Note: Within nine months of the publication of the mention of the grant of the European patent in the European PatentBulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with theImplementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has beenpaid. (Art. 99(1) European Patent Convention).

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(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention of the grant of the patent: 23.07.2014 Bulletin 2014/30

(21) Application number: 01941895.3

(22) Date of filing: 01.06.2001

(51) Int Cl.:A61K 51/08 (2006.01) A61K 51/04 (2006.01)

(86) International application number: PCT/US2001/018060

(87) International publication number: WO 2001/091807 (06.12.2001 Gazette 2001/49)

(54) ETHYLENEDICYSTEINE (EC)-glucose analog CONJUGATES

ETHYLENEDICYSTEINE (EC)-Glucose Analoge KONJUGATE

CONJUGUES D’ETHYLENEDICYSTEINE (EC) ET D’UN analogue de la glucose, COMPOSITIONS ET PROCEDES POUR L’IMAGERIE DE MALADIES A SPECIFICITE TISSULAIRE

(84) Designated Contracting States: AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

(30) Priority: 02.06.2000 US 58758321.06.2000 US 599152

(43) Date of publication of application: 05.03.2003 Bulletin 2003/10

(60) Divisional application: 10181884.7 / 2 316 494

(73) Proprietor: Board of Regents, The University of Texas SystemAustin, TX 78701 (US)

(72) Inventors: • YANG, David, J.

Sugarland, TX 77030 (US)• LIU, Chun-Wei

Sugarland, TX 77479 (US)• YU, Dong-Fang

Houston, TX 77030 (US)• KIM, E., Edmund

Houston, TX 77024 (US)

(74) Representative: Dehmel, AlbrechtDehmel & Bettenhausen Patentanwälte Herzogspitalstrasse 1180331 München (DE)

(56) References cited: • ZARENEYRIZI, FERESHTEH ET AL: "Synthesis of

[99mTc] ethylenedicysteine - colchicine for evaluation of antiangiogenic effect" ANTI-CANCER DRUGS (1999), 10(7), 685-692 , XP001073905

• YANG, DAVID J. ET AL: "In vivo and in vitro measurement of apoptosis in breast cancer cells usin 99mTc-EC-annexin V" CANCER BIOTHERAPY & RADIOPHARMACEUTICALS (2001), 16(1), 73-83 , XP001073912

• YANG, DAVID J. ET AL: "Noninvasive assessment of tumor hypoxia with 99mTc labeled metronidazole" PHARMACEUTICAL RESEARCH (1999), 16(5), 743-750 , XP001073914

• ILGAN, SEYFETTIN ET AL: "99mTc- ethylenedicysteine -folate: A new tumor imaging agent. Synthesis, labeling and evaluation in animals" CANCER BIOTHERAPY & RADIOPHARMACEUTICALS (1998), 13(6), 427-435 , XP001073907

• YANG, D. J. (1) ET AL: "Molecular imaging using 99m-Tc-EC- nitroimidazole, and 99mTc - EC -annexin V in tumor-bearing rodents." PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL MEETING, (MARCH, 2000) NO. 41, PP. 766. PRINT.. MEETING INFO.: 91ST ANNUAL MEETING OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH. SAN FRANCISCO, CALIFORNIA, USA APRIL 01-05, 2000 , XP002201455

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• YANG, D. J. (1) ET AL: "Imaging tumor folate receptors using radiolabeled folate and methotrexate." JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, (JUNE, 1999) VOL. 42, NO. SUPPL. 1, PP. S696-S697. MEETING INFO.: XIIITH INTERNATIONAL SYMPOSIUM ON RADIOPHARMACEUTICAL CHEMISTRY ST. LOUIS, MISSOURI, USA JUNE 27-JULY 1, 1999 , XP001073918

• YANG, D. J. (1) ET AL: "99mTc - EC - deoxyglucose: Synthesis, cellular uptake, biodistribution and scintigraphic imaging." JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, (MAY, 2001) VOL. 44, NO. SUPPLEMENT 1, PP. S513-S514. PRINT. MEETING INFO.: FOURTEENTH INTERNATIONAL SYMPOSIUM ON RADIOPHARMACEUTICAL CHEMISTRY INTERLAKEN, SWITZERLAND JUNE 10-15, 2001 , XP001073917

• E.E. KIM ET AL.: "Synthesis, biodistribution and imaging of mammary tumors using 99m Tc-EC-Polyglutamate ; a glutamate receptor peptide." JOURNAL OF NUCLEAR MEDICINE, vol. 41, no. 5 SUP, May 2000 (2000-05), page 231P XP002201456 NEW YORK US

• DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US 1973 CROWE C C ET AL: ’SISOMICIN EVALUATION IN-VITRO AND COMPARISON WITH GENTAMYCIN AND TOBRAMYCIN’ Database accession no. PREV197356012915

• DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US 1984 ITOH S ET AL: ’BIOSYNTHESIS OF ASTROMICIN AND RELATED ANTIBIOTICS 1. BIOSYNTHETIC STUDIES BY BIOCONVERSION EXPERIMENTS’ Database accession no. PREV198579085590 & JOURNAL OF ANTIBIOTICS (TOKYO), vol. 37, no. 12, 1984, pages 1664-1669, ISSN: 0021-8820

• DATABASE MEDLINE [Online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US July 1998 MAINA N W ET AL: ’The activity of aminoglycoside antibiotics against Trypanosoma brucei.’ Database accession no. NLM17581012 & AFRICAN JOURNAL OF HEALTH SCIENCES 1998 JUL-DEC, vol. 5, no. 3-4, July 1998 (1998-07), pages 126-128, ISSN: 1022-9272

• Chun-Bao Zhu ET AL: "Role of Aminoglycoside 6’-Acetyltransferase in a Novel Multiple Aminoglycoside Resistance of an Actinomycete Strain #8. Inactivation of Aminoglycosides with 6’-Amino Group Except Arbekacin and Neomycin.", The Journal of Antibiotics, vol. 52, no. 10, 1 January 1999 (1999-01-01), pages 889-894, XP055070182, ISSN: 0021-8820, DOI: 10.7164/antibiotics.52.889

• DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US March 1999 MEYER JOETTE M ET AL: ’In-vitro synergy of paromomycin with metronidazole alone or metronidazole plus hydroxymetronidazole against Helicobacter pylori’ Database accession no. PREV199900216678 & JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY, vol. 43, no. 3, March 1999 (1999-03), pages 403-406, ISSN: 0305-7453

• GRIFFEY RICHARD H ET AL: "Determinants of aminoglycoside-binding specificity for rRNA by using mass spectrometry", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 96, no. 18, 31 August 1999 (1999-08-31), pages 10129-10133, XP002387285, ISSN: 0027-8424, DOI: 10.1073/PNAS.96.18.10129

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Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

[0001] The present invention relates generally to the fields of labeling, radioimaging and chemical synthesis. Moreparticularly, it concerns a strategy for radiolabeling target ligands. It further concerns methods of using those radiolabeledligands in tumor imaging and tissue-specific disease imaging.

