WO2002056670A2 - Sondes d'imagerie activables - Google Patents

Sondes d'imagerie activables Download PDF

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WO2002056670A2
WO2002056670A2 PCT/US2002/000379 US0200379W WO02056670A2 WO 2002056670 A2 WO2002056670 A2 WO 2002056670A2 US 0200379 W US0200379 W US 0200379W WO 02056670 A2 WO02056670 A2 WO 02056670A2
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probe
chromophores
imaging
chromophore
attachment moiety
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PCT/US2002/000379
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WO2002056670A3 (fr
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Ralph Weissleder
Ching-Hsuan Tung
Umar Mahmood
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The General Hospital Corporation
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Priority to AU2002251739A priority Critical patent/AU2002251739A1/en
Priority to EP02720763A priority patent/EP1379284A4/fr
Publication of WO2002056670A2 publication Critical patent/WO2002056670A2/fr
Publication of WO2002056670A3 publication Critical patent/WO2002056670A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0082—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes
    • A61B5/0084—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes for introduction into the body, e.g. by catheters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0082—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes
    • A61B5/0084—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes for introduction into the body, e.g. by catheters
    • A61B5/0086—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes for introduction into the body, e.g. by catheters using infrared radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0093—Detecting, measuring or recording by applying one single type of energy and measuring its conversion into another type of energy
    • A61B5/0097—Detecting, measuring or recording by applying one single type of energy and measuring its conversion into another type of energy by applying acoustic waves and detecting light, i.e. acoustooptic measurements
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/41—Detecting, measuring or recording for evaluating the immune or lymphatic systems
    • A61B5/411—Detecting or monitoring allergy or intolerance reactions to an allergenic agent or substance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/41—Detecting, measuring or recording for evaluating the immune or lymphatic systems
    • A61B5/414—Evaluating particular organs or parts of the immune or lymphatic systems
    • A61B5/415—Evaluating particular organs or parts of the immune or lymphatic systems the glands, e.g. tonsils, adenoids or thymus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/41—Detecting, measuring or recording for evaluating the immune or lymphatic systems
    • A61B5/414—Evaluating particular organs or parts of the immune or lymphatic systems
    • A61B5/418—Evaluating particular organs or parts of the immune or lymphatic systems lymph vessels, ducts or nodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
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    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
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    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • A—HUMAN NECESSITIES
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    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/45—For evaluating or diagnosing the musculoskeletal system or teeth
    • A61B5/4528—Joints

Definitions

  • the invention relates to biochemistry, cell biology, and optical imaging.
  • Optically based biomedical imaging techniques have advanced over the past decade due to developments in laser technology, sophisticated reconstruction algorithms, and imaging software originally developed for non-optical, tomographic imaging modes such as CT and MRI. Visible wavelengths are used for optical imaging of surface structures by means of endoscopy and microscopy.
  • Near infrared wavelengths (approx. 600-1000 nm) have been used in optical imaging of internal tissues, because near infrared radiation exhibits tissue penetration of up to about fifteen centimeters. See, e.g., Wyatt, 1997, "Cerebral oxygenation and haemodynamics in the fetus and newborn infant," Phil. Trans. R. Soc. London B 352:701-706; and Tromberg et al., 1997, “Non-invasive measurements of breast tissue optical properties using frequency- domain photo migration,” Phil. Trans. R. Soc. London B 352:661-667.
  • near infrared imaging over other currently used clinical imaging techniques include the following: potential for simultaneous use of multiple, distinguishable probes (important in molecular imaging); high temporal resolution (important in functional imaging); high spatial resolution (important in in vivo microscopy); and safety (no ionizing radiation).
  • filtered light or a laser with a defined bandwidth is used as a source of excitation light.
  • the light may be continuous in intensity, pulsed, or may be modulated (for example by frequency or amplitude).
  • the excitation light travels through body tissues (but may remain near the surface, for example at the skin or at an endothelial surface).
  • contrast agent When the excitation light encounters a near infrared fluorescent molecule ("contrast agent”), the light is absorbed. The fluorescent molecule then emits light that has detectably different properties (i.e., spectral properties of the probe (slightly longer wavelength), e.g., fluorescence) from the excitation light.
  • properties i.e., spectral properties of the probe (slightly longer wavelength), e.g., fluorescence
  • conventional near infrared fluorescence probes are subject to many of the same limitations encountered with other contrast agents, including low target/background ratios.
  • the invention is based on the discovery of imaging probes that have altered optical properties after interaction with a target molecule, i.e., activation of the probe. This enables 1) detection of early disease, 2) a high target/background ratio for improved detection of subtle disease, and 3) non-invasive, imaging of internal molecular targets based on their biological activity.
  • the design of the new probes is based on various fluorescence activation strategies, e.g., fluorescence quenching/dequenching, wavelength shifts, polarization, and change in fluorescence lifetime.
  • the probes can be used to detect endogenous enzyme activity in disease, to monitor efficacy of inhibitors, to help guide surgical interventions, to determine therapeutic doses, and to image gene expression.
  • the invention features an imaging probe comprising a chromophore attachment moiety and one or more, e.g., a plurality of, chromophores, wherein the chromophores are chemically linked to the chromophore attachment moiety so that upon activation of the imaging probe, the optical properties of the chromophores are altered.
  • the probe is intramolecularly quenched.
  • the imaging probe includes one or more quencher molecules that quench the initial signal, wherein dequenching of the chromophores occurs upon activation of the probe.
  • two separate probes (which may be identical or may have different optical, biological, or chemical properties) become activated when they are in proximity to one another.
  • the probes can be activated by phosphorylation, dephosphorylation, pH mediated cleavage, conformation change, enzyme-mediated splicing, enzyme-mediated transfer of the one or more chromophores, hybridization of a nucleic acid sequence to a complementary target nucleic acid, binding of the probe to an analyte, chemical modification of the chromophore, or binding of the probe to a receptor.
  • the optical properties of the chromophores can be altered by dequenching, quenching, changes in wavelength, changes in fluorescence lifetime, changes in spectral properties, or changes in polarity or combinations thereof.
  • the chromophores can be fluorochromes, non-fluorescent chromophores, fluorescence quenchers, absorption chromophores, or combinations thereof.
  • the invention features a cell coupled to an imaging probe, where the imaging probe comprises a chromophore attachment moiety and one or more, e.g., a plurality of, chromophores wherein the chromophores are chemically linked to the chromophore attachment moiety so that upon activation of the imaging probe, a property of the chromophores are altered.
  • the cell may be a transformed cell or a transformed cell that expresses the imaging probe.
  • a “chromophore” includes, but is not limited to, a fluorocl rome, non-fluorochrome chromophore, fluorescence quencher, or absorption chromophore, including but not limited to organic and inorganic fluorochromes.
  • the imaging probe comprises a chromophore attachment moiety and a plurality of chromophores chemically linked to the chromophore attachment moiety so that upon activation, the optical properties of the chromophores are altered.
  • a "chromophore attachment moiety” is a biocompatible molecule, e.g., a backbone, to which two or more chromophores are chemically linked (directly or through a spacer) and maintained in spectral property altering permissive positions relative to one another.
  • chemically linked is meant connected by any attractive force between atoms strong enough to allow the combined aggregate to function as a unit. This includes, but is not limited to, chemical bonds such as covalent bonds (e.g., polar, or nonpolar), and non-covalent bonds such as ionic bonds, metallic bonds, and bridge bonds.
  • activation of an imaging probe is meant any change to the probe that alters a detectable property, e.g., an optical property, of the probe. This includes, but is not limited to, any modification, alteration, or binding (covalent or non-covalent) of the probe that results in a detectable difference in properties, e.g., optical properties of the probe, e.g., changes in the fluorescence signal amplitude (e.g., dequenching and quenching), change in wavelength, fluorescence lifetime, spectral properties, or polarity.
  • Optical properties include wavelengths, for example, in the visible, ultraviolet, near-infrared, and infrared regions of the electromagnetic spectrum.
  • Activation can be, without limitation, by enzymatic cleavage, enzymatic conversion, phosphorylation or dephosphorylation, conformation change due to binding, enzyme-mediated splicing, enzyme-mediated transfer of the cl romophore, hybridization of complementary DNA or RNA, analyte binding such as association with an analyte such as Na + , K + , Ca 2+ , Cl " , or another analyte, change in hydophobicity of the probe environment, and chemical modification of the chromophore.
  • Activation of the optical properties may or may not be accompanied by alterations in other detectable properties, such as (but not limited to) magnetic relaxation and bioluminescence.
  • an “activation site” is a site which, upon activation, confers a detectable, e.g., conformational, change to the probe.
  • an activation site can be a covalent bond within a probe, wherein said bond is: (1) cleavable by an enzyme present in a target tissue, and (2) located so that its cleavage liberates a chromophore from being held in an optical- quenching interaction-permissive position.
  • Optical-quenching interaction-permissive positions are the positions of two or more atoms to which chromophores can be chemically linked (directly or indirectly through a spacer) so that the chromophores are maintained in a position relative to each other that permits them to interact photochemically and quench each other's emitted signal.
  • a “protective chain” is a biocompatible moiety covalently linked to the chromophore attachment moiety to inhibit undesired biodegradation, clearance, or immunogenicity of the probe.
  • a “targeting moiety” is a moiety bound covalently or noncovalently to a probe, which moiety enhances the concentration of the probe in a target tissue relative to surrounding tissue.
  • the invention also features an activatable imaging probe that is activated by phosphorylation or dephosphorylation of the probe.
  • the phosphorylation can be mediated by a kinase
  • the dephosphorylation can be mediated by a phosphatase.
  • the probes can have one or more phophorylation sites, and these sites can be, or be part of the chromophore attachment moiety, or can be within a spacer between the chromophore attachment moiety and the chromophores.
  • the invention features an activatable imaging probe that includes a chromophore attachment moiety, a functional group, and one or more chromophores, wherein the chromophores are chemically linked to the chromophore attachment moiety so that upon activation of the probe the optical properties of the chromophores are altered, and wherein the probe is activated by enzyme-mediated removal of the functional group from the probe.
  • the functional group can be chemically linked to the chromophore attachment moiety or to a spacer between the chromophore attachment moiety and the chromophores.
  • the invention also includes an activatable imaging probe that has a chromophore attachment moiety and one or more chromophores, wherein chromophores are chemically linked to the chromophore attachment moiety so that upon activation of the imaging probe the optical properties of chromophores are altered, and wherein the probe is activated by enzyme-mediated splicing.
  • the probe can include a nucleic acid sequence specific for enzyme-mediated splicing.
  • the nucleic acid sequence specific for enzyme-mediated splicing can be, or be part of, the chromosome attachment moiety.
  • the nucleic acid sequence can be within a spacer between the chromophore attachment moiety and the chromophores.
  • the new probes can also include a transmembrane signal sequence, e.g., one derived from a TAT protein comprising a caspase-3 sensitive cleavage site or one having the sequence Gly-Arg-Lys-Lys-Arg-Gln-Arg-Arg (SEQ ID NO: 15) or Gly-Arg-Lys-Lys-Arg- Arg-Gln-Arg-Arg (SEQ ID NO: 16).
  • the invention also features in vivo optical imaging methods and probes for use in these methods.
  • the methods include: (a) delivering to the subject an imaging probe of claim 1; (b) allowing adequate time for the imaging probe to be activated within the target; (c) illuminating the target with light of a wavelength absorbable by the chromophores; (d) detecting a signal emitted by the chromophores; and (e) forming an optical image from the emitted signal.
  • steps (a) - (d) can be repeated at predetermined intervals to enable evaluation of the emitted signal of the chromophores in the subject over time.
  • steps (a) - (d) can be repeated at predetermined intervals to enable evaluation of the emitted signal of the chromophores in the subject over time.
  • These methods can be used to detect a disease in the subject, or to characterize a phenotype or genotype and/or severity of a disease in the subject.
  • the disease can be cancer, cardiovascular diseases, neurodegenerative diseases, immunologic diseases, autoimmune diseases, inherited diseases, infectious diseases, bone diseases, and environmental diseases.
  • the subject can be a mammal, including a human, or an animal model of a particular disease or disorder.
