Patent Publication Number: US-7582281-B2

Title: Ethylenedicysteine (EC)-drug conjugates compositions and methods for tissue specific disease imaging

Description:
This is a continuation application of application Ser. No. 10/672,763, filed Sep. 26, 2003, now U.S. Pat. No. 7,223,380, which is a continuation application of U.S. patent application Ser. No. 09/599,152, filed Jun. 21, 2000, now issued as U.S. Pat. No. 7,067,111, which is a continuation-in-part of Ser. No. 09/587,583, filed Jun. 2, 2000, now abandoned, which is a continuation-in-part of Ser. No. 09/434,313, filed Oct. 25, 1999, now issued as U.S. Pat. No. 6,692,724. 
    
    
     BACKGROUND OF THE INVENTION 
     The government does not own rights in the present invention. 
     1. Field of the Invention 
     The present invention relates generally to the fields of labeling, radioimaging and chemical synthesis. More particularly, it concerns a strategy for radiolabeling target ligands. It further concerns methods of using those radiolabeled ligands in tumor imaging and tissue-specific disease imaging. 
     2. Description of Related Art 
     Improvement of scintigraphic tumor imaging is extensively determined by development of more tumor specific radiopharmaceuticals. Due to greater tumor specificity, radiolabeled ligands as well as radiolabeled antibodies have opened 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; single photon emission computed tomography, SPECT) are diagnostic cross-sectional imaging techniques that map the location and concentration of radionuclide-labeled radiotracers. Although CT and MRI provide considerable anatomic information about the location and the extent of tumors, these imaging modalities cannot adequately differentiate invasive lesions from edema, radiation necrosis, grading or gliosis. PET and SPECT can be used to localize and characterize tumors by measuring metabolic activity. 
     The development of new tumor hypoxia agents is clinically desirable for detecting primary and metastatic lesions as well as predicting radioresponsiveness and time to recurrence. None of the contemporary imaging modalities accurately measures hypoxia since the diagnosis of tumor hypoxia requires pathologic examination. It is often difficult to predict 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 technique is invasive and needs a skillful operator. Additionally, this technique can only be used on accessible tumors (e.g., head and neck, cervical) and multiple readings are needed. Therefore, an accurate and easy method of measuring tumor hypoxia will be useful for patient selection. However, tumor to normal tissue uptake ratios vary depending upon the radiopharmaceuticals used. Therefore, it would be rational to correlate tumor to normal tissue uptake ratio with the gold standard Eppendorf electrode measures of hypoxia when new radiopharmaceuticals are introduced to clinical practice. 
     [ 18 F]FMISO has been used to diagnose head and neck tumors, myocardial infarction, inflammation, and brain ischemia (Matin et al. 1992; Yeh et al. 1994; Yeh et al. 1996; Liu et al. 1994). Tumor to normal tissue uptake ratio was used as a baseline to assess tumor hypoxia (Yet et al. 1996). Although tumor hypoxia using [ 18 F]FMISO was clearly demonstrated, introducing new imaging agents into clinical practice depends on some other factors such as easy availability and isotope cost. Although tumor metabolic imaging using [ 18 F]FDG was clearly demonstrated, introducing molecular imaging agents into clinical practice depends on some other factors such as easy availability and isotope cost. [ 18 F]fluorodeoxyglucose (FDG) has been used to diagnose tumors, myocardial infarction, and neurological disease. In addition, PET radiosynthesis must be rapid because of short half-life of the positron isotopes.  18 F chemistry is also complex. The  18 F chemistry is not reproducible in different molecules. Thus, it would be ideal to develop a chelator which could conjugate to various drugs. The preferred isotope would be  99m Tc due to low cost ($0.21/mCi vs. $50/mCi for  18 F) and low energy (140 Kev vs. 571 Kev for  18 F).  99m Tc is easily obtained from a  99 Mo generator. Due to favorable physical characteristics as well as extremely low price,  99m Tc has been preferred to label radiopharmaceuticals. 
     Several compounds have been labeled with  99 Tc using nitrogen and sulfur chelates (Blondeau et al., 1967; Davison et al., 1980). Bis-aminoethanethiol tetradentate ligands, also called diaminodithol compounds, are known to form very stable Tc(V)O complexes on the basis of efficient binding of the oxotechnetium group to two thiolsulfur and two amine nitrogen atoms.  99m Tc-L,L-ethylenedicysteine ( 99m Tc-EC) is a recent and successful example of N 2 S 2  chelates. EC can be labeled with  99m Tc easily and efficiently with high radiochemical purity and stability, and is excreted through the 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).  99m Tc-EC-neomycin and  99m Tc-EC-deoxyglucose were developed and their potential use in tumor characterization was-evaluated. 
     SUMMARY OF THE INVENTION 
     The present invention overcomes these and other drawbacks of the prior art by providing a new radiolabeling strategy to target tissues for imaging. The invention provides radiolabeled tissue-specific ligands, as well as methods for making the radiolabeled ligands and for using them to image tissue-specific diseases. 
     The present invention provides compositions for tissue specific disease imaging. The imaging compositions of the invention generally include a radionuclide label chelated with ethylenedicysteine and a tissue specific ligand conjugated to the ethylenedicysteine on one or both of its acid arms. The ethylenedicysteine forms an N 2 S 2  chelate with the radionuclide label. Of course, the chelated compound will include an ionic bond between the ranionuclide 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. 
     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 the oxotechnetium group to two thiol-sulphur and two amine-nitrogen atoms. The  99m Tc labeled diethylester ( 99m Tc-L,L-ECD) is known as a brain agent.  99m Tc-L,L-ethylenedicysteine ( 99m Tc-L,L-EC) is its most polar metabolite and was discovered to be excreted rapidly and efficiently in the urine. Thus,  99m Tc-L,L-EC has been used as a renal function agent. (Verbruggen et al. 1992). 
     A tissue specific ligand is a compound that, when introduced into the body of a mammal or patient, will specifically bind to a specific type of tissue. It is envisioned that the compositions of the invention may include virtually any known tissue specific compound. Preferably, the tissue specific ligand used in conjunction with the present invention will be an anticancer agent, DNA topoisomerase inhibitor, antimetabolite, tumor marker, folate receptor targeting ligand, tumor apoptotic cell targeting ligand, tumor hypoxia targeting ligand, DNA intercalator, receptor marker, peptide, nucleotide, organ specific ligand, antimicrobial agent, such as an antibiotic or an antifungal, glutamate pentapeptide or an agent that mimics glucose. The agents that mimic glucose may also be referred to as “sugars.” 
     Preferred anticancer agents include methotrexate, doxorubicin, tamoxifen, paclitaxel, topotecan, LHRH, mitomycin C, etoposide, tomudex, podophyllotoxin, mitoxantrone, captothecin, colchicine, endostatin, fludarabin and gemcitabine. Preferred tumor markers include PSA, ER, PR, AFP, CA-125, CA-199, CEA, interferons, BRCA1, cytoxan, p53, VEGF, integrins, enidostatin, HER-2/neu, antisense markers or a monoclonal antibody. It is envisioned that any other known tumor marker or any monoclonal antibody will be effective for use in conjunction with the invention. Preferred folate receptor targeting ligands include folate, methotrexate and tomudex. Preferred tumor apoptotic cell or tumor hypoxia targeting ligands include annexin V, colchicine, nitroimidazole, mitomycin or metronidazole. Preferred antimicrobials include ampicillin, amoxicillin, penicillin, cephalosporin, clidamycin, gentamycin, kanamycin, neomycin, natamycin, nafcillin, rifampin, tetracyclin, vancomycin, bleomycin, and doxycyclin for gram positive and negative bacteria and amphotericin B, amantadine, nystatin, ketoconazole, polymycin, acyclovir, and ganciclovir for ffingi. Preferred agents that mimic glucose, or sugars, include neomycin, kanamycin, gentamycin, paromycin, amikacin, tobramycin, netilmicin, ribostamycin, sisomicin, micromicin, lividomycin, dibekacin, isepamicin, astromicin, aminoglycosides, glucose or glucosamine. 
     In certain embodiments,-it will be necessary to include a linker between the ethylenedicysteine and the tissue specific ligand. A linker is typically used to increase drug solubility in aqueous solutions as well as to minimize alteration in the affinity of drugs. While virtually any linker which will increase the aqueous solubility of the composition is envisioned for use in conjunction with the present invention, the linkers will generally be 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, such as for estradiol, topotecan, paclitaxel, or raloxifen etoposide, 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 drug functional group is aliphatic or aromatic-NH 2  or peptide, such as in doxorubicin, mitomycin C, endostatin, annexin V, LHRH, octreotide, and VIP, 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), glutamic acid or aspartic acid. When the drug functional group is carboxylic acid or peptide, such as in methotrexate or folic acid, the linker may be ethylenediamine, or lysine. 
     While the preferred radionuclide for imaging is  99m Tc, it is envisioned that other radionuclides may be chelated to the EC-tissue specific ligand conjugates, or EC-drug conjugates of the invention, especially for use as therapeutics. For example, other useful radionuclides are  188 Re,  186 Re,  153 Sm,  166 Ho,  90 Y,  89 Sr,  67 Ga,  68 Ga,  111 In,  153 Gd, and  59 Fe. These compositions 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,  186/188 Re-EC-folate) and head and neck cancer (using for example,  186/188 Re-EC-nitroimidazole). 
     Specific embodiments of the present invention include  99m Tc-EC-annexin V,  99m Tc-EC-colchicine,  99m Tc-EC-nitroimidazole,  99m Tc-EC-glutamiate pentapeptide,  99m Tc-EC-metronidazole,  99m Tc-EC-folate,  99m Tc-EC-methotrexate,  99m Tc-EC-tomudex,  99m Tc-EC-neomycin,  99m Tc-EC-kanamycin,  99m Tc-EC-aminoglycosides, (glucosamine, EC-deoxyglucose),  99m Tc-EC-gentamycin, and  99m Tc-EC-tobramycin. 
     The present invention further provides a method of synthesizing a radiolabeled ethylenedicysteine drug conjugate or derivative for imaging or therapeutic use. The method includes obtaining a tissue specific ligand, 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. The radionuclide is chelated to the EC via an N 2 S 2  chelate. The tissue specific ligand is conjugated to one or both acid arms of the EC either directly or through a linker as described above. The reducing agent is preferably a dithionite ion, a stannous ion or a ferrous ion. 
     The present invention further provides a method for labeling a tissue specific ligand for imaging, therapeutic use or for diagnostic or prognostic use. The labeling method includes the steps of obtaining a tissue specific ligand, admixing the tissue specific ligand with ethylenedicysteine (EC) to obtain an EC-ligand drug conjugate, and reacting the drug conjugate with  99m Tc in the presence of a reducing agent to form an N 2 S 2  chelate between the ethylenedicysteine and the  99m Tc. 
     For purposes of this embodiment, the tissue specific ligand may be any of the ligands described above or discussed herein. The reducing agent may be any known reducing agent, but will preferably be a dithionite ion, a stannous ion or a ferrous ion. 
     In another embodiment, the present invention provides a method of imaging a site within a mammalian body. The imaging method includes the steps of administering an effective diagnostic amount of a composition comprising a  99m Tc labeled ethylenedicysteine-tissue specific ligand conjugate and detecting a radioactive signal from the  99m Tc localized at the site. The detecting step will typically be performed from about 10 minutes to about 4 hours after introduction of the composition into the mammalian body. Most preferably, the detecting step will be performed about 1 hour after injection of the composition into the mammalian body. 
     In certain preferred embodiments, the site will be an infection, tumor, heart, lung, brain, liver, spleen, pancreas, intestine or any other organ. The tumor or infection 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. 
     The invention also provides a kit for preparing a radiopharmaceutical preparation. The kit generally includes a sealed via or bag, or any other kind of appropriate container, containing a predetermined quantity of an ethylenedicysteine-tissue specific ligand conjugate composition and a sufficient amount of reducing agent to label the conjugate with  99m Tc. In certain cases, the ethylenedicysteine-tissue specific ligand conjugate composition will also include a linker between the ethylenedicysteine and the tissue specific ligand. The tissue specific ligand may be any ligand that specifically binds to any specific tissue type, such as those discussed herein. When a linker is included in the composition, it may be any linker as described herein. 
     The components of the kit may be in any appropriate form, such as in liquid, frozen or dry form. In a preferred embodiment, the kit components are provided in lyophilized form. The kit may also include an antioxidant and/or a scavenger. The antioxidant may be any known antioxidant but is preferably vitamin C. Scavengers may also be present to 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. This leftover glucoheptonate will go to a tumor and skew imaging results. Therefore, the inventors prefer to use EDTA as the scavenger as it is cheaper and reacts more completely. 
     Another aspect of the invention is a prognostic method for determining the potential usefulness of a candidate compound for treatment of specific tumors. Currently, most tumors are treated with the “usual drug of choice” in chemotherapy without any indication whether the drug is actually effective against that particular tumor until months, and many thousands of dollars, later. The imaging compositions of the invention are useful in delivering a particular drug to the site of the tumor in the form of a labeled EC-drug conjugate and then imaging the site within hours to determine whether a particular drug. 
     In that regard, the prognostic method of the invention includes the steps of determining the site of a tumor within a mammalian body, obtaining an imaging composition which includes a radionuclide chelated to EC which is conjugated to a tumor specific cancer chemotherapy drug candidate, administering the composition to the mammalian body and imaging the site to determine the effectiveness of the candidate drug against the tumor. Typically, the imaging step will be performed within about 10 minutes to about 4 hours after injection of the composition into the mammalian body. Preferably, the imaging step will be performed within about 1 hour after injection of the composition into the mammalian body. 
     The cancer chemotherapy drug candidate to be conjugated to EC in the prognostic compositions may be chosen from known cancer chemotherapy drugs. Such drugs appear in Table 2. There are many anticancer agents known to be specific for certain types of cancers. However, not every anticancer agent for a specific type of cancer is effective in every patient. Therefore, the present invention provides for the first time a method of determining possible effectiveness of a candidate drug before expending a lot of time and money on treatment. 
     Yet another embodiment of the-present invention is a reagent for preparing a scintigraphic imaging agent. The reagent of the invention includes a tissue specific ligand, having an affinity for targeted sites in vivo sufficient to produce a scintigraphically-detectable image, covalently linked to a  99m Tc binding moiety. The  99m Tc binding moiety is either directly attached to the tissue specific ligand or is attached to the ligand through a linker as described above. The  99m Tc binding moiety is preferably an N 2 S 2  chelate between  99m Tc in the +4 oxidation state and ethylenedicysteine (EC). The tissue specific ligand will be covalently linked to one or both acid arms of the EC, either directly or through a linker as described above. The tissue specific ligand may be any of the ligands as described above. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. 
         FIG. 1 . Synthetic scheme of  99m Tc-EC-folate. 
         FIG. 2 . Synthetic scheme of  99m Tc-EC-MTX (methotrexate). 
         FIG. 3 . Synthetic scheme of  99m Tc-EC-T)X (tomudex). 
         FIG. 4 . Biodistribution studies for  99m Tc-EC and  99m Tc-EC-folate. 
         FIG. 5 . Blocking studies for tumor/muscle and tumor/blood count ratios with  99m Tc-EC-folate. 
         FIG. 6A and 6B . Scintigraphic images of tumor in  99m Tc-EC-folate injected group as compared to  99m Tc-EC injected group. 
         FIG. 7 . Synthetic scheme of EC-MN (metronidazole) 
         FIG. 8A  and  FIG. 8B . For EC-NIM,  FIG. 8A  shows the synthetic scheme and  FIG. 8B  illustrates the  1 H-NMR confirmation of the structure. 
         FIG. 9 . Biodistribution studies (tumor/blood ratios) for  99m Tc-EC-MN, [ 18 F]FMISO and [ 131 I]IMSO. 
         FIG. 10 . Biodistribution studies (tumor/muscle ratios) for  99m Tc-EC, [ 18 F]FMISO and [ 131 I]IMISO. 
         FIG. 11A and 11B . Scintigraphic images of tumor in  99m Tc-EC-MN ( FIG. 11A ) and  99m Tc-EC ( FIG. 11B ) injected groups. 
         FIG. 12 . Autoradiograms performed at 1 hour after injection with  99m Tc-EC-MN. 
         FIG. 13 . Illustrates stability of  99m Tc-EC-NIM in dog serum samples. 
         FIG. 14A  and  FIG. 14B . Illustrates breast tumor uptake of  99m Tc-EC-NIM vs.  99m Tc-EC in rats ( FIG. 14A ) and in rats treated with paclitaxel compared to controls ( FIG. 14B ). 
         FIG. 15A ,  FIG. 15B ,  FIG. 15C , and  FIG. 15D . Illustrates ovarian tumor uptake of  99m Tc-EC-NIM vs.  99m Tc-EC in rats ( FIG. 15A ) The tumor uptake in rats treated with paclitaxel ( FIG. 15B ) was less than tumor uptake in rats not treated with paclitaxel ( FIG. 15A ). Also illustrated is tumor uptake of  99m Tc-EC-NIM in rats having sarcomas.  FIG. 15C  shows tumor uptake in sarcoma bearing rats treated with paclitaxel while  FIG. 15D  shows tumor uptake in rats not treated with paclitaxel. There was a decreased uptake of  99m Tc-EC-NIM after treatment with paclitaxel. 
         FIG. 16 . Synthetic scheme of EC-GAP (pentaglutamate). 
         FIG. 17 . Scintigraphic images of breast tumors in  99m Tc-EC-GAP injected group. 
         FIG. 18 . Scintigraphic images of breast tumors in  99m Tc-EC-ANNEX V injected group at different time intervals. 
         FIG. 19A  and  FIG. 19B . Comparison of uptake difference of  99 Tc-EC-ANNEX V between pre- ( FIG. 19A ) and post- ( FIG. 19B ) paclitaxel treatment in ovarian tumor bearing group. 
         FIG. 20A  and  FIG. 20B . Comparison of uptake difference of  99m Tc-EC-ANNEX V between pre- ( FIG. 20A ) and post- ( FIG. 20B ) paclitaxel treatment in sarcoma tumor bearing group. 
         FIG. 21 . Synthetic scheme of EC-COL (colchicine). 
         FIG. 22 . Illustration that no degradation products observed in EC-COL synthesis. 
         FIG. 23 . Ratios of tumor to muscle and tumor to blood as function of time for  99m Tc-EC-COL. 
         FIG. 24 . Ratios of tumor to muscle and tumor to blood as function of time for  99m Tc-EC. 
         FIG. 25 . In vivo imaging studies in breast tumor bearing rats with  99m Tc-EC-COL. 
         FIG. 26 . In vivo imaging studies in breast tumor bearing rats with  99m Tc-EC. 
         FIG. 27 . Computer outlined region of interest after injection of  99m Tc-EC-COL vs.  99m Tc-EC. 
         FIG. 28 . SPECT with  99m Tc-EC-MN of 59 year old male patient who suffered stroke. Images taken one hour post-injection. 
         FIG. 29 . MRI T1 weighted image of same patient as  FIG. 28 . 
         FIG. 30 . SPECT with  99m Tc-EC-MN of 73 year old male patient one day after stroke at one hour post-injection. 
         FIG. 31 . SPECT with  99m Tc-EC-MN of same 73 year old patient as imaged in  FIG. 30  twelve days after stroke at one hour post-injection. 
         FIG. 32 . CT of same 73 year old male stroke patient as imaged in  FIG. 30 , one day after stroke. 
         FIG. 33 . CT of same 73 year old male stroke patient as imaged in  FIG. 32 , twelve days after stroke. Note, no marked difference between days one and twelve using CT for imaging. 
         FIG. 34 . SPECT with 99mTc-EC-MN of 72 year old male patient who suffered a stroke at one hour post-injection. 
         FIG. 35 . CT of same 72 year old stroke patient as imaged in  FIG. 34 . Note how CT image exaggerates the lesion size. 
         FIG. 36 . Synthetic scheme of 99mTc-EC-neomycin. 
         FIG. 37A . Scintigraphic image of breast tumor-bearing rats after administration of  99m Tc-EC and  99m Tc-EC-neomycin (100 μCi/rat, iv.) showed that the tumor could be well visualized from 0.5-4 hours postinjection. 
         FIG. 37B . Scintimammography with  99m Tc-EC-neomycin (30 mCi, iv.) of a breast cancer patient. Images taken two hours post-injection. 
         FIG. 38A .  1 H-NMR of EC. 
         FIG. 38B .  1 H-NMR of neomycin. 
         FIG. 38C .  1 H-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  99m Tc-EC-neomycin. 
         FIG. 42 . HPLC analysis of  99m Tc-EC-neomycin (radioactive detector). 
         FIG. 43 . HPLC analysis of  99 Tc-EC-neomycin (UV 254 nm). 
         FIG. 44 . HPLC analysis of  18 F-FDG (radioactive detector). 
         FIG. 45 . HPLC analysis of  18 F-FDG (UV 254 nm). 
         FIG. 46 . In vitro cellular uptake assay of a series of  99m Tc-EC-drug conjugates in lung cancer cell line.  99m Tc-EC-neomycin showed highest uptake in the agents tested. 
         FIG. 47 . Effect of glucose on cellular (A549) uptake of  99m Tc-EC-neomycin and  18 F-FDG. 
         FIG. 48A  and  FIG. 48B . Effect of glucose on cellular (H1299) uptake of  99m Tc-EC-neomycin and  18 F-FDG illustrated as percent of drug uptake ( FIG. 48A ) and as percent of change with glucose loading ( FIG. 48B ). 
         FIG. 49 . Synthetic scheme of  99m Tc-EC-Glucosamine 
         FIG. 50 . Hexokinase assay of glucose. 
         FIG. 51 . Hexokinase assay of glucosamine. 
         FIG. 52 . Hexokinase assay of EC-glucosamine. 
         FIG. 53 . Hexokinase assay of EC-GAP-glucosamine. 
         FIG. 54 . Synthetic scheme of  99m Tc-EC-GAP-glucosamine. 
         FIG. 55A ,  FIG. 55B ,  FIG. 55C . In vitro cellular uptake assay of  99m Tc-EC ( FIG. 56A ),  99m Tc-EC-deoxyglucose-GAP ( FIG. 56B ), and  18 F-FDG ( FIG. 56C ) in lung cancer cell line (A549).  99m Tc-EC-DG showed similar uptake compared to  18 F-FDG. 
         FIG. 56 . Tumor-to-tissue count density ratios of  99m Tc-EC-GAP in breast tumor-bearing rats. 
         FIG. 57  In vitro cellular uptake of  18 PDG with glucose loading at 2 hours post-injection in breast cancer cell line (13762). 
         FIG. 58 . In vivo tissue uptake of  99m Tc-EC-neomycin in breast tumor-bearing mice. 
         FIG. 59 . Synthetic scheme of  99m Tc-EC-deoxyglucose. 
         FIG. 60 . Mass spectrometry of EC-deoxyglucose. 
         FIG. 61 .  1 H-NMR of EC-deoxyglucose (EC-DG). 
         FIG. 62 .  1 H-NMR of glucosamine. 
         FIG. 63 . Radio-TLC analysis of  99m Tc-EC-DG. 
         FIG. 64 . HPLC analysis of  99m Tc-EC-deoxyglucose and  99m Tc-EC-(radioactive detector). 
         FIG. 65 . HPLC analysis of  99m Tc-EC-deoxyglucose and  99m Tc-EC (radioactive detector, mixed). 
         FIG. 66 . Hexokinase assay of glucose. 
         FIG. 67 . Hexokinase assay of FDG. 
         FIG. 68 . Hexokinase assay of EC-DG. 
         FIG. 69 . In vitro cellular uptake assay of  99m Tc-EC-deoxyglucose,  99m Tc-EC and  18 F-FDG in lung cancer cell line (A549).  99m Tc-EC-DG showed similar uptake compared to  18 F-FDG. 
         FIG. 70 . Effect of d- and l-glucose on breast cellular (13762 cell line) uptake of  99m Tc-EC-DG. 
         FIG. 71 . Effect of d- and l-glucose on breast cellular (13762 cell line) uptake of  18 F-FDG. 
         FIG. 72 . Effect of d- and l-glucose on lung cellular (A549 cell line) uptake of  18 F-FDG. 
         FIG. 73 . Effect of d- and l-glucose on breast cellular (A549 cell line) uptake of  99m Tc-EC-DG. 
         FIG. 74 . Effect of in vivo blood glucose level induced by glucosamine and EC-DG (1.2 mmol/kg, i.v.). 
         FIG. 75 . Effect of in vivo blood glucose level induced by FDG (1.2 and 1.9 mmol/kg, i.v.) and insulin. 
         FIG. 76 . Tumor-to-tissue count density ratios of  99m Tc-EC-deoxyglucose in breast tumor-bearing rats. 
         FIG. 77 . In vivo biodistribution of  99m Tc-EC-deoxyglucose in breast tumor-bearing rats. 
         FIG. 78 . In vivo tissue uptake of  99m Tc-EC-deoxyglucose in lung tumor-bearing mice. 
         FIG. 79 . In vivo tissue uptake of  99m Tc-EC-neomycin in lung tumor-bearing mice. 
         FIG. 80 . In vivo tissue uptake of  18 F-FDG in lung tumor-bearing mice. 
         FIG. 81 . Planar image of breast tumor-bearing rats after administration of  99m Tc-EC and  99m Tc-EC-deoxyglucose (100 μCi/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  99m Tc-EC-DG of a patient with malignant astrocytoma. 
         FIG. 83A . MRI of a patient with hemorrhagic astrocytoma. 
         FIG. 83B . SPECT with  99m Tc-EC-DG of a patient with malignant astrocytoma. 
         FIG. 84A . MRI of a patient with benign meningioma. 
         FIG. 84B . SPECT with  99m Tc-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  99m Tc-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  99m Tc-EC-DG of a patient with lung cancer. 
         FIG. 86C . SPECT with  99m Tc-EC-DG of a patient with lung cancer, the tumor showed focal intensed uptake. 
     