2. Description of Related Art

[0002] Improvement of scintigraphic tumor imaging is extensively determined by development of more tumor specificradiopharmaceuticals. Due to greater tumor specificity, radiolabeled ligands as well as radiolabeled antibodies haveopened a new era in scintigraphic detection of tumors and undergone extensive preclinical development and evaluation.(Mathias et al., 1996, 1997a, 1997b). Radionuclide imaging modalities (positron emission tomography, PET; singlephoton emission computed tomography, SPECT) are diagnostic cross-sectional imaging techniques that map the locationand concentration of radionuclide-labeled radiotracers. Although CT and MRI provide considerable anatomic informationabout the location and the extent of tumors, these imaging modalities cannot adequately differentiate invasive lesionsfrom edema, radiation necrosis, grading or gliosis. PET and SPECT can be used to localize and characterize tumors bymeasuring metabolic activity.[0003] The development of new tumor hypoxia agents is clinically desirable for detecting primary and metastatic lesionsas well as predicting radioresponsiveness and time to recurrence. None of the contemporary imaging modalities accu-rately measures hypoxia since the diagnosis of tumor hypoxia requires pathologic examination. It is often difficult topredict the outcome of a therapy for hypoxic tumor without knowing at least the baseline of hypoxia in each tumor treated.Although the Eppendorf polarographic oxygen microelectrode can measure the oxygen tension in a tumor, this techniqueis invasive and needs a skillful operator. Additionally, this technique can only be used on accessible tumors (e.g., headand neck, cervical) and multiple readings are needed. Therefore, an accurate and easy method of measuring tumorhypoxia will be useful for patient selection. However, tumor to normal tissue uptake ratios vary depending upon theradiopharmaceuticals used. Therefore, it would be rational to correlate tumor to normal technique can only be used onaccessible tumors (e.g., head and neck, cervical) and multiple readings are needed. Therefore, an accurate and easymethod of measuring tumor hypoxia will be useful for patient selection. However, tumor to normal tissue uptake ratiosvary depending upon the radiopharmaceuticals used. Therefore, it would be rational to correlate tumor to normal tissueuptake ratio with the gold standard Eppendorf electrode measures of hypoxia when new radiopharmaceuticals areintroduced to clinical practice.[0004] [18F]FMISO has been used to diagnose head and neck tumors, myocardial infarction, inflammation, and brainischemia (Martin et al. 1992; Yeh et al. 1994; Yeh et al. 1996; Liu et al. 1994). Tumor to normal tissue uptake ratio wasused as a baseline to assess tumor hypoxia (Yet et al. 1996). Although tumor hypoxia using [18F]FMISO was clearlydemonstrated, introducing new imaging agents into clinical practice depends on some other factors such as easy avail-ability and isotope cost. Although tumor metabolic imaging using [18F]FDG was clearly demonstrated, introducing mo-lecular imaging agents into clinical practice depends on some other factors such as easy availability and isotope cost..[18F]fluorodeoxyglucose (FDG) has been used to diagnose tumors, myocardial infarction, and neurological disease. Inaddition, PET radiosynthesis must be rapid because of short half-life of the positron isotopes. 18F chemistry is alsocomplex. The 18F chemistry is not reproducible in different molecules. Thus, it would be ideal to develop a chelator whichcould conjugate to various drugs. The preferred isotope would be 99mTc due to low cost ($0.21/mCi vs. $50/mCi for 18F)and low energy (140 Kev vs. 571 Kev for 18F). 99mTc is easily obtained from a 99Mo generator. Due to favorable physicalcharacteristics as well as extremely low price, 99mTc has been preferred to label radiopharmaceuticals.[0005] Several compounds have been labeled with 99mTc using nitrogen and sulfur chelates (Blondeau et al., 1967;Davison et al., 1980). Bis-aminoethanethiol tetradentate ligands, also called diaminodithiol compounds, are known toform very stable Tc(V)O complexes on the basis of efficient binding of the oxotechnetium group to two thiolsulfur andtwo amine nitrogen atoms. 99mTc-L,L-ethylenedicysteine (99mTc-EC) is a recent and successful example of N2S2 chelates.EC can be labeled with 99mTc easily and efficiently with high radiochemical purity and stability, and is excreted throughthe kidney by active tubular transport (Surma et al., 1994; Van Nerom et al., 1990, 1993; Verbruggen et al., 1990, 1992).Other applications of EC would be chelated with galium-68 (a positron emitter, t1/2=68 min) for PET and gadolinium,iron or manganese for magnetic resonance imaging (MRI). 99mTc-EC-neomycin and 99mTc-EC-deoxyglucose weredeveloped and their potential use in tumor characterization was evaluated.[0006] Zareneyrizi F. et al., Anti-Cancer Drugs (1999), 10(7) 685-692 disclose the synthesis of [99mTc] ethylenedi-cysteine-colchicine for the evaluation of antiangiogenic effect.

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SUMMARY OF THE INVENTION

[0007] The present invention overcomes these and other drawbacks of the prior art by providing a new radiolabelingstrategy to target tissues for imaging. The invention provides a reagent for preparing a scintigraphic imaging agent asfurther specified in claim 1, a method of synthesizing such reagent, an imaging agent comprising such reagent, the useof the imaging agent, and a kit for preparing a radiopharmaceutical preparation.[0008] The present invention provides imaging agents for tissue-specific disease imaging. These agents include aradionuclide label chelated with ethylenedicysteine covalently linked to a tissue-specific ligand on one or both of its acidarms, wherein the tissue-specific ligand is an agent that mimics glucose selected from glucose, glucosamine, micromicin,and aminoglycosides.[0009] The ethylenedicysteine forms an N2S2 chelate with the radionuclide label. Of course, the chelate will includean ionic bond between the radionuclide and the chelating compound. The terms "EC-tissue-specific ligand conjugate,""EC-derivative" and "EC-drug conjugate" are used interchangeably herein to refer to the unlabeled ethylenedicysteine-tissue-specific ligand compound. As used herein, the term "conjugate" refers to a covalently bonded compound.[0010] Ethylenedicysteine is a bis-aminoethanethiol (BAT) tetradentate ligand, also known as diaminodithiol (DADT)compounds. Such compounds are known to form very stable Tc(V)O-complexes on the basis of efficient binding of theoxotechnetium group to two thiol-sulphur and two amine-nitrogen atoms. The 99mTc labeled diethylester (99mTc-L,L-ECD) is known as a brain agent. 99mTc-L,L-ethylenedicysteine (99mTc-L,L-EC) is its most polar metabolite and wasdiscovered to be excreted rapidly and efficiently in the urine. Thus, 99mTc-L,L-EC has been used as a renal functionagent. (Verbruggen et al. 1992).[0011] A tissue-specific ligand is a compound that, when introduced into the body of a mammal or patient, will specificallybind to a specific type of tissue. It is envisioned that the tissue-specific ligand according to the present either a poly-amino acid, a water-soluble peptide, or a single amino acid. For example, when the functional group on the tissue-specific ligand, or drug, is aliphatic or phenolic-OH, the linker may be poly-glutamic acid (MW about 750 to about 15,000),poly-aspartic acid (MW about 2,000 to about 15,000), bromo ethylacetate, glutamic acid or aspartic acid. When the drugfunctional group is aliphatic or aromatic-NH2 or peptide, the linker may be poly-glutamic acid (MW about 750 to about15,000), poly-aspartic acid (MW about 2,000 to about 15,000), glutamic acid or aspartic acid. When the drug functionalgroup is carboxylic acid or peptide, the linker may be ethylenediamine or lysine.[0012] While the preferred radionuclide for imaging is 99mTc, it is envisioned that other radionuclides may be chelatedto the EC-tissue-specific ligand conjugates or reagents of the invention, especially for use as therapeutics. For example,other useful radionuclides are 188Re, 186Re, 153Sm, 166Ho, 90Y, 89Sr, 67Ga, 68Ga, 111In, 153Gd, and 59Fe. The tissue-specific ligands are useful to deliver the therapeutic radionuclides to a specific lesion in the body, such as breast cancer,ovarian cancer, prostate cancer (using for example, 186/188Re-EC-folate) and head and neck cancer (using for example,186/188Re-EC-nitroimidazole).[0013] Specific embodiments of the present invention include 99mTc-EC-neomycin, 99mTc-EC-kanamycin, 99mTc-EC-aminoglycosides, (glucosamine, EC-deoxyglucose), 99mTc-EC-gentamy-cin, and 99mTc-EC-tobramycin.[0014] The present invention further provides a method of synthesizing a reagent according to the invention andradionuclide-labeling same for imaging or therapeutic use. The method includes obtaining a tissue-specific ligand asdefined in the claims, admixing the ligand with ethylenedicysteine (EC) to obtain an EC-tissue-specific ligand derivative,and admixing the EC-tissue-specific ligand derivative with a radionuclide and a reducing agent to obtain a radionuclide-labeled EC-tissue-specific ligand derivative, that is, an imaging agent according to the invention. The radionuclide ischelated to the EC via an N2S2 chelate. The tissue-specific ligand is conjugated to one or both acid arms of the ECeither directly or through a linker as described above. The reducing agent is preferably a dithionite ion, a stannous ion,or a ferrous ion.[0015] In another embodiment, the present invention provides a use of an imaging agent for the manufacture of apharmaceutical to image a tumor, an infection site, a heart, a lung, a brain, a liver, a spleen, a pancreas, or an intestine.The imaging agent can be use to image a site within a mammalian body. The imaging method includes the steps ofadministering an effective diagnostic amount of the imaging agent and detecting a radioactive signal from the radionuclide(e.g., 99mTc) localized at the site. The detecting step will typically be performed from about 10 minutes to about 4 hoursafter introduction of the imaging agent into the mammalian body. Most preferably, the detecting step will be performedabout 1 hour after injection of the imaging agent into the mammalian body.[0016] The site is an infection site, a tumor, a heart, a lung, a brain, a liver, a spleen, a pancreas, or an intestine. Thetumor or infection site may be located anywhere within the mammalian body but will generally be in the breast, ovary,prostate, endometrium, lung, brain, or liver. The site may also be a folate-positive cancer or estrogen-positive cancer.[0017] The invention also provides a kit for preparing a radiopharmaceutical preparation. The kit generally includes asealed vial or bag, or any other kind of appropriate container, containing a predetermined quantity of the reagent asfurther specified in claim 1 and a sufficient amount of a reducing agent to label the conjugate with 99mTc. In certaincases, the reagent also includes a linker, as defined above, between the ethylenedicysteine and the tissue-specific ligand.