  • the invention also features an in vivo method for selectively imaging two or more cells or tissue types simultaneously, and probes for use in these methods.
  • the method includes administering to a subject two or more activatable imaging probes, each of the two or more probes comprises a chromophore whose optical properties is distinguishable from that of the other chromophore, and each of the two or more probes contains a different activation site.
  • the method therefore, allows the recording of multiple events.
  • One or both of these probes (or different portions of the same probe) may be activatable or unchanged after target interaction, thereby providing local tissue concentration of probe delivery in addition to activation.
  • the methods of the invention can be used to dete ⁇ nine a number of indicia, including tracking the localization of the imaging probe in a subject over time and assessing changes in the level of the imaging probe in the subject over time.
  • the methods of the invention can also be used in the detection, characterization (i.e., genotype and phenotype) and/or determination of the localization of a disease, the severity of a disease or a disease-associated condition.
  • Such disease or disease-conditions include inflammation (e.g., inflammation that results in arthritis, for example, rheumatoid arthritis), all types of cancer, cardiovascular disease (e.g., atherosclerosis and inflammatory conditions of blood vessels), dermatologic disease (e.g, Kaposi's Sarcoma, psoriasis), ophthalmic disease (e.g., macular degeneration and diabetic retinopathy), infectious disease, immunologic disease (e.g., Acquired Immunodeficiency Syndrome, lymphoma, type I diabetes, and multiple sclerosis), neurodegenerative disease (e.g., Alzheimer's disease), and bone-related disease (e.g., osteoporosis and primary and metastatic bone tumors).
  • inflammation e.g., inflammation that results in arthritis, for example, rheumatoid arthritis
  • cardiovascular disease e.g., atherosclerosis and inflammatory conditions of blood vessels
  • dermatologic disease e.g, Kaposi's Sarcoma, psori
  • the methods of the invention can therefore be used, for example, to determine the presence of tumor cells and localization of tumor cells, the presence and localization of inflammation, the presence and localization of vascular disease including areas at risk for acute occlusion (vulnerable plaques) in coronary and peripheral arteries and regions of expanding aneurysms, and the presence and localization of osteoporosis.
  • the methods can also be used to follow therapy for such diseases by imaging molecular events modulated by such therapy, including but not limited to determining efficacy, optimal timing, optimal dosing levels (including for individual patients or test subjects), and synergistic effects of combinations of therapy.
  • animal models are available and known in the art that mimic the progression and symptoms of several different human diseases.
  • animal models for multiple sclerosis, congestive heart failure, Alzheimer's disease, and Parkinson's disease have been established (Smith AH et al., 2000, J. Pharmacol. Toxicol. Methods, 43(2): 125; Hilliard, B et al., 2000, J. Immunol. 166(2): 1314; Yamada, K etal, 2000, Pharmacol. Ther. 88(2):93; Bonn, MC et al, 2000, Novartis Found. Symp. 231(70), discussion 89-93).
  • transgenic mice for breast cancer Hutchinson, JN et al., 2000, Oncogene 19(53):6130.
  • transgenic mice for breast cancer Hutchinson, JN et al., 2000, Oncogene 19(53):6130.
  • the invention also features in vitro and in vivo optical imaging methods for assessing activity of an agent.
  • the probes of the present invention may be used to assess molecular targets in vitro (e.g., in cell culture) and in vivo (e.g., animals or humans).
  • the in vitro method for assessing the efficacy of an agent includes: (a) administering to the sample a new imaging probe; (b) allowing time for a molecule in the sample to activate the probe, if the molecule is present; (c) illuminating the sample with light of a wavelength absorbable by the chromophores; (d) detecting a signal emitted from the chromophores; (e) forming an optical image from the emitted signal; (f) administering to the sample the agent and repeating steps (a)-(e); and (g) comparing the emitted signals and images of steps (d) and (e) over time or at different agent doses to assess the activity of the agent.
  • the sample can include, without limitation, cells, cell culture, tissue section, cytospin samples, or the like.
  • the in vivo method for assessing the efficacy of an agent includes: (a) administering to the subject an imaging probe; (b) allowing time for a molecule in a target tissue to activate the probe, if the molecule is present; (c) illuminating the target tissue with light of a wavelength absorbable by the chromophores; (d) detecting a signal emitted by the chromophores; (e) forming an optical image from the emitted signal; (f) administering to the subject the agent and repeating steps (a)-(e); and (g) comparing the emitted signals and images of steps (d) and (e) over time or at a different agent dose to assess activity of the agent.
  • the subject may be a mammal, including a human.
  • the methods are performed at least twice, once with and once without administering to the subject the agent, thereby providing a comparison of the outcome of the two methods for assessing the activity of the agent.
  • the methods may also be performed prior to administration of the agent to determine whether a target (e.g., a drug target) is present and/or expressed, and therefore whether the agent should be administered to the subject.
  • administration of the agent can be performed throughout the method including, without limitation, prior to administering the probe.
  • a portion of the probe can be detected by other means (including second fluorescent wavelength, bioluminescence, changes in magnetic properties, or gamma radiation) or a second probe can be administered to determine the local concentration of the activatable probe, by any of the above means.
  • the invention also includes a method for determining the presence of a composition (e.g., a drug or a polypeptide expressed by a gene, such as a gene introduced into the subject by gene therapy techniques) in a subject.
  • a composition e.g., a drug or a polypeptide expressed by a gene, such as a gene introduced into the subject by gene therapy techniques
  • the agent can be any compound, including, but not limited to, therapeutic compounds.
  • the agent can be an enzyme inhibitor, e.g., a proteinase, kinase, transferase, or polymerase inhibitor, or their upstream regulators.
  • the methods can therefore be used to identify the efficacy of therapeutic drug candidates. These methods can also be used to assess drug levels in a subject.
  • the methods of the present invention may be used to optimize drug therapy, e.g., to optimize the dose, timing and/or administration route of a given therapeutic agent.
  • the methods of the present invention may further be used for high throughput testing of therapeutic drug candidates (e.g., combinatorially designed therapeutic drug candidates).
  • the methods can also be used to select drug candidates for clinical testing.
  • the invention also features in vivo optical imaging methods for guiding therapeutic, e.g., surgical, interventions by: (a) administering to a subject an imaging probe including a chromophore attachment moiety and a plurality of chromophores wherein the plurality of chromophores are chemically linked to the chromophore attachment moiety so that upon activation of the imaging probe, the optical properties of the chromophores are altered; (b) allowing time for molecules in a target tissue to activate the probe, if the molecules and/or target tissue are present; (d) iUurriinating the target tissue with light of a wavelength absorbable by the chromophores; and (e) detecting the optical signal emitted by the chromophores.
  • the subject can be a mammal, including a human.
  • the invention can be used to help a physician or surgeon to identify and characterize areas of disease, such as colon polyps or vulnerable plaque, to distinguish diseased and normal tissue, such as detecting tumor margins that are difficult to detect using an ordinary operating microscope, e.g., in brain surgery, and help dictate a therapeutic or surgical intervention, e.g., by deterrnining whether a lesion is cancerous and should be removed or non-cancerous and left alone.
  • Figs. 1A and IB are schematic diagrams indicating the chemical components, and their structural arrangement, in probes representing two embodiments of the invention.
  • Figs. 2A and 2B are spectrophotometer scans of the near infrared chromophore, Cy5.5, before (Fig. 2A) and after (Fig. 2B) covalent linkage to PL-MPEG.
  • Fig. 3 is a bar graph summarizing data on intramolecular quenching and probe activation. The data were obtained using Cy-PL-MPEG probes with different levels of chromophore loading.
  • Fig. 4 is a schematic diagram illustrating the use of an endoscope in the invention.
  • the invention features an imaging probe including a chromophore attachment moiety and one or more, e.g., a plurality of, chromophores wherein the chromophores are chemically linked to the chromophore attachment moiety so that upon activation of the imaging probe, the properties, e.g., optical properties, of the chromophores are altered.
  • the probe is intramolecularly quenched.
  • the imaging probe includes one or more quencher molecules that quench the initial signal, wherein dequenching of the chromophores occurs upon activation of the probe.
  • a chromophore attachment moiety can be any biocompatible backbone that allows a plurality of chromophores to be covalently linked thereto.
  • the chromophore attachment moiety is a polymer, for example, a polypeptide, a polysaccharide, a nucleic acid, or a synthetic polymer.
  • the chromophore attachment moiety is a monomeric, dimeric, or oligomeric molecule.
  • Polypeptides useful as the chromophore attachment moiety include, for example, polylysine, albumins, and antibodies. Poly(L- lysine) is a useful polypeptide chromophore attachment moiety.
  • the cl romophore attacliment moiety also can be a synthetic polymer such as polyglycolic acid, polylactic acid, polyglutamic acid, poly(glycolic-colactic) acid, polydioxanone, polyvalerolactone, poly- ⁇ - caprolactone, poly(3-hydroxybutyrate, poly(3-hydroxyvalerate) polytartronic acid, and poly( ⁇ -malonic acid).
  • Activation sites can be located in the chromophore attachment moiety, e.g., when the chromophores are linked directly to ⁇ -amino groups of polylysine.
  • each cliromophore can be linked to the chromophore attachment moiety by a spacer, e.g., a spacer containing a chromophore activation site.
  • the spacers can be oligopeptides.
  • Oligopeptide sequences useful as a spacer include: Arg-Arg; Arg-Arg-Gly; Gly-Pro-Ile- Cys-Phe-Phe-Arg-Leu-Gly (SEQ ID NO:l); His-Ser-Ser-Lys-Leu-Gln-Gly (SEQ ID NO:2); Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys(FITC)-Gly-Asp-Glu-Val-Asp-Gly-
  • the imaging probe can include one or more protective chains covalently linked to the chromophore attachment moiety.
  • Suitable protective chains include polyethylene glycol, methoxypolyethylene glycol, methoxypolypropylene glycol, copolymers of polyethylene glycol and methoxypolypropylene glycol, polylactic-polyglycolic acid, poloxamer, polysorbate 20, dextran and its derivatives, starch and starch derivatives, and fatty acids and their derivatives.
  • the chromophore attachment moiety is polylysine and the protective chains are methoxypolyethylene glycol.
  • Chromophores useful in the new probes include near infrared chromophores such as
  • the chromophores can be covalently linked to the chromophore attachment moiety including the spacers, using any suitable reactive group on the chromophore and a compatible functional group on the cliromophore attachment moiety or spacer.
  • a probe according to the present invention can also include a targeting moiety such as an antibody, antigen-binding antibody fragment, a receptor-binding polypeptide, a receptor-binding polysaccharide, or a hydrophobic region.
  • the invention features a cell coupled to an imaging probe, where the imaging probe includes a chromophore attachment moiety and one or more, e.g., a plurality of, chromophores wherein the chromophores are chemically linked to the chromophore attachment moiety so that upon activation of the imaging probe, the optical properties of the chromophores are altered.
  • the cell may be isolated from primary tissue, transformed, or genetically engineered to express the imaging probe.
  • the imaging probe coupled to the cell may be used in the non-invasive in vivo optical imaging methods of the present invention.
  • the invention also features methods of optical imaging including the steps of delivering to a subject an imaging probe that includes a chromophore attachment moiety and a plurality of chromophores wherein the plurality of chromophores are chemically linked to the chromophore attachment moiety so that upon activation of the imaging probe, the optical properties of the chromophores are altered, allowing adequate time for the imaging probe to be activated within the target tissue, illuminating the target tissue with light of a wavelength absorbable by the chromophores, and detecting the signal emitted by the chromophores. These steps can be repeated at predetermined intervals thereby allowing the evaluation of emitted signal from the chromophore in a subject over time.
  • the methods can be performed either in vivo or in vitro.
  • the probe can also be coupled to a cell.
  • a cell coupled to an imaging probe is a cell expressing an imaging probe on its surface (e.g., an antibody or antibody fragment, a receptor or a ligand) or a cell transfected with a heterologous genetic construct that encodes an imaging probe.
  • the cell can be prokaryotic or eukaryotic.
