and monomeric 64 Cu-DOTA-YSA monomer analogs at all time points examined.
  • the tumor uptake of YSA-TNYL-DOTA- 64 Cu could be partially blocked with an excess amount of cold TNYL, YSA, or mixture of TNYLRAW and YSA peptides.
  • the heterodimer YSA-TNYL- DOTA- Cu Compared with 64 Cu- DOTATNYL and 64 Cu-DOTA-YSA monomeric tracers, the heterodimer YSA-TNYL- DOTA- Cu also showed improved pharmacokinetics, resulting in a significantly higher target-to-background ratio.
  • this class of radiotracers directed at both EphA2 and EphB4 should be useful imaging probes for early tumor detection and noninvasive characterization of Ephrin receptors.
  • 64 Cu-DOTA and Cy5.5 dye were introduced to TNYL peptide to synthesize dual-tracer imaging probe. In vitro, dual labeled TNYL displayed significantly higher binding to U251 glioma cells over-expressing EphB4 than to U87 cells that express low level of EphB4.
  • this invention discloses new classes of peptide imaging agents suitable for noninvasive detection of tumor cells overexpressing EphB4 receptors (and in some cases EphA2 receptors).
  • the disclosed peptides can be potentially used for early cancer detection and monitoring of treatment response.
  • the concept can be potentially used for guiding cancer surgery under fluorescent imaging.
  • the same concept can be applied to the detection of other receptors in the Ephrin receptor family.
  • this invention discloses a novel 64 Cu-labeled peptide with high receptor binding affinity (i.e., low nanomolar 3 ⁇ 4 values) for PET imaging of EphB4 receptors.
  • the expression of EphB4 receptors can be noninvasively interrogated by ⁇ /CT using the disclosed peptide, 64 Cu-DOTA-TNYL-RAW.
  • Photothermal ablation is an emerging technique that uses near-infrared laser light-generated heat to destroy tumor cells.
  • complete tumor eradication by PTA therapy alone is difficult because heterogeneous heat distribution can lead to sub-lethal thermal dose in some areas of the tumor.
  • Successful PTA therapy requires selective delivery of photothermal conducting nanoparticles to mediate effective PTA of tumor cells, and the ability to combine PTA with other therapy modalities.
  • Multifunctional doxorubicin (DOX)- loaded hollow gold nanospheres were synthesized that target EphB4, a member of the Eph family of receptor tyrosine kinases overexpressed on the cell membrane of multiple tumors and angiogenic blood vessels.
  • T represents a cyclic peptide targeting EphB4 plus laser regressed completely with only residual scar tissue by 22 days following injection, and none of the treatment groups experienced a loss in body weight.
  • Concerted chemo-photothermal therapy with a single nanodevice capable of mediating simultaneous PTA and local drug release may have promise as a new anticancer therapy.
  • this invention discloses an optimized cyclic peptide with high stability and low background suitable for noninvasive detection of tumor cells overexpressing EphB4 receptors.
  • the disclosed peptides can be potentially used for early cancer detection and monitoring of treatment response.
  • the same peptide can also be used for targeted delivery nanoparticles and therapeutic agents.
  • the cyclic peptide can be labeled with any positron emitter or gamma emitter for PET and SPECT imaging, respectively.
  • the peptide can also be labeled with a fluorescent dye to guide surgery.
  • this invention discloses a method of nuclear imaging for reducing renal uptake of radiotracers.
  • the method can be potentially used to reduce renal toxicity of radiolabeled compounds used in radionuclide therapy and imaging.
  • Eph receptors are the largest family of receptor tyrosine kinases
  • Eph receptor tyrosine kinases and their ligands regulate a wide range of cell contact-dependent signaling that can effect cell proliferation, migration, morphology, adhesion, and invasion (Pitulescu et al. Genes & Dev., 24:2480-2492, 2010). This can occur through Eph signaling which alters the actin cytoskeleton organization and integrins and intercellular adhesion molecules processes. Eph-ephrin interactions are important for many biological roles including axon growth and maturation, cell positioning in the gastoinestinal tract, blood vessel morphogenesis and angiogenic sprouting, insulin secretion, bone remodeling, and immune function.
  • Eph receptors are divided into an EphA and an EphB class that bind to glycosylphosphatidylinositol-linked ephrin-A ligands and the transmembrane ephrin-B ligands, respectively.
  • EphB4 receptors play important roles in a variety of biological processes, including cell aggregation and migration, neural development, embryogenesis and angiogenesis, and vascular development (Dodelet and Pasquale, 2000; Noren et al, 2004; Erber et al, 2006; Wang et al, 1998).
  • EphB4 selectively binds to its endogenous ligand, ephrin-B2, to promote cell signaling required for cancer progression and angiogenesis and has been shown to be profoundly upregulated in numerous cancer types, such as prostate, colon, lung, gastric, bladder, ovarian, and breast cancers (Xia et al, 2006; Davalos et al, 2006; Xia et al, 2005; Takai et al, 2001; Kumar et al, 2009; Stephenson et al, 2001 ; Kumar et al, 2006).
  • Overexpression of EphB4 in cancer cells is associated with tumorigenesis and angiogenesis by stimulating reverse signaling through ephrin-B2.
  • EphB4 forward signaling has been shown to inhibit cellular proliferation (Noren et al, 2004).
  • Eph receptors are typically divided into a globular ligand-binding domain, a cysteine-rich region, and two fibronectin type III repeats in the extracellular region, a short transmembrane region with several conserved tyrosine residues and the tyrosine kinase domain, a sterile a motif (SAM) protein-protein interaction domain, and a C-terminal PDZ- binding motif in the intracellular region. Eph receptors are divided into two classes, EphA and EphB.
  • Eph ligands which are also cell-surface associated proteins, divided into two classes, GPI-anchored ephrin-A and transmembrane ephrin-B.
  • Ephrin-B molecules contain a cytoplasmic domain with several highly conserved tyrosine phosphorylation sites and a C-terminal PDZ motif.
  • EphA receptors bind to ephrin- A
  • EphB receptors bind to ephrin-B, but cross-signaling can occur.
  • Eph and ephrins are capable of bi-directional signaling through trans interactions, though interactions in cis (i.e., between molecules expressed in the same cell) appear to inhibit receptor activation.
  • a targeting peptide of the embodiments is conjugated to a radioisotope.
  • the targeting agent comprises a chelating moiety and a radionuclide chelate.
  • the peptide can be chelated to a radionuclide, such as a technetium ion, a copper ion, an indium ion, a thallium ion, a gallium ion, an arsenic ion, a rhenium ion, a holmium ion, a yttrium ion, a samarium ion, a selenium ion, a strontium ion, a gadolinium ion, a bismuth ion, an iron ion, a manganese ion, a lutetium ion, a cobalt ion, a platinum ion
  • radionuclides examples include, but are not limited to, 99m Tc, 188 Re, 186 Re, 153 Sm, 166 Ho, 90 Y, 89 Sr, 67 Ga, 68 Ga, m In, 183 Gd, 59 Fe, 225 Ac, 212 Bi, 211 At, 45 Ti, 177 Lu, 60 Cu, 61 Cu, 67 Cu, and 64 Cu.
  • Chelating moieties for use according to the invention include, but are not limited to, a acyclic polyamioncarboxylate, a diposphine, a Schiff base, a bis(thiosemicarbazone), a cyclic polyamine, a cyclic polyaminocarboxylate, a cross-bridged cyclic polyamine, a cross-bridged cyclicpolyamioncarboxylate, a l,3,5-cis,cis- triamioncyclohexane derivative, a sarcophagine, or a sepulchrate.
  • the chelating moiety can be l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), 1,4,7,10- tetraazacyclododecane-l,4,7, 10-tetraacetic acid (DOTA), diethylenetriaminetetraacetic acid (DTTA), Diethylenetriaminopentaacetic acid (DTP A), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,8,1 1-tetraazacyclotetradecane- 1,4,8,1 1 -tetraacetic acid (TETA), 1,4,8,1 l-tetraazacyclotetradecane-l,8-diacetic acid (TE2A) Mercaptoacetyltriglycine (MAG 3 ) or 4,5-bis(2-mercaptoacetamido)pentanoic acid, metformin, or phenformine.