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[0018] The components of the kit may be in any appropriate form, such as in liquid, frozen, or dry form. In a preferredembodiment, the kit components are provided in lyophilized form. The kit may also include an antioxidant and/or ascavenger. The antioxidant may be any known antioxidant but is preferably vitamin C. Scavengers may also be presentto bind leftover radionuclide. Most commercially available kits contain glucoheptonate as the scavenger. However,glucoheptonate does not completely react with typical kit components, leaving approximately 10-15% left over. Thisleftover glucoheptonate will go to a tumor and skew imaging results. Therefore, the inventors prefer to use EDTA as thescavenger as it is cheaper and reacts more completely.[0019] Further, a prognostic method for determining the potential usefulness of a candidate compound for treatmentof specific tumors is available. Currently, most tumors are treated with the "usual drug of choice" in chemotherapy withoutany indication whether the drug is actually effective against that particular tumor until months, and many thousands ofdollars, later. The imaging agents of the invention are useful in delivering a particular drug to the site of the tumor in theform of a labeled EC-drug conjugate and then imaging the site within hours to determine whether the particular drug iseffective.[0020] In that regard, the prognostic method includes the steps of determining the site of a tumor within a mammalianbody, obtaining an imaging agent which includes a radionuclide chelated to EC which is conjugated to a tumor-specificcancer chemotherapy drug candidate, administering the imaging agent to the mammalian body and imaging the site todetermine the effectiveness of the candidate drug against the tumor. Typically, the imaging step will be performed withinabout 10 minutes to about 4 hours after injection of the imaging agent into the mammalian body. Preferably, the imagingstep will be performed within about 1 hour after injection of the imaging agent into the mammalian body.[0021] Yet another embodiment of the present invention is a reagent for preparing a imaging agent, as defined in claim1. The reagent of the invention includes a tissuespecific ligand having an affinity for targeted sites in vivo sufficient toproduce a scintigraphically-detectable image, covalently linked to a radionuclide- (e.g.,99mTc-)binding moiety. The radi-onuclide-/99mTc-binding moiety is either directly attached to the tissuespecific ligand or is attached to the ligand througha linker as described above. The radionuclide-/99mTc-binding moiety is preferably an N2S2 chelate between 99mTc inthe +4 oxidation state and ethylenedicysteine (EC). The tissue-specific ligand will be covalently linked to one or bothacid arms of the EC, either directly or through a linker as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]

FIG. 24. Ratios of tumor to muscle and tumor to blood as function of time for 99mTc-Ec.FIG. 26. In vivo imaging studies in breast tumor bearing rats with 99mTc-EC.FIG. 36. Synthetic scheme of 99mTc-EC-neomycin.FIG. 37A. Scintigraphic image of breast tumor-bearing rats after administration of 99mTc-EC and 99mTc-EC-neomycin(100 mCi/rat, iv.) showed that the tumor could be well visualized from 0.5-4 hours postinjection.FIG. 37B. Scintimammography with 99mTc-EC- neomycin (30 mCi, iv.) of a breast cancer patient. Images taken twohours post-injection.FIG. 38A. 1H-NMR of EC.FIG. 38B. 1H-NMR of neomycin.FIG. 38C. 1H-NMR of EC-neomycin.FIG. 39. Mass spectrometry of EC-neomycin (M+ 1112.55).FIG. 40A. UV wavelength scan of EC.FIG. 40B. UV wavelength scan of neomycin.FIG. 40C. UV wavelength scan of EC-neomycin.FIG. 41. Radio-TLC analysis of 99mTc-EC-neomycin.FIG. 70. Effect of d- and 1-glucose on breast cellular (13762 cell line) uptake of 99mTc-EC- DG.FIG. 73. Effect of d- and 1-glucose on breast cellular (A549 cell line) uptake of 99mTc-EC- DG.FIG. 74. Effect of in vivo blood glucose level induced by glucosamine and EC-DG (1.2 mmol/kg, i.v.).FIG. 76. Tumor-to-tissue count density ratios of 99mTc-EC-deoxyglucose in breast tumor-bearing rats.FIG. 77. In vivo biodistribution of 99mTc-EC-deoxyglucose in breast tumor-bearing rats.FIG. 78. In vivo tissue uptake of 99mTc-EC-deoxyglucose in lung tumor-bearing mice.FIG. 79. In vivo tissue uptake of 99mTc-EC-neomycin in lung tumor-bearing mice.FIG. 81. Planar image of breast tumor-bearing rats after administration of 99mTc-EC and 99mTc-EC-deoxyglucose(100 mCi/rat, iv.) showed that the tumor could be well visualized from 0.5-4 hours postinjection.FIG. 82A. MRI of a patient with malignant astrocytoma.FIG. 82B. SPECT with 99mTc-EC-DG of a patient with malignant astrocytoma.FIG. 83A. MRI of a patient with hemorrhagic astrocytoma.

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FIG. 83B. SPECT with 99mTc-EC-DG of a patient with malignant astrocytoma.FIG. 84A. MRI of a patient with benign meningioma.FIG. 84B. SPECT with 99mTc-EC-DG of a patient with benign meningioma showed no focal intensed uptake.FIG. 85A. CT of a patient with TB in lung.FIG. 85B. SPECT with 99mTc-EC-DG of a patient with TB showed no focal intensed uptake.FIG. 86A. CT of patient with lung cancer.FIG. 86B. Whole body images of 99mTc-EC-DG of a patient with lung cancer.FIG. 86C. SPECT with 99mTc-EC-DG of a patient with lung cancer, the tumor showed focal intensed uptake.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0023] In the field of nuclear medicine, certain pathological conditions are localized, or their extent is assessed, bydetecting the distribution of small quantities of internally-administered radioactively labeled tracer compounds (calledradiotracers or radiopharmaceuticals). Methods for detecting these radiopharmaceuticals are known generally as imagingor radioimaging methods.[0024] In radioimaging, the radiolabel is a gamma-radiation emitting radionuclide and the radiotracer is located usinga gamma-radiation detecting camera (this process is often referred to as gamma scintigraphy). The imaged site isdetectable because the radiotracer is chosen either to localize at a pathological site (termed positive contrast) or, alter-natively, the radiotracer is chosen specifically not to localize at such pathological sites (termed negative contrast).[0025] A variety of radionuclides are known to be useful for radioimaging, including 67Ga, 99mTc, 111In 123I, 125I, 169Yb,or 186Re. Due to better imaging characteristics and lower price, attempts have been made to replace the 123I, 131I, 67Ga,and 111In labeled compounds with corresponding 99mTc labeled compounds when possible. Due to favorable physicalcharacteristics as well as extremely low price ($0.21/mCi), 99mTc has been preferred to label radiopharmaceuticals.Although it has been reported that a DTPA-drug conjugate could be labeled with 99mTc effectively (Mathias et al., 1997),the DTPA moiety does not chelate with 99mTc as stable as with 111In. (Goldsmith, 1997).[0026] A number of factors must be considered for optimal radioimaging in humans. To maximize the efficiency ofdetection, a radionuclide that emits gamma energy in the 100 to 200 keV range is preferred. To minimize the absorbedradiation dose to the patient, the physical half-life of the radionuclide should be as short as the imaging procedure willallow. To allow for examinations to be performed on any day and at any time of the day, it is advantageous to have asource of the radionuclide always available at the clinical site. 99mTc is a preferred radionuclide because it emits gammaradiation at 140 keV, it has a physical half-life of 6 hours, and it is readily available on-site using a molybdenum-99/technetium-99m generator.[0027] Bis-aminoethanethiol tetradentate ligands, also called diaminodithiol compounds, are known to form very stableTc(V)O-complexes on the basis of efficient binding of the oxotechnetium group to two thiolsulfur and two amine nitrogenatoms. (Davison et al., 1980;1981; Verbruggen et al., 1992). 99mTc-L,L-ethylenedicysteine (99mTc-EC) is the most recentand successful example of N2S2 chelates. (Verbruggen et al., 1992; Van Nerom et al., 1993; Surma et al., 1994). EC,a new renal imaging agent, can be labeled with 99mTc easily and efficiently with high radiochemical purity and stabilityand is excreted through kidney by active tubular transport. (Verbruggen et al., 1992; Van Nerom et al., 1993; Surma etal., 1994; Verbruggen et al., 1990;Van Nerom et al., 1990; Jamar et al., 1993). Other applications of EC would be chelatedwith gallium-68 (a positron emitter, tl/2 = 68 minutes) for PET and gadolinium, iron, or manganese for magnetic resonanceimaging (MRI).[0028] The present invention utilizes 99mTc- EC as a labeling agent to target ligands to specific tissue types for imaging.The advantage of conjugating the EC with tissue-targeting ligands is that the specific binding properties of the tissue-targeting ligand concentrates the radioactive signal over the area of interest. While it is envisioned that the use of 99mTc-EC as a labeling strategy can be effective with virtually any type of compound, some suggested preferred ligands areprovided herein for illustration purposes. It is contemplated that the 99mTc-EC-drug conjugates of the invention may beuseful to image not only tumors, but also other tissue-specific conditions, such as infection, hypoxic tissue (stroke),myocardial infarction, apoptotic cells, Alzheimer’s disease and endometriosis.[0029] Radiolabeled proteins and peptides have been reported in the prior art. (Ege et al., US. 4,832,940, Abrams etal., 1990; Bakker et al., 1990; Goldsmith et al., 1995, 1997; Olexa et al. 1982; Ranby et al. 1988; Hadley et al. 1988;Lees et al. 1989; Sobel et al. 1989; Stuttle, 1990; Maraganore et al. 1991; Rodwell et al. 1991; Tubis et al. 1968;Sandrehagen 1983). However, 99mTc-EC has not been used in conjunction with any ligands, other than as the diethylester(Kabasakal, 2000), prior to the present invention. The diethylester of EC was used as a cerebral blood flow agent(Kikukawa, et al., 2000).[0030] Although optimal for radioimaging, the chemistry of 99mTc has not been as thoroughly studied as the chemistryof other elements and for this reason methods of radiolabeling with 99mTc are not abundant. 99mTc is normally obtainedas 99mTc pertechnetate (TcO4

-; technetium in the +7 oxidation state), usually from a molybdenum-99/technetium-99m