  • Expression vectors containing a wide variety of regulatory elements are available and well known in the art. These vectors can be used to generate constructs capable of encoding an imaging probe. These constructs can be transiently transfected into a wide variety of cell types, including somatic cells, primary culture cells, and lymphoid cells. Alternatively, stable transfectants may be established from any number of well known cell lines, such as, but not limited to, HeLa, Daudi, K562, and COS cells.
  • Expression of the imaging probe in transfected cells can be regulated through the use of many different promoters known in the art.
  • Constitutively active promoters such as CMN (cytomegalovirus) or SN40 (Simian Virus 40) can be used.
  • inducible promoters such as the Tet system® and the Ecdysone-Inducible Expression System (with Ponasterone A)® (both available from Invitrogen, Inc.) can also be used and are commercially available and well known to those skilled in the art.
  • Probe architecture i.e., the particular arrangement of probe components, can vary as long as the probe retains a chromophore attachment moiety, and optionally spacers, and one or more, e.g., a plurality of, chromophores, e.g., near infrared chromophores, linked to the chromophore attachment moiety so that upon activation of the imaging probe, the optical properties of the chromophores are altered.
  • the activation sites can be in the backbone itself, as shown in Fig. 1A, or in side chains, as shown in Fig. IB. Although each chromophore in Figs. 1 A and IB is in a separate side chain, a pair of chromophores can be in a single side chain. In such an embodiment, an activation site is placed in the side chain between the pair of chromophores.
  • the probe comprises a polypeptide backbone containing only a small number of amino acids, e.g., 5 to 20 amino acids, with chromophores attached to amino acids on opposite sides of a protease cleavage (activation) site.
  • Guidance concerning various probe components, including backbone, protective side chains, chromophores, chromophore attachment moieties, spacers, activation sites and targeting moieties is provided in the paragraphs below.
  • the chromophore attachment moiety design will depend on considerations such as biocompatibility (e.g., toxicity and immunogenicity), serum half-life, useful functional groups (for conjugating chromophores, spacers, and protective groups), and cost.
  • backbones useful types of chromophore attachment moieties, also referred to herein as "backbones,” include polypeptides (polyamino acids), polyethyleneaticiannes, polysaccharides, aminated polysaccharides, aminated oligosaccharides, polyamidoamines, polyacrylic acids, and polyalcohols.
  • the backbone consists of a polypeptide formed from L- amino acids, D-amino acids, or a combination thereof.
  • Such a polypeptide can be, e.g., a polypeptide identical or similar to a naturally occurring protein such as albumin, a homopolymer such as polylysine, or a copolymer such as a D-Tyr-D-Lys copolymer.
  • a polypeptide identical or similar to a naturally occurring protein such as albumin, a homopolymer such as polylysine, or a copolymer such as a D-Tyr-D-Lys copolymer.
  • the ⁇ -amino "groups" on the side chains of the lysine residues can serve as convenient reactive groups for covalent linkage of chromophores and spacers (Figs. 1 A and IB).
  • the backbone is a polypeptide
  • the molecular weight of the probe can be from 2 kD to 1000 kD, e.g., from 4 kD to 500 kD.
  • the chromophore attacliment moieties can also be non-covalently associated complexes, such as liposomes. Chromophores may be attached to lipids before or after liposome formation. When these complexes interact with targets, the complexes can be activated, for example, without limitation, by quenching, de-quenching, wavelength shift, fluorescence energy transfer, fluorescence lifetime change, and polarity change.
  • the probes can be located entirely within such a liposome and released locally with disruption of the liposome (such as with acoustic resonance energy imparted at ultrasound frequencies), or can be attached at the lipid surface.
  • a chromophore attachment moiety can be chosen or designed to have a suitably long in vivo persistence (half-life). Therefore, protective chains are not necessary in some embodiments of the invention.
  • a rapidly biodegradable backbone such as polylysine can be used in combination with covalently linked protective chains.
  • useful protective chains include polyethylene glycol (PEG), methoxypolyethylene glycol (MPEG), methoxypolypropylene glycol, polyethylene glycol-diacid, polyethylene glycol monoamine, MPEG monoamine, MPEG hydrazide, and MPEG imidazole.
  • the protective chains can also be block-copolymers of PEG and a different polymer such as a polypeptide, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. Synthetic, biocompatible polymers are discussed generally in Holland et al., 1992, “Biodegradable Polymers,” Advances in Pharmaceutical Sciences, 6:101-164.
  • a useful backbone-protective chain combination is methoxypoly(ethylene)glycol- succinyl-N- ⁇ -poly-L-lysyine (PL-MPEG).
  • PL-MPEG methoxypoly(ethylene)glycol- succinyl-N- ⁇ -poly-L-lysyine
  • Modifications to the chromophore attachment moiety can also be made to improve delivery and activation.
  • graft copolymers can be modified to improve both the probes' biological properties and/or improve activation.
  • a 560 kD MPEG-PL graft copolymer randomly modified with Cy5.5 to yield a cathepsin B-sensitive probe (as described in the examples of U.S. Patent No. 6,083,486) was further modified to yield a succinilated probe, i.e., the positive charges on the probe were modified to neutral or negative charges by acetylation or succinilation, respectively, which demonstrated improved activation properties.
  • Membrane translocation signals can also be added to the imaging probes to improve deliverability. Since many graft copolymers can enter various cell types through fluid phase endocytosis, improvement of cellular uptake and assurance of cytoplasmic deposition of the imaging probe can be achieved by attaching membrane translocation (or transmembrane) signal sequences. These signal sequences can be derived from a number of sources including, without limitation, viruses and bacteria.
  • a Tat protein-derived peptide containing a caspase-3 sensitive cleavage site with the sequence — Gly-Arg-Lys-Lys- Arg-Arg-Gln-Arg-Arg-Arg-Lys(FITC)-Gly-Asp-GluNal-Asp-Gly-Cys(QSY7)-NH 2 - (SEQ ID NO: 3) has been shown to be efficiently internalized into cells for monitoring caspase-3 activity.
  • Gly-Arg-Lys-Lys-Arg-Gln-Arg-Arg SEQ ID NO: 15
  • Gly-Arg- Lys-Lys-Arg-Arg-Gln-Arg-Arg SEQ ID NO: 16
  • targeting and delivery approaches can also be used such as folate-mediated targeting (Leamon & Low, 2001, Drug Discovery Today, 6:44-51), liposomes, transferrin, vitamins, carbohydrates and the use of other ligands that target internalizing receptors, including, but not limited to, somatostatin, nerve growth factor, oxytocin, bombesin, calcitonin, arginine vasopressin, angiotensin II, atrial nati-uretic peptide, insulin, glucagons, prolactin, gonadotropin, and various opioids.
  • otlier ligands can be used that upon intracellular delivery, undergo an enzymatic conversion that leaves the resulting conversion product trapped within the cell, such as nitroheteroaromatic compounds that are irreversibly oxidized by hypoxic cells.
  • chromophores include the following: Cy5.5, Cy5 and Cy7 (Amersham, Arlington Hts., IL); IRD41 and IRD700 (LI- COR, Lincoln, NE); NIR-1 and IC5-OSu, (Dejindo, Kumamoto, Japan); Alexflour 660, Alexflour 680 (Molecular Probes, Eugene, OR), LaJoUa Blue (Diatron, Miami, FL); FAR- Blue, FAR-Green One, and FAR-Green Two (Innosense, Giacosa, Italy), ADS 790-NS and ADS 821-NS (American Dye Source, Montreal, Canada), indocyanine green (ICG) and its analogs (Licha et al., 1996, SPIE 2927:192-198; Ito et al., U.S.
  • ICG indocyanine green
  • Fluorescent lanthanide metals include europium and terbium. Fluorescence properties of lanthanides are described in Lackowicz, 1999, Principles of Fluorescence Spectroscopy, 2 nd Ed., Kluwar Academic, New York.
  • Imaging probes with excitation and emission wavelengths in the near infrared spectrum are preferred, i.e., 650-1300 nm. Use of this portion of the electromagnetic spectrum maximizes tissue penetration and minimizes absorption by physiologically abundant absorbers such as hemoglobin ( ⁇ 650 nm) and water (>1200 nm). Ideal near infrared chromophores for in vivo use exhibit the following characteristics: (1) narrow spectral characteristics, (2) high sensitivity (quantum yield), (3) biocompatibility, and (4) decoupled absorption and excitation spectra. Table 1 summarizes information on the properties of six commercially available near infrared chromophores.
  • chromophores can be used, it will be appreciated that the use of chromophores with excitation and emission wavelengths in other spectrums, such as the visible light spectrum, can also be employed in the compositions and methods of the present invention.
  • Intramolecular quenching by non-activated probes can occur by any of various quenching mechanisms.
  • Several mechanisms are known including resonance energy transfer between two chromophores.
  • the emission spectrum of a first chromophore should be very similar to the excitation of a second chromophore, which is in close proximity to the first cliromophore.
  • Efficiency of energy transfer is inversely proportional to r 6 , where r is the distance between the quenched chromophore and excited chromophore.
  • Self-quenching can also result from chromophore aggregation or excimer formation. This effect is concentration dependent. Quenching also can result from a non- polar-to-polar environmental change.
  • the chromophore can be covalently linked to a chromophore attachment moiety or spacer using any suitable reactive group on the chromophore and a compatible functional group on the chromophore attacliment moiety or spacer.
  • a carboxyl group (or activated ester) on a chromophore can be used to form an amide linkage with a primary amine such as the ⁇ -amino group of the lysyl side chain on polylysine.
  • chromophores are linked to the chromophore attachment moiety through spacers containing activation sites.
  • oligopeptide spacers can be designed to contain amino acid sequences recognized by specific proteases associated with target tissues.
  • two paired chromophores in quenching positions are in a single polypeptide side chain containing an activation site between the two chromophores.
  • a side chain can be synthesized as an activatable module that can be used as a probe er se, or linked to a backbone or targeting moiety, e.g., an albumin, antibody, receptor binding molecule, synthetic polymer or polysaccharide.
  • a useful conjugation strategy is to place a cysteine residue at the N-terminus or C-terrninus of the molecule and then employ SPDP for covalent linkage between the side chain of the terminal cysteine residue and a free amino group of the carrier or targeting molecule.
  • PSA Prostate Specific Antigen
  • prostatic epithelial cells a 33 kD chymotrypsin-like serine protease secreted exclusively by prostatic epithelial cells.
  • PSA concentrations are proportional to the volume of prostatic epithelium.
  • the release of PSA from prostate tumor cells is about 30-fold higher than that from normal prostate epithelium cells. Damage to basal membrane and deranged tissue architecture allow PSA to be secreted directly into the extracellular space and into the blood.
  • PSA protein-binding protein
  • PSA activity can be used as a marker for prostate tumor tissue.
  • prostate tumor tissue is highly enriched in PSA, therefore, spacers containing the amino acid sequence recognized by PSA can be used to produce an imaging probe that undergoes activation specifically in prostate tumor tissue.
  • An example of a PSA-sensitive spacer is His-Ser-Ser-Lys-Leu-Gln-Gly (SEQ ID NO:2).
  • PSA-sensitive spacers can be designed using information known in the art regarding the substrate specificity of PSA. See, e.g., 1997, Denmeade et al., Cancer Res. 57:4924-4930.
  • Cathepsin D an abundant lysosomal aspartic protease distributed in various mammalian tissues. In most breast cancer tumors, cathepsin D is found at levels from 2-fold to 50-fold greater than levels found in fibroblasts or normal mammary gland cells. Thus, cathepsin D can be a useful marker for breast cancer.
  • Spacers containing the amino acid sequence recognized by cathepsin D can be used to produce an imaging probe that undergoes activation specifically in breast cancer tissue.
  • cathepsin D-sensitive spacer is the oligopeptide: Gly-Pro-Ile-Cys-Phe-Phe-Arg-Leu-Gly (SEQ ID NO:l).
  • Other cathepsin D-sensitive spacers can be designed using information known in the art regarding the substrate specificity of cathepsin D. See, e.g., Gulnik et al., 1997, FEBSLet., 413:379-384.
  • Another example involves matrix metalloproteinases (MMPs).
  • MMPs matrix metalloproteinases
  • MMP-2 (gelatinase) in particular, has been identified as one of the key MMPs in these processes, being capable of degrading type IV collagen, the major component of basement membranes. Based on these observations, several companies have initiated the development of different MMP inhibitors to treat malignancies and other diseases involving pathologic angiogenesis.