    
    
     DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 
     In the field of nuclear medicine, certain pathological conditions are localized, or their extent is assessed, by detecting the distribution of small quantities of internally-administered radioactively labeled tracer compounds (called radiotracers or radiopharmaceuticals). Methods for detecting these radiopharmaceuticals are known generally as imaging or radioimaging methods. 
     In radioimaging, the radiolabel is a gamma-radiation emitting radionuclide and the radiotracer is located using a gamma-radiation detecting camera (this process is often referred to as gamma scintigraphy). The imaged site is detectable because the radiotracer is chosen either to localize at a pathological site (termed positive contrast) or, alternatively, the radiotracer is chosen specifically not to localize at such pathological sites (termed negative contrast). 
     A variety of radionuclides are known to be useful for radioimaging, including  67 Ga,  99m Tc,  111 In,  123 I,  125 I,  169 Yb or  186 Re. Due to better imaging characteristics and lower price, attempts have been made to replace the  123 I,  131 I,  67 Ga and  111 In labeled compounds with corresponding  99m Tc labeled compounds when possible. Due to favorable physical characteristics as well as extremely low price ($0.21/mCi),  99m Tc has been preferred to label radiopharmaceuticals. Although it has been reported that DTPA-drug conjugate could be labeled with  99m Tc effectively (Mathias et al., 1997), DTPA moiety does not chelate with  99m Tc as stable as with  111 In. (Goldsmith, 1997). 
     A number of factors must be considered for optimal radioimaging in humans. To maximize the efficiency of detection, a radionuclide that emits gamma energy in the 100 to 200 keV range is preferred. To minimize the absorbed radiation dose to the patient, the physical half-life of the radionuclide should be as short as the imaging procedure will allow. To allow for examinations to be performed on any day and at any time of the day, it is advantageous to have a source of the radionuclide always available at the clinical site.  99m Tc is a preferred radionuclide because it emits gamma radiation 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. 
     Bis-aminoethanethiol tetradentate ligands, also called diaminodithiol compounds, are known to form very stable Tc(V)O-complexes on the basis of efficient binding of the oxotechnetium group to two thiolsulfar and two amine nitrogen atoms. (Davison et al., 1980; 1981; Verbruggen et al., 1992).  99m Tc-L,L-ethylenedicysteine ( 99m Tc-EC) is the most recent and successful example of N 2 S 2  chelates. (Verbruggen et al., 1992; Van Nerom et al., 1993; Surma et al., 1994). EC, a new renal imaging agent, can be labeled with  99m Tc easily and efficiently with high radiochemical purity and stability and is excreted through kidney by active tubular transport. (Verbruggen et al., 1992; Van Nerom et al., 1993; Surma et al., 1994; Verbruggen et al., 1990; Van Nerom et al., 1990; Jamar et al., 1993). Other applications of EC would be chelated with galium-68 (a positron emitter, t1/2=68 minutes) for PET and gadolinium, iron or manganese for magnetic resonance imaging (MRI). 
     The present invention utilizes  99m Tc-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  99m Tc-EC as a labeling strategy can be effective with virtually any type of compound, some suggested preferred ligands are provided herein for illustration purposes. It is contemplated that the  99m Tc-EC mg conjugates of the invention may be useful to image not only tumors, but also other tissue-specific conditions, such as infection, hypoxic tissue (stroke), myocardial infarction, apoptotic cells, Alzheimer&#39;s disease and endometriosis. 
     Radiolabeled proteins and peptides have been reported in the prior art. (Ege et al., U.S. Pat. No. 4,832,940, Abrams et al., 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,  99m Tc-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). 
     Although optimal for radioimaging, the chemistry of  99m Tc has not been as thoroughly studied as the chemistry of other elements and for this reason methods of radiolabeling with  99m Tc are not abundant.  99m Tc is normally obtained as  99m Tc pertechnetate (TcO 4   − ; technetium in the +7 oxidation state), usually from a molybdenum-99/technetium-99m generator. However, pertechnetate does not bind well with other compounds. Therefore, in order to radiolabel a compound,  99m Tc pertechnetate must be converted to another form. Since technetium does not form a stable ion in aqueous solution, it must be held in such solutions in the form of a coordination complex that has sufficient kinetic and thermodynamic stability to prevent decomposition and resulting conversion of  99m Tc either to insoluble technetium dioxide or back to pertechnetate. 
     For the purpose of radiolabeling, it is particularly advantageous for the  99m Tc complex to be formed as a chelate in 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  99m Tc to be covalently bound to a tissue specific ligand either directly or through a single linker between the ethylenedicysteine and the ligand. 
     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 present invention, it is important that the Tc be in the +4 oxidation state. This oxidation state is ideal for forming the N 2 S 2  chelate with EC. Thus, in forming a complex of radioactive technetium with the drug conjugates of the invention, the technetium complex, preferably a salt of  99m Tc pertechnetate, is reacted with the drug conjugates of the invention in the presence of a reducing agent. 
     The preferred reducing agent for use in the present invention is stannous ion in the form of stannous chloride (SnCl 2 ) to reduce the Tc to its +4 oxidation state. However, it is contemplated that other reducing agents, such as dithionate ion or ferrous ion may be useful in conjunction with the present invention. It is also contemplated that the reducing agent may be a solid phase reducing agent. The amount of reducing agent can be important as it is necessary to avoid the formation of a colloid. It is preferable, for example, to use from about 10 to about 100 μg SnCl 2  per about 100 to about 300 mCi of Tc pertechnetate. The most preferred amount is about 0.1 mg SnCl 2  per about 200 mCi of Tc pertechnetate and about 2 ml saline. This typically produces enough Tc-EC-tissue specific ligand conjugate for use in 5 patients. 
     It is often also important to include an antioxidant in the composition to prevent oxidation of the ethylenedicysteine. 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. 
     In general, the ligands for use in conjunction with the present invention will possess either amino or hydroxy groups 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  99m Tc using the methods of the invention by adding a linker, such as ethylenediamine, amino propanol, diethylenetriamine, aspartic acid, polyaspartic acid, glutamic acid, polyglutamic acid, or lysine. Ligands contemplated for use in the present invention include, but are not limited to, angiogenesis/antiangiogenesis ligands, DNA topoisomerase inhibitors, glycolysis markers, antimetabolite ligands, apoptosis/hypoxia ligands, DNA intercalators, receptor markers, peptides, nucleotides, antimicrobials such as antibiotics or antifungals, organ specific ligands and sugars or agents that mimic glucose. 
     EC itself is water soluble. It is necessary that the EC-drug conjugate 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 will increase the solubility of the ligand may be used. Linkers may attach to an aliphatic or aromatic alcohol, amine or peptide or to a carboxylic and or peptide. Linkers may be either poly amino acid (peptide) or amino acid such as glutamic acid, aspartic acid or lysine. Table 1 illustrates desired linkers for specific drug functional groups. 
     
       
         
           
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Drug Functional Group 
                 Linker 
                 Example 
               
               
                   
               
             
            
               
                 Aliphatic or phenolio-OH 
                 EC-Poly (glutamic acid) 
                 A 
               
               
                   
                 (MW. 750-15,000) or EC. 
               
               
                   
                 poly(aspertic acid) (MW. 
               
               
                   
                 2000-15,000) or bromo 
               
               
                   
                 ethylacetate or EC-glutamic 
               
               
                   
                 acid or EC-aspertic acid. 
               
               
                 Aliphatic or aromatic-NH 2   
                 EC-poly(glutamic acid) 
                 B 
               
               
                 or peptide 
                 (MW. 750-15,000) or EC- 
               
               
                   
                 poly(aspertic acid) (MW. 
               
               
                   
                 2000-15,000) or EC- 
               
               
                   
                 glutamic acid (mono- or 
               
               
                   
                 diester) or EC-aspartic acid. 
               