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generator. However, pertechnetate does not bind well with other compounds. Therefore, in order to radiolabel a com-pound, 99mTc pertechnetate must be converted to another form. Since technetium does not form a stable ion in aqueoussolution, it must be held in such solutions in the form of a coordination complex that has sufficient kinetic and thermo-dynamic stability to prevent decomposition and resulting conversion of 99mTc either to insoluble technetium dioxide orback to pertechnetate.[0031] For the purpose of radiolabeling, it is particularly advantageous for the 99mTc complex to be formed as a chelatein which all of the donor groups surrounding the technetium ion are provided by a single chelating ligand - in this case,ethylenedicysteine. This allows the chelated 99mTc to be covalently bound to a tissue-specific ligand either directly orthrough a single linker between the ethylenedicysteine and the ligand.[0032] Technetium has a number of oxidation states: +1, +2, +4, +5, +6 and +7. When it is in the +1 oxidation state,it is called Tc MIBI. Tc MIBI must be produced with a heat reaction. (Seabold et al. 1999). For purposes of the presentinvention, it is important that the Tc be in the +4 oxidation state. This oxidation state is ideal for forming the N2S2 chelatewith EC. Thus, in forming a complex of radioactive technetium with the drug conjugates (reagents) of the invention, thetechnetium complex, preferably a salt of 99mTc pertechnetate, is reacted with the drug conjugates (reagents) of theinvention in the presence of a reducing agent.[0033] The preferred reducing agent for use in the present invention is stannous ion in the form of stannous chloride(SnCl2) to reduce the Tc to its +4 oxidation state. However, it is contemplated that other reducing agents, such as thedithionite or ferrous ion may be useful in conjunction with the present invention. It is also contemplated that the reducingagent may be a solid phase reducing agent. The amount of reducing agent can be important as it is necessary to avoidthe formation of a colloid. It is preferable, for example, to use from about 10 to about 100 mg SnCl2 per about 100 toabout 300 mCi of Tc pertechnetate. The most preferred amount is about 0.1 mg SnCl2 per about 200 mCi of Tc pertech-netate and about 2 ml saline. This typically produces enough Tc-EC-tissue-specific ligand conjugate for use in 5 patients.[0034] It is often also important to include an antioxidant in the composition to prevent oxidation of the ethylenedi-cysteine. The preferred antioxidant for use in conjunction with the present invention is vitamin C (ascorbic acid). However,it is contemplated that other antioxidants, such as tocopherol, pyridoxine, thiamine, or rutin, may also be useful.[0035] In general, the ligands for use in conjunction with the present invention will possess either amino or hydroxygroups that are able to conjugate to EC on either one or both acid arms. If amino or hydroxy groups are not available(e.g., acid functional group), a desired ligand may still be conjugated to EC and labeled with 99mTc using the methodsof the invention by adding a linker, such as ethylenediamine, aminopropanol, diethylenetriamine, aspartic acid, pol-yaspartic acid, glutamic acid, polyglutamic acid, or lysine.[0036] EC itself is water-soluble. It is necessary that the EC-drug conjugate (reagent) of the invention also be water-soluble. Many of the ligands used in conjunction with the present invention will be water-soluble, or will form a water-soluble compound when conjugated to EC. If the tissue-specific ligand is not water-soluble, however, a linker which willincrease the solubility of the ligand may be used. Linkers may attach to an aliphatic or aromatic alcohol, amine or peptideor to a carboxylic and/or peptide. Linkers may be either polyamino acids (peptides) or amino acids such as glutamicacid, aspartic acid, or lysine. Table 1 illustrates desired linkers for specific drug functional groups.

[0037] It is also envisioned that the EC-tissue-specific ligand drug conjugates (reagents) of the invention may bechelated to radionuclides and used for radionuclide therapy. Generally, it is believed that virtually any α, β-emitter, γ-emitter, or β, γ-emitter can be used in conjunction with the invention. Preferred β, γ-emitters include 166Ho, 188Re, 186Re,153Sm, and 89Sr. Preferred β-emitters include 90Y and 225Ac. Preferred γ-emitters include 67Ga, 68Ga, 64Cu, 62Cu, and111In. Preferred α-emitters include 211At and 212Bi. It is also envisioned that paramagnetic substances such as Gd, Mn,

Table 1

Drug Functional Group Linker Example

Aliphatic or phenolic -OH EC-Poly (glutamic acid) (MW. 750-15,000) or EC. poly(aspertic acid) (MW. 2000-15,000) or bromo ethylacetate or EC-glutamic acid or EC-aspertic acid.

A

Aliphatic or aromatic -NH2 or peptide

EC-poly(glutamic acid) (MW. 750-15,000) or EC-poly(aspertic acid) (MW. 2000-15,000) or EC-glutamic acid (mono- or diester) or EC-aspartic acid.

B

Carboxylic acid or peptide Ethylene diamine, lysine C

Examples:A. estradiol, topotecan, paclitaxel, raloxlfen etoposideB. doxorubicin, mitomycin C, endostatin, annexin V. LHRH, octreotide, VIPC. methotrexate, folic acid

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and Fe can be chelated with EC for use in conjunction with the present invention.[0038] Complexes and means for preparing such complexes are conveniently provided in a kit form including a sealedvial containing a predetermined quantity of an EC-tissue-specific ligand conjugate (reagents) of the invention to belabeled and a sufficient amount of reducing agent to label the conjugate with 99mTc. 99mTc-labeled scintigraphic imagingagents according to the present invention can be prepared by the addition of an appropriate amount of 99mTc or 99mTccomplex into a vial containing the reagent and reducing agent and reaction under conditions described in Example 1hereinbelow. The kit may also contain conventional pharmaceutical adjunct materials such as pharmaceutically accept-able salts to adjust the osmotic pressure, buffers, preservatives, antioxidants, and the like. The components of the kitmay be in liquid, frozen, or dry form. In a preferred embodiment, kit components are provided in lyophilized form.[0039] Radioactively labeled reagents or conjugates provided by the present invention are provided having a suitableamount of radioactivity. In forming 99mTc radioactive complexes, it is generally preferred to form radioactive complexesin solutions containing radioactivity at concentrations of about 0.01 millicurie (mCi) to about 300 mCi per mL.[0040] 99mTc labeled scintigraphic imaging agents provided by the present invention can be used for visualizing sitesin a mammalian body. In accordance with this invention, the 99mTc labeled scintigraphic imaging agents are administeredin a single unit injectable dose. Any of the common carriers known to those with skill in the art, such as sterile salinesolution or plasma, can be utilized after radiolabeling for preparing the injectable solution to diagnostically image variousorgans, tumors and the like in accordance with this invention. Generally, the unit dose to be administered has a radioactivityof about 0.01 mCi to about 300 mCi, preferably 10 mCi to about 200 mCi. The solution to be injected at unit dosage isfrom about 0.01 mL to about 10 mL. After intravenous administration, imaging of the organ or tumor in vivo can takeplace, if desired, in hours or even longer, after the radiolabeled reagent is introduced into a patient. In most instances,a sufficient amount of the administered dose will accumulate in the area to be imaged within about 0.1 of an hour topermit the taking of scintiphotos. Any conventional method of scintigraphic imaging for diagnostic or prognostic purposescan be utilized in accordance with this invention.[0041] The 99mTc-EC labeling strategy of the invention may also be used for prognostic purposes. It is envisioned thatEC may be conjugated to known drugs of choice for cancer chemotherapy. These EC-drug conjugates may then beradiolabeled with 99mTc and administered to a patent having a tumor. The labeled EC-drug conjugates (reagents) willspecifically bind to the tumor. Imaging may be performed to determine the effectiveness of the cancer chemotherapydrug against that particular patient’s particular tumor. In this way, physicians can quickly determine which mode oftreatment to pursue, which chemotherapy drug will be most effective. This represents a dramatic improvement overcurrent methods which include choosing a drug and administering a round of chemotherapy. This involves months ofthe patient’s time and many thousands of dollars before the effectiveness of the drug can be determined.[0042] The 99mTc labeled EC-tissue-specific ligand conjugates provided by the invention may be administered intra-venously in any conventional medium for intravenous injection such as in aqueous saline or in blood plasma medium.Such medium may also contain conventional pharmaceutical adjunct materials such as pharmaceutically acceptablesalts to adjust the osmostic pressure, buffers, preservatives, antioxidants and the like. Among the preferred media arenormal saline and plasma.[0043] Specific, preferred targeting strategies are discussed in more detail below.

Tumor Glycolysis Targeting

[0044] The radiolabeled ligands, such as polysaccharide (neomycin, kanamycin, tobramycin) and monosaccharide(glucosamine) bind to cell glucose transporter, followed by phosphorylation which are overexpressed on tumor cells(Rog-ers et al., 1968; Fanciulli et al., 1994; Popovici et al., 1971; Jones et al., 1973; Hermann et al., 2000). Polysaccharide(neomycin, kanamycin, tobramycin) and monosaccharide (glucosamine) induced glucose level could be suppressed byinsulin (Harada et al., 1995; Moller et al., 1991; Offield et al., 1996; Shankar et al., 1998; Yoshino et al., 1999; Villevalois-Cam et al., 2000) Since these ligands are not immunogenic and are cleared quickly from the plasma, metabolic imagingwould seem to be more promising compared to antibody imaging.