  • Chemotherapeutic agents
  • a targeting peptide of the embodiments is conjugated to or associated with a chemotherapeutic agent.
  • chemotherapeutic agents of use as conjugates (or for loading in peptide-conjugated nanoparticles) include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin;
  • nanoparticle refers to any particles having dimensions in the 1-1,000 nm range.
  • nanoparticles have dimensions in the 2-200 nm range, preferably in the 5-150 nm range, and even more preferably in the 10- 100 nm range.
  • the nanoparticles may be conjugated to a targeting peptide of the embodiments.
  • Example nanoparticles include for example polymeric micelles, polyethylene glycol, carbon nanotubes, gold nanostructures, such as gold nanoshells, gold nanorods, gold nanocages, and hollow gold nanospheres, as detailed herein, and copper sulfide nanoparticles (Haram et al, 1996; Xu et al, 2009; Huang et al, 2010).
  • CuS NPs have been synthesized and demonstrated for photothermal destruction of tumor cells in vitro using a NIR laser beam centered at 808 nm.
  • Targeting agents of the embodiments may be used in an imaging or detection method for diagnosis or localization of tumor or angiogenic tissues. Any optical or nuclear imaging method may be contemplated, such as PET, SPECT, CT, or photoacoustic tomography.
  • the integrated radioactive isotope in the nanoparticle may exert a radiotherapeutic effect on the tissue incorporating such nanoparticle.
  • a photothermal ablation therapy may be administered to the tissue having the targeting agents to enhance the therapeutic effect.
  • Targeting agents may be used in PET.
  • Positron emission tomography PET is a powerful and widely used diagnostic tool that has the advantages of high sensitivity (down to the picomolar level) and ability to provide quantitative imaging analyses of in vivo abnormalities (Scheinin et al, 1999; Eckelman, 2003; Welch et al, 2009).
  • PET may be used in certain aspects to trace nanoparticles in vivo.
  • Certain targeting agents may also be used in SPET.
  • Single photon emission computed tomography SPECT, or less commonly, SPET
  • SPET is a nuclear medicine tomographic imaging technique using gamma rays. It is very similar to conventional nuclear medicine planar imaging using a gamma camera. However, it is able to provide true 3D information. This information is typically presented as cross-sectional slices through the patient, but can be freely reformatted or manipulated as required.
  • the SPET basic technique requires injection of a gamma-emitting radioisotope called radionuclide) into the bloodstream of the patient.
  • a radioisotope is conjugated to a targeting agent, which allow it to be concentrated in ways of medical interest for disease detection.
  • a targeting agent comprising a marker radioisotope, which is of interest for its radioactive properties, has been attached to a targeting ligand, which is of interest for its chemical binding properties to certain types of tissues.
  • This marriage allows the combination of ligand and radioisotope (the radiopharmaceutical) to be carried and bound to a place of interest in the body, which then (due to the gamma-emission of the isotope) allows the ligand concentration to be seen by a gamma-camera.
  • Targeting agents may also be used in MRI. Magnetic resonance imaging
  • MRI Magnetic resonance Imaging
  • contrast materials such as intravenous DOTA-Gd contrast are used. This is useful to highlight structures such as blood vessels that otherwise would be difficult to delineate from their surroundings. Using contrast material can also help to obtain functional information about tissues.
  • Certain targeting agents may also be used in photoacoustic tomography.
  • Photoacoustic tomography or photoacoustic computed tomography (PACT) is a materials analysis technique based on the reconstruction of an internal photoacoustic source distribution from measurements acquired by scanning ultrasound detectors over a surface that encloses the source under study.
  • the PA source is produced inside the object by the thermal expansion that results from a small temperature rise, which is caused by the absorption of externally applied radiation of pulsed electromagnetic (EM) waves.
  • EM pulsed electromagnetic
  • This technique has great potential for applications in the biomedical field because of the advantages of ultrasonic resolution in combination with EM absorption contrast.
  • PAT is also called optoacoustic tomography (OAT).
  • each temporal PA signal measured at various detection positions, provides one-dimensional radial information about the PA source relative to the detector position; 2D surface scans offer other 2D lateral information about the PA source. Combining the temporal and spatial measurements affords sufficient information for a complete reconstruction of a 3D PA source. Because the PA signal received by each ultrasound detector is the integral of the ultrasound waves over the sensing aperture of the detector, the reconstruction algorithms depend on the detector apertures as well as the scanning geometries. Small-aperture detectors are often used to approximate point detectors, which receive PA signals originating from spherical shells, centered at each point detector, with radii determined by the acoustic times of flight.
  • Targeting agents may also be used in photothermal ablation therapy.
  • Photothermal ablation (PTA) therapy has gained increasing attention in recent years as a minimally invasive alternative to conventional approaches to cancer treatment such as surgery and chemotherapy (Amin et ah, 1993; Nolsoe et ah, 1993; Fiedler et ah, 2001 ; Vogeland Venugopalan, 2003).
  • NPs with unique optical properties primarily gold nanostructures, such as gold nanoshells (Hirsch et ah, 2003; Loo e ah, 2005), gold nanorods (Dickerson et ah, 2008; Park et ah, 2010), gold nanocages (Chen et ah, 2007; Au et ah, 2008), and hollow gold nanospheres (Lu et ah, 2010; Melancon et al obsession 2008; (Lu et ah, 2009), but also carbon nanotubes (Chakravarty et ah, 2008 Burke et ah, 2009)— have been investigated as photothermal coupling agents to enhance the efficacy of PTA therapy.
  • gold nanostructures such as gold nanoshells (Hirsch et ah, 2003; Loo e ah, 2005), gold nanorods (Dickerson et ah, 2008; Park et ah, 2010), gold nanocages
  • plasmonic nanomaterials exhibit strong absorption in the near-infrared (MR) region (wavelength 700-1 100 nm) and offer an opportunity to convert optical energy to thermal energy, enabling deposition of otherwise benign optical energy into tumors for thermal ablation of tumor cells.
  • MR near-infrared
  • EphB4 receptor a member of the Ephrin receptor tyrosine kinase family.
  • Noninvasive imaging of EphB4 could potentially increase early detection rates, monitor response to therapy directed again EphB4, and improve patient outcomes.
  • Disclosed herein is a series of peptide-based imaging agents with high receptor binding affinity for nuclear imaging of EphB4 receptors.
  • the EphB4-binding peptide TN YLF SPNGPIARA W TNYL-RAW; SEQ ID NO: 1 was conjugated with l,4,7, 10-tetraazadodecane-N,N',N",N" '-tetraacetic acid (DOTA).
  • DOTA-TNYL-RAW was labeled with 64 Cu with high labeling efficiency.
  • EphB4 receptors can be noninvasively interrogated by ⁇ /CT using 64 Cu-DOTA -TNYL-RAW.
  • Pasquale and colleagues identified, using phage display technology, several 12-mer peptides that selectively bind to individual Eph receptors. Tyr- Asn-Tyr-Leu-Phe-Ser-Pro-Asn-Gly-Pro-Ile-Ala (TNYLFSPNGPIA; SEQ ID NO: 2), an EphB4 binding peptide from the initial screening, was further modified to include a RAW moiety at the carboxyl terminus.
  • TNYLFSPNGPIARAW Tyr-Asn-Tyr-Leu-Phe-Ser-Pro-Asn- Gly-Pro-Ile-Ala-Arg-Ala-Trp
  • TNYL-RAW can be used as a receptor ligand for the noninvasive imaging of EphB4.
  • the present disclosure shows that 64 Cu-labeled TNYL-RAW is a promising radiotracer for PET imaging of EphB4 receptor expression in both human prostate and colon cancer xenograft models.
  • l,4,7,10-Tetraazadodecane-N,N',N",N"'-tetraacetic acid was obtained from Macrocyclics (Dallas, TX).