               
                 Carboxylic acid or peptide 
                 Ethylene diamine, lysine 
                 C 
               
               
                   
               
               
                 Examples: 
               
               
                 A estradiol, topotecan, paclitaxel, raloxlfen etoposide 
               
               
                 B doxorubicin, mitomycin C, endostatin, annexin V. LHRH, octreotide, VIP 
               
               
                 C methotrexate, folic acid 
               
            
           
         
       
     
     It is also envisioned that the EC-tissue specific ligand drug conjugates of the invention may be chelated to other 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  166 Ho,  188 Re,  186 Re,  153 Sm, and  89 Sr. Preferred β-emitters include  90 Y and  225 Ac. Preferred γ-emitters include  67 Ga,  68 Ga,  64 Cu,  62 Cu and  111 In. Preferred α-emitters include  211 At and  212 Bi. It is also envisioned that para-magnetic substances, such as Gd, Mn and Fe can be chelated with EC for use in conjunction with the present invention. 
     Complexes and means for preparing such complexes are conveniently provided in a kit form including a sealed vial containing a predetermined quantity of an EC-tissue specific ligand conjugate of the invention to be labeled and a sufficient amount of reducing agent to label the conjugate with  99m Tc.  99m Tc labeled scintigraphic imaging agents according to the present invention can be prepared by the addition of an appropriate amount of  99m Tc or  99m Tc complex into a vial containing the EC-tissue specific ligand conjugate and reducing agent and reaction under conditions described in Example 1 hereinbelow. The kit may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives, antioxidants, and the like. The components of the kit may be in liquid, frozen or dry form. In a preferred embodiment, kit components are provided in lyophilized form. 
     Radioactively labeled reagents or conjugates provided by the present invention are provided having a suitable amount of radioactivity. In forming  99m Tc radioactive complexes, it is generally preferred to form radioactive complexes in solutions containing radioactivity at concentrations of from about 0.01 millicurie (mCi) to about 300 mCi per mL. 
       99m Tc labeled scintigraphic imaging agents provided by the present invention can be used for visualizing sites in a mammalian body. In accordance with this invention, the  99m Tc labeled scintigraphic imaging agents are administered in a single unit injectable dose. Any of the common carriers known to those with skill in the art, such as sterile saline solution or plasma, can be utilized after radiolabeling for preparing the injectable solution to diagnostically image various organs, tumors and the like in accordance with this invention. Generally, the unit dose to be administered has a radioactivity of about 0.01 mCi to about 300 mCi, preferably 10 mCi to about 200 mCi. The solution to be injected at unit dosage is from about 0.01 mL to about 10 mL. After intravenous administration, imaging of the organ or tumor in vivo can take place, 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 to permit the taking of scintiphotos. Any conventional method of scintigraphic imaging for diagnostic or prognostic purposes can be utilized in accordance with this invention. 
     The  99m Tc-EC labeling strategy of the invention may also be used for prognostic purposes. It is envisioned that EC may be conjugated to known drugs of choice for cancer chemotherapy, such as those listed in Table 2. These EC-drug conjugates may then be radio labeled with  99m Tc and administered to a patent having a tumor. The labeled EC-drug conjugates will specifically bind to the tumor. Imaging may be performed to determine the effectiveness of the cancer chemotherapy drug against that particular patient&#39;s particular tumor. In this way, physicians can quickly determine which mode of treatment to pursue, which chemotherapy drug will be most effective. This represents a dramatic improvement over current methods which include choosing a drug and administering a round of chemotherapy. This involves months of the patient&#39;s time and many thousands of dollars before the effectiveness of the drug can be determined. 
     The  99m Tc labeled EC-tissue specific ligand conjugates and complexes provided by the invention may be administered intravenously in any conventional medium for intravenous injection such as an aqueous saline medium, or in blood plasma medium. Such medium may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmostic pressure, buffers, preservatives, antioxidants and the like. Among the preferred media are normal saline and plasma. 
     Specific, preferred targeting strategies are discussed in more detail below. 
     Tumor Folate Receptor Targeting 
     The radiolabeled ligands, such as pentetreotide and vasoactive intestinal peptide, bind to cell receptors, some of which are overexpressed on tumor cells (Britton and Granowska, 1996; Krenning et al., 1995; Reubi et al., 1992; Goldsmith et al., 1995; Virgolini et al., 1994). Since these ligands are not immunogenic and are cleared quickly from the plasma, receptor imaging would seem to be more promising compared to antibody imaging. 
     Folic acid as well as antifolates such as methotrexate enter into cells via high affinity folate receptors (glycosylphosphatidylinositol-linked membrane folate-binding protein) in addition to classical reduced-folate carrier system (Westerhof et al., 1991; Orr et al., 1995; Hsueh and Dolnick, 1993). Folate receptors (FRs) are overexposed on many neoplastic cell types (e.g., lung, breast, ovarian, cervical, colorectal, nasopharyngeal, renal adenocarcinomas, malign melanoma and ependymomas), but primarily expressed only several normal differentiated tissues (e.g., choroid plexus, placenta, thyroid and kidney) (Orr et al., 1995; Weitman et al., 1992a; Campbell et al., 1991; Weitman et al., 1992b; Hohm et al., 1994; Ross et al., 1994; Franklin et al., 1994; Weitman et al., 1994). FRs have been used to deliver folate-conjugated protein toxins, drug/antisense oligonucleotides and liposomes into tumor cells overexpressing the folate receptors (Ginobbi et al., 1997; Leamon and Low, 1991; Leamon and Low, 1992; Leamon et al., 1993; Lee and Low, 1994). Furthermore, bispecific antibodies that contain anti-FR antibodies linked to anti-T cell receptor-antibodies have been used to target T cells to FR-positive tumor cells and are currently in clinical trials for ovarian carcinomas (Canevari et al., 1993; Bolhuis et al., 1992; Patrick et al., 1997; Coney et al., 1994; Kranz et al., 1995). Similarly, this property has been inspired to develop radiolabeled folate-conjugates, such as  67 Ga-deferoxamine-folate and  111 In-DTPA-folate for imaging of folate receptor positive tumors (Mathias et al., 1996; Wang et al., 1997; Wang et al., 1996; Mathias et al., 1997b). Results of limited in vitro and in vivo studies with these agents suggest that folate receptors could be a potential target for tumor imaging. In this invention, the inventors developed a series of new folate receptor ligands. These ligands are  99m Tc-EC-folate,  99m Tc-EC-methotrexate ( 99m Tc-EC-MTX),  99m Tc-EC-tomudex ( 99m Tc-EC-TDX). 
     Tumor Hypoxia Targeting 
     Tumor cells are more sensitive to conventional radiation in the presence of oxygen than in its absence; even a small percentage of hypoxic cells within a tumor could limit the response to radiation (Hall, 1988; Bush et al., 1978; Gray et al., 1953). Hypoxic radioresistance has been demonstrated in many animal tumors but only in few tumor types in humans (Dische, 1991; Gatenby et al., 1988; Nordsmark et al., 1996). The occurrence of hypoxia in human tumors, in most cases, has been inferred from histology findings and from animal tumor studies. In vivo demonstration of hypoxia requires tissue measurements with oxygen electrodes and the invasiveness of these techniques has limited their clinical application. 
     Misonidazole (MISO) is a hypoxic cell sensitizer, and labeling MISO with different radioisotopes (e.g.,  18 F,  123 I,  99m Tc) may be useful for differentiating a hypoxic but metabolically active tumor from a well-oxygenated active tumor by PET or planar scintigraphy. [ 18 F]Fluoromisonidazole (FMISO) has been used with PET to evaluate tumors hypoxia. Recent studies have shown that PET, with its ability to monitor cell oxygen content through [ 18 F]FMISO, has a high potential to predict tumor response to radiation (Koh et al., 1992; Valk et al., 1992; Martin et al., 1989; Rasey et al., 1989; Rasey et al., 1990; Yang et al., 1995). PET gives higher resolution without collimation, however, the cost of using PET isotopes in a clinical setting is prohibitive. Although labeling MISO with iodine was the choice, high uptake in thyroid tissue was observed. Therefore, it is desirable to develop compounds for planar scintigraphy that the isotope is less expensive and easily available in most major medical facilities. In this invention, the inventors present the synthesis of  99m Tc-EC-2-nitroimidazole and  99m Tc-EC-metronidazole and demonstrate their potential use as tumor hypoxia markers. 
     Peptide Imaging of Cancer 
     Peptides and amino acids have been successfully used in imaging of various types of tumors (Wester et al., 1999; Coenen and Stocklin, 1988; Raderer et al., 1996; Lambert et al., 1990; Bakker et al., 1990; Stella and Mathew, 1990; Butterfield et al., 1998; Piper et al., 1983; Mochizuki et al., Dickinson and Hiltner, 1981). Glutamic acid based peptide has been used as a drug carrier for cancer treatment (Stella and Mathew, 1990; Butterfield et al., 1998; Piper et al., 1983; Mochizuki et al., 1985; Dickinson and Hiltner, 1981). It is known that glutamate moiety of folate degraded and formed polyglutamate in vivo. The polyglutamate is then re-conjugated to folate to form folyl polyglutamate, which is involved in glucose metabolism. Labeling glutamic acid peptide may be useful in differentiating the malignancy of the tumors. In this invention, the inventors report the synthesis of EC-glutamic acid pentapeptide and evaluate its potential use in imaging tumors. 
     Imaging Tumor Apoptotic Cells 
     Apoptosis occurs during the treatment of cancer with chemotherapy and radiation (Lennon et al., 1991; Abrams et al., 1990; Blakenberg et al., 1998; Blakenberg et al., 1999; Tait and Smith, 1991) Annexin V is known to bind to phosphotidylserin, which is overexpressed by tumor apoptotic cells (Blakenberg et al., 1999; Tait and Smith, 1991). Assessment of apoptosis by annexin V would be useful to evaluate the efficacy of therapy such as disease progression or regression. In this invention, the inventors synthesize  99m Tc-EC-annexin V (EC-ANNEX) and evaluate its potential use in imaging tumors. 
     Imaging Tumor Angiogenesis 
     Angiogenesis is in part responsible for tumor growth and the development of metastasis. Antimitotic compounds are antiangiogenic and are known for their potential use as anticancer drugs. These compounds inhibit cell division during the mitotic phase of the cell cycle. During the biochemical process of cellular functions, such as cell division, cell motility, secretion, ciliary and flagellar movement, intracellular transport and the maintenance of cell shape, microtubules are involved. It is known that antimitotic compounds bind with high affinity to microtubule proteins (tubulin), disrupting microtubule assembly and causing mitotic arrest of the proliferating cells. Thus, antimitotic compounds are considered as microtubule inhibitors or as spindle poisons (Lu, 1995). 
     Many classes of antimitotic compounds control microtubule assembly-disassembly by binding to tubulin (Lu, 1995; Goh et al., 1998; Wang et al., 1998; Rowinsky et al., 1990; Imbert, 1998). Compounds such as colchicinoids interact with tubulin on the colchicine-binding sites and inhibit microtubule assembly (Lu, 1995; Goh et al., 1998; Wang et al., 1998). Among colchicinoids, colchicine is an effective anti-inflammatory drug used to treat prophylaxis of acute gout. Colchicine also is used in chronic myelocytic leukemia Although colchicinoids are potent against certain types of tumor growth, the clinical therapeutic potential is limited due to inability to separate the therapeutic and toxic effects (Lu, 1995). However, colchicine may be useful as a biochemical tool to assess cellular functions. In this invention, the inventors developed  99m Tc-EC-colchicine (EC-COL) for the assessment of biochemical process on tubulin functions. 
     Imaging Tumor Apoptotic Cells 
     Apoptosis occurs during the treatment of cancer with chemotherapy and radiation. Annexin V is known to bind to phosphotidylserin, which is overexpressed by tumor apoptotic cells. Assessment of apoptosis by annexin V would be useful to evaluate the efficacy of therapy such as disease progression or regression. Thus,  99m Tc-EC-annexin V (EC-ANNEX) was developed. 
     Imaging Tumor Hypoxia 
     The assessment of tumor hypoxia by an imaging modality prior to radiation therapy would provide rational means of selecting patients for treatment with radiosensitizers or bioreductive drugs (e.g., tirapazamine, mitomycin C). Such selection of patients would permit more accurate treatment patients with hypoxic tumors. In addition, tumor suppressor gene (P53) is associated with multiple drug resistance. To correlate the imaging findings with the overexpression of P53 by histopathology before and after chemotherapy would be useful in following-up tumor treatment response.  99m Tc-EC-2-nitroiniidazole and  99m Tc-EC-metronidazole were developed. 
     Imaging Tumor Angiogenesis 
     Angiogenesis is in part responsible for tumor growth and the development of metastasis. Antimitotic compounds are antiangiogenic and are known for their potential use as anticancer drugs. These compounds inhibit cell division during the mitotic phase of the cell cycle. During the biochemical process of cellular functions, such as cell division, cell motility, secretion, ciliary and flagellar movement, intracellular transport and the maintenance of cell shape, microtubules are involved. It is known that antimitotic compounds bind with high affinity to microtubule proteins (tubulin), disrupting microtubule assembly and causing mitotic arrest of the proliferating cells. Thus, antimitotic compounds are considered as microtubule inhibitors or as spindle poisons. Colchicine, a potent antiangiogenic agent, is known to inhibit microtubule polymerization and cell arrest at metaphase. Colchicine (COL) may be useful as a biochemical tool to assess cellular functions.  99m Tc-EC-COL was then developed. 
     Imaging Hypoxia Due to Stroke 
     Although tumor cells are more or less hypoxic, it requires an oxygen probe to measure the tensions. In order to mimic hypoxic conditions, the inventors imaged 11 patients who had experienced stroke using  99m Tc-EC-metronidazole ( 99m Tc-EC-MN). Metronidazole is a tumor hypoxia marker. Tissue in the area of a stroke becomes hypoxic due to lack of oxygen. The SPECT images were conducted at 1 and 3 hours post injection with  99m Tc-EC-MN. All of these imaging studies positively localized the lesions. CT does not show the lesions very well or accurately. MRI and CT in some cases exaggerate the lesion size. The following are selected cases from three patients. 
     Case 1. A 59 year-old male patient suffered a stroke in the left basal ganglia. SPECT  99m Tc-EC-MN identified the lesions at one hour post-injection ( FIG. 28 ), which corresponds to MRI T1 weighted image ( FIG. 29 ). 
     Case 2. A 73 year old male patient suffered a stroke in the left medium cerebral artery (MCA) territory. SPECT  99m Tc-EC-MN was obtained at day 1 and day 12 ( FIGS. 30 and 31 ) at one hour post-injection. The lesions showed significant increased uptake at day 12. CT showed extensive cerebral hemorrhage in the lesions. No marked difference was observed between days 1 and 12 ( FIGS. 32 and 33 ). The findings indicate that the patient symptoms improved due to the tissue viability (from anoxia to hypoxia). SPECT  99m Tc-EC-MN provides functional information which is better than CT images. 
     Case 3. A 72 year old male patient suffered a stroke in the right MCA and PCA area SPECT  99m Tc-EC-MN identified the lesions at one hour post-injection ( FIG. 34 ). CT exaggerates the lesion size. ( FIG. 35 ). 
     Tumor Glycolysis Targeting 
     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 (Rogers 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 by insulin (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 imaging would seem to be more promising compared to antibody imaging. 
     The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. 
     EXAMPLE 1 
     Tumor Folate Receptor Targeting 
     Synthesis of EC 
     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 structure was confirmed by  1 H-NMR and fast-atom bombardment mass spectroscopy (FAB-MS). 
     Synthesis of Aminoethylamido Analogue of Methotrexate (MTX-NH 2 ) 
     MIX (227 ma, 0.5 mmol) was dissolved in 1 ml of HCl solution (2N). The pH value was &lt;3. To this stirred solution, 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 was stirred overnight and the solvent was evaporated in vacuo. The raw solid material was washed with diethyl ether (10 ml), acetonitrile (10 ml) and 95% ethyl alcohol (50 ml) to remove the unreacted EEDQ and EDA. The product was then dried 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);  1 H-NMR (D 2 O) δ 2.98-3.04 (d, 8H, —(CH 2 ) 2 CONH(CH 0 ) 2 NH 2 ), 4.16-4.71 (m, 6H, —CH 2 -pteridinyl, aromatic-NCH 3 , NH—CH—COOH glutamate), 6.63-6.64 (d, 2H, aromatic-CO), 7.51-753 (d, 2H, aromatic-N), 8.36 (s, 1H, pteridinyl). FAB MS m/z calcd for C 22 H 28 ,N 10 ,O 4 (M) +  496.515, found 496.835. 
     Synthesis of Aminoethylamido Analogue of Folate (Folate-NH 2 ) 
     Folic acid dihydrate (1 g, 2.0 mmol) was added in 10 ml of water. The pH value was adjusted to 2 using HCl (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 room temperature. The solvent was evaporated in vacuo. The product was precipitated in methanol (50 ml) and further washed with acetone (100 ml) to remove the unreacted EEDQ and EDIT. The product was then freeze-dried and used without further purification. Ninhydrin (2% in methanol) spray indicated the positivity of amino group. The product weighed 0.6 g (yield 60%) as a yellow powder. m.p. of product: 250° (dec).  1 H-NMR D 2 O) δ1.97-2.27 (m, 2H, —CH 2  glutamate of folate), 3.05-3.40 (d, 6H, —CH 2 CONH(CH 2 ) 2 NH 2 ), 4.27-4.84 (m, 3H, —CH 2 -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 C 21 H 25 N 9 ,O 5 (M) +  483, found 483.21. 
     Synthesis of Ethylenedicysteine-folate (EC-Folate) 
     To dissolve EC, NaOH (2N, 0.1 ml) was added to a stirred solution of EC (114 ma, 0.425 mmol) in water (1.5 ml). To this colorless solution, sulfo-NHS (92.3 mg, 0.425 mmol) and EDC (81.5 mg, 0.425 mmol) were added. Folate-NH 2  (205 mg, 0.425 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 molecule cut-off at 500 (Spectrum Medical Industries Inc., Houston, Tex.). After dialysis, the product was freeze dried. The product weighed 116 mg (yield 35%). m.p. 195° (dec);  1 H-NMR (D 2 O) δ1.98-2.28 (m, 2H, —CH2 glutamate of folate), 2.60-2.95 (m, 4H and —CH 2 —SH of EC). 3.24-3.34 (m, 10H, —CH 2 —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 C29H37N 11 S 2 O 8  Na 2 (8H 2 O), 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  99m Tc 
     Radiosynthesis of  99m Tc-EC-folate was achieved by adding required amount of  99m Tc-pertechnetate into home-made kit containing the lyophilized residue of EC-folate (3 mg), SnCl 2  (100 μg), Na 2 HPO 4  (13.5 mg), ascorbic acid (0.5 mg) and NaEDTA (0.5 mg). Final pH of preparation was 7.4.  99m Tc-EC was also obtained by using home-made kit containing the lyophilized residue of EC (3 mg), SnCl 2  (100 μg), Na 2 , IPO 4  (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 (ITLC SG, Gelman Sciences, Ann Arbor, Mich.) eluted with, respectively, acetone (system A) and ammonium acetate (1M in water):methanol (4:1) (system B). From radio-TLC (Bioscan, Washington, D.C.) analysis, the radiochemical purity was &gt;95% for both radiopharmaceuticals. Radio-TLC data are summarized in Table 2. Synthesis of  99m Tc-EC-folate is shown in  FIG. 1 . 