EXAMPLE 1: TUMOR FOLATE RECEPTOR TARGETING

Synthesis of EC

[0045] EC was prepared in a two-step synthesis according to the previously described methods (Ratner and Clarke,1937; Blondeau et al., 1967; each incorporated herein by reference). The precursor, L-thiazolidine-4-carboxylic acid,was synthesized (m.p. 195°, reported 196-197°). EC was then prepared (m.p. 237°, reported 251-253°). The structurewas confirmed by 1H-NMR and fast-atom bombardment mass spectroscopy (FAB-MS).

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Synthesis of aminoethylamido analogue of methotrexate (MTX- NH2)

[0046] MIX (227 ma, 0.5 mmol) was dissolved in 1 ml of HCI solution (2N). The pH value was <3. To this stirredsolution, 2 ml of water and 4 ml of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 6.609% in methanol, 1 mmol)were added at room temperature. Ethylenediamine (EDA, 0.6 ml, 10 mmol) was added slowly. The reaction mixture wasstirred overnight and the solvent was evaporated in vacuo. The raw solid material was washed with diethyl ether (10ml), acetonitrile (10 ml) and 95% ethyl alcohol (50 ml) to remove the unreacted EEDQ and EDA. The product was thendried by lyophilization and used without further purification. The product weighed 210 mg (84.7%) as a yellow powder.m.p. of product: 195-198 °C (dec, MIX); 1H-NMR (D2O) δ 2.98-3.04 (d, 8H, - (CH2)2CONH(CH0)2NH2), 4.16-4.71 (m,6H, -CH2- pteridinyl, aromatic-NCH3, NH-CH-COOH glutamate), 6.63-6.64 (d, 2H, aromatic-CO), 7.51-753 (d, 2H. aro-matic-N), 8.36 (s, 1H, pteridinyl). FAB MS m/z calcd for C22H28,N10,O4(M)+ 496.515, found 496.835.

Synthesis of aminoethylamido analogue of folate (Folate- NH2)

[0047] Folic acid dihydrate (1 g, 2.0 mmol) was added in 10 ml of water. The pH value was adjusted to 2 using HCI(2 N). To this stirred solution, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 1 g in 10 ml methanol, 4.0 mmol)and ethylenediamine (EDA, 1.3 ml, 18 mmol) were added slowly. The reaction mixture was stirred overnight at roomtemperature. The solvent was evaporated in vacuo. The product was precipitated in methanol (50 ml) and further washedwith acetone (100 ml) to remove the unreacted EEDQ and EDIT. The product was then freeze-dried and used withoutfurther purification. Ninhydrin (2% in methanol) spray indicated the positivity of amino group. The product weighed 0.6g (yield 60%) as a yellow powder. m.p. of product: 250° (dec). 1H-NMR (D2O) δ1.97-2.27 (m, 2H, -CH2 glutamate offolate), 3.05-3.40 (d, 6H, -CH2CONH(CH2)2NH2), 4.27-4.84 (m, 3H, -CH2-pteridinyl, NH-CH-COOH glutamate), 6.68-6.70(d, 2H, aromatic-CO), 7.60-7.62 (d, 2H, aromatic-N), 8.44 (s, 1H, pteridinyl). FAB MS m/z calcd for C21H25N9,O5(M)+

483, found 483.21.

Synthesis of ethylenedicysteine- folate (EC- Folate)

[0048] To dissolve EC, NaOH (2N, 0.1 ml) was added to a stirred solution of EC (114 ma, 0.425 mmol) in water (1.5ml). To this colorless solution, sulfo-NHS (92.3 mg, 0.425 mmol) and EDC (81.5 mg, 0.425 mmol) were added. Folate-NH2 (205 mg, 0.425 mmol) was then added. The mixture was stirred at room temperature for 24 hours. The mixture wasdialyzed for 48 hours using Spectra/POR molecular porous membrane with molecule cut-off at 500 (Spectrum MedicalIndustries Inc., Houston, TX). After dialysis, the product was freeze dried. The product weighed 116 mg (yield 35%).m.p. 195° (dec); 1H-NMR (D2O) δ1.98-2.28 (m, 2H, -CH2 glutamate of folate), 2.60-2.95 (m, 4H and -CH2-SH of EC).3.24-3.34 (m, 10H, -CH2-CO, ethylenediamine of folate and ethylenediamine of EC), 4.27-4.77 (m, 5H, -CH-pteridinyl,NH-CH-COOH glutamate of folate and NH-CH-COOH of EC), 6.60-6.62 (d, 2H, aromatic-CO), 7.58-7.59 (d, 2H. aromatic-N), 8.59 (s, 1H, pteridinyl). Anal. calcd for C29H37N11S2O8 Na2(8H2O), FAB MS m/z (M)+ 777.3 (free of water). C, 37.79;H. 5.75; N, 16.72; S, 6.95. Found: m/z (M)+ 777.7 (20), 489.4 (100). C, 37.40; H, 5.42; N. 15.43; S, 7.58.

Radiolabeling of EC-folate and EC with 99mTc

[0049] Radiosynthesis of 99mTc- EC-folate was achieved by adding required amount of 99mTc-pertechnetate into home-made kit containing the lyophilized residue of EC-folate (3 mg), SnCl2 (100 mg), Na2HPO4 (13.5 mg), ascorbic acid (0.5mg) and NaEDTA (0.5 mg). Final pH of preparation was 7.4. 99mTc-EC was also obtained by using home-made kitcontaining the lyophilized residue of EC (3 mg), SnCl2 (100 mg), Na2,IPO4 (13.5 mg), ascorbic acid (0.5 mg) and NaEDTA(0.5 mg) at pH 10. Final pH of preparation was then adjusted to 7.4. Radiochemical purity was determined by TLC (ITLCSG, Gelman Sciences, Ann Arbor, MI) eluted with, respectively, acetone (system A) and ammonium acetate (1M inwater):methanol (4:1) (system B). From radio-TLC (Bioscan, Washington, DC) analysis, the radiochemical purity was>95% for both radiopharmaceuticals.

TUMOR GLYCOLYSIS TARGETING

EXAMPLE 2: DEVELOPMENT OF 99mTc-EC-NEOMYCIN

Synthesis of EC

[0050] EC was prepared in a two-step synthesis according to the previously described methods (Ratner and Clarke,1937; Blondeau et al., 1967). The precursor, L-thiazolidine-4-carboxylic acid, was synthesized (m.p. 195°, reported196-197°). EC was then prepared (m.p. 237°, reported 251-253°). The structure was confirmed by 1H-NMR and fast-

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atom bombardment mass spectroscopy (FAB-MS).

Synthesis of Ethylenedicysteine-neomycin (EC-neomycin)

[0051] Sodium hydroxide (2N, 0.2 ml) was added to a stirred solution of EC (134 mg, 0.50 mmol) in water (5 ml). Tothis colorless solution, sulfo-NHS (217 mg, 1.0 mmol) and EDC (192 mg, 1.0 mmol) were added. Neomycin trisulfatesalt (909 mg, 1.0 mmol) was then added. The mixture was stirred at room temperature for 24 hours. The mixture wasdialyzed for 48 hours using Spectra/POR molecular porous membrane with cut-off at 500 (Spectrum Medical IndustriesInc., Houston, TX). After dialysis, the product was frozen dried using lyophilizer (Labconco, Kansas City, MO). Theproduct weighed 720 mg (yield 83%). The synthetic scheme of EC-neomycin is shown in FIG. 36. The structure isconfirmed by 1H-NMR (FIGS. 38A-B), mass spectrometry (FIGS. 39A-B) and elemental analysis (Galbraith Laboratories,Inc. Knoxville, TN). Elemental analysis C39H75N10S4O19.15H2O (C,H,N,S), Calc. C:33.77, H:7.58, N:10.11, S:9.23; foundC:32.44, H:5.90, N:10.47, S:10.58. UV wavelength of EC-neomycin was shifted to 270.5 nm when compared to EC andneomycin (FIGS. 40A-C)

Radiolabeling of EC-MN and EC-neomycin with 99mTc

[0052] Radiosynthesis of 99mTc-EC and 99mTc-EC-neomycin were achieved by adding required amount of 99mTc-pertechnetate into home-made kit containing the lyophilized residue of EC or EC-neomycin (10 mg), SnCl2 (100 mg),Na2HPO4 (13.5 mg) and ascorbic acid (0.5 mg). NaEDTA (0.5 mg) in 0.1 ml of water was then added. Final pH ofpreparation was 7.4. Radiochemical purity was determined by TLC (ITLC SG, Gelman Sciences, Ann Arbor, MI) elutedwith ammonium acetate (1M in water):methanol (4:1). From radio-TLC (Bioscan, Washington, DC) analysis (FIG. 41)and HPLC analysis (FIGS. 42-45), the radiochemical purity was >95% for both radiotracers.

Stability assay of 99mTc-EC and 99mTc-EC-neomycin

[0053] Stability of labeled 99mTc-EC and 99mTc-EC-neomycin were tested in dog serum samples. Briefly, 740 KBq of1 mg 99mTc-EC and 99mTc-EC-neomycin were incubated in dog serum (200 ml) at 37°C for 4 hours. The serum sampleswere diluted with 50% methanol in water and radio-TLC repeated at 0.5, 2 and 4 hours as described above.