  • the BIACore sensor chip CM5 amine coupling kit, HBSEP running buffer (0.01 M HEPES [4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid], pH 7.4, 0.15 M NaCl, 3 mM ethylenediaminetetraacetic acid [EDTA], and 0.005% [v/v] surfactant P20 solution), and regeneration buffer were purchased from BIACORE, Inc. (Piscataway, NJ).
  • 4',6-Diamidino-2-phenylindole (DAPI) was obtained from Sigma-Aldrich (St. Louis, MO).
  • the 64 Cu-labeled peptide was further purified, if necessary, by reverse-phase high- performance liquid chromatography (RP-HPLC) on an Agilent 1 100 system (C-18, Vydac, 4.6 x 250 mm, 10 ⁇ ) eluted with a linear gradient of 10%-90% acetonitrile in a 0.1% aqueous TFA solution over 35 min at a flow rate of 1.0 mL/min.
  • the solvent was then removed, reconstituted in saline, and passed through a 0.22- ⁇ filter for use in the animal experiments.
  • Natural copper chloride (“ ⁇ CuC ⁇ ) was used to synthesize nat Cu-DOTA-TNYL- RAW under identical conditions, and its identity was confirmed by high-resolution electrospray ionization mass spectrometry (HRMS-ESI). 64 Cu-DOTA-TNYL-RAW was co- injected with nat Cu-DOTA-TNYL-RAW into the above-mentioned HPLC system equipped with both ultraviolet and radiodetectors to confirm its identity.
  • HRMS-ESI high-resolution electrospray ionization mass spectrometry
  • the stock solution (100 ⁇ g/mL) of EphB4/Fc in phosphate-buffered saline (PBS) was diluted to 25, 12.5, and 6.25 ⁇ g/mL with 10 mM sodium acetate buffer at pH 4.5 and immobilized to a CM5 sensor chip using the amine coupling reaction following manufacturer-provided procedures (BIACORE). Briefly, the surfaces of the chips in flow cells (FC)-l, -2, -3, and -4 were activated by exposing them to a mixture of 200 mM N-ethyl-N'-dimethylaminopropyl carbodiimide and 50 mM N-hydroxysuccinimide for 7 min.
  • FC flow cells
  • FC-1 was used as a reference surface and was directly deactivated by injecting 1 M ethanolamine at pH 8.5 for 7 min.
  • the other three flow cells were injected with 25 ⁇ g/mL, 12.5 ⁇ g/mL, and 6.25 ⁇ g/mL EphB4/Fc, respectively, followed by injection of 1 M ethanolamine to block the remaining activated ester groups on the surface.
  • the chip was allowed to stabilize for at least 2 h in HBSEP running buffer before injecting test analytes.
  • SPR Surface plasmon resonance
  • the CT26 murine colon cancer cell line and A549 human lung adenocarcinoma epithelial cell line were purchased from the American Type Culture Collection (Manassas, VA).
  • the PC-3M human prostate cancer cell line was obtained from Professor Dominic Fan (MD Anderson Cancer Center, Houston, TX).
  • PC-3M, CT26, or A549 cells were seeded (1 x 10 5 /well) in Lab-Tek II chambered slides (Nalge Nunc International, Naperville, IL) supplemented with RPMI-1640 medium plus 10% FBS one day before the experiment.
  • the cells were incubated with 100 ⁇ ⁇ of phenol-free RPMI-1640 culture medium containing 10 ⁇ of FITC-TNYL-RAW or scrambled FITC-scTNYL-RAW (AGPFNTYLRTNAWSP; SEQ ID NO: 3) for 20 min at room temperature.
  • 10 ⁇ of FITC-TNYL-RAW and 1 mM of TNYL-RAW were added to the cells.
  • cells were incubated with 10 of PE-conjugated rat anti-human anti-EphB4 monoclonal antibody (R&D Systems, Minneapolis, MN) in 100 ⁇ ⁇ of phenol red-free RPMI-1640 medium.
  • the cells were washed and fixed with 4% paraformaldehyde for 15 min at room temperature.
  • the cell nuclei were counterstained with DAPI.
  • the slides were mounted and visualized under a Zeiss Axiovert Z.1 fluorescent microscope (Zeiss, Jena, Germany).
  • the tubes were briefly vortexed and 100 ⁇ ⁇ of the cell suspension was transferred into a microcentrifuge tube containing 500 ⁇ ⁇ of a 75:25 mixture of silicon oil (density 1.05; Sigma-Aldrich) and mineral oil (density 0.872; Acros, Geel, Belgium). The mixture was centrifuged at 14,000 rpm for 5 min. After the tubes were frozen in liquid nitrogen, the bottom tips containing the cell pellet were cut off. The cell pellets and the supernatants were counted with Packard Cobra Quantum ⁇ -counter (GMI, Ramsey, Minnesota). The protein content in a 100- ⁇ , cell suspension was quantified using the Bio-Rad protein assay kit according to the manufacturer's protocol. The radioactivity in the cell pellets and media was counted, and the data were expressed as activity ratios of the cell pellet to the medium ([cpm ⁇ ig protein in pellet]/[cpm ⁇ g medium]). The experiments were performed in pentaplicate.
  • the spatial resolution of the PET system is approximately 1.4 mm.
  • Tumor-bearing mice were anesthetized with isoflurane (2% in oxygen) and placed in a prone position.
  • the CT imaging parameters were as follows: X-ray voltage, 80 kVp; anode current, 500 mA; exposure time of each of the 360 rotational steps, 300-350 ms. Images were acquired at 1, 4, and 24 h after intravenous administration of 64 Cu-DOTA-TNYL-RAW. Images were reconstructed using the two-dimensional ordered subsets expectation maximization algorithm. PET and CT image fusion and image analysis were performed using Inveon Research Workplace (Siemens Preclinical Solutions, Knoxville, TN).
  • VOI 3- dimensional region of interest
  • a circular VOI was drawn on the muscle of the legs.
  • VOI was also drawn on a standard (radiotracer solution containing 1% of the injection dose) placed along with the animals.
  • the mean activities within the VOI of the tumor and muscle were calculated in IRW workstation (Siemens).
  • the organs of interest were excised and weighed and their radioactivity counted using an automatic gamma counter. (GMI, Ramsey, Minnesota). The stomach and intestines were not emptied prior to radioactivity measurements. The percentage of injected dose per gram of tissue (%ID/g) was calculated by dividing the %ID/organ by the weight of the organ. Values were expressed as mean ⁇ standard deviation (SD).
  • DOTA-TNYL-RAW is shown in FIG. 1.
  • the reaction scheme for the synthesis of nat Cu/ 64 Cu- DOTA-TNYL-RAW is shown in FIG. 7A.
  • 64 Cu-DOTA-TNYL-RAW used in the in vitro and in vivo experiments was typically 7.4-14.8 MBq/nmol (0.2-0.4 Ci/ ⁇ ) at the end of synthesis.
  • 64 Cu-DOTA-TNYL-RAW was stable in DMEM culture medium containing 10% FBS for up to 24 h at 37°C. Approximately 30% of the ⁇ Cu-DOTA-TNYL- RAW was degraded after 2 h of incubation in mouse serum at 37°C.
  • TNYL-RAW peptides bound to EphB4 receptors with nM affinity.
  • FIG. 2 shows representative sensorgrams obtained from SPR analyses of nat Cu-DOTA- TNYL-RAW and a scrambled peptide, with fitting curves obtained using a global 1 : 1 mass transfer model (gray lines).
  • SPR sensorgrams of TNYL-RAW and DOTA-TNYL-RAW peptides are presented in FIG. 8.
  • the corresponding binding kinetics and affinity data are summarized in Table 1.
  • TNYL had an equilibrium dissociation constant (KD) of 3.06 nM. Conjugation of DOTA to the N-terminus of the peptide increased the KD value to 23.3 nM.
  • KD equilibrium dissociation constant
  • EphB4 in vitro Two EphB4-positive cell lines (PC-3M and CT26) and one EphB4-negative cell line (A549) were used for an in vitro binding study. Immunohistostaining with PE- conjugated rat anti-human EphB4 monoclonal antibody confirmed the expression of EphB4 on the surface of PC-3M, CT26, and A549 cells.