                     TABLE 2                  DRUGS OF CHOICE FOR CANCER CHEMOTHERAPY       The tables that follow list drugs used for treatment of cancer in the USA and       Canada and their major adverse effects. The Drugs of Choice listing based on the       opinions of Medical Letter consultants. Some drugs are listed for indications for which       they have not been approved by the US Food and Drug Administration. Anticancer drugs       and their adverse effects follow. For purposes of the present invention, these lists are       meant to be exemplary and not exhaustive.               DRUGS OF CHOICE                         Cancer   Drugs of Choice   Some alternatives               Adrenocortical**   Mitotane   Doxorubicin, streptozocin,           Cisplatin   etoposide       Bladder*   Local: Instillation of BCG   Instillation of mitomycin,           Systemic: Methotrexate + vinblastine + doxorubicin + claplatin   doxorubicin or thiotape           (MVAC)   Pecitaxel, substitution of           Claplatin + Methotrexate + vinblastine   carboplatin for claplatin in           (CMV)   combinations       Brain       Anaplastic astrocytoma*   Procarbazine + lamuatine + vincristine   Carmustine, Claplatin       Anaplastic oligodendro-   Procarbazine + lamustine + vincristine   Carmustine, Claplatin       Giloma*       Gilabiastome**   Carmustine or lamustine   Procarbazine, claplatin       Medulloblastoma   Vincristine + carmustine ± mechiorethamine ± methotrexate   Etoposide           Mechiorethamine + vincristine + procarbazine + prednisone           (MOPP)           Vincristine + claplatin ± cyclophosphamide       Primary central nervous   Methotrexate (high dose Intravenous and/or       system lymphoma   Intrathecal) ± cytarabine (Intravenous and/or           Intrathecal)           Cyclophosphamide + Doxorubicin + vincristine + prednisone           (CHOP)       Breast   Adjuvant 1 : Cyclophosphamide + methotrexate + fluorouracil           (CMF);           Cyclophosphamide + Doxorubicin ± fluorouracil           (AC or CAF); Tamoxifen           Metastatic: Cyclophosphamide + methotrexate + fluorouracil   Paclitaxel; thiotepa +           (CMF) or   Doxorubicin + vin-blastine;           Cyclophosphamide + duxorubicin ± fluorouracil   mitomycin + vinblastine;           (AC or CAF) for receptor-   mitomycin + methotrexate +           negative and/or hormone-refractory;   mitoxantrone; fluorouracil by           Tamoxifen for receptor-positive and/or   continuous infusion; Bone           hormone-sensitive 2     marrow transplant 3         Cervix**   Claplatin   Chlorambucil, vincristine,           Ifosfamide with means   fluorouracil, Doxorubicin,           Bleomycin + ifosfamide with means + claplatin   methotrexate, altretamine       Chorlocarcinoma   Methotrexate ± leucovorin   Methotrexate + dactinomycin +           Dactinomycin   cyclophosphamide (MAC)               Etoposide + methotrexate +               dactinomycin +               cyclophosphamide + vincristine       Colorectal*   Adjuvant colon 4 : Fluorouracil + levam-isole;   Hepatic metastases:           fluorouracil + leucovorin   Intrahepatic-arterial floxuridine           Metastatic: fluorouracil + leucovorin   Mitomycin       Embryonal rhabdomyosar-coma 5     Vincristine + dectinomycin ± cyclophasphamide   Same + Doxorubicin           Vincristine + ifosfamide with means + etoposide       Endometrial**   Megastrol or another progestin   fluorouracil, tamoxifen,           Doxorubicin + claplatin ± cyclophosphamide   altretamine       Esophageal*   Claplatin + fluorouracil   Doxorubicin, methotraxate,               mitomycin       Ewing&#39;s sarcoma 5     Cyclophosphamide (or ifosfamide with   CAV + etoposide           means) + Doxorubicin + vincristine (CAV) ± dactinomycin       Gastric**   Fluorouracil ± leucavorin   Claplatin Doxorubicin,               etoposide, methotrexate + leucovorin,               mitomycin       Head and neck squambus cell* 6     Claplatin + fluorouracil   Blomycin, carboplatin, paclitaxel           Methotrexate       Islet cell**   Streptozocin + Doxorubicin   Streptozocin + fluorouracil;               chlorozotocin † ; octreotide       Kaposi&#39;s sarcoma* (Aids-related)   Etoposide or interferon alfa or vinblastine   Vincristine, Doxorubicin,           Doxorubicin + bleomycin + vincristine or   bleomycin           vinblastine (ABV)       Leukemia       Acute lymphocytic leukemia   Induction: Vincristine + prednisone + asparaginase ± daunorubicin   Induction: same ± high-dose       (ALL) 7     CNS prophylaxis: Intrathecal methotrexate ± systemic   methotrexate ± cyterabine;           high-dose methotrexate with   pegaspargase instead of           leutovorin ± Intrathecal cytarabine ± Intrathecal   asparaginese           hydrocortisone   Teniposide or etoposide               High-dose cytarabine           Maintenance: Methotrexate + mercaptopurine   Maintenance: same + periodic           Bone marrow transplant. 3 8     vincristine + prednisone       Acute myeloid leukemia (AML) 9     Induction: Cytsrabine + either daunorubicin   Cytarabine + mitoxentrone           or idarubicin   High-dose cyterabine           Post Induction: High-dose cytarabine ± other           drugs such as etoposide           Bone marrow transplant 3 .       Chronic lymphocytic leukemia   Chlorambucil ± prednisone   Cladribine, cyclophosphamide,       (CLL)   Fludarabin   pentostatin, vincristine,               Doxorubicin       Chronic myeloid leukemia       (CML) 10         Chronic phase   Bone marrow transplant 3     Busulfan           Interferon alfa           Hydroxyures       Accelerated 11     Bone marrow transplant 3     Hydroxyures, busulfen       Blast crisis 11     Lymphoid: Vincristine + prednisone + L-   Tretinoln †             separaginess + intrathecal methotrexate (± maintenance   Amsecrine,  † azacitidine           with methotrexate + 8-   Vincristine ± plicamycin           marcaptopurine)       Hairy cell Leukemia   Pentostatin or cladribine   Interferon alfa, chlorambucil,               fludarabin       Liver**   Doxorubicin   Intrahepatic-arterial floxuridine           Fluorouracil   or claplatin       Lung, small cell (cat cell)   Claplatin + etoposide (PE)   Ifosfamide with means +           Cyclophosphamide + doxorubicin + vincristine   carboplatin + etoposide (ICE)           (CAV)   Daily oral etoposide           PE alternated with CAV   Etoposide + ifosfamide with           Cyclophosphamide + etoposide + claplatin   means + claplatin (VIP           (CEP)   Paclitaxel           Duxorubicin + cyclophosphamide + etoposide           (ACE)       Lung   Claplatin + etoposide   Claplatin + fluorouracil + leucovorin       (non-small cell)**   Claplatin + Vinblastine ± mitomycin   Carboplatin + paclitaxel           Claplatin + vincrisine       Lymphomas       Hodgkin&#39;s 12     Doxorubicin + bleomycin + vinblastine + dacarbazine   Mechlorethamine + vincristine +           (ABVD)   procarbazine + prednisone (MOPP)           ABVD alternated with MOPP   Chlorambusil + vinblastine +           Mechlorethamine + vincristine + procarbazine   procarbazine + prednisone ±           (± prednisone) + doxorubicin + bleomycin + vinblastine   carmustine           (MOP[P]-ABV)   Etoposide + vinblastine +               doxorubicin               Bone marrow transplant 3         Non-Hodgkin&#39;s       Burkitt&#39;s lymphoma   Cyclophosphamide + vincristine + methotrexate   Ifosfamide with means           Cyclophosphamide + high-dose cytarabine ± methotrexate   Cyclophosphamide +           with leutovorin   doxorubicin + vincrletine +           Intrathecal methotrexate or cytarabine   prednisone (CHOP)       Difuse large-cell lymphoma   Cyclophosphamide + doxorubicin + vincristine + prednisone   Dexamethasone sometimes           (CHOP)   substituted for prednisone               Other combination regimens,               which may include methotrexate,               etoposide, cytarabine,               bleomycin, procarbazine,               ifosfamide and mitoxantrone               Bone marrow transplant 3         Follicular lymphoma   Cyclophosphamide or chlorambusil   Same ± vincristine and               prednisone, ± etoposide               Interferon alfa, cladribine,               fludarabin               Bone marrow transplant 3                 Cyclophosphamide +               doxorubicin + vincristine +               prednisone (CHOP)       Melanoma**   Interferon Alfa   Carmustine, lomustine, cisplatin           Dacarbazine   Dacarbazine + clapletin +               carmustine + tamoxifen               Aldesleukin       Mycosis fungoides*   PUVA (psoralen + ultraviolet A)   Isotretinoin, topical carmustine,           Mechlorethamine (topical)   pentosistin, fludarabin,           Interferon alfa   cladribine, photopheresis (extra-           Electron beam radiotherapy   corporeal photochemitherapy),           Methotrexate   chemotherapy as in non-               Hodgkin&#39;s lymphoma       Mysloma*   Melphelan (or cyclophosphamide) + prednisons   Interferon alfa           Melphalan ± carmustine + cyclophosphamide + prednisons + vincristine   Bone marrow transplant 3             Dexamethasone + doxorubicin + vincristine   High-dose dexamethasons           (VAD)           Vincristine + carmustine + doxorubicin + prednisons           (VBAP)       Neuroblestoma*   Doxorubicin + cyclophosphamide + claplatin + teniposide   Carboplatin, etoposide           or etoposide   Bone marrow transplant 3             doxorubicin + cyclophosphamide           Claplatin + cyclophosphamide       Osteogenic sarcoma 5     Doxorubicin + claplatin ± etopside ± ifosfamide   Ifosfamide with means,               etoposide, carboplatin, high-               dose methotrexate with               leucovorin               Cyclophosphamide + etoposide       Ovary   Claplatin (or carboplatin) + paclitaxel   Ifosfamide with means,           Claplatin (or carboplatin) + cyclophosphamide   paclitaxel, tamoxifen,           (CP) ± doxorubicin   melphalan, altretamine           (CAP)       Pancreatic**   Fluoroutacil ± leucovorin   Gemoltabinet       Prostate   Leuprolide (or goserelln) ± flutamide   Estramustine ± vinblastine,               aminoglutethimide + hydrocortleone,               estramustine + etoposide,               diethylstllbestrol, nilutamide       Renal**   Aldesleukin   Vinblastine, floxuridine           Inteferon alfa       Retinoblestoma 5 *   Doxorubicin + cyclophosphamide ± claplatin ± etoposide ± vincristina   Carboplatin, etoposide,               Ifosfamide with means       Sarcomas, soft tissue, adult*   Doxorubicin ± decarbazine ± cyclophosphamide ± Ifosfamide   Mitornyeln + doxorubicin + claplatin           with   Vincristina, etoposide           means       Testicular   Claplatin + etoposide ± bleomycin   Vinblestine (or etoposide) + Ifosfamide           (PEB)   with means + claplatin               (VIP)               Bone marrow transplant 3         Wilms&#39; tumor 5     Dectinomycln + vincriatine ± doxorubicin ± cyclophosphamide   Ifosfamide with means,               etoposide, carboplatin                         *Chemotherapy has only moderate activity.       **Chemotherapy has only minor activity.         1 Tamoxifen with or without chemotherapy is generally recommended for postmenopausal estrogen-receptor-positive, mode-positive patients       and chemotherapy with or without tamoxlfen for premenopausal mode-positive patients. Adjuvant treatment with chemotherapy and/or       tamoxifen is recommended for mode-negative patients with larger tumors or other adverse prognostic indicators.         2 Megastrol and other hormonal agents may be effective in some patients with tamoxifen fails.         3 After high-dose chemotherapy (Medical Letter, 34: 79, 1982).         4 For rectal cancer, postoperative adjuvant treatment with fluoroutacil plus radiation, preceded and followed by treatment with fluorouracil alone.         5 Drugs have major activity only when combined with surgical resection, radiotherapy or both.         6 The vitamin A analog lactratinoln (Acgutana) can control pre-neoplastic lesions (leukoplakla) and decreases the rate of second primary       tumors (SE Banner et al, J Natl Cancer Inst, 88: 140 1994).         † Available in the USA only for investigational use.         7 High-risk patients (e.g., high counts, cytogenetic abnormalities, adults) may require additional drugs for induction, maintenance       and “Intensificiation” (use of additional drugs after achievement of remission). Additional drugs include       cyclophosphamida, mitoxantrone and thloguanine. The results of one large controlled trial in the United Kingdom suggest that       Intensificiation may improve survival in all children with ALL (JM Chasselle et al, Lancet, 34B: 143, Jan. 21, 1995).         8 Patients with a poor prognosis initially or those who relapse after remission.         9 Some patients with acute promyelocytic leukemia have had complete responses to tratinoin. Such treatment can       cause a toxic syndrome characterized primarily by fever and respiratory distress (RP Warrell, Jr et al, N Engl       J Med. 328: 177, 1993).         10 Allogeheic HLA-identical sibling bone marrow transplantation can cure 40% to 70% of patients with CML       in chronic phase, 18% to 28% of patients with accelerated phase CML, and &lt;15% patients in blast crisis.       Disease-free survival after bone marrow transplantations adversely influenced by age &gt;50 years, duration       of disease &gt;3 years from diagnosis, and use of one-antigen-mismatched or matched-unrelated donor marrow.       Interferon also may be curative in patients with chronic phase CML who achieve a complete cytogenetic response       (about 10%); it is the treatment of choice for patents &gt;80 years old with newly diagnosed chronic phase CML and       for all patients who are not candidates for an allgensic bone marrow transplant. Chemotherapy alone is palliative.         11 If a second chronic phase is achieved with any of these combinations, allogeneic bone marrow transplant       should be considered. Bone marrow transplant in second chronic phase may be curative for 30% to 35% of patients with CML.         12 Limited-stage Hodgkin&#39;s disease (stages 1 and 2) is curable by radiotherapy. Disseminated disease (stages 3b and 4)       require chemotherapy. Some intermediate stages and selected clinical situations may benefit from both.       + Available in the USA only for investigational use.                         ANTICANCER DRUGS AND HORMONES                                         Drug   Acute Toxicity‡   Delayed toxicity‡                       Aldesleukin (Interleukin-2;   Fever; fluid retention; hypertension;   Neuropsychiatric disorders;           Proleukin - Cetus   respiratory distress; rash; anemia;   hypothyrldiam; nephrotic           Oncology)   thrombocytophenia; nausea and   syndrome; possibly acute               vomiting; diarrhea; capillary leak   leukoencaphalopathy;               syndrome; naphrotoxlolty; myocardial   brachial plexopathy; bowel               toxicity; hepatotoxicity; erytherna   perforation               nodosum; neutrophil chemotactic defects           Altretamine (hexamethyl-   Nausea and vomiting   Bone marrow depression;           melamine; Hexalen - U       CNS depression; peripheral           Bioscience)       neuropathy; visual                   hallucinations; stexis;                   tremors, alopecia; rash           Aminogiutethimide (Cytadren -   Drowsiness; nausea; dizziness; rash   Hypothryroidism (rare); bone           Ciba)       marrow depression; fever;                   hypotension; mascullinization           †Amsacrine (m-AMSA;   Nausea and vomiting; diarrhea; pain or   Bone marrow depression;           amaidine; AMSP P-D-   phlebitis on infuelon; anaphylaxia   hepactic injury; convulsions;           Parke-Davis, Amsidyl-       stomatitle; ventricular           Warner-Lambert)       fibrillation; alopecia;                   congestive heart failure; renal                   dysfunction           Asparaginase (Elspar-merck;   Nausea and vomiting; fever; chills;   CNS depression or           Kidrolase in Canada)   headache; hypersensitivity, anaphylexia;   hyperexcitability; acute               abdominal pain; hyperglycemia leading   hemorrhagic pancreatitis;               to coma   coagulation defects;                   thromboals; renal damage;                   hepactic damage           Cervix**   Claplatin Ifosfamide with means   Chlorambucil, vincristine,               Bleomycin patin   fluoroutacil, doxorubicin,               Ifosfamide with means   methotrexete, altretamine           Chorlocarcinoma   Methotrexete ± leucovorin   Methotrexete + dectinomycin +               Dactinomyclin   cyclophosphamide (MAC)                   Etoposide + methotrexate +                   dactinomycin + cyclophosphamide +                   vincrlatine           Colorectal*   Adjuvant colon 4 : Fluoroutacil + lavamleole;   Hepatic metastases:               fluoroutacil + leucovarin   Intrahepactic-arterial               Metastatic: Fluoroutacil + leucvarin   floxuridine                   Mitomyclin           Embryonal   Vincriatine + dectinomycin ± cyclophosphamide   Same + doxorubicin           rhebdomyosarcoma 6     Vincristine + Ifosfamide with means + etoposide           Endometrial**   Megastrol or another progeetin   Fluoroutacil, tamoxifen,               Doxorubicin + claplatin ± cyclophosphamide   altretamine                       Cancer   Drugs of Choice   Some alternatives                       Esophageal*   Claplatin + Fluoroutacil   Doxorubicin, methotrexete,           Ewing&#39;s sarcoma 5     Cyclophosphamide (or ifosfamide with   mitomycin               means) + doxorubicin + vincrietine   CAV + etoposide               (CAV) ± dectinomycin           Gastric**   Fluoroutacil ± leucovoin   Claplatin, doxorubicin,                   etoposide, methotrexete + leucovorin,                   mitomycin           Head and neck squamous   Claplatin + fluoroutacil   Blaonycin, carboplatin,           cell* 5     Methotrexete   paciltaxel           Islet call**   Streptozocin + doxorubicin   Streptozocln + fluoroutacil;                   chlorozotocin; actreatide           Kaposal&#39;s sercoma*   Etoposide or Interferon alfa or   Vincristine, doxorubicin,           (AIDS-related)   vinbleomycin stine   bleomycln               Doxorubicin + bleomycin + vincristine               or vinbleomycin stine (ABV)           Leukemias   Induction: Vincristine + prednisone + asparaginase ± daunorubieln   Industion: same ± high-dose           Acute lymphocytic leukemia   CNS prophylaxia; Intrathecal   methotrexete ± cyterabine;           (ALL) 7     methotrexete ± systemic high-dose   pegaspargase instead of               methotrexete with leucovorin ± Intrethecal   aspareginese               cytarabine ± Intrathecal   Teniposide or etoposide               hydrocortisone   High-dose cytarabine               Maintenance: methotrexete ± mercaptopurine   Maintenance: same + periodic               Bone marrow transplant 3     vincristine + prednisone           Acute myeloid leukemia   Induction: Cytarabine + either   Cytarabine + mitoxantrone           (AML) 9     daunbrublein or idarubieln   High-dose cytarabine               Post Induction: High-dose cytarabine ± other               drugs such as etoposide               Bone marrow transplant 3             Chronic lymophocytic   Chlorambuell ± prednisone   Claplatin, cyclophosphamide,           leukemia (CLL)   Fludarabin   pentostatin, vinorlstine,                   doxorubicin                             †Available in the USA only for investigational use.       ‡Dose-limiting effects are in bold type. Cutaneous reactions (sometimes severe), hyperpigmentation, and ocular toxicity have       been reported with virtually all nonhormonal anticancer drugs. For adverse interactions with other drugs, see the Medical Letter       Handbook of Adverse Drug Interactions, 1995.         1 Available in the USA only for investigational use.         2 Megestrol and other hormonal agents may be effective in some pateients when tamoxifen fails.         3 After high-dose chemotherapy (Medical Letter, 34: 78, 1992).         4 For rectal cancer, postoperative adjuvant treatment with fluoroutacil plus radiation, preceded and followed by treatment with       fluoroutacil alone.         5 Drugs have major activity only when combined with surgical resection, radiotherapy or both.         6 The vitamin A analog isotretinoin (Accutane) can control pre-neoplastic isions (leukoplaka) and decreases the rats of second       primary tumors (SE Senner et al., J Natl Cancer Inst. 88: 140, 1994).         7 High-risk patients (e.g., high counts, cytogenetic abnormalities, adults) may require additional drugs for Induction, maintenance       and “Intensification” (use of additional drugs after achievement of remission). Additional drugs include cyclophosphamide,       mitoxantrone and thioguamine. The results of one large controlled trial in the United Kingdom suggest that intensilibation may       improve survival in all children with ALL (jm Chassella et al., Lancet, 348: 143, Jan. 21. 1998).         8 Patients with a poor prognosis initially or those who relapse after remission         9 Some patients with acute promyclocytic leukemia have had complete responses to tretinoin. Such treatment can cuase a toxic       syndrome characterized primarily by fever and respiratory distress (RP Warrell, Jr et al. N Eng J. Med, 329: 177, 1993).         10 Allogenaic HLA Identical sibling bone marrow transplantation can cure 40% to 70% of patients with CML in chroni phase, 15%       to 25% of patients with accelerated phase CML, and &lt;15% patients in blast crisis. Disease-free survival after bone marrow       transplantation is adversely influenced by age &gt;50 years, duration of disease &gt;3 years from diagnosis, and use of one antigen       mismatched or matched-unrelated donor marrow. Inteferon alfa may be curative in patients with chronic phase CML who       achieve a complete cytogenetic resonse (about 10%); It is the treatment of choices for patients &gt;50 years old with newly       diagnosed chronic phase CML and for all patients who are not candidates for an allogenic bone marrow transplant.       Chemotherapy alone is palliative.                    
Radiolabeling of EC-MTX and EC-TDX with  99m Tc
 