Tissue distribution studies of 99mTc-EC-neomycin

[0054] Female Fischer 344 rats (150625 g) (Harlan Sprague-Dawley, Indianapolis, IN) were innoculated subcutane-ously with 0.1 ml of mammary tumor cells from the 13762 tumor cell line suspension (106 cells/rat, a tumor cell linespecific to Fischer rats) into the hind legs using 25-gauge needles. Studies performed 14 to 17 days after implantationwhen tumors reached approximately 1 cm diameter. Rats were anesthetized with ketamine (10-15 mg/rat, intraperito-neally) before each procedure.[0055] In tissue distribution studies, each animal was injected intravenously with 10-20 mCi of 99mTc-EC or 99mTc-EC-neomycin (n=3/time point). The injected mass of 99mTc-EC-neomycin was 200 mg per rat. At 0.5, 2 and 4 hours followingadministration of the radiotracers, the rats were sacrificed and the selected tissues were excised, weighed and countedfor radioactivity. The biodistribution of tracer in each sample was calculated as percentage of the injected dose per gramof tissue wet weight (%ID/g). Tumor/nontarget tissue count density ratios were calculated from the corresponding %ID/gvalues. When compared to 99mTc-EC and free technetium (Table 3), tumor-to tissue ratios increased as a function oftime in 99mTc-EC-neomycin group (Table 2).

Scintigraphic imaging studies

[0056] Scintigraphic images, using a gamma camera (Siemens Medical Systems, Inc., Hoffman Estates, IL) equippedwith low-energy, parallel-hole collimator, were obtained 0.5, 2 and 4 hours after i.v. injection of 100 mCi of each radiotracer.Compare to 99mTc-EC, high uptake in the tumors was observed (FIG. 37A). Preliminary clinical imaging studies wereconducted in a patient with breast cancer. The tumor was visualized well at 2 hours post-administration of 99mTc-EC-neomycin (FIG. 37B).

TABLE 2Biodistribution of 99mTc-EC-neomycin in Breast Tumor Bearing Rats

30 Min. 1 Hour 2 Hour 4 HourBlood 0.46360.007 0.26260.040 0.13960.016 0.08560.004

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In vitro cellular uptake of 99mTc-EC-drug conjugates

[0057] To evaluate the cellular uptake of 99mTc-EC-drug conjugates, each well containing 80,000 cells (A549 lungcancer cell line) was added with 2 mCi of 99mTc-EC-neomycin and 18F-FDG. After incubation at 0.5-4 hours, the cellswere washed with phosphate-buffered saline 3 times, followed by trypsin treatment to lose the cells. The cells were thencounted by a gamma counter. 99mTc-EC-neomycin showed highest uptake among those agents tested in human lungcancer cell line (FIG. 46).

(continued)

Biodistribution of 99mTc-EC-neomycin in Breast Tumor Bearing Rats

30 Min. 1 Hour 2 Hour 4 Hour

Lung 0.34460.011 0.20260.030 0.11460.014 0.08060.003Liver 0.33760.012 0.26960.013 0.22160.020 0.19560.012Stomach 0.27960.039 0.14760.001 0.06160.008 0.05460.008Spleen 0.15960.008 0.11460.013 0.09560.007 0.08960.003Kidney 8.39160.395 8.80460.817 8.35660.408 8.63860.251Thyroid 0.34960.008 0.20260.028 0.11460.007 0.08660.001

Muscle 0.09360.001 0.04960.010 0.02160.006 0.01060.001Intestine 0.15960.004 0.09360.014 0.06160.004 0.26660.200Urine 25.40268.621 21.78662.690 0.22460.000 2.60962.377Tumor 0.41960.023 0.27960.042 0.16660.023 0.13160.002

Brain 0.02260.001 0.01460.003 0.01060.001 0.00760.001

Heart 0.14760.009 0.08160.012 0.04060.004 0.02960.002

Tumor/Blood 0.90660.039 1.07060.028 1.19660.061 1.53660.029

Tumor/Muscle 4.51260.220 5.85560.458 8.36461.469 12.70660.783

Tumor/Brain 19.49561.823 20.00160.890 17.51562.035 20.25561.693

Values shown represent the mean 6 standard deviation of data from 3 animals.

TABLE 3Biodistribution of 99mTc Pertechnetate in Breast Tumor Bearing Rats

30 Min. 2 Hour 4 Hour

Blood 1.21860.328 0.66660.066 0.71560.052Lung 0.64660.291 0.63260.026 0.38760.024Liver 0.54160.232 0.30460.026 0.50160.081

Spleen 0.33160.108 0.18760.014 0.22560.017Kidney 0.63860.197 0.48960.000 0.93260.029Thyroid 24.82165.181 11.907615.412 17.23265.002Muscle 0.13060.079 0.07660.002 0.06360.003Intestine 0.15360.068 0.18660.007 0.34460.027Tumor 0.59160.268 0.32860.016 0.42360.091

Brain 0.03860.014 0.02260.002 0.03160.009

Heart 0.27560.089 0.14560.015 0.16660.012

Tumor/Blood 0.47260.093 0.49760.073 0.59760.144

Tumor/Muscle 4.78860.833 4.30260.093 6.68961.458

Tumor/Liver 1.08460.023 1.08460.115 0.86560.270

Values shown represent the mean 6 standard deviation of data from 3animals.

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Effect of glucose on cellular uptake of 99mTc-EC-neomycin and 18F-FDG

[0058] Neomycin is known to influence glucose absorption (Rogers et al., 1968; Fanciulli et al., 1994). Previousexperiments have shown that 99mTc-EC-neomycin has higher uptake than 18F-FDG in human lung cancer cell line(A549). To determine if uptake of 99mTc-EC-neomycin is mediated via glucose-related mechanism, glucose (0.1 mg-2.0mg) was added to each well containing either 50,000 (breast) cells or 80,000 cells (lung) along with 2 mCi of 99mTc-EC-neomycin and 18F-FDG. After incubation, the cells were washed with phosphate buffered saline 3 times, followed bytrypsin treatment to lose the cells. The cells were then counted by a gamma counter.[0059] By adding glucose at the concentration of 0.1-2.0 mg/well, decreased uptake of 99mTc-EC-neomycin in twolung cancer cell lines and one breast cell line was observed. Similar results were observed in 18F-FDG groups. 99mTc-EC (control) showed no uptake. The findings suggest that the cellular uptake of 99mTc-EC-neomycin may be mediatedvia glucose-related mechanism (FIGS. 47, 48A and 48B).

EXAMPLE 3: TUMOR METABOLIC IMAGING WITH 99mTc-EC-DEOXYGLUCOSE

Synthesis of EC-deoxyglucose (EC-DG)

[0060] Sodium hydroxide (1N, 1 ml) was added to a stirred solution of EC (110 mg, 0.41 mmol) in water (5 ml). Tothis colorless solution, sulfo-NHS (241.6 mg, 1.12 mmol) and EDC (218.8 mg, 1.15 mmol) were added. D-Glucosaminehydrochloride salt (356.8 mg, 1.65 mmol) was then added. The mixture was stirred at room temperature for 24 hours.The mixture was dialyzed for 48 hours using Spectra/POR molecular porous membrane with cut-off at 500 (SpectrumMedical Industries Inc., Houston, TX). After dialysis, the product was frozen dried using lyophilizer (Labconco, KansasCity, MO). The product in the salt form weighed 568.8 mg. The synthetic scheme is shown in Figure 59. The structurewas confirmed by mass spectrometry (FIG. 60) and proton NMR (FIGS. 61 and 62). Radiochemical purity of 99mTc-EC-DG was 100% as determined by radio-TLC (FIG. 63) and HPLC (FIGS. 64 and 65) analysis.

Hexokinase assay

[0061] To determine if EC-DG mimics glucose phosphorylation, a hexokinase assay was conducted. Using a readymade kit (Sigma Chemical Company), EC-DG, glucosamine, and glucose (standard) were assayed at UV wavelength340 nm. Glucose, EC-DG, and glucosamine showed positive hexokinase assay (FIGS. 66-68).

In vitro cellular uptake assay

[0062] In vitro cellular uptake assay was conducted by using a human lung cancer cell line (A549). Two mCi of 99mTc-EC-DG and 18F-FDG were added to wells containing 80,000 cells each. After incubation at 0.5-4 hours, the cells werewashed with phosphate buffered saline 3 times, followed by trypsin treatment to lose the cells. The cells were thencounted by a gamma counter. The uptake of 99mTc-EC-DG was comparable to FDG (FIG. 69).

Effect of d- and 1-glucose on cellular uptake of 99mTc-EC-deoxyglucose and 18F-FDG

[0063] To evaluate if the uptake of 99mTc-EC-deoxyglucose is mediated via d-glucose mechanism, d- and 1-glucose(1 mg and 2.0 mg) were added to each well containing either breast or lung cancer cells (50,000/0.5 ml/well), along with2mCi of 99mTc-EC-deoxyglucose and 18F-FDG. After 2 hours incubation, the cells were washed with phosphate bufferedsaline 3 times, followed by trypsin treatment to lose the cells. The cells were counted by a gamma counter.[0064] By adding glucose at the concentration of 1-2.0 mg/well, a decreased uptake of 99mTc-EC-deoxyglucose and18F-FDG by d-glucose in breast and lung cancer cells was observed. However, there was no influence on both agentsby 1-glucose (FIG. 70-73). The findings suggest that the cellular uptake of 99mTc-EC-deoxyglucose is mediated via d-glucose mechanism.