  • MMP inhibitors may also be more effective when used in combination with chemotherapeutic agents.
  • a specific molecular target-based pharmacodynamic assessment of each therapeutic approach would therefore be highly desirable (for estimating the relative contributions of each agent and resulting synergies). For the reasons outlined above there is a need to directly detect and monitor proteinase activities in vivo in an intact tumor environment.
  • Spacers containing the amino acid sequence recognized by MMP -2 can be used to produce an imaging probe that undergoes activation specifically in cancer tissue expressing MMP -2.
  • An example of a MMP-2-sensitive spacer is the oligopeptide: GREG RGK(FITC)C-NH 2 (SEQ ID NO: 10).
  • Otlier MMP-2-sensitive spacers can be designed using information known in the art regarding the substrate specificity of MMP-2. In addition, other MMP probes can be designed accordingly.
  • Protease cleavage sites can be determined and designed using information and techniques known in the art including using various compound and peptide libraries and associated screening techniques (Turk et al., 2001, Nature Biotech., 19:661-667).
  • probe activation may be by cleavage of the backbone.
  • High chromophore loading of the backbone can interfere with backbone cleavage by activating enzymes such as cathepsins. Therefore, a balance between signal quenching and accessibility of the backbone by probe-activating enzymes is important. For any given backbone-chromophore combination (when activation sites are in the backbone) probes representing a range of cliromophore loading densities can be produced and tested in vitro to determine the optimal chromophore loading percentage.
  • chromophores When the chromophores are linked to the backbone through activation site-containing spacers, accessibility of the backbone by probe-activating moieties is unnecessary. Therefore, high loading of the backbone with spacers and chromophores does not significantly interfere with probe activation. For example, in such a system, every lysine residue of polylysine can carry a spacer and chromophore, and every chromophore can be released by activating enzymes. Accumulation of a probe in a target tissue can be achieved or enhanced by binding a tissue-specific targeting moiety to the probe. The binding can be covalent or non-covalent.
  • targeting moieties include a monoclonal antibody (or antigen-binding antibody fragment) directed against a target-specific marker, a receptor-binding polypeptide directed to a target-specific receptor, and a receptor-binding polysaccharide directed against a target- specific receptor.
  • Antibodies or antibody fragments can be produced and conjugated to probes of this invention using conventional antibody technology (see, e.g., Folli et al., 1994, "Antibody- Indocyanin Conjugates for Immunophotodetection of Human Squamous Cell Carcinoma in Nude Mice," Cancer Res., 54:2643-2649; Neri et al., 1997, “Targeting By Affinity-Matured Recombinant Antibody Fragments of an Angiogenesis Associated Fibronectin Isoform,” Nature Biotechnology, 15:1271-1275).
  • receptor-binding polypeptides such as somatostatin peptide, and receptor-binding polysaccharides can be produced and conjugated to probes of this invention using known techniques.
  • Other targeting and delivery approaches can also be used such as folate-mediated targeting approaches (Leamon & Low, 2001, Drug Discovery Today, 6:44-51), liposomes, transferrin, vitamins, carbohydrates and use of other ligands that target internalizing receptors including but not limited to nerve growth factor, oxytocin, bombesin, calcitonin, arginine vasopressin, angiotensin II, atrial nati-uretic peptide, insulin, glucagons, prolactin, gonadotropin, and various opioids.
  • ligands can be used that upon intracellular delivery, undergo an enzymatic conversion that leaves the resulting conversion product trapped in the cell, such as nitroheteroaromatic compounds that are irreversibly oxidized by hypoxic cells.
  • activation of the imaging probe can be achieved through phosphorylation or dephosphorylation of the probe. Phosphorylation is mediated through enzymes such as kinases, which are abundantly involved in signal transduction and function by adding a phosphate group to either serine, threonine or tyrosine amino acids.
  • kinases There are a number of different types of kinases including, without limitation, receptor tyrosine kinases, the Src family of tyrosine kinases, serine/threonine kinases and the Mitogen- Activated Protein (MAP) kinases. In addition, many of these molecules are associated with various disease states. Examples of kinases useful in the present invention and their associated diseases are listed in Table 3.
  • Receptor Tyrosine Kinases 1. Epidermal Growth Factor 1. cancers of the digestive tract, Receptor (EGFR) breast and colorectal cancer
  • VEGF angiogenesis Factor
  • PLC Protein Kinase C
  • MAP Mitogen- Activated Protein
  • phosphorylation is used to activate the probe.
  • the phosphorylation of the serine, threonine, or tyrosine amino acids will cause attraction of the negatively charged phosphate groups to the positively charged groups on the opposite molecule, thus bringing the chromophores into an interactive permissive position, causing changes in their optical parameters, e.g., quenching, dequenching, wavelength shift, fluorescence energy transfer, fluorescence life time change, or polarity change.
  • the molecules can be fluorescence dyes, quenchers, and/or inducers (i.e., a compound which causes fluorescence lifetime change or polarity change).
  • Phosphorylation may also increase the local hydrophilicity, thus decreasing the fluorescent resonance energy transfer between fluorochromes that is dependent upon local solvent concentration (e.g., resulting in decreased quenching).
  • activation can be accomplished by utilizing an enzyme that removes or modifies a functional group (e.g., a phosphate group) located on the spacer of the probe.
  • the probe is thus modified to incorporate a target sequence or chemical structure into a spacer that is then modified or removed from the spacer in order to activate the probe.
  • a phosphate-ester metabolizing enzyme such as an alkaline or acid phosphatase is used. These enzymes hydrolyze phosphate monoesters to an alcohol and inorganic phosphate.
  • enzymes useful in the present invention include conjugates of calf intestinal alkaline (CIP) phosphatase and PTP1B and PTEN phosphatase inhibitors, both of which have been currently developed for diabetes and gliomas, respectively.
  • Methylase enzymes covalently link methyl groups to adenine or cysteine nucleotides within restriction enzyme target sequences, thus rendering them resistant to cleavage by restriction enzymes.
  • a methylation enzyme such as S-adenosylmethionine may therefore be used to methylate a spacer of the imaging probe, thus rendering a quencher molecule resistant to restriction enzyme cleavage.
  • a demethylase such as purified 5-MeC-DNA glycosylase may be used to demethylate a spacer, thus allowing restriction enzyme cleavage of a quenching molecule and the subsequent dequenching of the chromophore.
  • probes containing mismatches or mutations in their sequence are provided wherein the function of specific DNA repair enzymes is used to activate the probe. For example, a mismatch within the spacer of the imaging probe, results in the signal being quenched. Upon the correction of this mismatch by the appropriate DNA enzyme, a conformational change occurs allowing the dequenching of the signal.
  • DNA repair There are several enzymes involved in DNA repair, including, without limitation, poly ADP-ribose polymerase (PARP), DNA polymerases a, ⁇ , and ⁇ and DNA ligase.
  • PARP poly ADP-ribose polymerase
  • DNA polymerases a Several human diseases are a result of deficiencies in DNA repair, including Ataxia- Telangiectasia, Xeroderma Pigmentosum, Cockayne Syndrome, and Santis-Caccione Syndrome.
  • the loss of mismatch repair enzyme function has also been associated with the early development of many cancers. , ' Mutations can be inserted into the probe DNA in several different ways.
  • some methods of mutagenesis include: (1) utilizing degenerate oligonucleotides to create numerous mutations in a small DNA sequence; (2) spacer-scanning using nested deletions and complementary nucleotides to insert point mutations throughout a sequence of interest; (3) spacer-scanning using oligonucleotide-directed mutagenesis; and (4) utilizing the polymerase chain reaction (PCR) to generate specific point mutations.
  • PCR polymerase chain reaction
  • ubiquitin-specific target sequences can be added to the probe wherein the ubiquination of the target sequence allows for the chromophores to be brought into close proximity, permitting energy transfer between the chromophores, thus activating the probe through any of thee mechanisms listed herein.
  • Ubiquination is an important process in the regulation of many biological processes, including angiogenesis and oxygen sensing.
  • VHL von Hippel-Lindau
  • pVHL tumor suppressor gene
  • HIF Hypoxia-Indicuble- Factor
  • Inhibitors of the ubiquination pathway include Lactocystin and the Calpain I inhibitor LLnL (N-acetyl-Leu-Leu-Norleucinal) (J. Biomol. Screen, 2000, 5(5):319-328).
  • specific target binding sites can be incorporated into the probe. These can include, without limitation, peptide substrates, enzyme binding sites, peptide sequences, sugars, RNA or DNA sequences, or other specific target binding sites or moieties.
  • the probe is activated upon the binding of the target binding site, e.g., a change in the spectral properties of the cliromophore occurs, for example, by adequate separation between the spacer and quencher. This is commonly referred to as a "molecular beacon.” Tyagi, 1998, Nature Biotech., 16:49.
  • cathepsin B-specific substrates include RRK(FITC)C-NH 2 (SEQ ID NO:4), GRRK(FITC)C-NH 2 (SEQ ID NO:5), GRRRRK(FITC)C-NH 2 (SEQ ID NO:6), GRRGRRK(FITC)C-NH 2 (SEQ ID NO:7), GFGSVQ:FAGK(FITC)C-NH 2 (SEQ ID NO:8) (Bioconjugate Chem 1999, 553), and GFLGGK(FITC)C-NH 2 (SEQ ID NO:9), (Bioconjugate Chem 2000, 132).
  • MMP substrate Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys(FITC)-Cys-NH 2 (SEQ ID NO: 10).
  • a monoclonal antibody (or antigen-binding antibody fragment) directed against a target-specific marker or a receptor-binding polypeptide or polysaccharide directed against a target-specific receptor may also be used to activate the probe.
  • Specific proteins include, but are not limited to, G protein coupled receptors, nuclear hormone receptors such as estrogen receptors, and receptor tyrosine kinases.
  • enzymes that are capable of transferring the chromophore are used to activate the probe.
  • Specific target sequences that are recognized by enzymes involved in recombination of DNA (recombinases) are incorporated into the probe.
  • the chromophore is transferred to another molecule (recombination) resulting in altered spectral properties of the chromophore or removal or alteration of the quencher from the spacer.
  • Enzymes involved in recombination are well known in the art.
  • recombinases are involved in immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements, a process involving the recombination of non-homologous gene segments, which occurs in immature B and T cells.
  • Ig immunoglobulin
  • TCR T cell receptor
  • the genes that encode these recombinases have been cloned and identified as RAG-1 and RAG-2.
  • the probes can be activated by incorporating into the probe target sequences for enzymes involved in RNA splicing.
  • This embodiment involves incorporating an RNA splicing sequence (e.g., an intron segment) on the spacer portion of the probe, resulting in the alteration of the spacer length. Activation is accomplished by either changing the spectral properties of the chromophore or by the removal or alteration of the quencher from the spacer of the probe.
  • RNA splicing sequence e.g., an intron segment
  • Activation is accomplished by either changing the spectral properties of the chromophore or by the removal or alteration of the quencher from the spacer of the probe.
  • snRNAs small nuclear RNAs
  • RNA molecules by precisely breaking sugar-phosphate bonds at the boundaries of introns and rejoining the free ends generated by intron removal into a continuous mRNA molecule.
  • rnRNAs that in turn encode for different but related proteins.
  • the thyroid hormone calcitonin and the calcitonin gene-related polypeptide found in hypothalamus cells are derived from the same pre-mRNA species, but due to alternative splicing, result in two different, but related proteins.
  • the invention features a fluorescent probe including a fluorochrome attachment moiety and a plurality of fluorochromes wherein the plurality of fluorochromes are chemically linked to the fluorochrome attachment moiety so that upon "activation" of the fluorescent probe by an analyte, the spectral properties of the fluorochromes are altered.
  • An “analyte” refers to a molecule or ion that binds to and activates fluorescent probes.
  • Such analytes include, but are not limited to H + , Ca 2+ , Na + , Mg 2+ , Mn 2+ , Cl " , Zn 2+ , O 2 , NO, Fe + , K + , and H 2 O 2 .