     Use the same method described for the synthesis of EC-folate, EC-MTX and EC-TDX were prepared. The labeling procedure is the same as described for the preparation of  99m Tc-EC-folate except EC-MTX and EC-TDX were used. Synthesis of  99m Tc-EC-MTX and  99m Tc-EC-TDX is shown in  FIG. 2  and  FIG. 3 . 
     Stability Assay of  99m Tc-EC-folate,  99m Tc-EC-MTX and  99m Tc-EC-TDX 
     Stability of  99m Tc-EC-Folate,  99m Tc-EC-MTX and  99m Tc-EC-TDX was tested in serum samples. Briefly, 740 KBq of 1 mg  99m Tc-EC-Folate,  99m Tc-EC-MIX and  99m Tc-EC-TDX was incubated in dog serum (200 μl) at 37° C. for 4 hours. The serum samples was diluted with 50% methanol in water and radio-TLC repeated at 0.5, 2 and 4 hours as described above. 
     Tissue Distribution Studies 
     Female Fischer 344 rats (150±25 g) (Harlan Sprague-Dawley, Indianapolis, Ind.) were inoculated subcutaneously with 0.1 ml of mammary tumor cells from the 13762 tumor cell line suspension (10 6  cells/rat, a tumor cell line specific to Fischer rats) into the hind legs using 25-gauge needles. Studies performed 14 to 17 days after implantation when tumors reached approximately 1 cm diameter. Animals were anesthetized with ketamine (10-15 mg/rat, intraperitoneally) before each procedure. 
     In tissue distribution studies, each animal injected intravenously with 370-550 KBq of  99m Tc-EC-folate or  99m Tc-EC (n=3/time point). The injected mass of each ligand was 10 μg per rat. At 20 min, 1, 2 and 4 h following administration of the radiopharmaceuticals, the anesthetized animals were sacrificed and the tumor and selected tissues were excised, weighed and counted for radioactivity by a gamma counter (Packard Instruments, Downers Grove, Ill.). The biodistribution of tracer in each sample was calculated as percentage of the injected dose per gram of tissue wet weight (% ID/g). Counts from a diluted sample of the original injectate were used for reference. Tumor/nontarget tissue count-density ratios were calculated from the corresponding % ID/g values. Student-t test was used to assess the significance of differences between two groups. 
     In a separate study, blocking studies were performed to determine receptor-mediated process. In blocking studies, for  99m Tc-EC-folate was co-administrated (iv.) with 50 and 150 μmol/kg folic acid to tumor bearing rats (n=3/group). Animals were killed 1 h post-injection and data was collected. 
     Scintigraphic Imaging and Autoradiography Studies 
     Scintigraphic images, using a gamma camera (Siemens Medical Systems, Inc., Hoffman Estates, Ill.) equipped with low-energy, parallel-hole collimator, were obtained 0.5, 2 and 4 hrs after i.v. injection of 18.5 MBq of  99m Tc-labeled radiotracer. 
     Whole-body autoradiogram were obtained by a quantitative image analyzer (Cyclone Storage Phosphor System, Packard, Meridian, CI.). Following i.v. injection of 37 MBq of  99m Tc-EC-folate, animal killed at 1 h and body was fixed in carboxymethyl cellulose (4%). The frozen body was mounted onto a cryostat (LKB 2250 cryomicrotome) and cut into 100 μm coronal sections. Each section was thawed and mounted on a slide. The slide was then placed in contact with multipurpose phosphor storage screen (MP, 7001480) and exposed for 15 h  99m Tc-labeled). The phosphor screen was excited by a red laser and resulting blue light that is proportional with previously absorbed energy was recorded. 
     Results 
     Chemistry and Stability of  99m Tc-EC-Folate 
     A simple, fast and high yield aminoethylamido and EC analogues of folate, MTX and TDX were developed. The structures of these analogues were confirmed by NMR and mass spectroscopic analysis. Radiosynthesis of EC-folate with  99m Tc was achieved with high (&gt;95%) radiochemical purity.  99m Tc-EC-folate was found to be stable at 20 min. 1, 2 and 4 hours in dog serum samples. 
     Biodistribution of  99m Tc-EC-folate 
     Biodistribution studies showed that tumor/blood count density ratios at 20 min-4 h gradually increased for  99m Tc-EC-folate, whereas these values decreased for  99m Tc-EC in the same time period ( FIG. 4 ). % ID/g uptake values, tumor/blood and tumor/muscle ratios for  99m Tc-EC-folate and  99m Tc-EC were given in Tables 3 and 4, respectively. 
                     TABLE 3                  Biodistribution of  99m Tc-EC-folate in Breast Tumor-Bearing Rats                         % of injected  99m Tc-EC-folate dose per organ or tissue                                     20 min   1 h   2 h   4 h                                             Blood   0.370 ± 0.049   0.165 ± 0.028   0.086 ± 0.005   0.058 ± 0.002       Lung   0.294 ± 0.017   0.164 ± 0.024   0.092 ± 0.002   0.063 ± 0.003       Liver   0.274 ± 0.027   0.185 ± 0.037   0.148 ± 0.042   0.105 ± 0.002       Stomach   0.130 ± 0.002   0.557 ± 0.389   0.118 ± 0.093   0.073 ± 0.065       Kidney   4.328 ± 0.896   4.052 ± 0.488   5.102 ± 0.276   4.673 ± 0.399       Thyroid   0.311 ± 0.030   0.149 ± 0.033   0.095 ± 0.011   0.066 ± 0.011       Muscle   0.058 ± 0.004   0.0257 ± 0.005    0.016 ± 0.007    0.008 ± 0.0005       Intestine   0.131 ± 0.013   0.101 ± 0.071   0.031 ± 0.006   0.108 ± 0.072       Urine   12.637 ± 2.271    10.473 ± 3.083    8.543 ± 2.763   2.447 ± 0.376       Tumor   0.298 ± 0.033   0.147 ± 0.026   0.106 ± 0.029   0.071 ± 0.006       Tumor/Blood   0.812 ± 0.098   0.894 ± 0.069   1.229 ± 0.325   1.227 ± 0.129       Tumor/Muscle   5.157 ± 0.690   5.739 ± 0.347   6.876 ± 2.277   8.515 ± 0.307               Values shown represent the mean ± standard deviation of data from 3 animals            
Scintigraphic Imaging and Autoradiography Studies
 