Effect of EC-deoxyglucose loading on blood glucose level in normal rats

[0065] Previous experiments have shown that cellular uptake of 99mTc-EC-deoxyglucose is similar to FDG. For in-stance, the hexokinase assay (glucose phosphorylation) was positive. The uptake of 99mTc-EC-deoxyglucose is mediatedvia d-glucose mechanism. This study is to determine whether blood glucose level could be induced by either FDG orEC-deoxyglucose and suppressed by insulin.[0066] Normal healthy Fischer 344 rats (weight 145-155 g) were fasting overnight prior to the experiments. The con-centration of glucosamine hydrochloride, FDG and EC-deoxyglucose prepared was 60% and 164% (mg/ml). The blood

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glucose level (mg/dl) was determined by a glucose meter (Glucometer DEX, Bayer Corporation, Elkhart, IN). Prior tothe study, the baseline of blood glucose level was obtained. Each rat (n=3/group) was administered 1.2 mmol/kg ofglucosamine, FDG and EC-deoxyglucose. In a separate experiment, a group of rats was administered EC-deoxyglucoseand FDG. Insulin (5 units) was administered after 30 minutes. Blood samples were collected from the tail vein every 30minutes up to 6 hours post-administration.[0067] Blood glucose level was induced by bolus intravenous administration of glucosamine, FDG and EC-deoxyglu-cose. This increased blood glucose level could be suppressed by coadministration of EC-deoxyglucose or FDG andinsulin (FIGS. 74 and 75).

Tissue distribution studies of 99mTc-EC-DG

[0068] For breast tumor-bearing animal model, female Fischer 344 rats (150625 g) (Harlan Sprague-Dawley, Indian-apolis, IN) were innoculated subcutaneously with 0.1 ml of mammary tumor cells from the 13762 tumor cell line suspension(106 cells/rat, a tumor cell line specific to Fischer rats) into the hind legs using 25-gauge needles. Studies were performed14 to 17 days after implantation when tumors reached approximately 1 cm diameter. Rats were anesthetized withketamine (10-15 mg/rat, intraperitoneally) before each procedure.[0069] For lung tumor-bearing animal model, each athymic nude mouse (20-25g) was innoculated subcutaneouslywith 0.1 ml of human lung tumor cells from the A549 tumor cell line suspension (106 cells/mouse) into the hind legsusing 25-gauge needles. Studies were performed 17 to 21 days after implantation when tumors reached approximately0.6 cm diameter.[0070] In tissue distribution studies, each animal was injected intravenously with 10-20 mCi (per rat) or 1-2 mCi (permouse) of 99mTc-EC or 99mTc-EC-DG (n=3/time point). The injected mass of 99mTc-EC-DG was 1 mg per rat. At 0.5, 2and 4 hours following administration of the radiotracers, the rodents were sacrificed and the selected tissues wereexcised, weighed and counted for radioactivity. The biodistribution of tracer in each sample was calculated as percentageof the injected dose per gram of tissue wet weight (%ID/g). Tumor/nontarget tissue count density ratios were calculatedfrom the corresponding %ID/g values. When compared to 99mTc-EC and free technetium (Table 3), tumor-to tissue ratiosincreased as a function of time in 99mTc-EC-DG group (FIGS. 76-80).

Scintigraphic Imaging Studies

[0071] Scintigraphic images, using a gamma camera equipped with low-energy, parallel-hole collimator, were obtained0.5, 2 and 4 hours after i.v. injection of 100 mCi of the radiotracer. The animal model used was breast tumor-bearingrats. Tumor could be visualized well when compared to 99mTc-EC (control group) (FIG. 81). Preliminary clinical studieswere conducted in 5 patients (3 brain tumors and 2 lung diseases). The images were obtained at 1-2 hours post-administration. 99mTc-EC-DG was able to differentiate benign versus malignant tumors. For instance, malignant astro-cytoma showed high uptake (FIGS. 82A, 82B, 83A and 83B). Benign meningioma showed poor uptake compared tomalignant meningioma (Figs 84A and B). Poor uptake was observed in patient with TB (FIG. 85A and FIG. 85B), buthigh uptake was observed in lung tumor (FIG. 86A, FIG. 86B, and FIG. 86C).

REFERENCES

[0072]

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Claims

1. A reagent for preparing a scintigraphic imaging agent,the reagent comprising a tissue-specific ligand covalently linked to ethylenedicysteine (EC), wherein the tissue-specific ligand is an agent that mimics glucoseselected from glucose, glucosamine, micromicin, and aminoglycosides.

2. The reagent of claim 1, wherein the aminoglycoside is neomycin, kanamycin, gentamycin, paromomycin, amikacin,tobramycin, netilmicin, ribostamycin, sisomicin, lividomycin, dibekacin, isepamicin, astromicin.

3. The reagent of claim 1, wherein said tissue-specific ligand is covalently linked to EC on one or both acid arms of EC.

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4. The reagent of claim 1, wherein the reagent is EC-glucosamine.

5. The reagent of claim 1, the reagent further comprising a linker between EC and the tissue-specific ligand.

6. The reagent of claim 5, wherein the linker is a water-soluble peptide, an amino acid such as glutamic acid, asparticacid, and lysine; a polyamino acid such as polyglutamic acid and polyaspartic acid; bromoethylacetate; or ethylen-ediamine.

7. An imaging agent comprising the reagent of claim 1 and further comprising a radionuclide, wherein the EC that iscovalently linked to the tissue-specific ligand forms an N2S2 chelate with said radionuclide.

8. The imaging agent of claim 7, wherein said radionuclide is 99mTc, 188Re, 186Re, 183Sm, 166Ho, 90Y, 89Sr, 67Ga,68Ga, 111In, 183Gd, 59Fe, 225Ac, 212Bi, 211At, 64Cu, or 62Cu.

9. The imaging agent of claim 8, wherein said radionuclide is 99mTc.

10. The imaging agent of claim 9, wherein the imaging agent (i.e., the radionuclide-labeled reagent) is 99mTc-EC-glucosamine or 99mTc-EC-GAP-glucosamine (GAP = pentaglutamate).

11. The imaging agent of claim 7, wherein the linker is a water-soluble peptide, an amino acid such as glutamic acid,aspartic acid, and lysine; a polyamino acid such as polyglutamic acid and polyaspartic acid; bromoethylacetate; orethylenediamine.

12. A composition comprising an imaging agent as set forth in claim 9, a medium for intravenous injection, and one ormore pharmaceutically acceptable adjunct materials.

13. A method of synthesizing a reagent as defined in claim 1 and radionuclide-labeling same, the method comprisingthe steps of:

a) obtaining the tissue-specific ligand as defined in claim 1;b) mixing said tissue-specific ligand with EC to obtain a reagent as defined in claim 1; andc) mixing said reagent, a radionuclide, and a reducing agent to obtain an imaging agent as defined in claim 7,wherein the EC that is covalently linked to the tissue-specific ligand forms an N2S2 chelate with the radionuclide.

14. The method of claim 13, wherein said reducing agent is a dithionite ion, a stannous ion, or a ferrous ion.

15. Use of an imaging agent as defined in claim 9 for the manufacture of a pharmaceutical to image a tumor, an infectionsite, a heart, a lung, a brain, a liver, a spleen, a pancreas, or an intestine in a mammal.

16. The use of claim 15, wherein the tumor is a breast cancer, an ovarian cancer, a prostate cancer, a folate (+) cancer,or an ER (+) cancer.

17. A kit for preparing a radiopharmaceutical preparation, said kit comprising a sealed container such as a bag, thecontainer including a predetermined quantity of a reagent as defined in claim 1 and a reducing agent to label thereagent with 99mTc.

18. The kit of claim 17, wherein the reducing agent is a dithionite ion, a stannous ion, or a ferrous ion.

19. The kit of claim 17, wherein the components of the kit are in liquid form, in frozen form, or in a dry form such as alyophilized form.

20. The kit of claim 17, wherein the kit further comprises one or more pharmaceutical adjunct materials, in particularpharmaceutically acceptable salts, buffers, preservatives, antioxidants such as vitamin C, and a scavenger such asglucoheptonate or EDTA.

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Patentansprüche

1. Ein Reagens zum Herstellen eines szintigrafischen Bildgebungsagens,wobei das Reagens einen gewebespezifischen Liganden kovalent verknüpft an Ethylendicystein (EC) umfasst,wobei der gewebespezifische Ligand ein Agens ist, das Glukose imitiert ausgewählt aus Glucose, Glucosamin,Micromicin und Aminoglykosiden.

2. Das Reagens nach Anspruch 1, wobei die Aminoglykoside Neomycin, Kanamycin, Gentamycin, Paromomycin,Amikacin, Tobramycin, Netilmicin, Ribostamycin, Sisomicin, Lividomycin, Dibekacin, Isepamcin, Astromicin.

3. Das Reagens nach Anspruch 1, wobei der gewebespezifische Ligand kovalent an EC an einen oder beide Armevon EC verknüpft ist.

4. Das Reagens nach Anspruch 1, wobei das Reagens EC-Glucosamin ist.

5. Das Reagens nach Anspruch 1, wobei das Reagens weiter einen Linker zwischen EC und dem gewebespezifischenLiganden umfasst.