  • analyte binding is used to activate the probe.
  • the binding of the analyte to the activation site causes an analyte-induced conformational change, thus bringing the fluorochromes into an interaction permissive position, causing changes in their optical parameters, e.g., quenching, dequenching, wavelength shift, fluorescence energy transfer, fluorescence life time change, or polarity change.
  • the molecules can be fluorescent dyes, quenchers, and/or inducers (t.e., a compound which causes a fluorescence lifetime change or polarity change).
  • Peptides and polypeptides that selectively bind to analytes and undergo analyte- induced conformational changes are known, including peptides based on zinc fmger domains and calcium binding EF-hand domains (See, e.g., Berg and Merckle, J. Am. Chem. Soc, 1989, 111:3759-3761; Krizek et al., Inorg. Chem., 1993, 32:937-940; Krizek and Berg, Inorg. Chem., 1992, 31:2984-2986; Kim et al., J. Biol. Inorg. Chem., 2001, 6:173-81; and U.S. Patent No. 6,197,928).
  • a single zinc finger domain is 25-30 amino acids in length and has the consensus sequence (F/Y)-X-C-X 2 - 4 -C-X 3 -F-X 5 -L-X 2 -H-X 3 - 5 -H-X 2 - 6 (SEQ ID NO: 1
  • a single EF-domain is a helix-loop-helix motif that usually has 12 residues with the pattern, X-A-X-A-X-A-X-A-X-A-A-X (SEQ ID NO: 14), where X is an amino acid that participates in metal coord nation, e.g., histidine, glutamic acid, or aspartic acid, and A represents the intervening amino acids, which can be any amino acid (Bently, A.L. and Rety,
  • probe activation can be achieved by using the fluorochrome itself as a molecule that changes spectral properties after interaction with and/or binding to a specific analyte.
  • fluorochrome molecules that exhibit altered spectral properties after interaction with a specific analyte are commercially available and are well known (See Tsien R.Y., 1992, Probe of dynamic biochemical signals inside living cells.
  • fluorochrome sensors/indicators molecules examples include but are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to these fluorochrome molecules.
  • succimidyl esters that can be easily conjugated to primary amine groups, e.g., of peptides or other biologically compatible molecules.
  • near-infrared fluorochromes are useful, it will be appreciated that the use of fluorochromes with excitation and emission wavelengths in other spectrums, such as the visible light spectrum, can also be employed in the compositions and methods of the present invention.
  • BCECF has been used in vivo to measure the pH of gastrointestinal mucosa, which is an important factor in the detection of hypoxia-induced dysfunctions (Marechal et al., Photochem. Photobiol., 1999, 70:813-819) as well as for intracellular pH measurement during cerebral ischemia and reperfusion (Itoh et al., Keio J. Med., 1998, 47:37-41) and for non-invasively monitoring the in vivo pH in conscious mice (Russell et al., Photochem. Photobiol, 1994, 59:309-313).
  • probes can be activated by changes in H + ion concentration or pH changes.
  • Probes can be designed to contain spacers that are cleaved when physiological pH values are lowered. Examples of such spacers include alkylhydrazones, acylhydrazones, arylhydrazones, sulfonylhydrazones, imines, oximes, acetals, ketals, and orthoesters.
  • analyte activation described herein can be used to detect and/or evaluate many diseases or disease-associated conditions.
  • the redistribution of analytes such as potassium, sodium, and calcium is often indicative of certain physiological processes and diseases including hypoxia and ischemia (e.g., cerebro-vascular ischemia due to stroke, embolism or thrombosis; ischemia of the colon, vascular ischemia due to coronary artery disease of heart disease, ischemia due to physical trauma, poisons, ischemia associated with encephalopathy; and renal ischemia).
  • hypoxia and ischemia e.g., cerebro-vascular ischemia due to stroke, embolism or thrombosis; ischemia of the colon, vascular ischemia due to coronary artery disease of heart disease, ischemia due to physical trauma, poisons, ischemia associated with encephalopathy; and renal ischemia.
  • tumors are characterized by low pH values by comparison to normal tissue as well as inflammation, particularly inflammation caused by foreign pathogens
  • a quencher molecule is used to quench the initial signal. Prior to activation, the quencher molecule is situated such that it quenches the optical properties of the reporter molecule (t.e., cliromophore). Upon activation, the reporter molecule is de-quenched.
  • the reporter molecule and quencher molecule located on the probe will exhibit different signal intensities when the probe is active or inactive. It is therefore possible to determine whether the probe is active or inactive in a living organism by identifying a change in the signal intensity of the reporter molecule, the quencher molecule, or a combination thereof.
  • the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not activated, the probe can be designed such that the reporter molecule exhibits limited signal until the probe is either hybridized or digested.
  • the quencher DABCYL was utilized to record apoptosis associated caspase-3 activity using a near infrared chromophore (NIRM image at 700 NM). There was a significantly lower signal when caspace-3 inhibitor was present.
  • quenchers there are a number of quenchers available and known to those skilled in the art including, but not limited to, DABCYL, QSY-7 (Molecular probe), QSY-33 (Molecular probe), Fluorescence dyes such as Cy5 and Cy5.5 pare (Schobel, Bioconjugate 1999, 10, 1107), Fluorescent Isothiocyanates (FITC) and Rhodamine pair (Molecular Probes, Inc., OR).
  • An additional method of detection includes two distinct fluorochromes (fluorochrome 1 and fluorochrome2) that are spatially near one another such that fluorescent resonance energy transfer (FRET) takes place.
  • FRET fluorescent resonance energy transfer
  • Activation of the probe can be determined in this embodiment as loss of signal at the fluoiOchrome2 emission wavelength with excitation at fluorochromel excitation wavelength.
  • Signal increase at the fluorochromel emission wavelength after excitation at the fluorochromel excitation wavelength may aide the dete ⁇ nination of activation in this case.
  • Emission at the fluorochrome2 emission wavelength after excitation at the fluorochrome2 wavelength can also be used to determine local probe concentration.
  • the FRET method can be used to determine activation of probes when two components are brought into proximity after enzymatic activity (e.g., ubiquination), such that fluorochromel and fluorochrome2, which are initially spatially separated, are subsequently spatially near enough to each other so that FRET can take place.
  • enzymatic activity e.g., ubiquination
  • fluorochromel and fluorochrome2 which are initially spatially separated, are subsequently spatially near enough to each other so that FRET can take place.
  • activation is detected by exciting at the fluorochromel excitation wavelength and recording at the fluorochrome2 emission wavelength.
  • an imaging probe After an imaging probe is designed and synthesized, it can be tested routinely in vitro to verify a requisite level of signal before activation. Preferably, this is done by obtaining a signal value for the quenching, de-quenching, wavelength shift, fluorescence energy transfer, fluorescence life time change, polarity change of the fluorochrome-containing probe, etc. in a dilute, physiological buffer. This value is then compared to the signal value obtained from an equimolar concentration of free chromophore in the same buffer, under the same chromophore-measuring conditions. Preferably, this comparison will be done using a series of dilutions, to verify that the measurements are taking place on a linear portion of the signal value vs. cliromophore concentration curve.
  • the molar amount of a chromophore on a probe can be determined by one of ordinary skill in the art using any suitable technique.
  • the molar amount can be determined readily by near infrared absorption measurements.
  • the molar amount can be determined readily by measuring the loss of reactive linking groups on the backbone or spacer, e.g., decrease in ninhydrin reactivity due to loss of amino groups.
  • the chromophore signal emittance is measured before and after treatment with an activating agent, e.g., an enzyme. If the probe has activation sites in the backbone (as opposed to in spacers), de-quenching should be tested at various levels of chromophore loading, where "loading" refers to the percentage of possible chromophore linkage sites on the backbone actually occupied by chromophores.
  • an activating agent e.g., an enzyme
  • probe molecules free in cell culture medium should be non-detectable by fluorescence microscopy.
  • Cellular uptake should result in probe activation and a fluorescence signal from probe-containing cells.
  • Microscopy of cultured cells thus can be used to verify that activation takes place upon cellular uptake of a probe being tested.
  • Microscopy of cells in culture is also a convenient means for determining whether activation occurs in one or more subcellular compartments. It will be appreciated that the compositions and methods of the present invention may be used in combination with other imaging compositions and methods.
  • the methods of the present invention may be used in combination with traditional imaging modalities such as CT, PET/SPECT or MRI, and probes used in these methods can contain components, such as iodine, gadolinium atoms or radioactive isotopes, which change imaging characteristics of tissues when imaged using CT, PET, SPECT, or MR.
  • the probes of the present invention may be constructed using a plurality of chromophores chemically linked to chromophore attachment moieties with various magnetic properties, such as crosslinked iron oxide nanoparticle (CLIO).
  • CLIO crosslinked iron oxide nanoparticle
  • the imaging methods of the present invention can be combined with therapeutic methods.
  • an immediate anti-tumor therapy can be employed.
  • the probes themselves can contain a component that is therapeutic or becomes therapeutic after target interaction.
  • An imaging system useful in the practice of this invention typically includes three basic components: (1) a near infrared light source, (2) a means for separating or distinguishing emissions from light used for chromophore excitation, and (3) a detection system.
  • the light source provides monochromatic (or substantially monochromatic) near infrared light.
  • the light source can be a suitably filtered white light, i.e., bandpass liglit from a broadband source.
  • a suitably filtered white light i.e., bandpass liglit from a broadband source.
  • light from a 150- watt halogen lamp can be passed through a suitable bandpass filter commercially available from Omega Optical (Brattleboro, VT).
  • the light source is a laser. See, e.g., Boas et al., 1994, Proc. Natl. Acad. Sci. USA 91 :4887-4891 ; Ntziachristos et al., 2000, Proc. Natl. Acad. Sci. USA 97:2767-2772; Alexander, 1991, J. Clin. Laser Med. Surg. 9:416-418.
  • a high pass or bandpass filter 700 nm can be used to separate optical emissions from excitation light.
  • a suitable high pass or bandpass filter is commercially available from Omega Optical.
  • quantum dots a single excitation wavelength can be used to excite multiple different fluorochromes on a single probe or multiple probes (with different activation sites), and spectral separation with a series of bandpass filters, diffraction grating, or other means may be used to independently read the different activations.
  • the light detection system can be viewed as including a light gathering/image forming component and a light detection/image recording component.
  • a recording device may simply record a single (time varying) scalar intensity instead of an image.
  • a catheter- based recording device can record information from multiple sites simultaneously (i.e., an image), or may report a scalar signal intensity that is correlated with location by other means (such as a radio-opaque marker at the catheter tip, viewed by fluoroscopy).
  • a particularly useful light gathering/image forming component is an endoscope.
  • Endoscopic devices and techniques that have been used for in vivo optical imaging of numerous tissues and organs, including peritoneum (Gahlen et al., 1999, J. Photochem.
  • FIG. 4 shows a schematic representation of an endoscope for use with in new methods and probes.
  • catheter-based devices including fiber optics devices.
  • fiber optics devices are particularly suitable for intravascular imaging. See, e.g., Tearney et al., 1997, Science 276:2037-2039; Proc. Natl. Acad. Sci. USA 94:4256-4261.
  • Still other imaging technologies including phased array technology (Boas et al., 1994, Proc. Natl. Acad. Sci. USA 91:4887-4891; Chance, 1998, Ann. NY Acad. Sci. 838:29- 45), diffuse optical tomography (Cheng et al., 1998, Optics Express 3:118-123; Siegel et al., 1999, Optics Express 4:287-298), intravital microscopy (Dellian et al., 2000, Br. J. Cancer 82:1513-1518; Monsky et al, 1999, Cancer Res.
  • phased array technology Boas et al., 1994, Proc. Natl. Acad. Sci. USA 91:4887-4891; Chance, 1998, Ann. NY Acad. Sci. 838:29- 45
  • diffuse optical tomography Choeng et al., 1998, Optics Express 3:118-123; Siegel et al., 1999, Optics Express 4:287-298
  • Any suitable light detection/image recording component e.g., charge coupled device (CCD) systems, photomultiplier tubes, or photographic film, can be used in the invention.