  • PC-3M, CT26, and A549 cells were treated with FITC-TNYL-RAW (10 ⁇ ) or a scrambled peptide FITC-sc-TNYL-RAW (10 ⁇ ) for 20 min at room temperature.
  • FITC-TNYL- RAW 10 ⁇ was co-incubated with TNYL-RAW (1 mM). The cell nuclei were counterstained with DAPI. Stained cells were imaged by fluorescence microscopy. PC-3M and CT26 cells, but not A549 cells, were readily stained with FITC-TNYL-RAW. A FITC- labeled scrambled TNYL-RAW peptide (FITC-sc-TNYL-RAW) did not show detectable binding to PC-3M and CT26 cells. The binding of FITC-TNYL-RAW to PC-3M and CT26 cells was efficiently blocked by an excess amount of unlabeled TNYL-RAW peptide.
  • 64 Cu-DOTA-TNYL-RAW peptide selectively binds to EphB4-positive ceUs.
  • 64 Cu-DOTA-TNYL-RAW had increased uptake with time in EphB4-positive PC-3M (FIG. 3B) and CT26 (FIG. 3 A) cell lines, but not in EphB4-negative A549 cells (FIG. 3C).
  • Co-incubation with cold TNYL-RAW peptide completely abolished the binding of 64 Cu- DOTA-TNYL-RAW to PC-3M at all time points tested (FIG. 3B) and reduced its binding to CT26 cells by ⁇ 10-fold (FIG. 3A).
  • uPET/CT imaging FIG.
  • the uptake values of ⁇ Cu-DOTA-TNYL- RAW in PC-3M tumors were 1.4, 3.2, and 3.6 %ID/g at 1, 4, and 24 h postinjection, respectively.
  • the accumulation of 64 Cu-DOTA-TNYL-RAW in A549 tumor was low at all time points examined, reaching a level of 1.7, 1.5, and 1.2 %ID/g at 1, 4, and 24 h after radiotracer administration. These values were only slightly higher than those recorded for muscle tissue in the same animals.
  • Ex vivo immunohistochemical staining confirmed the expression of EphB4 throughout the CT26 and PC-3M tumors, whereas A549 tumors did not express EphB4 receptors.
  • FIG. 5 compares ⁇ images obtained in the presence and absence of a large excess of cold TNYL-RAW at 4 h after radiotracer injection in a CT26 tumor-bearing mouse and at 24 h after radiotracer injection in a PC-3M tumor-bearing mouse.
  • CT26 and PC-3M tumor models the co-administration of cold TNYL-RAW caused a 77% and 81% reduction in 64 Cu-DOTA-TNYL-RAW accumulation in tumors, respectively.
  • the tumor-to-muscle ratio was reduced 56.7% in CT26 tumors at 4 h postinjection and 47.6% in PC-3M tumors at 24 h postinjection when ⁇ Cu-DOTA-TNYL-RAW was co-injected with cold TNYL-RAW peptide (FIG. 6B).
  • the results of tissue sampling study corroborated with the pattern of 64 Cu-DOTA -TNYL-RAW biodistribution determined by non-invasive in vivo PET/CT imaging.
  • TNYL-RAW peptide had a dissociation constant (K D ) of 3.09 nM, which is comparable to that reported in the literature (KD 1 ⁇ 2 nM) (Koolpe et al, 2005). TNYL-RAW also had a slow dissociation rate (-1.3 x 10 "3 [s-1]), which is a better indicator than simple binding affinity for in vivo molecular imaging applications (Berezov et al, 2001). Previous studies showed that the N-terminal residues of the peptide could be modified without affecting the stability of the binding complex with EphB4 (Chrencik et al, 2006).
  • the level of EphB4 receptor expression should be the predominant factor influencing the magnitude of Cu-DOTA-TNYL-RAW accumulation and retention in tumor tissue.
  • the difference in the degradation of 64 Cu-DOTA-TNYL-RAW peptide-based radiotracer in different tumor microenvironments may also contribute to the difference in tumor retention of the radiotracer.
  • the stability of radioactive compounds is critical because the molecular integrity of the radiopharmaceutical must be maintained for an adequate time in the blood circulation during biodistribution and imaging studies.
  • ⁇ Cu-DOTA-TNYL-RAW was stable in DMEM with 10% FBS for at least 24 h and was stable in mouse serum for up to 2 h, after which slow degradation was observed. Nevertheless, these ⁇ /CT studies revealed that 64 Cu-DOTA-TNYL-RAW possess sufficient in vivo stability for tumor imaging.
  • the 64 Cu-DOTA-TNYL-RAW biodistribution data obtained by both non-invasive PET/CT imaging and by tissue sampling indicate that the liver and the kidney were the major organs for physiological uptake and clearance of this radiotracer.
  • hepatobiliary clearance is a major route for hydrophobic peptides (Rusckowski et ah, 2001).
  • TNYL-RAW peptide contains several hydrophobic amino acids (Asn, He, Leu, Phe, Pro, Ala, Trp) that could contribute to the high liver uptake of 64 Cu-DOTA-TNYL-RAW.
  • Photothermal ablation (PTA) therapy is a recently developed technique that uses near-infrared (MR) laser light-generated heat to destroy tumor cells.
  • MR near-infrared
  • PTA has gained popularity recently because a specific amount of photo-energy is delivered directly into the tumor mass without causing systemic effects, thus promising minimally invasive intervention as an alternative to surgery (Bardhan et al, 2011 ; Melancon et al, 2009).
  • PTA therapy alone is unlikely to kill all tumor cells because the heat distribution is non-uniform, especially in areas peripheral to large blood vessels where heat can be rapidly dissipated by circulating blood.
  • light-absorbing photothermal conducting nanoparticles are introduced.
  • MR laser-modulated photothermal effects can not only enable PTA of tumor cells but also trigger release of anticancer agents. Such a multimodal approach, which permits simultaneous PTA therapy and chemotherapy, should provide an opportunity for complete eradication of tumor cells.
  • HAuNS Hollow gold nanospheres
  • DOX doxorubicin
  • DOX@HAuNS DOX-loaded HAuNS
  • EphB4 is a particularly promising target for tumor-specific delivery of DOX@HAuNS.
  • DOX@HAuNS were selectively targeted to EphB4-positive tumors, and concerted chemo-photothermal therapy mediated by EphB4-targeting DOX@HAuNS induced remarkable antitumor efficacy with reduced systemic toxicity. These results support the concept of integrating multiple functions into a single nanodevice to mediate simultaneous PTA and local drug release.
  • Methoxy-polyethylene glycol (PEG)-SH (molecular weight, 5,000) and NH 2 -PEG-COOH (molecular weight, 5,000) were purchased from Nektar (San Francisco, CA).
  • Sodium citrate (>99%), cobalt chloride hexahydrate (99.99%), sodium borohydride (99%), and chloroauric acid trihydrate (American Chemical Society reagent grade) were purchased from Thermo Fisher Scientific (Waltham, MA) and were used as received. All amino acid derivatives and coupling reagents were purchased from EMD Chemicals (Philadelphia, PA), Bachem Americas (Torrance, CA), and Chem-Impex International (Wood Dale, IL).
  • PL-DMA resin was obtained from EMD Chemicals. DOX, (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4',6-diamidino-2- phenylindole (DAPI), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Reagent-grade solvents were used without further purification unless otherwise specified.
  • the BIACore sensor chip CM5 For the surface plasmon resonance (SPR) assay, the BIACore sensor chip CM5, amine-coupling kit, HBSEP running buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.005% [v/v] surfactant P20 solution), and regeneration buffer were purchased from GE Healthcare (Waukesha, WI). 3 H-labeled DOX was purchased from Moravek Biochemicals Inc. (Brea, CA).