     Scintigraphic images obtained at different time points showed visualization of tumor in  99m Tc-EC-folate injected group. Contrary, there was no apparent tumor uptake in  99m Tc-EC injected group ( FIG. 6 ). Both radiotracer showed evident kidney uptake in all images. Autoradiograms performed at 1 h after injection of  99m Tc-EC-folate clearly demonstrated tumor activity. 
     EXAMPLE 2 
     Tumor Hypoxia Targeting 
     Synthesis of 2-(2-methyl-5-nitro- 1 H imidazolyl)ethylamine (amino analogue of metronidazole, MN-NH 2 ) 
     Amino analogue of metronidazole was synthesized according to the previously described methods (Hay et al., 1994) Briefly, metronidazole was converted to a mesylated analogue (m.p. 149-150° C., reported 153-154° C., TLC:ethyl acetate, Rf=0.45), yielded 75%. Mesylated metronidazole was then reacted with sodium azide to afford azido analogue (TLC:ethyl acetate, Rf=0.52), yielded 80%. The azido analogue was reduced by triphenyl phosphine and yielded (60%) the desired amino analogue (m.p. 190-192° C., reported 194-195° C., TLC:ethyl acetate, Rf=0.15). Ninhydrin (2% in methanol) spray indicated the positivity of amino group of MN-NH 2 . The structure was confirmed by  1 H-NMR and mass spectroscopy (FAB-MS) m/z 171(M + H, 100). 
     Synthesis of Ethylenedicysteine-Metronidazole (EC-MN) 
     Sodium hydroxide (2N, 0.2 ml) was added to a stirred solution of EC (134 ma, 0.50 mmol) in water (5 ml). To this colorless solution, sulfo-NHS (217 mg, 1.0 mmol) and 1˜)C (192 ma 1.0 mmol) were added. MN-NH: dihydrochloride salt (340 mg, 2.0 mmol) was then added. The mature was stirred at room temperature for 24 hours. The mixture was dialyzed for 48 hrs using Spectra/POR molecular porous membrane with cut-off at 500 (Spectrum Medical Industries Inc., Houston, Tex.). After dialysis, the product was frozen dried using lyophilizer (Labconco, Kansas City, Mo.). The product weighed 315 mg (yield 55%).  1 H-NMR (D 2 O) δ 2.93 (s, 6H, nitroimidazole- CH   3 ), 2.60-2.95 (m, 4H and — CH   2 —SH of EC), 3.30-3.66 (m, 8H, ethylenediamine of EC and nitromidazole-CH 2 — CH   2 —NH 2 ), 3.70-3.99 (t, 2H, NH—CH—CO of EC), 5.05 (t, 4H, metronidazole- CH   2 -CH 2 —NH 2 ) (s, 2H, nitroimidazole C═ CH ). FAB MS m/z 572 (M + , 20). The synthetic scheme of EC-MN is shown in  FIG. 7 . 
     Synthesis of 3-(2-nitro- 1 H-imidazolyl)propylamine (amino analogue of nitroimidazole, NIM-NH 2 ) 
     To a stirred mixture containing 2-nitroimidazole (1 g, 8.34 mmol) and Cs 2 ,CO 3  (2.9 g, 8.90 mmol) in dimethylformaide (DMF, 50 ml), 1,3-ditosylpropane (3.84 g, 9.99 mmol) was added. The reaction was heated at 80° C. for 3 hours. The solvent was evaporated under vacuum and the residue was suspended in ethylacetate. The solid was filtered, the solvent was concentrated, loaded on a silica gel-packed column and eluted with hexane:ethylacetate (1:1). The product, 3-tosylpropyl-(2-nitroimidazole), was isolated (1.67 g, 57.5%) with m.p. 108-111° C.  1 H-NMR (CDCl 3 ) δ 2.23 (m, 2H), 2.48 (S, 3H), 4.06 (t, 2H, J=5.7 Hz), 4.52 (t, 2H, J=6.8 Hz), 7.09 (S, 1H), 7.24 (S, 1H), 7.40 (d, 2H, J=8.2 Hz), 7.77 (d, 2H, J=8.2 Hz). 
     Tosylated 2-nitroimidazole (1.33 g, 4.08 mmol) was then reacted with sodium azide (Q29 g, 4.49 mmol) in DMF (10 ml) at 100° C. for 3 hours. After cooling, water (20 ml) was added and the product was extracted from ethylacetate (3×20 ml). The solvent was dried over MgSO 4  and evaporated to dryness to afford azido analogue (0.6 g, 75%, TLC: hexane:ethyl acetate; 1:1, Rf=0.42).  1 H-NMR (CDCl 3 ) δ 2.14 (m, 2H), 3.41 (t, 2H, J=6.2 Hz), 4.54 (t, 2H, J=6.9 Hz), 7.17 (S, 2H). 
     The azido analogue (0.57 g, 2.90 mmol) was reduced by taphenyl phosphine (1.14 g, 4.35 mmol) in tetrahydrofuran (PHI; ) at room temperature for 4 hours. Concentrate HCl (12 ml) was added and heated for additional 5 hours. The product was extracted from ethylacetate and water mixture. The ethylacetate was dried over MgSO 4  and evaporated to dryness to afford amine hydrochloride analogue (360 ma, 60%). Ninhydrin (2% in methanol) spray indicated the positivity of amino group of NIM-NH.  1 H-NMR (D 2 O) δ 2.29 (m, 2H), 3.13 (t, 2H, J=7.8 Hz), 3.60 (br, 2H), 4.35 (t, 2H, J=7.4 Hz), 7.50 (d, 1H, J=2.1 Hz), 7.63 (d, 1H, J=2.1 Hz). 
     Synthesis of Ethylenedicysteine-nitroimidazole (EC-NIM) 
     Sodium hydroxide (2N, 0.6 ml) was added to a stirred solution of EC (134 ma, 0.50 mmol) in water (2 ml). To this colorless solution, sulfo-NHS (260.6 mg, 1.2 mmol), EDC (230 ma, 1.2 mmol) and sodium hydroxide (2N, 1 ml) were added. NIM-NH 2  hydrochloride salt (206.6 mg, 1.0 mmol) was then added. The mixture was stirred at room temperature for 24 hours. The mixture was dialyzed for 48 hrs using Spectra/POR molecular porous membrane with cut-off at 500 (Spectrum Medical Industries Inc., Houston, Tex.). After dialysis, the product was frozen dried using lyophilizer (Labconco, Kansas City, Mo.). The product weighed 594.8 mg (yield 98%). The synthetic scheme of EC-NIM is shown in  FIG. 8A . The structure is confirmed by  1 H-NMR (D 2 O) ( FIG. 8B ). 
     Radiolabeling of EC-MN and EC-NIM with  99m Tc 
     Radiosynthesis of  99m Tc-EC-MN and  99m Tc-EC-NIM were achieved by adding required amount of pertechnetate into home-made kit containing the lyophilized residue of EC-MN or EC-NIM (3 mg), SnCl 2 , (100 μg), Na 2 HPO 4  (13.5 mg), ascorbic acid (0.5 mg) and NaEDTA (0.5 mg). Final pH of preparation was 7.4. Radiochemical purity was determined by TLC (ITLAC SG, Gelman Sciences, Ann Arbor, Mich.) eluted with acetone (system A) and ammonium acetate (1M in water):methanol (4:1) (system B), respectively. From radio-TLC (Bioscan, Washington, D.C.) analysis, the radiochemical purity was &gt;96% for both radiotracers. 
     Synthesis of [ 18 F]FMISO and [ 131 I]IMISO 
     [Should this be  18 ?][FI]uoride was produced by the cyclotron using proton irradiation of enriched  18 O-water in a small-volume silver target. The tosyl MISO (Hay et al., 1994) (20 mg) was dissolved in acetonitrile (1.5 ml), added to the kryptofix-fluoride complex. After heating, hydrolysis and column purification, A yield of 25-40% (decay corrected) of pure product was isolated with the end of bombardment (EOB) at 60 min. HPLC was performed on a C-18 ODS-20T column, 4.6×25 mm (Waters Corp., Milford, Mass.), with water/acetonitrile, (80/20), using a flow rate of 1 ml/min. The no-carrier-added product corresponded to the retention time (6.12 min) of the unlabeled FMISO under similar conditions. The radiochemical purity was greater than 99%. Under the UV detector (310 nm), there were no other impurities. The specific activity of [ 18 F]FMISO determined was 1 Ci/μmol based upon UV and radioactivity detection of a sample of known mass and radioactivity. 
     [13I]IMISO was prepared using the same precursor (Cherif et al., 1994), briefly, 5 mg of tosyl MISO was dissolved in acetonitrile (1 ml), and Na  131 I (1 mCi in 0.1 ml IN NaOH) (Dupont New England Nuclear, Boston, Mass.) was added. After heating and purification, the product (60-70% yield) was obtained. Radio-TLC indicated the Rf values of 0.01 for the final product using chloroform methanol (7:3) as an eluant. 
     Stability assay of  99m Tc-EC-MN and  99m Tc-EC-NIM 
     Stability of labeled  99m Tc-EC-MN and  99m Tc-EC-NIM were tested in serum samples. Briefly, 740 KBq of 1 mg  99m Tc-EC-MN and  99m Tc-EC-NIM were incubated in dog serum (200 μl) at 37° C. for 4 hours. The serum samples were diluted with 50% methanol in water and radio-TLC repeated at 0.5, 2 and 4 hours as described above. 
     Tissue Distribution Studies of  99m Tc-EC-MN 
     Female Fischer 344 rats (150±25 g) (Harlan Sprague-Dawley, Indianapolis, Ind.) were inoculated subcutaneously with 0.1 ml of mammary tumor cells from the 13762 tumor cell line suspension (10 6  cells/rat, a tumor cell line specific to Fischer rats) into the hind legs using 25-gauge needles. Studies performed 14 to 17 days after implantation when tumors reached approximately 1 cm diameter. Rats were anesthetized with ketamine (10-15 mg/rat, intraperitoneally) before each procedure. 
     In tissue distribution studies, each animal was injected intravenously with 370-550 KBq of  99m Tc-EC-MN or  99m Tc-EC (n=3/time point). The injected mass of  99m Tc-EC-MN was 10 μg per rat. At 0.5, 2 and 4 hrs following administration of the radiotracers, the rats were sacrificed and the selected tissues were excised, weighed and counted for radioactivity. The biodistribution of tracer in each sample was calculated as percentage of the injected dose per gram of tissue wet weight (% ID/g). Tumor/nontarget tissue count density radios were calculated from the corresponding % ID/g values. The data was compared to [ 18 F]FMISO and [ 131 I]IMISO using the same animal model. Student t-test was used to assess the significance of differences between groups. 
     Scintigraphic Imaging and Autoradiography Studies 
     Scintigraphic images, using a gamma camera (Siemens Medical Systems, Inc., Hoffman Estates, Ill.) equipped with low-energy, parallel-hole collimator, were obtained 0.5, 2 and 4 hrs after i.v. injection of 18.5 MBq of each radiotracer. 
     Whole-body autoradiogram was obtained by a quantitative image analyzer (Cyclone Storage Phosphor System, Packard, Meridian, Conn.). Following i.v. injection of 37 MBq of  99m Tc-EC-MN, the animals were killed at 1 h and the body were fixed in carboxymethyl cellulose (4%) as previously described (Yang et al., 1995). The frozen body was mounted onto a cryostat (LKB 2250 cryomicrotome) and cut into 100 μm coronal sections. Each section was thawed and mounted on a slide. The slide was then placed in contact with multipurpose phosphor storage screen (MP, 7001480) and exposed for 15 hrs. 
     To ascertain whether  99m Tc-EC-NIM could monitor tumor response to chemotherapy, a group of rats with tumor volume 1.5 cm and ovarian tumor-bearing mice were treated with paclitaxel (40 mg/kg/rat, 80 mg/kg/mouse, i.v.) at one single dose. The image was taken on day 4 after paclitaxel treatment. Percent of injected dose per gram of tumor weight with or without treatment was determined. 
     Polarographic Oxygen Microelectrode pO 2  Measurements 
     To confirm tumor hypoxia, intratumoral pO 2  measurements were performed using the Eppendorf computerized histographic system. Twenty to twenty-five pO 2  measurements along each of two to three linear tracks were performed at 0.4 mm intervals on each tumor (40-75 measurements total). Tumor pO measurements were made on three tumor-bearing rats. Using an on-line computer system, the pot measurements of each track were expressed as absolute values relative to the location of the measuring point along the track, and as the relative frequencies within a pO 2  histogram between 0 and 100 mmHg with a class width of 2.5 mm. 
     Results 
     Radiosynthesis and Stability of  99m Tc-EC-MN and  99m Tc-EC-NIM 
     Radiosynthesis of EC-MN and EC-NIM with  99m Tc were achieved with high (&gt;95%) radiochemical purity Radiochemical yield was 100%.  99m Tc-EC-MN and  99m Tc-EC-NIM ( FIG. 13 ) were found to be stable at 0.5, 2 and 4 hrs in dog serum samples. There was no degradation products observed. Radiofluorination and radioiodination of MISO were achieved easily using the same precursor. In both labeled MISO analogues, the radiochemical purity was greater than 99%. 
     In vivo Tissue Distribution Studies 
     The tissue distribution of  99m Tc-EC-MN and  99m Tc-EC in the tumor-bearing rats is shown in Tables 4 and 5. Due to high affinity for ionic  99m Tc, there was no significant and consistent thyroid uptake, suggesting the in vivo stability of  99m Tc-EC-MN (Table 5). 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Biodistribution of  99m Tc-EC in Breast Tumor-Bearing Rats 
               
            
           
           
               
               
            
               
                   
                 % of injected  99m Tc-EC dose per organ or tissue 
               
            
           
           
               
               
               
               
               
            
               
                   
                 20 min 
                 1 h 
                 2 h 
                 4 h 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Blood 
                 0.435 ± 0.029 
                 0.273 ± 0.039 
                 0.211 ± 0.001 
                 0.149 ± 0.008 
               
               
                 Lung 
                 0.272 ± 0.019 
                 0.187 ± 0.029 
                 0.144 ± 0.002 
                 0.120 ± 0.012 
               
               
                 Liver 
                 0.508 ± 0.062 
                 0.367 ± 0.006 
                 0.286 ± 0.073 
                 0.234 ± 0.016 
               
               
                 Stomach 
                 0.136 ± 0.060 
                 0.127 ± 0.106 
                 0.037 ± 0.027 
                 0.043 ± 0.014 
               
               
                 Kidney 
                 7.914 ± 0.896 
                 8.991 ± 0.268 
                 9.116 ± 0.053 
                 7.834 ± 1.018 
               
               
                 Thyroid 
                 0.219 ± 0.036 
                 0.229 ± 0.118 
                 0.106 ± 0.003 
                 0.083 ± 0.005 
               
               
                 Muscle 
                 0.060 ± 0.006 
                 0.043 ± 0.002 
                 0.028 ± 0.009 
                 0.019 ± 0.001 
               
               
                 Intestine 
                 0.173 ± 0.029 
                 0.787 ± 0.106 
                 0.401 ± 0.093 
                 0.103 ± 0.009 
               
               
                 Urine 
                 9.124 ± 0.808 
                 11.045 ± 6.158  
                 13.192 ± 4.505  
                 8.693 ± 2.981 
               
               
                 Tumor 
                 0.342 ± 0.163 
                 0.149 ± 0.020 
                 0.115 ± 0.002 
                 0.096 ± 0.005 
               
               
                 Tumor/Blood 
                 0.776 ± 0.322 
                 0.544 ± 0.004 
                 0.546 ± 0.010 
                 0.649 ± 0.005 
               
               
                 Tumor/Muscle 
                 5.841 ± 3.253 
                 3.414 ± 0.325 
                 4.425 ± 1.397 
                 5.093 ± 0.223 
               
               
                   
               
               
                 Values shown represent the mean ± standard deviation of data from 3 animals 
               
            
           
         
       
     
     In blocking studies, tumor/muscle and tumor/blood count density ratios were significantly decreased (p&lt;0.01) with folic acid co-administrations ( FIG. 5 ). 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Biodistribution of  99m Tc-EC-metronidazole 
               
               
                 conjugate in breast tumor bearing rats 1   
               
            
           
           
               
               
               
               
            
               
                   
                 30 Min. 
                 2 Hour 
                 4 Hour 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Blood 
                 1.46 ± 0.73 
                 1.19 ± 0.34 
                 0.76 ± 0.14 
               
               
                   
                 Lung 
                 0.79 ± 0.39 
                 0.73 ± 0.02 
                 0.52 ± 0.07 
               
               
                   
                 Liver 
                 0.83 ± 0.36 
                 0.91 ± 0.11 
                 0.87 ± 0.09 
               
               
                   
                 Spleen 
                 0.37 ± 0.17 
                 0.41 ± 0.04 
                 0.37 ± 0.07 
               
               
                   
                 Kidney 
                 4.30 ± 1.07 
                 5.84 ± 0.43 
                 6.39 ± 0.48 
               
               
                   
                 Muscle 
                 0.08 ± 0.03 
                 0.09 ± 0.01 
                 0.07 ± 0.01 
               
               
                   
                 Intestine 
                 0.27 ± 0.12 
                 0.39 ± 0.24 
                 0.22 ± 0.05 
               
               
                   
                 Thyroid 
                 0.051 ± 0.16  
                 0.51 ± 0.09 
                 0.41 ± 0.02 
               
               
                   
                 Tumor 
                 0.034 ± 0.13  
                 0.49 ± 0.02 
                 0.50 ± 0.09 
               
               
                   
                   
               
               
                   
                   1 Each rat received 99m Tc-EC-metronidazole (10 μCi, iv). 
               
               
                   
                 Each value is percent of injected dose per gram weight (n = 3)/time interval. 
               
               
                   
                 Each data represents mean of three measurements with standard deviation. 
               
            
           
         
       