6. Das Reagens nach Anspruch 5, wobei der Linker ein wasserlösliches Peptid, eine Aminosäure wie Glutaminsäure,Asparginsäure und Lysin; eine Polyaminosäure wie Polyglutaminsäure und Polyasparaginsäure; Bromethylacetat;oder Ethylendiamin ist.

7. Ein Bildgebungsagens umfassend das Reagens nach Anspruch 1 und weiter umfassend ein Radionuklid, wobeidas EC, das kovalent an dem gewebespezifischen Liganden verknüpft ist, ein N2S2 Chelat mit dem Radionuklid bildet.

8. Das Bildgebungsagens nach Anspruch 7, wobei das Radionuklid 99mTc, 188Re, 186Re, 183Sm, 166Ho, 90Y, 89Sr,67Ga, 68Ga, 111In, 183Gd, 59Fe, 225Ac, 212Bi, 211At, 64Cu oder 62Cu.

9. Das Bildgebungsagens nach Anspruch 8, wobei das Radionuklid 99mTc ist.

10. Das Bildgebungsagens nach Anspruch 9, wobei das Bildgebungsagens (d.h. das Radionuklid markierte Reagens)99mTc-EC-Glucosamin oder 99mTc-EC-GAP-Glucosamin (GAP=Pentaglutamat) ist.

11. Das Bildgebungsagens nach Anspruch 7, wobei der Linker ein wasserlösliches Peptid, eine Aminosäure wie Glut-aminsäure, Asparaginsäure und Lysin; eine Polyaminsäure wie Polyglutaminsäure und Polyasparaginsäure; Brom-ethylacetat; oder Ethylendiamin ist.

12. Eine Zusammensetzung umfassend ein Bildgebungsagens wie in Anspruch 9 dargelegt, ein Medium zur intrave-nösen Injektion und ein oder mehrere pharmazeutische verträgliche Zusatzmaterialien.

13. Ein Verfahren zum Synthetisieren eines Reagens wie in Anspruch 1 definiert und Radionuklid-Markieren des selben,wobei das Verfahren die Schritte umfasst von:

a) Erhalten des gewebespezifischen Liganden wie in Anspruch 1 definiert;b) Mischen des gewebespezifischen Liganden mit EC, um ein Reagens wie in Anspruch 1 definiert zu erhalten;undc) Mischen des Reagens, eines Radionuklids und eines reduzierenden Agens, um ein Bildgebungsagens zuerhalten wie in Anspruch 7 definiert, wobei das EC, das kovalent an den gewebespezifischen Liganden verknüpftist, ein N2S2 Chelat bildet mit dem Radionuklid.

14. Das Verfahren nach Anspruch 13, wobei das reduzierende Agens ein Dithionition, ein Zinnion oder ein Eisenion ist.

15. Verwendung eines Bildgebungsagens wie in Anspruch 9 definiert zur Herstellung eines Arzneimittels, um einenTumor, eine Infektionsstelle, ein Herz, eine Lunge, ein Gehirn, eine Leber, eine Milz, einen Pankreas oder einenDarm in einem Säugetier abzubilden.

16. Die Verwendung nach Anspruch 15, wobei der Tumor ein Brustkrebs, ein Ovarialkrebs, ein Prostatakrebs, ein Folat(+)-Krebs oder ein ER (+)-Krebs ist.

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17. Ein Kit zur Herstellung eines radiopharmazeutischen Präparats, wobei das Kit einen versiegelten Behälter wie eineTasche umfasst, wobei der Behälter eine vorbestimmte Menge an Reagens wie in Anspruch 1 definiert beinhaltetund ein reduzierendes Agens, um das Reagens mit 99mTc zu markieren.

18. Das Kit nach Anspruch 17, wobei das reduzierende Agens ein Dithionition, ein Zinnion oder Eisenion ist.

19. Das Kit nach Anspruch 17, wobei die Komponenten des Kits in flüssiger Form, in gefrorener Form oder in einertrockenen Form wie einer lyophilisierten Form vorliegen.

20. Das Kit nach Anspruch 17, wobei das Kit weiter einen oder mehrere pharmazeutische Zusatzmaterialien umfasst,insbesondere pharmazeutisch verträgliche Salze, Puffer, Konservierungsstoffe, Antioxidantien wie Vitamin C undeinen Scavenger wie Glucoheptonat oder EDTA.

Revendications

1. Réactif pour la préparation d’un agent d’imagerie scintigraphique :

le réactif comprenant un ligand à spécificité tissulaire lié de manière covalente à de l’éthylènedicystéine (EC),dans lequel le ligand à spécificité tissulaire est un agent qui mime le glucose choisi parmi le glucose, la gluco-samine, la micromycine et des aminoglycosides.

2. Réactif selon la revendication 1, dans lequel l’aminoglycoside est la néomycine, la kanamycine, la gentamycine, laparomomycine, l’amikacine, la tobramycine, la nétilmicine, la ribostamycine, la sisomycine, la lividomycine, la dibé-kacine, l’isépamycine, l’astromicine.

3. Réactif selon la revendication 1, dans lequel ledit ligand à spécificité tissulaire est lié de manière covalente à del’EC sur l’un ou les deux bras acide de l’EC.

4. Réactif selon la revendication 1, dans lequel le réactif est l’EC-glucosamine.

5. Réactif selon la revendication 1, le réactif comprenant en outre un lieur entre l’EC et le ligand à spécificité tissulaire.

6. Réactif selon la revendication 5, dans lequel le lieur est un peptide hydrosoluble, un acide aminé tel que l’acideglutamique, l’acide aspartique, et la lysine ; un polyacide aminé tel qu’un polyacide glutamique et un polyacideaspartique ; l’acétate de bromoéthyle ; ou l’éthylènediamine.

7. Agent d’imagerie comprenant le réactif selon la revendication 1 et comprenant en outre un radionucléide, danslequel l’EC qui est liée de manière covalente au ligand à spécificité tissulaire forme un chélate N2S2 avec leditradionucléide.

8. Agent d’imagerie selon la revendication 7, dans lequel ledit radionucléide est le 99mTc, 188Re, 186Re, 183Sm, 166Ho,90Y, 89Sr, 67Ga, 68Ga, 111In, 183Gd, 59Fe, 225Ac, 212Bi, 211At, 64Cu, ou 62Cu.

9. Agent d’imagerie selon la revendication 8, dans lequel ledit radionucléide est le 99mTc.

10. Agent d’imagerie selon la revendication 9, dans lequel l’agent d’imagerie (c’est-à-dire, le réactif marqué par unradionucléide) est la 99mTc-EC-glucosamine ou la 99mTc-EC-GAP-glucosamine (GAP = pentaglutamate).

11. Agent d’imagerie selon la revendication 7, dans lequel le lieur est un peptide hydrosoluble, un acide aminé tel quel’acide glutamique, l’acide aspartique, et la lysine ; un polyacide aminé tel qu’un polyacide glutamique et un polyacideaspartique ; l’acétate de bromoéthyle ; ou l’éthylènediamine.

12. Composition comprenant un agent d’imagerie tel que présenté dans la revendication 9, un milieu pour injectionintraveineuse, et un ou plusieurs matériaux complémentaires pharmaceutiquement acceptables.

13. Méthode de synthèse d’un réactif tel que défini dans la revendication 1 et de marquage par un radionucléide decelui-ci, le procédé comprenant les étapes suivantes :

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a) l’obtention du ligand à spécificité tissulaire tel que défini dans la revendication 1 ;b) le mélange dudit ligand à spécificité tissulaire avec de l’EC pour obtenir un réactif tel que défini dans larevendication 1 ; etc) le mélange dudit réactif, d’un radionucléide, et d’un agent réducteur pour obtenir un agent d’imagerie tel quedéfini dans la revendication 7, dans lequel l’EC qui est liée de manière covalente au ligand à spécificité tissulaireforme un chélate N2S2 avec le radionucléide.

14. Méthode selon la revendication 13, dans laquelle ledit agent réducteur est un ion dithionite, un ion stanneux, ou union ferreux.

15. Utilisation d’un agent d’imagerie tel que défini dans la revendication 9 pour la fabrication d’un produit pharmaceutiquepour l’imagerie d’une tumeur, d’un site infectieux, d’un coeur, d’un poumon, d’un cerveau, d’un foie, d’une rate, d’unpancréas, ou d’un intestin chez un mammifère.

16. Utilisation selon la revendication 15, dans laquelle la tumeur est un cancer du sein, un cancer ovarien, un cancerde la prostate, un cancer folates+, ou un cancer ER+.

17. Kit pour la préparation d’une préparation radiopharmaceutique, ledit kit comprenant un récipient scellé comme unepoche, le récipient comprenant une quantité prédéterminée d’un réactif tel que défini dans la revendication 1 et unagent réducteur pour marquer le réactif avec du 99mTc.

18. Kit selon la revendication 17, dans lequel l’agent réducteur est un ion dithionite, un ion stanneux, ou un ion ferreux.

19. Kit selon la revendication 17, dans lequel les composants du kit sont sous forme liquide, sous forme congelée, ousous une forme sèche telle qu’une forme lyophilisée.

20. Kit selon la revendication 17, dans lequel le kit comprend en outre un ou plusieurs matériaux complémentairespharmaceutiques, en particulier des sels pharmaceutiquement acceptables, des tampons, des conservateurs, desantioxydants tels que la vitamine C, et un piégeur tel que le glucoheptonate ou l’EDTA.

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