  • HAuNS were synthesized according to a previously reported method (You et ah, 2010). Briefly, cobalt nanoparticles were first synthesized by deoxygenating deionized water containing 4.5 mL of 1 M sodium borohydride, 2.8 mL of 0.1 M sodium citrate, and 1.0 mL of 0.4 M cobalt chloride. After chloroauric acid was added into the solution containing cobalt nanoparticles, the cobalt immediately reduced the gold ions onto the surface of the nanoparticles and was simultaneously oxidized to cobalt oxide. Any remaining cobalt was further oxidized by air, resulting in the final product, HAuNS.
  • the size of the HAuNS was determined using dynamic light scattering on a Brookhaven 90Plus particle size analyzer (Holtsville, NY). The UV-visible spectra were recorded on a Beckman Coulter DU-800 UV- visible spectrometer (Brea, CA). The morphology of the HAuNS was examined using a JEM 1010 transmission electron microscope (JEOL USA, Peabody, MA).
  • DOX loading For DOX loading, free DOX (5 mg) in water (5 mL) was added to c(TNYL-RAW)-HAuNS (40 OD) in 5 mL of 2.8 mM citrate solution, and the mixture was stirred at room temperature for 24 h. The resulting DOX-loaded c(TNYL-RAW)-HAuNS (T- DOX@HAuNS) were purified by 3 repeated centrifugation and washing steps.
  • Fluorenylmethyloxycarbonyl chloride (Fmoc)-Asp-Wang resin)OAll was formed by esterification with DIPCDI (3 equiv.) and 4-dimethylaminopyridine (DMAP) (0.5 equiv.).
  • Fmoc-Lys(Boc)-Thr(tBu)-Asn(Trt)-Tyr(tBu)-Leu-Phe-Ser(tBu)-Pro-Asn(Trt)-Gly- Pro-ILe-Ala-Arg(Pbf)-Ala-Trp(Boc)-Asp(resin)OAll was synthesized using an Fmoc solid- phase strategy.
  • EphB4/Fc extracellular domain of human EphB4 fused to the carboxy -terminal Fc region of human IgGl was immobilized to CM5 sensor chips using the amine coupling reaction according to the manufacturer's instructions (GE Healthcare). Briefly, the surfaces of the chips in flow cells 1, 2, 3, and 4 were activated by exposing the chips to a mixture of 200 mM N-ethyl-N'- dimethylaminopropyl carbodiimide and 50 mM N-hydroxysuccinimide (NHS) for 7 min. Flow cell 1 was used as a reference surface and was directly deactivated by injecting 1 M ethanolamine at pH 8.5 for 7 min.
  • EphB4/Fc solutions of different concentrations were obtained by diluting a stock solution of EphB4/Fc (100 ⁇ g/mL) in phosphate-buffered saline (PBS) with 10 mM sodium acetate buffer (pH 4.5).
  • PBS phosphate-buffered saline
  • pH 4.5 10 mM sodium acetate buffer
  • Binding assays were performed at 25°C in HBSEP running buffer.
  • c(TNYL-RAW) was diluted in HBSEP buffer, filtered, degassed, and injected at concentrations between 1.6 nM and 800 nM at a flow rate of 30 ⁇ / ⁇ .
  • Peptides were injected into the HBSEP buffer over 4 min, and injection was followed by a 4-min dissociation period.
  • the chips were regenerated using a 1-min pulse of 10 mM glycine (pH 2.2) after each binding circle. Each cycle consisted of a 1-min waiting period to allow monitoring of the baseline binding stability. For subtraction of bulk effects caused by changes in the buffer composition or nonspecific binding, a double-referencing technique was employed.
  • Radiolabeling of HAuNS with m In To conjugate a radiometal chelator to c(TNYL-RAW)-HAuNS, 4-aminobenzyl-diethylenetriaminepentaacetic acid thioctamide (DTPA-TA, 10 mg/mL; 5.0 ⁇ ) (Lu et al, 2007) was mixed with 1.0 mL of aqueous solution of HAuNS (200 OD/mL) for 6 h at room temperature. SH-PEG-c(TNYL- RAW) was then added to the DTPA-TA-conjugated HAuNS as described in the previous section.
  • DTPA-TA 4-aminobenzyl-diethylenetriaminepentaacetic acid thioctamide
  • m In-labeled c(TNYL- RAW)-HAuNS were loaded with DOX to create 1 "in-labeled T-DOX@HAuNS.
  • the radiolabeling efficiency and the stability of m In-labeled T-DOX@HAuNS were analyzed using instant thin-layer chromatography.
  • the labeling efficiency was >95%.
  • MDA-MB-231 human breast carcinoma
  • Hey human ovarian carcinoma
  • A549 human lung adenocarcinoma
  • the cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Life Technologies, Inc., Carlsbad, CA) at 37°C in a humidified atmosphere containing 5% CO 2 .
  • A2780 human ovarian carcinoma cells were kindly provided by Dr. Stephen J. Williams (Fox Chase Cancer Center, Philadelphia, PA). These cells were maintained at 37°C in RPMI-1640 medium containing 10% fetal bovine serum and insulin (0.25 units/mL).
  • the cells were scraped off the dish, suspended in PBS, and the radioactivity of the cell suspension was then measured with a gamma counter. Protein concentration in cell suspension was quantified using the Bio-Rad protein assay kit (Richmond, CA). The data are expressed as radioactivity (dpm per ⁇ g protein).
  • EphB4 expression was probed with mouse anti-EphB4 antibody and Alexa Fluor 680-conjugated goat anti-mouse IgG (Invitrogen).
  • ⁇ - Actin was used as a control to indicate the loading and transfer efficiency. Protein bands were visualized with a LI-COR Odyssey system (Lincoln, NE).
  • a separate experiment was carried out to optically observe the binding between the antibody against EphB4 and the studied cell lines. Cells were cultured on 20-mm glass cover slips and incubated with primary antibody (mouse anti-EphB4 antibody) for 1 h at 4 °C.
  • the cells were then washed with PBS three times and incubated with secondary antibody (Alexa Fluor 680-conjugated goat anti-mouse IgG).
  • secondary antibody Alexa Fluor 680-conjugated goat anti-mouse IgG.
  • the cell-coated cover slip was then repeatedly rinsed with PBS and mounted for microscopic examination using an Axio Observer Zl fluorescence microscope (Carl Zeiss Microimaging GmbH, Germany).
  • Cytotoxicity was measured using an MTT assay according to the manufacturer's suggested procedures. EphB4-positive A2780 cells and EphB4-negative A549 cells were exposed to free DOX, DOX@HAuNS, or T-DOX@- HAuNS for 72 h. The data are expressed as the percentage of surviving cells and are reported as the mean values of three measurements.
  • the percentage of the injected dose per gram of blood was calculated.
  • the blood pharmacokinetic parameters for the radiotracer were analyzed using a noncompartmental model with WinNonlin 5.0.1 software (Pharsight, Sunnyvale, CA).
  • mice Female nude mice (Harlan, Indianapolis, IN) bearing 6-8 mm subcutaneous A2780, MDA-MB-231, or Hey tumors were intravenously injected with U 1 ln-labeled T-DOX@-HAuNS or m In- labeled DOX@HAuNS (20 ⁇ / ⁇ in 0.2 mL).
  • the mice were intravenously injected with a mixture of lu In-labeled T-DOX@-HAuNS (20 ⁇ / ⁇ in 0.2 mL) and an excess of free c(TNYL-RAW) (0.3 ⁇ ). Mice were killed at 24 h after injection.
  • SPECT Single photon emission computed tomography
  • mice bearing subcutaneous Hey tumors were intravenously injected with m In-labeled T- DOX@HAuNS or DOX@HAuNS (8.0 mCi/kg, 0.525 mg/mL [25 OD]).
  • mice were injected intravenously with a mixture of m In-labeled DOX@c(TNYL- RAW)-HAuNS and free c(TNYL-RAW) (0.3 ⁇ ).
  • the mice were placed in a prone position and anesthetized with 0.5%-2.0% isoflurane gas (Iso-Thesia, Rockville, NY) in oxygen.