     
     Biodistribudon studies showed that tumor/blood and tumor/muscle count density ratios at 0.54 hr gradually increased for  99m Tc-EC-MN, [ 18 F]FMISO and [ 131 ]IMSO, whereas these values did not alter for  99m Tc-EC in the same time period ( FIG. 9  and  FIG. 10 ). [ 18 F]FMISO showed the highest tumor-to-blood uptake ratio than those with [ 131 ]MISO and  99m Tc-EC-MN at 30 min, 2 and 4 hrs post-injection. Tumor/blood and tumor/muscle ratios for  99m Tc-EC-MN and [ 131 I]IMISO at 2 and 4 hrs postinjection were not significantly different (p&lt;0.05). 
     Scintigraphic Imaging and Autoradiographic Studies 
     Scintigraphic images obtained at different time points showed visualization of tumor in  99m Tc-EC-MN and  99m Tc-EC-NIM groups. Contrary, there was no apparent tumor uptake in  99m Tc-EC injected group ( FIG. 11 ). Autoradiograms performed at 1 hr after injection of  99m Tc-EC-MN clearly demonstrated tumor activity ( FIG. 12 ). Compare to  99m Tc-EC-NM,  99m Tc-EC-NIM appeared to provide better scintigraphic images due to higher tumor-to-background ratios. In breast tumor-bearing rats, tumor uptake was markedly higher in  99m Tc-EC-NIM group compared to  99m Tc-EC ( FIG. 14A ). Data obtained from percent of injected dose of  99m Tc-EC-NIM per gram of tumor weight indicated that a 25% decreased uptake in the rats treated with paclitaxel when compared to control group ( FIG. 14B ). 
     In ovarian tumor-bearing mice, there was a decreased tumor uptake in mice treated with paclitaxel ( FIG. 15A  and  FIG. 15B ). Similar results were observed in sarcoma-bearing ( FIG. 15C  and  FIG. 15D ). Thus,  99m Tc-EC-NIM could be used to assess tumor response to paclitaxel treatment. 
     Polarographic Oxygen Microelectrode pO 2  measurements 
     Intratumoral PO 2  measurements of tumors indicated the tumor oxygen tension ranged 4.6±1.4 mmHg as compared to normal muscle of 35±10 mmHg. The data indicate that the tumors are hypoxic. 
     EXAMPLE 3 
     Peptide Imaging of Cancer 
     Synthesis of Ethylenedicysteine-Pentaglutamate (EC-GAP) 
     Sodium hydroxide (1N, 1 ml) was added to a stirred solution of EC (200 mg, 0.75 mmol) in water (10 ml). To this colorless solution, sulfo-NHS (162 mg, 0.75 mmol) and EDC (143 mg, 0.75 mmol) were added. Pentaglutamate sodium salt (M.W. 750-1500, Sigma Chemical Company) (500 mg, 0.67 mmol) was then added. The mixture was stirred at room temperature for 24 hours. The mixture was dialyzed for 48 hrs using Spectra/POR molecular porous membrane with cut-off at 500 (Spectrum Medical Industries Inc., Houston, Tex.). After dialysis, the product was frozen dried using lyophilizer (Labconco, Kansas City, Mo.). The product in the salt form weighed 0.95 g. The synthetic scheme of EC-GAP is shown in  FIG. 16 . 
     Stability Assay of  99m Tc-EC-GAP 
     Radiolabeling of EC-GAP with  99m Tc was achieved using the same procedure described previously. The radiochemical purity was 100%. Stability of labeled  99m Tc-EC-GAP was tested in serum samples. Briefly, 740 KBq of 1 mg  99m Tc-EC-GAP was incubated in dog serum (200 μl) at 37° C. for 4 hours. The serum samples were diluted with 50% methanol in water and radio-TLC repeated at 0.5, 2 and 4 hours as described above. 
     Scintigraphic Imaging Studies 
     Scintigraphic images, using a gamma camera equipped with low-energy, parallel-hole collimator, were obtained 0.5, 2 and 4 hrs after i.v. injection of 18.5 MBq of each radiotracer. 
     Results 
     Stability Assay of  99m Tc-EC-GAP 
       99m Tc-EC-GAP found to be stable at 0.5, 2 and 4 hrs in dog serum samples. There was no degradation products observed. 
     Scintigraphic Imaging Studies 
     Scintigraphic images obtained at different time points showed visualization of tumor in  99m Tc-EC-GAP group. The optimum uptake is at 30 min to 1 hour post-administration ( FIG. 17 ). 
     EXAMPLE 4 
     Imaging Tumor Apoptotic Cells 
     Synthesis of Ethylenedicysteine-Annexin V (EC-ANNEX) 
     Sodium bicarbonate (1N, 1 ml) was added to a stirred solution of EC (5 mg, 0.019 mmol). To this colorless solution, sulfo-NHS (4 mg, 0.019 mmol) and EDC (4 mg, 0.019 mmol) were added. Annexin V (M.W. 33 kD, human, Sigma Chemical Company) (0.3 mg) was then added. The mixture was stirred at room temperature for 24 hours. The mixture was dialyzed for 48 hrs using Spectra/POR molecular porous membrane with cut-off at 10,000 (Spectrum Medical Industries Inc., Houston, Tex.). After dialysis, the product was frozen dried using lyophilizer (Labconco, Kansas City, Mo.). The product in the salt form weighed 12 mg. 
     Stability Assay of  99m Tc-EC-ANNEX 
     Radiolabeling of EC-ANNEX with  99m Tc was achieved using the same procedure described in EC-GAP. The radiochemical purity was 100%. Stability of labeled  99m Tc-EC-ANNEX was tested in serum samples. Briefly, 740 KBq of 1 mg  99m Tc-EC-ANNEX was incubated in dog serum (200 μl) at 37° C. for 4 hours. The serum samples were diluted with 50% methanol in water and radio-TLC repeated at 0.5, 2 and 4 hours as described above. 
     Scintigraphic Imaging Studies 
     Scintigraphic images, using a gamma camera equipped with low-energy, parallel-hole collimator, were obtained 0.5, 2 and 4 hrs after i.v. injection of 18.5 MBq of the radiotracer. The animal models used were breast, ovarian and sarcoma. Both breast and ovarian-tumor bearing rats are known to overexpress high apoptotic cells. The imaging studies were conducted on day 14 after tumor cell inoculation. To ascertain the tumor treatment response, the pre-imaged mice were administered paclitaxel (80 mg/Kg, iv, day 14) and the images were taken on day 18. 
     Results 
     Stability Assay of  99m Tc-EC-ANNEX 
       99m Tc-EC-ANNEX found to be stable at 0.5, 2 and 4 hrs in dog serum samples. There was no degradation products observed. 
     Scintigraphic Imaging Studies 
     Scintigraphic images obtained at different time points showed visualization of tumor in  99m Tc-EC-ANNEX group ( FIGS. 18-20 ). The images indicated that highly apoptotic cells have more uptake of  99m Tc-EC-ANNEX. There was no marked difference of tumor uptake between pre- and post-[aclitaxel treatment in the high apoptosis (ovarian tumor-bearing) group ( FIG. 19A  and  FIG. 19B ) and in the low apoptosis (sarcoma tumor-bearing) group ( FIG. 20A  and  FIG. 20B ). 
     EXAMPLE 5 
     Imaging Tumor Angiogenesis 
     Synthesis of (Amino Analogue of Colchcine, COL-NH 2 ) 
     Demethylated amino and hydroxy analogue of colchcine was synthesized according to the previously described methods (Orr et al., 1995). Briefly, colchicine (4 g) was dissolved in 100 ml of water containing 25% sulfuric acid The reaction mixture was heated for 5 hours at 100° C. The mixture was neutralized with sodium carbonate. The product was filtered and dried over freeze dryer, yielded 2.4 g (70%) of the desired amino analogue (m.p. 153-155° C., reported 155-157° C.). Ninhydrin (2% in methanol) spray indicated the positivity of amino group of COL-NH 2 . The structure was confirmed by  1 H-NMR and mass spectroscopy (FAB-MS).  1 H-NMR (CDCl 3 )δ 8.09 (S, 1H), 7.51 (d, 1H, J=12 Hz), 7.30 (d, 1H, J=12 Hz), 6.56 (S, 1H), 3.91 (S, 6H), 3.85 (m, 1H), 3.67 (S, 3H), 2.25-2.52 (m, 4H). m/z 308.2(M + , 20), 307.2 (100). 
     Synthesis of Ethylenedicysteine-Colchcine (EC-COL) 
     Sodium hydroxide (2N, 0.2 ml) was added to a stirred solution of EC (134 mg, 0.50 mmol) in water (5 ml). To this colotiess solution, sulfo-NHS (217 mg, 1.0 mmol) and EDC (192 mg, 1.0 mmol) were added. COL-NH 2  (340 mg, 2.0 mmol) was then added. The mixture was stirred at room temperature for 24 hours. The mixture was dialyzed for 48 hrs using Spectra/POR molecular porous membrane with cut-off at 500 (Spectrum Medical Industries Inc., Houston, Tex.). After dialysis, the product was frozen dried using lyophilizer (Labconco, Kansas City, Mo.). The product weighed 315 mg (yield 55%/,).  1 H-NMR (D 2 O) δ 7.39 (S, 1H), 7.20 (d, 1H, J=12 Hz), 7.03 (d, 1H, J=12 Hz), 6.78 (S, 1H), 4.25-4.40 (m, 1H), 3.87 (S, 3H, —OCH 3 ), 3.84 (S, 3H, —OCH 3 ), 3.53 (S, 3H, —OCH 3 ), 3.42-3.52 (m, 2H), 3.05-3.26 (m, 4H), 2.63-2.82 (m, 4H), 2.19-2.25 (m, 4H). FAB MS m/z 580 (sodium salt, 20). The synthetic scheme of EC-COL is shown in  FIG. 21 . 
     Radiolabeling of EC-COL and EC with  99m Tc 
     Radiosynthesis of  99m Tc-EC-COL was achieved by adding required amount of  99m Tc-pertechnetate into home-made kit containing the lyophilized residue of EC-COL (5 mg), SnCl 2  (100 μg), Na 2 HPO 4  (13.5 mg), ascorbic acid (0.5 mg) and NaEDTA (0.5 mg). Final pH of preparation was 7.4.  99m Tc-EC was also obtained by using home-made kit containing the lyophilized residue of EC (5 mg), SnCl 2  (100 μg), Na 2 HPO 4  (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 (ITLC SG, Gelman Sciences, Ann Arbor, Mich.) eluted with ammonium acetate (1M in water):methanol (4:1). Radio-thin layer chromatography (TLC, Bioscan, Washington, D.C.) was used to analyze the radiochemical purity for both radiotracers. 
     Stability Assay of  99m Tc-EC-COL 
     Stability of labeled  99m Tc-EC-COL was tested in serum samples. Briefly, 740 KBq of 5 mg  99m Tc-EC-COL was incubated in the rabbinate serum (500 μl) at 37° C. for 4 hours. The serum samples was diluted with 50% methanol in water and radio-TLC repeated at 0.5, 2 and 4 hours as described above. 
     Tissue Distribution Studies 
     Female Fischer 344 rats (150±25 g) (Harlan Sprague-Dawley, Indianapolis, Ind.) were inoculated subcutaneously with 0.1 ml of mammary tumor cells from the 13762 tumor cell line suspension (10 cells/rat, a tumor cell line specific to Fischer rats) into the hind legs using 25-gauge needles. Studies performed 14 to 17 days after implantation when tumors reached approximately 1 cm diameter. Rats were anesthetized with ketamine (10-15 mg/rat, intraperitoneally) before each procedure. 
     In tissue distribution studies, each animal was injected intravenously with 370-550 KBq of  99m Tc-EC-COL or  99m Tc-EC (n=3/time point). The injected mass of  99m Tc-EC-COL was 10 μg per rat. At 0.5, 2 and 4 hrs following administration of the radiotracers, the rats were sacrificed and the selected tissues were excised, weighed and counted for radioactivity. The biodistribution of tracer in each sample was calculated as percentage of the injected dose per gram of tissue wet weight (% ID/g). Tumor/nontarget tissue count density ratios were calculated from the corresponding % ID/g values. Student t-test was used to assess the significance of differences between groups. 
     Scintigraphic Imaging Studies 
     Scintigraphic images, using a gamma camera (Siemens Medical Systems, Inc., Hoffman Estates, Ill.) equipped with low-energy, parallel-hole collimator, were obtained 0.5, 2 and 4 hrs after i.v. injection of 300 μCi of  99m Tc-EC-COL and  99m Tc-EC. Computer outlined region of interest (ROI) was used to quantitate (counts per pixel) the tumor uptake versus normal muscle uptake. 
     Results 
     Radiosynthesis and Stability of  99m Tc-EC-COL 
     Radiosynthesis of EC-COL with  99m Tc was achieved with high (&gt;95%) radiochemical purity ( FIG. 21 ).  99m Tc-EC-COL was found to be stable at 0.5, 2 and 4 hrs in rabbit serum samples. There was no degradation products observed ( FIG. 22 ). 
     In Vivo Biodistribution 
     In vivo biodistribution of  99m Tc-EC-COL and  99m Tc-EC in breast-tumor-bearing rats are shown in Tables 4 and 6. Tumor uptake value (% ID/g) of  99m Tc-EC-COL at 0.5, 2 and 4 hours was 0.436±0.089, 0.395±0.154 and 0.221±0.006 (Table 6), whereas those for  99m Tc-EC were 0.342±0.163, 0.115±0.002 and 0.097±0.005, respectively (Table 4). Increased tumor-to-blood (0.52±0.12 to 0.72±0.07) and tumor-to-muscle (3.47±0.40 to 7.97±0.93) ratios as a function of time were observed in  99m Tc-EC-COL group ( FIG. 23 ). Conversely, tumor-to-blood and tumor-to-muscle values showed time-dependent decrease with  99m Tc-EC when compared to  99m Tc-EC-COL group in the same time period ( FIG. 24 ). 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Biodistribution of  99m Tc-EC-Colchicine 
               
               
                 in Breast Tumor Bearing Rats 
               
            
           
           
               
               
               
               
            
               
                   
                 30 Min. 
                 2 Hour 
                 4 Hour 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                 Blood 
                 0.837 ± 0.072 
                 0.606 ± 0.266 
                 0.307 ± 0.022 
               
               
                 Lung 
                 0.636 ± 0.056 
                 0.407 ± 0.151 
                 0.194 ± 0.009 
               
               
                 Liver 
                 1.159 ± 0.095 
                 1.051 ± 0.213 
                 0.808 ± 0.084 
               
               
                 Spleen 
                 0.524 ± 0.086 
                 0.559 ± 0.143 
                 0.358 ± 0.032 
               
               
                 Kidney 
                 9.705 ± 0.608 
                 14.065 ± 4.007  
                 11.097 ± 0.108  
               
               
                 Muscle 
                 0.129 ± 0.040 
                 0.071 ± 0.032 
                 0.028 ± 0.004 
               
               
                 Stomach 
                 0.484 ± 0.386 
                 0.342 ± 0.150 
                 0.171 ± 0.123 
               
               
                 Uterus 
                 0.502 ± 0.326 
                 0.343 ± 0.370 
                 0.133 ± 0.014 
               
               
                 Thyroid 
                 3.907 ± 0.997 
                 2.297 ± 0.711 
                 1.709 ± 0.776 
               
               
                 Tumor 
                 0.436 ± 0.089 
                 0.395 ± 0.154 
                 0.221 ± 0.006 
               
               
                   
               
               
                 * Each rat received  99m Tc-EC-Colchicine (10 μCi, iv.). 
               
               
                 Each value is the percent of injected dose per gram tissue weight (n = 3)/time interval. 
               
               
                 Each data represents mean of three measurements with standard deviation. 
               
            
           
         
       
     
                     TABLE 7                  Rf Values Determined by Radio-TLC (ITLC-SG) Studies                             System A*   System B†                                               99m Tc-EC-folate   0   1(&gt;95%)             99m Tc-EC-   0   1(&gt;95%)           Free  99m Tc   1   1           Reduced  99m Tc   0   0                       *Acetone           †Ammonium Acetate (1M in water):Methanol (4:1)            
Gamma Scintigraphic Imaging of  99m Tc-EC-COL in Breast Tumor-Bearing Rats
 
     In vivo imaging studies in three breast-tumor-bearing rats at 1 hour post-administration indicated that the tumor could be visualized well with  99m Tc-EC-COL group ( FIG. 25 ), whereas, less tumor uptake in the  99m Tc-EC group was observed ( FIG. 26 ). Computer outlined region of interest (ROI) showed that tumor/background ratios in  99m Tc-EC-COL group were significantly higher than  99m Tc-EC group ( FIG. 27 ). 
     Tumor Glycolysis Targeting 
     EXAMPLE 6 
     Development of  99m Tc-EC-Neomycin 
     Synthesis of EC 
     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°, reported 196-197°). EC was then prepared (m.p. 237°, reported 251-253°). The structure was confirmed by  1 H-NMR and fast-atom bombardment mass spectroscopy (FAB-MS). 
     Synthesis of Ethylenedicysteine-neomycin (EC-neomycin) 
     Sodium hydroxide (2N, 0.2 ml) was added to a stirred solution of EC (134 mg, 0.50 mmol) in water (5 ml). To this colorless solution, sulfo-NHS (217 mg, 1.0 mmol) and EDC (192 mg, 1.0 mmol) were added. Neomycin trisulfate salt (909 mg, 1.0 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 (Spectrum Medical Industries Inc., Houston, Tex.). After dialysis, the product was frozen dried using lyophilizer (Labconco, Kansas City, Mo.). The product weighed 720 mg (yield 83%). The synthetic scheme of EC-neomycin is shown in  FIG. 36 . The structure is confirmed by  1 H-NMR ( FIGS. 38A-B ), mass spectrometry ( FIGS. 39A-B ) and elemental analysis (Galbraith Laboratories, Inc. Knoxville, Tenn.). Elemental analysis C 39 H 75 N 10 S 4 O 19 .15H 2 O (C,H,N,S), Calc. C, 33.77; H, 7.58; N, 10.11; S, 9.23; found C, 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 and neomycin ( FIGS. 40A-C ) 
     Radiolabeling of EC-MN and EC-neomycin with  99m Tc 
     Radiosynthesis of  99m Tc-EC and  99m Tc-EC-neomycin were achieved by adding required amount of  99m Tc-pertechnetate into home-made kit containing the lyophilized residue of EC or EC-neomycin (10 mg), SnCl 2  (100 μg), Na 2 HPO 4  (13.5 mg) and ascorbic acid (0.5 mg). NaEDTA (0.5 mg) in 0.1 ml of water was then added. Final pH of preparation was 7.4. Radiochemical purity was determined by TLC (ITLC SG, Gelman-Sciences, Ann Arbor, Mich.) eluted with ammonium acetate (1M in water):methanol (4:1). From radio-TLC (Bioscan, Washington, D.C.) analysis ( FIG. 41 ) and HPLC analysis ( FIGS. 42-45 ), the radiochemical purity was &gt;95% for both radiotracers. 
     Stability Assay of  99m Tc-EC and  99m Tc-EC-neomycin 
     Stability of labeled  99m Tc-EC and  99m Tc-EC-neomycin were tested in dog serum samples. Briefly, 740 KBq of 1 mg  99m Tc-EC and  99m Tc-EC-neomycin were incubated in dog serum (200 μl) at 37° C. for 4 hours. The serum samples were diluted with 50% methanol in water and radio-TLC repeated at 0.5, 2 and 4 hours as described above. 
     Tissue Distribution Studies of  99m Tc-EC-neomycin 
     Female Fischer 344 rats (150±25 g) (Harlan Sprague-Dawley, Indianapolis, Ind.) were innoculated subcutaneously with 0.1 ml of mammary tumor cells from the 13762 tumor cell line suspension (10 6  cells/rat, a tumor cell line specific to Fischer rats) into the hind legs using 25-gauge needles. Studies performed 14 to 17 days after implantation when tumors reached approximately 1 cm diameter. Rats were anesthetized with ketamine (10-15 mg/rat, intraperitoneally) before each procedure. 
     In tissue distribution studies, each animal was injected intravenously with 10-20 μCi of  99m Tc-EC or  99m Tc-EC-neomycin (n=3/time point). The injected mass of  99m Tc-EC-neomycin was 200 μg per rat. At 0.5, 2 and 4 hours following administration of the radiotracers, the rats were sacrificed and the selected tissues were excised, weighed and counted for radioactivity. The biodistribution of tracer in each sample was calculated as percentage of the injected dose per gram of tissue wet weight (% ID/g). Tumor/nontarget tissue count density ratios were calculated from the corresponding % ID/g values. When compared to  99m Tc-EC (Table 4) and free technetium C(able 9), tumor-to tissue ratios increased as a function of time in  99m Tc-EC-neomycin group (Table 8). 
     Scintigraphic Imaging Studies 
     Scintigraphic images, using a gamma camera (Siemens Medical Systems, Inc., Hoffman Estates, Ill.) equipped with low-energy, parallel-hole collimator, were obtained 0.5, 2 and 4 hours after i.v. injection of 100 μCi of each radiotracer. Compare to  99m Tc-EC, high uptake in the tumors was observed ( FIG. 37A ). Preliminary clinical imaging studies were conducted in a patient with breast cancer. The tumor was visualized well at 2 hours post-administration of  99m Tc-EC-neomycin ( FIG. 37B ). 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Biodistribution of  99m Tc-EC-neomycin in Breast Tumor Bearing Rats 
               