  • CCD charge coupled device
  • the choice of light detection/image recording will depend on factors including type of light gathering/image forming component being used. Selecting suitable components, assembling them into a near infrared imaging system, and operating the system is within ordinary skill in the art.
  • an additional chromophore that emits light at a different near infrared wavelength is attached to the probe that is not in an optical- quenching interaction-permissive position.
  • two chemically similar probes, one activatable and one non-activatable, each labeled with a different chromophore can be used.
  • the activity of enzymes can be determined in a manner which is corrected for the ability of tissues to accumulate variable amounts of these probes. Both of these approaches can be used to monitor delivery of the probe, to track the probe, to calculate doses, and to serve as an internal standard for calibration purposes.
  • Pharmaceutically acceptable carriers, adjuvants, and vehicles may be used in the composition or pharmaceutical formulation of this invention. Included carriers, adjuvants, or and vehicles include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as albumin, buffer substances such as phosphate, glycine, sorbic acid, potassium sorbate, TRIS (tris(hydroxymethyl)amino methane), partial glyceride mixtures of fatty acids, water, salts or electrolytes, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polypropylene block polymers, sugars such as glucose, and suitable cryoprotectants.
  • ion exchangers alumina, aluminum stearate, lecithin, serum
  • the pharmaceutical compositions of the invention may be in the form of a sterile injectable preparation.
  • This preparation can be prepared by those skilled in the art of such preparations according to techniques known in the art.
  • the possible vehicles or solvents that can be used to make injectable preparations include water, Ringer's solution, and isotonic sodium chloride solution, and D5W.
  • oils such as mono- or di-glycerides and fatty acids such as oleic acid and its derivatives can be used.
  • the probes and pharmaceutical compositions of the present invention can be administered orally, parentally, by inhalation, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir.
  • parental administration includes intravenous, intramuscular, intra-articular, intra synovial, intrasternal, intrathecal, intraperitoneal, intracisternal, intrahepatic, intralesional, and intracranial injection or infusion techniques.
  • the probes may also be administered via catheters or through a needle to any tissue.
  • the pharmaceutical composition of the invention may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline.
  • the compositions can be formulated in ointments such as petrolatum.
  • the new pharmaceutical compositions can also be formulated in a suitable ointment, such as pefrolatum.
  • Transdermal patches can also be used. Topical application for the lower intestinal tract or vagina can be achieved by a suppository formulation or enema formulation.
  • the formulation of the probe can also include an antioxidant or some other chemical compound that prevents or reduces the degradation of the baseline fluorescence, or preserves the fluorescence properties, included but not limited to, quantum yield, fluorescence lifetime, excitation and emission wavelengths.
  • antioxidants or chemical compounds may include, but are not limited to, melatonin, dithiotreitol (dTT), defroxamine (DFX), methionine and N-acetyl cysteine. Dosing of the invention will depend on a number of factors including instrumentation sensitivity as well as a number of subject-related variables including animal species, age, body weight, mode of administration, sex, diet, time of administration, and rate of excretion.
  • the subject may be treated with an agent or regimen to enhance the imaging process.
  • an agent or regimen to enhance the imaging process.
  • a subject may be put on a special diet prior to imaging to reduce any auto- fluorescence or interference from ingested food, such as a low pheophorbide diet to reduce interference from fluorescent pheophorbides that are derived from some foods, such as green vegetables.
  • a cleansing regimen may be used prior to imaging, such as those cleansing regimens that are used prior to colonoscopies and include use of agents such as Visiciol.
  • the subject (patient or animal), may be treated with pharmacological modifiers to improve image quality.
  • enzymatic inhibitors may decrease background signal relative to target signal (secondary to proportionally lowering enzymatic activity of already low-enzymatic activity normal tissues to a greater extent than enzymatically-active pathological tissues) may improve the target to background ratio during disease screening.
  • pretreatment with methotrexate to relatively increase uptake in abnormal tissue i.e., metabolically active cancers
  • folate based targeted delivery may be employed.
  • Cy-PL-MPEG Cy5.5 was linked directly to the ⁇ -amino group of the polylysine side chains at various densities, which ranged from 0.1 % to 70% derivatization of the ⁇ -amino groups.
  • Cy-RRG-PL-MPEG Cy5.5 fluorochrome was linked to the polylysine by a spacer consisting of Arg-Arg-Gly.
  • Cy-GPICFFRLG-PL-MPEG the Cy 5.5 fluorochrome was linked to the polylysine by a spacer consisting of Gly-Pro-Ile-Cys- Phe-Phe-Arg-Leu-Gly (SEQ ID NO: 1). Trypsin and trypsin-like proteases are capable of cleaving the polylysine backbone of Cy-PL-MPEG, when it is only partially derivatized.
  • Probes Cy-RRG-PL-MPEG and Cy-GPICFFRLG-PL-MPEG were designed to allow fluorochrome cleavage of the spacer, but not necessarily the backbone.
  • the peptide spacer RRG sensitive to trypsin cleavage, was used to derivatize the PL-MPEG, and then Cy5.5 was linked to the N-terminus of the RRG spacers.
  • the cathepsin D sensitive peptide spacer, GPICFFRLG (SEQ ID NO:l) was similarly used to derivatize the PL- MPEG.
  • Cy5.5 commercially available as a monofunctional NHS-ester (Amersham, Arlington Heights, IL), was used according to the vendor's instructions, to label free ⁇ -amino groups of the polylysine backbone in PL-MPEG. Cy5.5 was added to a pre- ixed MPEG-PL solution (0.2 mg PL-MPEG in 1 ml 80 mM sodium bicarbonate solution) to a final concentration of 17 ⁇ M. After three hours, the reaction mixture was applied to a Sephadex® G25 (Pharmacia) column (12 cm) for separation of the reaction product (Cy-PL-MPEG) from the unreacted fluorochrome and other low-molecular weight components of the reaction mixture. Average fluorochrome loading was about 20%, i.e., 11 out of 55 free amino groups on the PL-MPEG labeled with Cy5.5 (based on TNBS assay and absorption measurement).
  • Fig. 2 A shows the excitation and emission spectra of Cy5.5 free in solution.
  • Fig. 2B shows the excitation and emission spectra of Cy5.5 fluorochrome of Cy-PL-MPEG.
  • the excitation and emission wavelengths of Cy5.5 are 675 nm and 694 nm, respectively.
  • the fluorescence level of the Cy-MPEG-PL was approximately 30-fold lower than that of the unbound Cy5.5.
  • chromophore loading i.e., percentage of ⁇ -amino groups on the polylysine backbone occupied by chromophore
  • Fig. 3 shows the relative fluorescent signal of Cy(n)-MPEG- PL (white bars) as a function of percentage of ⁇ -amino groups on the polylysine backbone occupied by fluorochrome.
  • 20% loading 11 of 55 groups
  • intramolecular quenching was observed, and the fluorescence signal was lowered in comparison to probes with lower fluorochrome loading.
  • fluorescence signal was recovered, as shown by the black bars in Fig. 3.
  • the next step in testing the imaging probe was to perform cell culture experiments.
  • Data obtained using amelanotic B16 melanoma cells confirmed our prediction and showed that: (1) the non-activated probe is non-fluorescent, (2) the probe is taken up by this cell line, and (3) cellular uptake results in activation of the probe and fluorescence signal detection.
  • a fiber optic light bundle with a 150 W halogen bulb (Fiberlite high intensity illuminator series 180, Dolan-Jennen Industries) provided broad spectrum white light.
  • a sharp cut off band pass optical filter (Omega Filter Corp., Brattleboro, VT) was mounted at the end of the fiber optic bundle to create a uniform excitation source in the 610-650 nm range. The light was placed approximately 15 cm above the imaging platform to provide homogenous illumination of the entire mouse.
  • Fluorescent (emission) photons were selected using a low pass filter with a sharp cut off at 700 nm (Omega Filter Corp.), although as stated above, laser sources and/or bandpass emission filters may alternatively be employed.
  • Cy5.5 dye has an excitation peak at approximately 670 nm, with a broad shoulder extending below 610 nm. Peak emission is at 694 nm.
  • Sharp cut-off filters with more than 5 OD attenuation combined with widely spaced frequencies for the filter set markedly decreased "cross talk" of incident excitation photons recorded as fluorescent emission signal. The narrow angle between light source and recording device ensured that only fluorescent emission photons or scattered photons that interacted with the mouse tissue reached the low pass filter.
  • the low-pass filter was mounted on a low power microscope
  • Example I To demonstrate the ability of the probes to image tumors, we tested the near intramolecularly-quenched infrared imaging probe (Cyn -PL-MPEG; 20% fluorochrome loading) in tumor-bearing mice. Nude mice bearing tumor line 9L or LX1 received 2 nmol of Cyi i -PL-MPEG intravenously. The mice were imaged by near infrared light immediately, and up to 36 hours after intravenous administration of the probe. The tumor was visible as an area of intense fluorescence, in contrast to the surrounding tissue. An increase in fluorescence signal within tumor was observed as a function of time, as the probe was internalized into tumor cells and became activated by endosomal hydrolases.
  • NIR fluorescence signal activation was linear over at least four orders of magnitude and specific when compared to scrambled nonsense substrates.
  • matched rodent tumor model cells implanted into nude mice expressing or lacking the targeted protease it could be shown that the former generated sufficient NIR signal to be directly detectable and that signal was significantly different compared to negative control tumors.
  • Representative optical images of the lower abdomen of a nude mouse implanted with a CaD+ and CaD- tumor were taken.
  • the CaD+ tumor emits fluorescence while the CaD- tumor has a significantly lower signal.
  • a thresholded false color map can be generated by superimposing a white light image with a fluorescence image.
  • the present invention may therefore be useful in detecting and evaluating cancers, and delineating tumor margins, wherein the probe is directed to tumor tissue.
  • Detection methods include, but are not limited to, reflective devices such as endoscopes, cameras, infrared goggles, and operating microscopes; and diffuse optical tomographic devices such as employed in Ntziachristos et al., 2000, Proc. Natl. Acad. Sci. USA 97:2767-2772.
  • a partial list of tumors include, but are not limited to tumors of the breast, prostate, colon, bronchi, lung, brain, ovary, muscle, fat, esophagus, head and neck, skin, small bowel, stomach, liver, adrenal gland, kidneys, bladder, pancreas, bone, ureters, blood vessels, and resultant metastases to lymph nodes and elsewhere.
  • Example 2 To demonstrate the ability of fluorescent probes to image colonic polyps, malignant and benign Apc-Min (C57 L/6J-Apc Mm ) mice, a strain highly susceptible to spontaneous intestinal adenoma fo ⁇ nation, were evaluated after the intravenous injection of 2 nmol per mouse of cathepsin B sensitive probe. Twenty-four hours after probe injection, animals were sacrificed and colons resected. White light and fluorescent images demonstrated the marked difference in fluorescent signal intensity in the polyps as compared to adjacent normal epithelium.
  • the resulting marked increase in contrast between normal and abnormal tissue may be exploited during colonoscopy (or endoscopy) to aid in lesion detection.
  • Example 3 To demonstrate the ability of the probes of the current invention to image ovarian cancer, very small peritoneal tumor deposits using CaD- and CaD+ cell lines (transfected 3Y1 rat embryonic tumor cell line) were implanted into mice intraperitoneally. The Cathepsin D probe described in more detail previously was then administered IN and the peritoneal surfaces were imaged 24 hours later using white light (i.e. as in conventional endoscopy) or at 700 nm ( ⁇ IRF imaging). Microscopic deposits of 300 ⁇ m could be readily detected by ⁇ IRF imaging that were not visible by white light imaging. The resulting marked increase in detection of minimal residual disease in ovarian cancer may be exploited during laproscopy (or endoscopy) to aid in lesion detection and to monitor therapy.
  • CaD- and CaD+ cell lines transfected 3Y1 rat embryonic tumor cell line
  • Example 4 To demonstrate the ability of the probes to image atherosclerosis, especially active or vulnerable plaques, control mice (C57BL/6) and Apoe-def ⁇ cient
  • mice which spontaneously develop arterial fatty streaks and atheromatous plaques, were evaluated after the intravenous injection of 2 nmol per mouse of a cathepsin B-sensitive probe. Twenty-four hours after probe injection, animals were sacrificed, and aortas were resected in toto from aortic root to beyond the iliac bifurcation. Using the previously described imaging system, white light and NIR fluorescent images of control and ApoE Mouse aortas were acquired. Plaque burden, as well as degree of plaque activity, was revealed in the fluorescent images, and was markedly different in control (minimal fluorescence) and ApoE mice (highly fluorescent). Fluorescent images were acquired under identical conditions, and were displayed using identical brightness parameters.