  • SPECT images were generated at 24 h after injection. After imaging, the mice were killed, and their tumors were removed. The tumors were snap-frozen and cut into 5- ⁇ slices that were then used for autoradiography analysis on a Fujifilm FLA-5100 imaging system (Stamford, CT).
  • mice bearing Hey tumors were injected intravenously with saline (5.0 mL/kg) or T-DOX@-HAuNS (5.0 mL/kg of 50 OD HAuNS).
  • the tumor was irradiated with an NIR laser (3 W/cm 2 ) for 5 min; Diomed 15 Plus, UK) through the skin surface.
  • Temperature was measured with two thermocouples inserted into the tumor. Care was taken to ensure the thermocouple was not directly exposed to the laser beam.
  • the DOX release mediated by the photothermal effect in the Hey tumors was studied using m In- and 3 H-labeled T-DOX@-HAuNS, in which the HAuNS were labeled with U 1 ln and the DOX with 3 H.
  • Tumors were irradiated by NIR laser light (3 W/cm 2 for 5 min) at 1 h after intratumoral injection of the dual-labeled nanoparticles ( 3 H: 10 ⁇ ; m In: 20 ⁇ ) into the center of the tumor.
  • the mice were killed 5 min after laser irradiation, and the tumors were removed, snap-frozen, and sliced into 10- ⁇ sections. Injected tumors that did not receive NIR laser treatment were similarly prepared and used as controls.
  • Fujifilm FLA-5100 imaging system Briefly, the sections of the tumors were exposed to phosphorous screen film (an SR imaging plate) for 15 min at -10°C, and the m In autoradiograph was obtained by scanning the film. After the m In was completely decayed (stored at -80°C for 5 weeks), the same sections were exposed to phosphorous screen film (a TR imaging plate) for 3 days at -10°C, and the 3 H autoradiograph was obtained by scanning the film. Concurrently, control specimens from tumors that were not laser-treated were subjected to the same procedures. The autoradiographic distribution of m In-HAuNS and 3 H- DOX was compared by overlaying the two autoradiograms.
  • Hey tumors were generated by subcutaneous injection of Hey cells (5.0 x 10 6 cells/mouse). When the mean tumor volume reached -200 mm 3 , mice were divided into four groups consisting of 6-8 mice each.
  • mice were killed by CO 2 asphyxiation, and the tumors were collected and weighed. Parts of the tumors were fixed in formalin and cut into 5- ⁇ slices for hematoxylin and eosin staining. Body weight was measured weekly to assess systemic toxicity.
  • the targeting ligand cyclic peptide c(TNYL-RAW) is a second- generation EphB4-binding antagonist.
  • the peptide had an equilibrium dissociation constant (Ka) of 4.4 nM as determined by surface plasmon resonance sensorgram (FIG. 15 A).
  • Ka equilibrium dissociation constant
  • No degradation of ⁇ Cu-labeled c(TNYL-RAW) was observed by high-performance liquid chromatography after incubation of the peptide in mouse plasma over a period of 24 h, whereas ⁇ Cu-labeled linear TNYL-RAW was degraded as soon as 2 h after incubation (FIG. 15B).
  • c(TNYL-RAW) was linked to SATA-PEG-NHS through an activated ester.
  • SH-PEG-c(TNYL-RAW) was conjugated to HAuNS in an aqueous solution via S-Au bonding (FIG. 9).
  • the amount of c(TNYL-RAW) conjugated to the HAuNS was determined by quantitative amino acid analysis after complete dissolution of c(TNYL-RAW)-conjugated HAuNS.
  • the conjugation efficiency was 13.7% and there were about 880 molecules of c(TNYL-RAW) on each HAuNS nanoparticle.
  • DOX was readily loaded into c(TNYL-RAW)-conjugated HAuNS using a previously reported method to give T-DOX@HAuNS (You et al, 2010). DOX loading efficiency was over 90%, and DOX content was 30% (w/w).
  • FIG. 10A shows representative photomicrographs of fluorescence and dark-field images of Hey cells incubated with T-DOX@HAuNS.
  • the nanoparticles were readily taken up by the tumor cells.
  • the fluorescence signal from the DOX was colocalized with the signal from the HAuNS, indicating that the DOX remained associated with the HAuNS after T-DOX@HAuNS were internalized.
  • significantly more T-DOX@HAuNS was internalized in the cells with high EphB4 receptor expression (Hey) than in the cells with low EphB4 receptor expression (A549) (P ⁇ 0.05, FIG. 10B).
  • FIG. 1 1A shows the mean blood activity time profile of U 1 ln-labeled T-DOX@HAuNS and DOX@HAuNS.
  • the pharmacokinetic parameters are summarized in Table 2.
  • FIG. 1 IB shows the biodistribution of T-DOX@HAuNS, T-DOX@HAuNS with blocking by free c(TNYL-RAW), and DOX@HAuNS in nude mice at 24 h after injection. Most nanoparticles were taken up by the liver, spleen, and kidney.
  • C maximum blood concentration
  • AUCo- ⁇ area under the blood drug concentration-time curve extrapolated to infinite time
  • CL systemic clearance
  • Vd volume of distribution
  • Vss volume of distribution at steady-state
  • MRT mean residence time.
  • microSPECT/CT images showed significant blood activity in the liver and spleen for both U 1 ln-labeled T-DOX@HAuNS and DOX@HAuNS in nude mice bearing Hey tumors after intravenous injection of m In-labeled T-DOX@HAuNS, m In-labeled T- DOX@HAuNS plus free c(TNYL-RAW), and m In-labeled DOX@HAuNS.
  • TNYL-RAW free c
  • thermocouple In vivo antitumor activity.
  • the temperature measured by the thermocouple within the tumor reached ⁇ 53°C after 5 min of MR laser exposure on the tumor surface at an output power of 3 W/cm 2 in mice injected with T-DOX@HAuNS (FIG. 12). No change in temperature was noted under the same conditions in the tumors of control mice.
  • Dual-tracer autoradiography showed that immediately after NIR laser irradiation, 3 H- DOX was released and dispersed into the area surrounding the site where T-DOX@HAuNS was introduced. Conversely, 3 H-DOX was mostly colocalized with u l In-HAuNS in mice that did not undergo NIR laser treatment.
  • FIG. 13A shows the Hey tumor growth curves after intravenous injections of saline, HAuNS (5.0 mL/kg of 1.25 mg HAuNS/mL saline [50 OD], no DOX, no targeting), DOX@HAuNS (10 mg equivalent DOX/kg, 5.0 mL/kg of 1.25 mg HAuNS/mL), and T-DOX@HAuNS (10 mg equivalent DOX/kg, 5.0 mL/kg of 1.25 mg HAuNS/mL).
  • Mice in each group received NIR laser treatment (2.0 W/cm 2 for 3 min) 24 h after injection.
  • mice in the saline-plus-laser group were killed on day 9 after injection because most of the tumors in this group were -1500 mm 3 at that time. Mice in the other three groups were killed on day 22.
  • Treatment with T-DOX@HAuNS-plus-laser showed significantly enhanced antitumor activity compared with saline-plus-laser, HAuNS-plus-laser, and DOX@HAuNS- plus-laser.
  • the tumors in this group of mice became whitish immediately after treatment, suggesting disruption of blood perfusion.
  • the tumors in six of the eight mice treated with T-DOX@HAuNS-plus-laser regressed completely and became scar tissue by 22 days after the injection. Histological analysis showed the presence of scar tissue and a lack of residual tumor cells in mice treated with T-DOX@HAuNS-plus-laser (FIG. 13C). Tumors in the nontargeted DOX@HAuNS-plus-laser group became loose and discrete, suggesting damage to tumor cells. However, tumors could not be completely eradicated without targeting ligand. Tumors in the saline-plus-laser group appeared to be intact.
  • T-DOX@HAuNS had significantly greater cytotoxic effects in tumor cells with high EphB4 receptor expression than DOX@HAuNS and free DOX did in vitro. Moreover, T-DOX@HAuNS demonstrated enhanced antitumor activity when combined with NIR laser irradiation than DOX@HAuNS plus laser treatment in vivo. These findings indicate that c(TNYL-RAW) peptide is highly effective for selective delivery of T-DOX@HAuNS to tumors with high expression of EphB4.
  • T-DOX@HAuNS The significantly enhanced antitumor activity of T-DOX@HAuNS could be attributed to 1) increased accumulation of the nanoparticles in tumors, 2) controlled release of DOX mediated by NIR laser irradiation, and 3) synergistic interaction between chemotherapy and PTA therapy, both of which were activated concurrently by NIR laser.
  • c(TNYL-RAW) peptide with high EphB4 binding affinity and high plasma stability was conjugated to HAuNS through a PEG linker, which ensured availability of the peptide to the target receptor (FIG. 9). Additional monofunctional SH-PEG chains were introduced together with SH-PEG-c(TNYL-RAW) to ensure that all available gold surface was covered by PEG. This process was used to create DOX@HAuNS and ligand conjugated T-DOX@HAuNS, both of which had high colloidal stability.
  • DOX@HAuNS could be partially blocked by free c(TNYL-RAW) peptide, confirming that cell uptake of T-DOX@HAuNS was mediated by EphB4 (FIGS. 10 and 1 1).
  • T- DOX@HAuNS displayed significantly higher accumulation than nontargeted DOX@HAuNS in all three tumor models evaluated.
  • DOX@HAuNS and T-DOX@HAuNS exhibited similar pharmacokinetic behaviors (FIG. 1 1A, Table 2), the difference in tumor uptake between targeted and nontargeted HAuNS is unlikely a result of enhanced permeability and retention effect.
  • these data support successful EphB4 receptor- mediated targeted delivery of T-DOX@HAuNS after intravenous injection.
  • T-DOX@HAuNS The temperature in tumors of mice that received an intravenous injection of T-DOX@HAuNS reached ⁇ 53°C after 5 min of continuous -wave NIR laser exposure at 3 W/cm 2 (FIG. 12). This temperature is sufficient for causing irreversible damage to cancer cells (Melancon et ah, 201 1). As expected, there was no temperature change in the tumors of mice that did not receive the nanoparticle injection followed by NIR irradiation. Therefore, T-DOX@HAuNS medicated efficient photothermal effect. In aqueous solution, the release of DOX from DOX@HAuNS could be activated by NIR laser irradiation (You et ah, 2010).
  • DOX@HAuNS after a single dose at 60 mg equivalent DOX/kg had no cardiotoxicity compared to liposomal DOX (two doses at a total dose of 30 mg DOX/kg) and free DOX (single dose of 15 mg/kg).
  • 100% of both liposomal DOX- and free DOX -treated mice had a vacuolar cardiomyopathy.
  • the histopathologic features in the heart were similar to those observed in the saline-treated control mice and no abnormal features were observed.
  • EphA2 and EphB4 receptors are over-expressed in a variety of solid tumors, including ovarian, breast, colorectal, brain, and prostate. These receptors are also expressed in angiogenic blood vessels. Therefore, members of the Ephrin receptor family are attractive targets for cancer imaging and therapy (Pasquale, 2010).
  • EphA2 and EphB4 are attractive targets for cancer imaging and therapy (Pasquale, 2010).
  • peptidyl heterodimers with high receptor binding affinity to EphA2 and EphB4 suitable for molecular imaging of Ephrin receptors.
  • the heterodimers exemplified by YSA- TNYL-DOTA - 64 Cu had significantly higher target-to-background ratio than its corresponding monomeric imaging probes 64 Cu-DOTA-TNYL-RAW and 64 Cu-DOTA-YSA targeting EphB4 and EphA2, respectively.
  • imaging probes that simultaneously bind to both EphA2 and EphB4 can potentially increase early detection rates and be used to monitor response to therapy directed against EphA2 and EphB4.
  • a peptidyl heterodimer, YSAYPDSVPMMS(SEQ ID NO: 4)-PEG- TNYLFSPNGPIARAW(SEQ ID NO: 1) was synthesized by linking the two peptides YSAYPDSVPMMS (YSA; SEQ ID NO: 4) targeting EphA2 and TNYLF SPNGPIARA W (TNYL; SEQ ID NO: 1) targeting EphB4 together with a polyethylene glycol (PEG) linker.
  • YSA-TNYL was then labeled with the positron emitter ⁇ Cu through 1,4,7, 10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) chelator (FIG. 19).
  • the receptor-binding characteristics and tumor-targeting efficacy of heterodimer YSA-TNYL were evaluated in vitro with Surface Plasmon Resonance (SPR) sensor chip technology (Tables 3-5) and in vivo using ⁇ imaging.
  • SPR Surface Plasmon Resonance
  • the KD decreased from about 10 ⁇ 9 to about 10 "11 when both EphB4 and EphA2 receptors were coated on the sensor chip, indicating that the binding affinity of the heterodimer increased when both EphB4 and EphA2 receptors were coated on the sensor chip.
  • Binding of the YSA-TNYL heterodimer to EphB4 and EphA2 was blocked by their natural ligands.
  • Surface competition assays were performed with YSA- TNYL-RAW dimer and ephrinB2 with the surface coated with EphB4 using an increasing concentration of heterodimer and a constant concentration of ephrinB2, the natural ligand of EphB4 (FIG. 20A, highest concentration is the bottom line on the sensorgram; lowest is the top).
  • YSA-TNYL-RAW dimer and EphA2 were also performed with YSA-TNYL-RAW dimer and EphA2 with the surface coated with ephrinAl, the natural ligand of EphA2, using an increasing concentration of heterodimer and a constant concentration of EphA2 (FIG. 20B, highest concentration is the bottom line on the sensorgram; lowest is the top).
  • YSA-TNYL-DOTA- 64 Cu dimer showed high uptake in tumor cells over a period of 2-hr incubation (FIG. 21). The tumor uptake of YSA-TNYL-DOTA- 64 Cu could be partially blocked with an excess amount of cold TNYL, YSA, or mixture of TNYL-RAW and YSA peptides.
  • the inventors also performed ⁇ imaging of EphA2 and EphB4 receptors in ovarian cancer xenografts using 64 Cu-labeled bispecific YSA-TNYL-RAW heterodimer.
  • the dimeric radiotracer exhibited higher tumor-to-target ratio (FIG. 22) and improved imaging properties as compared to its corresponding monomeric radiotracers.
  • KD 2 which represents binding between the bispecific peptide with EphB4
  • EphA2 was two orders of magnitude lower when both EphB4 and EphA2 were coated onto the sensor chip than when only EphB4 was coated onto the sensor chip, suggesting that binding of EphA2 enhanced the binding of the specific heterodimer to EphB4.
  • Example 4 Dual Labeled Peptides for Targeting of EphB4 Receptors
  • a dual labeling approach by introducing both a radionuclide and a near-infrared dye to an EphB4-targeting peptide that allows for dual modal imaging of the receptors.
  • imaging probes can provide increased information content and are useful for both diagnostic imaging and guiding surgery intraoperatively.
  • TNYL peptide 64 Cu-DOTA and Cy5.5 dye were introduced to TNYL peptide to synthesize a dual-tracer imaging probe.
  • dual labeled TNYL displayed significantly higher binding to U251 glioma cells over-expressing EphB4 (FIG. 23) than to U87 cells that express low levels of EphB4.
  • the binding of dual labeled TNYL-RAW to U251 cells could be blocked by a large excess of unlabeled peptide.
  • a cyclic peptide with high receptor binding affinity for EphB4 with high in vivo stability suitable for molecular imaging of Epherin receptors and for targeted drug delivery was disclosed.
  • the purpose of this study was to design, synthesis, and evaluate peptidomimetics with enhanced in vivo stability and low background through conformation constrain.
  • the lactam formation of the side chain to side chain peptide cyclization (FIG. 25) was utilized to improve stability of peptides in the presence of peptidases and proteinases.
  • SPR surface plasma resonance
  • the lead cyclic peptide was conjugated with 1,4,7-triazacyclononanetriacetic acid (NOTA) and labeled with 68 Ga.