  • the present invention may therefore be useful in detecting and evaluating cardiovascular disease and helping guide surgical interventions, wherein the probe is directed to vascular tissue.
  • One method of administering the probes of the present invention to vascular tissue is via catheters or by disruption of probe-containing microbubbles by local deposition of resonant energy at ultrasound frequencies, both well known procedures.
  • Example 5 To demonstrate the ability of the probes to image inflammatory (rheumatoid) arthritis, arthritic and non-arthritic littermates were evaluated after the intravenous injection of 2 nmol per mouse of cathepsin B sensitive NIRF probe.
  • the K/BxN T cell receptor (TCR) transgenic mouse line derived from a cross of KRN/C57B1/6 TCR with the NOD strain (Matsumoto, et. al., Science, 286:1732-1735 (1999)), which develops a disease very similar to human rheumatoid arthritis in 50% of animals, while 50% of animals remain unaffected, was used.
  • White light and fluorescent images were acquired 24 hours after probe injection.
  • the foot of a non-arthritic mouse and of an arthritic mouse demonstrate: 1) the marked overall increased fluorescent signal intensity in affected joints in arthritic animals, and 2) the non-invasive visualization of the heterogeneous distribution of phenotypic (clinical) disease in inflammatory arthritis.
  • Probes of different polymer lengths were also used.
  • An approximately 120 kD cathepsin B sensitive probe was injected into arthritic mice. Fluorescent imaging at 24 hours again revealed the marked heterogeneity in distribution of disease, in this case between right and left feet in the same animal.
  • the present invention may therefore be useful in detecting and evaluating inflammatory diseases such as rheumatoid arthritis, wherein the probe is directed to inflammation. It may also be useful for measuring therapeutic efficacy against such diseases.
  • One method of a ⁇ ninistering the probes of the present invention to arthritis areas is via intrarticular injections, a well known procedure.
  • Example 6 Imaging of specific enzymes in osteoporosis development and its treatment are useful for drug development and/or clinical use.
  • Several proteases have been implicated in osteoporosis development, in particular cathepsin K, which is produced by osteoclasts. Numerous osteoclast inhibitors are in clinical use.
  • Specific peptide substrates for cathepsin K that can be utilized in the probes of the present invention include, but are no limited to, Z-Leu-Arg-AMC, Z-Pro-Arg-AMC, Z-Phe-Arg-AMC, and Z-Phe-Arg-pNA ((1999) Biochemistry, 38:13594-13583; (2000) Biochemistry, 39:529-536).
  • Example 7 To illustrate the ability of the probes of the present invention to image thrombosis, a thrombin probe was synthesized.
  • the design of the protease activatable NIRF probe was based on a long circulating graft copolymer as a delivery vehicle, the peptide substrate and a near infrared fluorochrome.
  • the biological fate of the long circulating polymer (a partially pegylated polylysine copolymer) has been extensively studied in animals and humans.
  • the circulation time of the polymer is over 20 hours in human and is thus ideally suited for vascular imaging application.
  • the synthesized 11-amino-acid peptide Gly-D-Phe-Pip-Arg-Ser-Gly-Gly-Gly-Gly-Gly- Lys(FITC)-Cys-NH2
  • the thrombin substrate sequence, D-Phe-Pip- Arg had a D-phenylanaline at the P3 position and an unusual amino acid pipecolic acid at the P2 position.
  • the substrate has a reported kcat/K of 3.94 x IO7M-1S-1.
  • the peptide was coupled to the polymer (PGC) using biofunctional iodoacetic anhydride as the connecting linker.
  • the unpegylated free amino groups on the PGC backbone were capped with iodoacetic anhydride, converting all amino groups into tliiol reactive groups, which were subsequently reacted with peptides.
  • monoreactive indocyanine fluorochrome Cy5.5 was conjugated to the Nterminus of each peptide.
  • each polymer molecule contained 23 reporter substrate/fluorochromes. With this high number of reporters, fluorescence was efficiently quenched in the inactivated state. Similar conjugation efficiency and optical characteristics were obtained for the control probe.
  • the prepared probes were first tested with purified thrombin in PBS buffer as the NIRF signal was recorded over time. Initially both probes showed low NIR fluroescence (150 arbitrary units (AU)) (Fig. 3A). Following addition of thrombin, NIRF signal increased from 150 AU to 4100 AU within 20 minutes (27 fold increase). This was significantly greater activation compared to the control probes, with only a 1-fold increase in NIRF signal within the same time frame. There was a clear dose response when the probe was incubated with different amounts of thrombin.
  • hirudin a direct thrombin inhibitor used in the clinical treatment of vascular thrombosis.
  • hirudin a direct thrombin inhibitor used in the clinical treatment of vascular thrombosis.
  • thrombin was added to solutions containing the thrombin probe and hirudin, significantly less NIRF signal was detected compared to hirudin-free solutions.
  • hirudin did not destroy or alter the optical probe, we added additional thrombin, which overcame hirudin activation, releasing a strong NIRF signal.
  • a home-built imaging system which has a bandpass excitation filter at 610-650 nm and an emission filter at 680- 720 nm was used to acquire NIRF image of activation with various probes.
  • Thrombin, control, cathepsin B and cathepsin D probes were incubated with thrombin, individually.
  • the NIRF and bright field images were acquired 10 min after incubation. Without thrombin, there was no detectable fluorescent signal in any of the probes. Within 10 min after thrombin addition however, NIR fluorescence signal was selectively generated by the thrombin probe.
  • Thrombosis is a central pathophysiologic feature of many cardiovascular diseases such as unstable angina and myocardial infarction, as well as deep venous thrombosis and pulmonary embolism. Rapid diagnosis of these potentially life-threatening conditions is necessary to minimize the associated morbidity and mortality.
  • Current diagnostic imaging methods are fiowbased (x-ray angiography, computed tomography angiography, magnetic resonance angiography, doppler ultrasound) or perfusion-based (nuclear medicine perfusion scans) and suffer from two important limitations. First, these methods do not directly image thrombus, and therefore cannot reliably distinguish between a thrombotic or nonthrombotic (e.g. cholesterol, lipid) obstruction to flow.
  • a thrombotic or nonthrombotic e.g. cholesterol, lipid
  • mice Twenty four hours after the intravenous injection of 2 nmol per mouse of cathepsin B sensitive probe, mice were imaged using white light, filter combinations sensitive to the cathepsin B probe, and filter combinations sensitive to GFP fluorescence. By reviewing the cathepsin B and GFP images, one can obtain a ratio image of the GFP image divided by the cathepsin B image. The difference in relative gene expression levels between the two tumors (GFP and cathepsin B expression), are revealed in this ratio image, which illustrates the utility of multi-channel imaging.
  • the major advantages of imaging different biological targets simultaneously and independently include the ability to 1) co-localize targets, 2) probe for differential expression levels of multiple targets, 3) analyze the combination of expression levels of particular importance in cancer, where one target alone is rarely overexpressed, 4) develop mini-arrays for in vivo target assessment, 5) image the temporal and spatial correlation of distinct biological pathways in disease, and 6) image the effects of therapy on different biological targets simultaneously, and 7) evaluate tissue characteristics by exogenous probe administration combined with intrinsic chromophore gene expression, such as intrinsic bioluminescence ⁇ i.e., tissues transfected to express luciferase) with exogenous activatable probe administration.
  • intrinsic chromophore gene expression such as intrinsic bioluminescence ⁇ i.e., tissues transfected to express luciferase
  • Example 9 The following example illustrates the ability of the probes of the present invention to image to identify the efficacy of therapeutic drug candidates and measure a dose response and to assess drug levels in a subject.
  • the synthesized probes contain a preferential MMP-2 peptide substrate.
  • Two different peptide substrates were used in this study, an MMP-2 cleavable peptide
  • the relative fluorescence increase at equimolar conditions for the different MMP's were (scaled to active MMP-2 set to 100%): MMP-1: 19%, MMP-7: 12%, MMP-8: 28% and MMP-9: 19%.
  • the increase in near infrared fluorescence following enzyme activation occurred over at least 4 orders of magnitude of enzyme concentration using a constant amount of MMP-2 probe.
  • fluorescence activation could be completely blocked by 1 mM of 1,10 phenanthroline, a broad-spectrum experimental MMP inhibitor that acts as a Zinc chelator.
  • MMP inhibitor that potently inhibits critical MMPs, such as MMP-2, MMP-3, MMP-9, MMP-13 and MMP-14, at picomolar concentrations.
  • MMP-2 5 U of MMP-2 and 19 pmol of imaging probe, we performed a dose response study of mediated MMP inhibition up to 0.1 mM of inhibitor. At the highest dose tested, the inhibitory effect was 80%.
  • Ki was 0.1 nM, similar to the 0.05 ⁇ 0.02 nM value described in the literature.
  • inhibitors e.g., 1,10 phenanthroline, complete inhibition was observed.
  • the HT1080 human fibrosarcoma tumor model was chosen because of its reported high MMP-2 production and the MMP-2 sensitivity of the developed probe; HT1080 cells also produce MMP-1, MMP-7, MMP-14, MMP-15, and MMP16 and to a lesser degree MMP-9.
  • the BT20 tumor model was chosen because of its relative lack of MMP-2 (confirmed by RT-PCR).
  • zymography was used to probe for MMP-2 activity. These experiments confirmed enzymatic activity both in conditioned medium as well as in tumor tissue (435 U MMP-2/g tumor tissue) of HT1080 cells.
  • the MMP-2 peptide substrate Gly-Pro-Leu-Gly-Val-Arg-Gly- Lys(FITC)-Cys-NH 2 (SEQ ID NO: 10) (the italicized amino acids correspond to the MMP-2 substrate) and the scrambled control peptide, Gly-Val-Arg-Leu-Gly-Pro-Gly-Lys(FITC)- Cys-NH 2 (SEQ ID NO: 13) were synthesized on an automatic peptide synthesizer (PS3, Rainin, Woburn, MA) and purified by reverse phase HPLC.
  • PS3, Rainin, Woburn, MA automatic peptide synthesizer
  • the molecular weight of peptides was confirmed by MALDI-MS and was 1275.59 ( ⁇ M + H ⁇ + , 1275.45(calc.)) for the substrate peptide and 1275.96 ( ⁇ M + H ⁇ + , 1275.45 (calc.)) for the control peptide.
  • the NIRF probes were prepared according to a previously optimized method in which cathepsin D was targeted.
  • a protected graft copolymer consisting of a 35 kD poly-L- lysine backbone and multiple 5 kD methoxy-polyethylene glycol side chains (MW 500 kD) was reacted with a large excess of iodoacetyl anhydride to convert all remaining amino groups into iodol groups.
  • Specific peptides were then attached to the iodoacetylated PGC through thiol specific reactions.
  • the monoreactive Cy5.5 dye (Amersham-Pharmacia, Piscataway, NJ) was attached to the N-terminus of the enzyme peptide substrate.
  • One unit is the activity that hydrolyzes 1 ⁇ g of type IV collagen within 1 hour using a commercially available assay (gelatinase 72 kD, Boehringer Mannheim, Indianapolis, IN). Reverse phase HPLC (Brownlee, Spheri-5, ODS, 30 x 4.6 mm), using 0.1% TFA and acetonitrile as elution buffers was performed (Rainin Instruments, Woburn, MA).
  • MMP-2 To test for the ability of MMP-2 to activate the entirely assembled imaging probe, a constant amount (26.6 pmol of imaging probe corresponding to 320 pmol Cy 5.5) was incubated with 6 U of activated MMP-2 (Boehringer-Mannheim, IN; activation was achieved with 2.5 mM of p-aminophenyl mercuric acid; APMA) and fluorescence was determined over time at ⁇ ex 675nm / ⁇ em 694 nm at multiple time points (Hitachi, U4500, Tokyo, Japan).