  • NOTA 1,4,7-triazacyclononanetriacetic acid
  • Biodistribution, and small-animal PET/CT studies were performed in nude mice bearing EphB4-positive human melanoma.
  • the inventors identified candidate peptides with binding affinity ( 3 ⁇ 4) in the lower nanomolar range (4.4 nM; FIG. 26) with significantly improved stability in mouse plasma (FIG. 27) and reduced retention in the liver and the spleen (FIG. 28B).
  • ⁇ /CT studies demonstrated clear visualization of EphB4 expressing tumors (FIGS. 28A).
  • 68 Ga labeled cyclic(TNYL-RAW) peptide can be used as a PET/CT tracer to image tumor expression of EphB4, with a high tumor-to-background ratio.
  • DOX@HAuNS doxorubicin-loaded hollow gold nanospheres
  • the inventors demonstrated targeted delivery of nanoparticles conjugated to the cyclic(TNYL-RAW) speptide that targets EphB4, T- DOX@HAuNS. Increased uptake of targeted nanoparticles T-DOX@HAuNS was observed in three EphB4-positive tumors both in vitro and in vivo.
  • Treatment with T- DOX@HAuNS followed by near-infrared laser irradiation resulted in significantly decreased tumor growth when compared to treatments with non-targeted DOX@HAuNS plus laser or HAuNS plus laser.
  • the tumors in six of the eight mice treated with T-DOX@HAuNS plus laser regressed completely with only residual scar tissue by 22 days following injection, and none of the treatment groups experienced a loss in body weight.
  • the inventors have found a more than 3 -fold reduction in renal uptake of 68 Ga-labeled cyclic(TNYL-RAW) 68 Ga-NOTA-c(TNYL-RAW) when the radiotracer was co- injected with metformin (FIG. 29). These data indicate that metformin can be potentially used with radiolabeled compounds in radiotherapy and imaging to reduce rental toxicity.
  • EphB4 controls blood vascular morphogenesis during postnatal angiogenesis.
  • EphB receptor-binding peptides identified by phage display enable design of an antagonist with ephrin-like affinity. J. Biol. Chem., 280: 17301-1731 1, 2005.
  • EphB4 receptor suppresses breast cancer cell tumorigenicity through an
  • Receptor protein tyrosine kinase EphB4 is up-regulated in colon cancer.
  • EphB4 receptor tyrosine kinase is expressed in bladder cancer and provides signals for cell survival. Oncogene, 25:769-780, 2006.

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Abstract

L'invention concerne des agents ciblant un peptide qui peuvent être utilisés en tant qu'agents d'imagerie et/ou thérapeutiques pour des cellules tumorales surexprimant les récepteurs des éphrines. En particulier, les agents peuvent être utilisés pour cibler des cellules exprimant les récepteurs EphB4 et/ou EphA2. L'invention concerne également des procédés pour l'imagerie et le traitement contre le cancer non invasifs utilisant les agents ciblants.
PCT/US2013/021434 2012-01-13 2013-01-14 Agents ciblant les récepteurs des éphrines Ceased WO2013106824A1 (fr)

Applications Claiming Priority (4)

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US201261586282P 2012-01-13 2012-01-13
US61/586,282 2012-01-13
US201261666474P 2012-06-29 2012-06-29
US61/666,474 2012-06-29

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WO2013106824A1 true WO2013106824A1 (fr) 2013-07-18

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Publication number Priority date Publication date Assignee Title
CN105713075A (zh) * 2014-12-04 2016-06-29 北京睿德欧生物科技有限公司 一种EphB4 受体靶向多肽及其应用
CN105906691A (zh) * 2016-07-05 2016-08-31 珠海诺贝尔国际生物医药研究院有限公司 一种Eph激酶多肽抑制剂及其应用
US9974774B2 (en) 2013-07-26 2018-05-22 Race Oncology Ltd. Combinatorial methods to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof
CN113727737A (zh) * 2019-01-17 2021-11-30 卡斯西部储备大学 肿瘤细胞外基质中癌蛋白特异性肽pet/spect探针
CN114262362A (zh) * 2021-10-14 2022-04-01 齐鲁工业大学 一种靶向EphA2受体的68Ga-NODAGA-环状多肽FG01及制备方法与应用
US11964948B2 (en) 2022-06-07 2024-04-23 Actinium Pharmaceuticals, Inc. Bifunctional chelators and conjugates
US12398179B2 (en) 2019-03-14 2025-08-26 Sanford Burnham Prebys Medical Discovery Institute Nanomolar peptides and derivatives to differentially modulate ephrin receptors

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US20050147612A1 (en) * 2001-06-20 2005-07-07 Avner Yayon Antibodies that block receptor protein tyrosine kinase activation, methods of screening for and uses thereof
WO2008122993A1 (fr) * 2007-04-09 2008-10-16 Panacea Biotec Limited Formulation de microparticules enrobées à libération contrôlée
US7582438B2 (en) * 2005-01-27 2009-09-01 Burnham Institute For Medical Research EphB receptor-binding peptides
US20110091562A1 (en) * 2008-06-16 2011-04-21 Georgia Tech Research Corporation Nanogels for cellular delivery of therapeutics

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US20050147612A1 (en) * 2001-06-20 2005-07-07 Avner Yayon Antibodies that block receptor protein tyrosine kinase activation, methods of screening for and uses thereof
US7582438B2 (en) * 2005-01-27 2009-09-01 Burnham Institute For Medical Research EphB receptor-binding peptides
WO2008122993A1 (fr) * 2007-04-09 2008-10-16 Panacea Biotec Limited Formulation de microparticules enrobées à libération contrôlée
US20110091562A1 (en) * 2008-06-16 2011-04-21 Georgia Tech Research Corporation Nanogels for cellular delivery of therapeutics

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10548876B2 (en) 2013-07-26 2020-02-04 Race Oncology Ltd. Compositions to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof
US9974774B2 (en) 2013-07-26 2018-05-22 Race Oncology Ltd. Combinatorial methods to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof
US9993460B2 (en) 2013-07-26 2018-06-12 Race Oncology Ltd. Compositions to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof
US11147800B2 (en) 2013-07-26 2021-10-19 Race Oncology Ltd. Combinatorial methods to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof
US10500192B2 (en) 2013-07-26 2019-12-10 Race Oncology Ltd. Combinatorial methods to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof
US11135201B2 (en) 2013-07-26 2021-10-05 Race Oncology Ltd. Compositions to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof
CN105713075A (zh) * 2014-12-04 2016-06-29 北京睿德欧生物科技有限公司 一种EphB4 受体靶向多肽及其应用
CN105906691B (zh) * 2016-07-05 2019-09-13 珠海诺贝尔国际生物医药研究院有限公司 一种Eph激酶多肽抑制剂及其应用
CN105906691A (zh) * 2016-07-05 2016-08-31 珠海诺贝尔国际生物医药研究院有限公司 一种Eph激酶多肽抑制剂及其应用
CN113727737A (zh) * 2019-01-17 2021-11-30 卡斯西部储备大学 肿瘤细胞外基质中癌蛋白特异性肽pet/spect探针
US12398179B2 (en) 2019-03-14 2025-08-26 Sanford Burnham Prebys Medical Discovery Institute Nanomolar peptides and derivatives to differentially modulate ephrin receptors
CN114262362A (zh) * 2021-10-14 2022-04-01 齐鲁工业大学 一种靶向EphA2受体的68Ga-NODAGA-环状多肽FG01及制备方法与应用
CN114262362B (zh) * 2021-10-14 2023-05-26 齐鲁工业大学 一种靶向EphA2受体的68Ga-NODAGA-环状多肽FG01及制备方法与应用
US11964948B2 (en) 2022-06-07 2024-04-23 Actinium Pharmaceuticals, Inc. Bifunctional chelators and conjugates
US11975081B2 (en) 2022-06-07 2024-05-07 Actinium Pharmaceuticals, Inc. Bifunctional chelators and conjugates

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