  • the control probe contained the scrambled peptide.
  • MMP-1 human rheumatoid synovial fibroblast, Calbiochem
  • MMP-2 human recombinant protein purified from mammalian cells, Calbiochem
  • MMP-7 human recombinant, E. Coli, Calbiochem
  • MMP-8 human neutrophil granulocyte, Calbiochem
  • MMP-9 human recombinant protein purified from mammalian cells, Oncogene Research Products. Seven pmole of each APMA activated enzyme were incubated for 10 minutes with 10 pmole of the probe at 37 ° and fluorescence was then determined. Fluorescence activation was scaled to that of APMA activated MMP-2 which was set as 100% (4.7 AU).
  • HT1080 fibrosarcoma and BT20 mammary adenocarcinoma cells obtained from the American Type Culture Collection (ATCC, Manassas, VA) were cultured in MEM medium with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 gL sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate and 10% heat inactivated fetal bovine serum. Cells were used for zymographic MMP-2 determinations when they were about 60% confluent.
  • MMP-2 enzyme activity of conditioned medium and tumor tissue was measured by zymography. Briefly, aliquots of the concentrated conditioned medium or tumor homogenate respectively were applied to a 7.5% SDS-PAGE containing 1 mg/ml gelatin. After protein separation, SDS was removed by washing of the gel in 2.5 % Triton x-100® (Sigma, St. Louis, MO). The gel was then incubated at 37°C in 50 mM Tris- HCL (pH 7.6) containing 0.2 M NaCl, 5 mM CaCl 2 and 0.02% Brij-35 for 8-16 hours and stained with 1% Coomassie brilliant blue in 30% methanol /10% glacial acetic acid.
  • HT1080 and BT20 cells were injected subcutaneously in the mammary fat pad of athymic nude mice (nu/nu, 5-6 weeks old). Tumors were allowed to grow to 2-3 mm in size.
  • the system consisted of the light-tight chamber equipped with a 150 W halogen white light source and an excitation bandpass filter (610-650 nm, Omega Optical, Brattlebore, NT). Light was homogeneously distributed over the field of view (FON) by light diffusers. Fluorescence was detected by a 12 bit monochrome CCD camera (Kodak, Rochester, ⁇ Y) equipped with a f/1.2 12.5-75 mm zoom lens and an emission long-pass filter at 700 nm (Omega Optical, Brattlebore, VT). Images were acquired over 30 seconds at 610- 640 nm excitation and 700 nm emission wavelength.
  • Image analysis was performed using commercially available software (Kodak Digital Science ID software, Rochester, ⁇ Y). Regions of interest (> 200 pixels) were placed over the tumor, the adjacent skin and a reference standard containing 10 nM free Cy 5.5 fluorescent dye imaged in identical position adjacent to each animal. Fluorescence signal was adjusted to this standard and expressed as described previously.
  • Tumors were excised, fixed for 24 hours in 10% phosphate buffered formalin, paraffin embedded and sectioned into 7 ⁇ m slices. Immunohistochemistry was performed using a primary polyclonal goat - antibody against human MMP-2 (Santa Cruz Biotechnology, Santa Cruz, CA). An alkaline phosphatase labeled rabbit anti-goat antibody was used to reveal binding of the primary antibody. Endogenous alkaline phosphatase (AP) activity was eliminated by heating (65 ° C for 30 minutes) and specific AP activity was visualized using ⁇ BT/BCIP substrate (Boehringer-Mannheim, IN). Sections were counter- stained with nuclear fast red. Control sections were processed identically however without the primary antibody.
  • AP endogenous alkaline phosphatase
  • NIRF fluorescence microscopy tumors were snap frozen and cryosectioned into 8-10 ⁇ m slices. Air dried sections were then viewed in phase contrast or fluorescence mode using an inverted epifluorescence microscope (Zeiss Axiovert, Thornwood, NY). Excitation wavelength was 650 nm. A cooled CCD camera (Sensys, Photometries, Arlington, AZ) adapted with a broad band filter (> 700 nm) was used for image capture.
  • the present invention therefore provides compositions and methods for recording native enzyme activities in tumors. This represents an invaluable in vivo tool for elucidation of the functional contribution of specific agents in tumorigenesis, metastagenesis and angiogenesis. Indeed, such measurements can be performed at different resolutions ranging from the microscopic cellular level (e.g., using intravital, confocal, or two photon microscopy) to the macroscopic whole tumoral level (e.g., near infrared diffuse optical tomography, phase array detection, or reflectance imaging). The methods of the present invention may also be used to image dose responses.
  • the microscopic cellular level e.g., using intravital, confocal, or two photon microscopy
  • the macroscopic whole tumoral level e.g., near infrared diffuse optical tomography, phase array detection, or reflectance imaging.
  • the methods of the present invention may also be used to image dose responses.
  • Tissue inhibitors of metalloproteinases (TIMP) Hydroxamates
  • Epoxide inhibitor Chomittry & Biology, 2000, 7, 569

Abstract

L'invention concerne des sondes d'imagerie activables, qui comprennent un fragment de liaison au chromophore, et un ou plusieurs chromophores, tels que des chromophores du proche infrarouge, chimiquement liés au fragment de liaison au chromophore, de sorte que, après activation de la sonde d'imagerie, les propriétés optiques de la pluralité de chromophores soient modifiées. La sonde comprend, facultativement, des chaînes de protection et/ou des espaceurs de chromophores. L'invention concerne en outre des procédés d'utilisation des sondes d'imagerie pour réaliser une imagerie optique.
PCT/US2002/000379 2001-01-05 2002-01-07 Sondes d'imagerie activables WO2002056670A2 (fr)

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WO2006027738A1 (fr) * 2004-09-10 2006-03-16 Philips Intellectual Property & Standards Gmbh Composes et procedes pour combinaison imagerie optique-imagerie par ultrasons
EP1685795A1 (fr) * 2005-01-26 2006-08-02 Fuji Photo Film Co., Ltd Dispositif d'acquisition d'image tomographique par fluorescence modulée par ultrasons
WO2006082434A1 (fr) * 2005-02-04 2006-08-10 Ge Healthcare Limited Nouveaux agents d'imagerie
WO2008005942A2 (fr) * 2006-06-30 2008-01-10 The Govt. Of The Usa As Represented By The Secretary Of The Dept. Of Health And Human Services. Sondes susceptibles d'être activées et procédés d'utilisation
US7354568B1 (en) 1997-10-27 2008-04-08 California Institute Of Technology Magnetic resonance imaging agents for the detection of physiological agents
WO2008104271A3 (fr) * 2007-02-28 2008-11-06 Sanofi Aventis Sondes d'imagerie
WO2009092062A2 (fr) * 2008-01-18 2009-07-23 Visen Medical, Inc. Agents d'imagerie fluorescents
US7727739B2 (en) 2002-04-19 2010-06-01 Ge Healthcare Uk Limited Methods for measuring enzyme activity
WO2010092114A1 (fr) 2009-02-13 2010-08-19 Guerbet Utilisation de tampons pour la complexation de radionucléides
WO2011012646A2 (fr) 2009-07-28 2011-02-03 F. Hoffmann-La Roche Ag Procédé non invasif d'imagerie optique in vivo
WO2011138462A1 (fr) 2010-05-07 2011-11-10 F. Hoffmann-La Roche Ag Procédé de diagnostic pour la détection de cellules ex vivo
US8190241B2 (en) 2000-11-27 2012-05-29 The General Hospital Corporation Fluorescence-mediated molecular tomography
WO2012075075A2 (fr) 2010-12-01 2012-06-07 Lumicell Diagnostics, Inc. Procédés et systèmes destinés à identifier spatialement des cellules anormales
WO2012084981A1 (fr) 2010-12-20 2012-06-28 Guerbet Nanoemulsion de chelate pour irm
WO2012119999A1 (fr) 2011-03-07 2012-09-13 F. Hoffmann-La Roche Ag Moyens et procédés destinés aux tests in vivo d'anticorps thérapeutiques
WO2012120004A1 (fr) 2011-03-07 2012-09-13 F. Hoffmann-La Roche Ag Sélection in vivo d'anticorps thérapeutiquement actifs
WO2012131345A2 (fr) 2011-03-25 2012-10-04 Almac Sciences (Scotland) Limited Essais enzymatiques
CN102985825A (zh) * 2011-05-24 2013-03-20 戴立军 一种异相生物分析试剂及其使用方法
WO2013045333A1 (fr) 2011-09-26 2013-04-04 Guerbet Nanoemulsions et leur utilisation comme agents de contraste
US8486373B2 (en) 1998-05-14 2013-07-16 The General Hospital Corporation Intramolecularly-quenched near infrared fluorescent probes
WO2014041546A1 (fr) * 2012-09-13 2014-03-20 Ben-Gurion University Of The Negev Research And Development Authority Agents de diagnostic à sensibilité/spécificité améliorées
WO2014114724A1 (fr) 2013-01-23 2014-07-31 Guerbet Magneto-emulsion vectorisee
US8926945B2 (en) 2005-10-07 2015-01-06 Guerbet Compounds comprising a biological target recognizing part, coupled to a signal part capable of complexing gallium
US8986650B2 (en) 2005-10-07 2015-03-24 Guerbet Complex folate-NOTA-Ga68
US9032965B2 (en) 2010-12-08 2015-05-19 Lumicell, Inc. Methods and system for image guided cell ablation with microscopic resolution
US9763577B2 (en) 2013-03-14 2017-09-19 Lumicell, Inc. Imaging agent for detection of diseased cells
WO2018118902A1 (fr) * 2016-12-19 2018-06-28 The Regents Of The University Of California Peptides sensibles à deux enzymes
EP3375353A1 (fr) * 2017-03-16 2018-09-19 Universität Zürich Imagerie photoacoustique d'un tissu enflammé
WO2020007822A1 (fr) 2018-07-02 2020-01-09 Conservatoire National Des Arts Et Metiers (Cnam) Nanoparticules de bismuth métallique (0), procédé de fabrication et utilisations de celles-ci
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US8486373B2 (en) 1998-05-14 2013-07-16 The General Hospital Corporation Intramolecularly-quenched near infrared fluorescent probes
US8190241B2 (en) 2000-11-27 2012-05-29 The General Hospital Corporation Fluorescence-mediated molecular tomography
US7727739B2 (en) 2002-04-19 2010-06-01 Ge Healthcare Uk Limited Methods for measuring enzyme activity
US20050265922A1 (en) * 2004-04-20 2005-12-01 Emory University Multimodality nanostructures, methods of fabrication thereof, and methods of use thereof
WO2006027738A1 (fr) * 2004-09-10 2006-03-16 Philips Intellectual Property & Standards Gmbh Composes et procedes pour combinaison imagerie optique-imagerie par ultrasons
CN101019028A (zh) * 2004-09-10 2007-08-15 皇家飞利浦电子股份有限公司 用于组合的光学－超声成像的化合物和方法
EP1685795A1 (fr) * 2005-01-26 2006-08-02 Fuji Photo Film Co., Ltd Dispositif d'acquisition d'image tomographique par fluorescence modulée par ultrasons
US7620445B2 (en) 2005-01-26 2009-11-17 Fujifilm Corporation Apparatus for acquiring tomographic image formed by ultrasound-modulated fluorescence
WO2006082434A1 (fr) * 2005-02-04 2006-08-10 Ge Healthcare Limited Nouveaux agents d'imagerie
US8926945B2 (en) 2005-10-07 2015-01-06 Guerbet Compounds comprising a biological target recognizing part, coupled to a signal part capable of complexing gallium
US8986650B2 (en) 2005-10-07 2015-03-24 Guerbet Complex folate-NOTA-Ga68
WO2008005942A3 (fr) * 2006-06-30 2008-07-31 Govt Of The Usa As Represented Sondes susceptibles d'être activées et procédés d'utilisation
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WO2011012646A2 (fr) 2009-07-28 2011-02-03 F. Hoffmann-La Roche Ag Procédé non invasif d'imagerie optique in vivo
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WO2012131345A2 (fr) 2011-03-25 2012-10-04 Almac Sciences (Scotland) Limited Essais enzymatiques
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