            
           
           
               
               
               
               
               
            
               
                   
                 30 Min. 
                 1 Hour 
                 2 Hour 
                 4 Hour 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Blood 
                 0.463 ± 0.007 
                 0.262 ± 0.040 
                 0.139 ± 0.016 
                 0.085 ± 0.004 
               
               
                 Lung 
                 0.344 ± 0.011 
                 0.202 ± 0.030 
                 0.114 ± 0.014 
                 0.080 ± 0.003 
               
               
                 Liver 
                 0.337 ± 0.012 
                 0.269 ± 0.013 
                 0.221 ± 0.020 
                 0.195 ± 0.012 
               
               
                 Stomach 
                 0.279 ± 0.039 
                 0.147 ± 0.001 
                 0.061 ± 0.008 
                 0.054 ± 0.008 
               
               
                 Spleen 
                 0.159 ± 0.008 
                 0.114 ± 0.013 
                 0.095 ± 0.007 
                 0.089 ± 0.003 
               
               
                 Kidney 
                 8.391 ± 0.395 
                 8.804 ± 0.817 
                 8.356 ± 0.408 
                 8.638 ± 0.251 
               
               
                 Thyroid 
                 0.349 ± 0.008 
                 0.202 ± 0.028 
                 0.114 ± 0.007 
                 0.086 ± 0.001 
               
               
                 Muscle 
                 0.093 ± 0.001 
                 0.049 ± 0.010 
                 0.021 ± 0.006 
                 0.010 ± 0.001 
               
               
                 Intestine 
                 0.159 ± 0.004 
                 0.093 ± 0.014 
                 0.061 ± 0.004 
                 0.266 ± 0.200 
               
               
                 Urine 
                 25.402 ± 8.621  
                 21.786 ± 2.690  
                 0.224 ± 0.000 
                 2.609 ± 2.377 
               
               
                 Tumor 
                 0.419 ± 0.023 
                 0.279 ± 0.042 
                 0.166 ± 0.023 
                 0.131 ± 0.002 
               
               
                 Brain 
                 0.022 ± 0.001 
                 0.014 ± 0.003 
                 0.010 ± 0.001 
                 0.007 ± 0.001 
               
               
                 Heart 
                 0.147 ± 0.009 
                 0.081 ± 0.012 
                 0.040 ± 0.004 
                 0.029 ± 0.002 
               
               
                 Tumor/Blood 
                 0.906 ± 0.039 
                 1.070 ± 0.028 
                 1.196 ± 0.061 
                 1.536 ± 0.029 
               
               
                 Tumor/Muscle 
                 4.512 ± 0.220 
                 5.855 ± 0.458 
                 8.364 ± 1.469 
                 12.706 ± 0.783  
               
               
                 Tumor/Brain 
                 19.495 ± 1.823  
                 20.001 ± 0.890  
                 17.515 ± 2.035  
                 20.255 ± 1.693  
               
               
                   
               
               
                 Values shown represent the mean ± standard deviation of data from 3 animals. 
               
            
           
         
       
     
                     TABLE 9                  Biodistribution of  99m Tc Pertechnetate in Breast Tumor Bearing Rats                                 30 Min.   2 Hour   4 Hour                                         Blood   1.218 ± 0.328   0.666 ± 0.066   0.715 ± 0.052       Lung   0.646 ± 0.291   0.632 ± 0.026   0.387 ± 0.024       Liver   0.541 ± 0.232   0.304 ± 0.026   0.501 ± 0.081       Spleen   0.331 ± 0.108   0.187 ± 0.014   0.225 ± 0.017       Kidney   0.638 ± 0.197   0.489 ± 0.000   0.932 ± 0.029       Thyroid   24.821 ± 5.181    11.907 ± 15.412   17.232 ± 5.002        Muscle   0.130 ± 0.079   0.076 ± 0.002   0.063 ± 0.003       Intestine   0.153 ± 0.068   0.186 ± 0.007   0.344 ± 0.027       Tumor   0.591 ± 0.268   0.328 ± 0.016   0.423 ± 0.091       Brain   0.038 ± 0.014   0.022 ± 0.002   0.031 ± 0.009       Heart   0.275 ± 0.089   0.145 ± 0.015   0.166 ± 0.012       Tumor/Blood   0.472 ± 0.093   0.497 ± 0.073   0.597 ± 0.144       Tumor/Muscle   4.788 ± 0.833   4.302 ± 0.093   6.689 ± 1.458       Tumor/Liver   1.084 ± 0.023   1.084 ± 0.115   0.865 ± 0.270               Values shown represent the mean ± standard deviation of data from 3 animals.            
In vitro Cellular Uptake of  99m Tc-EC-drug Conjugates
 
     To evaluate the cellular uptake of  99m Tc-EC-drug conjugates, each well containing 80,000 cells (A549 lung cancer cell line) was added with 2 μCi of  99m Tc-EC-neomycin and  18 F-FDG. After incubation at 0.5-4 hours, the cells were washed with phosphate buffered saline 3 times and followed by trypsin to lose the cells. The cells were then counted by a gamma counter.  99m Tc-EC-neomycin showed highest uptake among those agents tested in human lung cancer cell line ( FIG. 46 ). 
     Effect of Glucose on Cellular Uptake of  99m Tc-EC-neomycin and  18 F-FDG 
     Neomycin is known to influence glucose absorption (Rogers et al., 1968; Fanciulli et al., 1994). Previous experiments have shown that  99m Tc-EC-neomycin has higher uptake than  18 F-FDG in human lung cancer cell line (A549). To determine if uptake of  99m Tc-EC-neomycin is mediated via glucose-related mechanism, glucose (0.1 mg-2.0 mg) was added to each well containing either 50,000 (breast) cells or 80,000 cells (lung) along with 2 μCi of  99m Tc-EC-neomycin and  18 F-FDG. After incubation, the cells were washed with phosphate buffered saline 3 times and followed by trypsin to lose the cells. The cells were then counted by a gamma counter. 
     By adding glucose at the concentration of 0.1-2.0 mg/well, decreased uptake of  99m Tc-EC-neomycin in two lung cancer cell lines and one breast cell line was observed. Similar results were observed in  18 F-FDG groups.  99m Tc-EC (control) showed no uptake. The findings&#39;suggest that the cellular uptake of  99m Tc-EC-neomycin may be mediated via glucose-related mechanism ( FIGS. 47 ,  48 A and  48 B). 
     EXAMPLE 7 
     Tumor Metabolic Imaging with  99m Tc-EC-Deoxyglucose 
     Synthesis of EC-deoxyglucose (EC-DG) 
     Sodium hydroxide (1N, 1 ml) was added to a stirred solution of EC (110 mg, 0.41 mmol) in water (5 ml). To this colorless solution, sulfo-NHS (241.6 mg, 1.12 mmol) and EDC (218.8 mg, 1.15 mmol) were added. D-Glucosamine hydrochloride 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 (Spectrum Medical Industries Inc., Houston, Tex.). After dialysis, the product was frozen dried using lyophilizer (Labconco, Kansas City, Mo.). The product in the salt form weighed 568.8 mg. The synthetic scheme is shown in  FIG. 59 . The structure was confirmed by mass spectrometry ( FIG. 60 ) and proton NMR ( FIGS. 61 and 62 ). Radiochemical purity of  99m Tc-EC-DG was 100% as determined by radio-TLC ( FIG. 63 ) and HPLC ( FIGS. 64 and 65 ) analysis. 
     Hexokinase Assay 
     To determine if EC-DG mimics glucose phosphorylation, a hexokinase assay was conducted. Using a ready made kit (Sigma Chemical Company), EC-DG, glucosamine and glucose (standard) were assayed at UV wavelength 340 nm. Glucose, EC-DG and glucosamine showed positive hexokinase assay ( FIGS. 66-68 ). 
     In vitro Cellular Uptake Assay 
     In vitro cellular uptake assay was conducted by using a human lung cancer cell line (A549). Two μCi of  99m Tc-EC-DG and  18 F-FDG were added to wells containing 80,000 cells each. After incubation at 0.54 hours, the cells were washed with phosphate buffered saline 3 times and followed by trypsin to lose the cells. The cells were then counted by a gamma counter. The uptake of  99m Tc-EC-DG was comparable to FDG ( FIG. 69 ). 
     Effect of d- and l-glucose on Cellular Uptake of  99m Tc-EC-deoxyglucose and  18 F-FDG 
     To evaluate if the uptake of  99m Tc-EC-deoxyglucose is mediated via d-glucose mechanism, d- and l-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 with 2 μCi of  99m Tc-EC-deoxyglucose and  18 F-FDG. After 2 hours incubation, the cells were washed with phosphate buffered saline 3 times and followed by trypsin to lose the cells. The cells were counted by a gamma counter. 
     By adding glucose at the concentration of 1-2.0 mg/well, a decreased uptake of  99m Tc-EC-deoxyglucose and  18 F-FDG by d-glucose in breast and lung cancer cells was observed. However, there was no influence on both agents by l-glucose ( FIG. 70-73 ). The findings suggest that the cellular uptake of  99m Tc-EC-deoxyglucose is mediated via d-glucose mechanism. 
     Effect of EC-deoxyglucose Loading on Blood Glucose Level in Normal Rats 
     Previous experiments have shown that cellular uptake of  99m Tc-EC-deoxyglucose is similar to FDG. For instance, the hexokinase assay (glucose phosphorylation) was positive. The uptake of  99m Tc-EC-deoxyglucose is mediated via d-glucose mechanism. This study is to determine whether blood glucose level could be induced by either FDG or EC-deoxyglucose and suppressed by insulin. 
     Normal healthy Fischer 344 rats (weight 145-155 g) were fasting overnight prior to the experiments. The concentration of glucosamine hydrochloride, FDG and EC-deoxyglucose prepared was 60% and 164% (mg/ml). The blood glucose level (mg/dl) was determined by a glucose meter (Glucometer DEX, Bayer Corporation, Elkhart, Ind.). Prior to the study, the baseline of blood glucose level was obtained. Each rat (n=3/group) was administered 1.2 mmol/kg of glucosamine, FDG and EC-deoxyglucose. In a separate experiment, a group of rats was administered EC-deoxyglucose and FDG. Insulin (5 units) was administered after 30 minutes. Blood samples were collected from the tail vein every 30 minutes up to 6 hours post-administration. 
     Blood glucose level was induced by bolus intravenous administration of glucosamine, FDG and EC-deoxyglucose. This increased blood glucose level could be suppressed by co-administration of EC-deoxyglucose or FDG and insulin ( FIGS. 74 and 75 ). 
     Tissue Distribution Studies of  99m Tc-EC-DG 
     For breast tumor-bearing animal model, female Fischer 344 rats (150±25 g) (Harlan Sprague-Dawley, Indianapolis, Ind.) were innoculated subcutaneously with 0.1 ml of mammary tumor cells from the 13762 tumor cell line suspension (10 6  cells/rat, a tumor cell line specific to Fischer rats) into the hind legs using 25-gauge needles. Studies were performed 14 to 17 days after implantation when tumors reached approximately 1 cm diameter. Rats were anesthetized with ketamine (10-15 mg/rat, intraperitoneally) before each procedure. 
     For lung tumor-bearing animal model, each athymic nude mouse (20-25 g) was innoculated subcutaneously with 0.1 ml of human lung tumor cells from the A549 tumor cell line suspension (10 6  cells/mouse) into the hind legs using 25-gauge needles. Studies were performed 17 to 21 days after implantation when tumors reached approximately 0.6 cm diameter. 
     In tissue distribution studies, each animal was injected intravenously with 10-20 μCi (per rat) or 1-2 μCi (per mouse) of  99m Tc-EC or  99m Tc-EC-DG (n=3/time point). The injected mass of  99m Tc-EC-DG was 1 mg per rat. At 0.5, 2 and 4 hours following administration of the radiotracers, the rodents were sacrificed and the selected tissues were excised, weighed and counted for radioactivity. The biodistribution of tracer in each sample was calculated as percentage of the injected dose per gram of tissue wet weight (% ID/g). Tumor/nontarget tissue count density ratios were calculated from the corresponding % ID/g values. When compared to  99m Tc-EC (Table 4) and free technetium (Table 9), tumor-to tissue ratios increased as a function of time in  99m Tc-EC-DG group ( FIGS. 70-80 ). 
     Scintigraphic Imaging Studies 
     Scintigraphic images, using a gamma camera equipped with low-energy, parallel-hole collimator, were obtained 0.5, 2 and 4 hours after i.v. injection of 100 μCi of the radiotracer. The animal model used was breast tumor-bearing rats. Tumor could be visualized well when compared to  99m Tc-EC (control group)  FIG. 81 ). Preliminary clinical studies were conducted in 5 patients (3 brain tumors and 2 lung diseases). The images were obtained at 1-2 hours post-administration.  99m Tc-EC-DG was able to differentiate benign versus malignant tumors. For instance, malignant astrocytoma showed high uptake ( FIGS. 82A ,  82 B,  83 A and  83 B). Benign meningioma showed poor uptake compared to malignant meningioma ( FIGS. 84A  and B). Poor uptake was observed in patient with TB ( FIG. 85A  and  FIG. 85B ), but high uptake was observed in lung tumor ( FIG. 86A ,  FIG. 86B , and  FIG. 86C ). 
     All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. 
     REFERENCES 
     The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
         Abrams, Juweid, Tenkate, “Technetium-99m-human polyclonal IgG radiolabeled via the hydrazino nicotinamide derivative for imaging focal sites of infection in rats,”  J. Nucl. Med.,  31:2022-2028, 1990.   Bakker, Krenning, Breeman, Kiper, Kooij, Reubi, Klijn, Visser, Docter, Lamberts, “Receptor scintigraphy with a radioiodinated somatostatin analogue: radiolabeling, purification, biologic activity and in vivo application in animals,”  J. Nucl. Med.,  31:1501-1509, 1990.   Blakenberg, Katsikis, Tait et al., “In vivo detection and imaging of phosphatidylserine expression during programmed cell death,”  Proc Natl. Acad. Sci USA,  95:6349-6354, 1998.   Blakenberg, Katsikis, Tait, Davis, Naumovski, Ohtsuki, Kopiwoda, Abrams, Strauss, “Imaging of apoptosis (programmed cell death) with  99m Tc annexin V.,”  J. Nucl. Med.,  40:184-191, 1999.   Blondeau, Berse, Gravel, “Dimerization of an intermediate during the sodium in liquid ammonia reduction of L-thiazolidine-4-carboxylic acid,”  Can J. Chem,  45:49-52, 1967.   Bolhuis, Lamers, Goey et al., “Adoptive immunotherapy of ovarian carcinoma with Bs-MAb targeted lymphocytes. A multicenter study,”  Int J Cancer,  7:78-81, 1992.   Britton and Granowska, “Imaging of tumors, in tomography in nuclear medicine,”  Proceedings of an International Symposium , Vienna, Austria, IAEA, 91-105, 1996.   Bush, Jenkins, Allt, Beale, Bena, Dembo, Pringle, “Definitive evidence for hypoxic cells influencing cure in cancer therapy,”  Br J Cancer , (Suppl. III) 37:302-306, 1978.   Butterfield, Fuji, Ladd, Snow, Tan, Toner, “Segmented chelating polymers as imaging and therapeutic agents,” U.S. Pat. No. 4,730,968, Mar. 24, 1998.   Campbell, Jones, Foulkes, Trowsdale, “Folate-binding protein is a marker for ovarian cancer,”  Cancer Res,  51:5329-5338, 1991.   Canevari, Miotti, Bottero, Valota, Colnaghi, “Ovarian carcinoma therapy with monoclonal antibodies,”  Hybridoma,  12:501-507, 1993.   Cherif, Yang, Tansey, Kim, Wallace, “Synthesis of [ 18 F]fluoromisonidazole,”  Pharm Res.,  11:466-469, 1994.   Coenen and Stocklin, “Evaluation of radiohalogenated amino acid analogues as potential tracers for PET and SPECT studies of protein synthesis,”  Radioisot Klinik Forschung,  18:402440, 1988.   Coney, Mezzanzanica, Sanborn, Casalini, Colnaghi, Zurawski, “Chimeric munne-human antibodies directed against folate binding receptor are eff˜cient mediators of ovarian carcinoma cell killing,”  Cancer Res,  54:2448-2455, 1994.   Davison, Jones, Orvig, Sohn, “A new class of oxotechnetium(+5) chelate complexes containing a TcON 2 S 2  Core,”  Inorg Chem,  20:1629-1632, 1980.   Dickinson and Hiltner, “Biodegradation of poly(χ-amino acid) hydrogel. II. In vitro”  J. Biomed Mater Res.,  15:591, 1981.   Dische, “A review of hypoxic-cell radiosensitizadon,”  Int J Radiat Oncol Biol Phys,  20:147-152, 1991.   Fanciulli, Paggi, Bruno, et al., “Glycolysis and growth rate in normal and in hexokinase-transfected NIH-3T3 cells,”  Oncol Res.  6(9):405-9, 1994.   Franklin, Waintrub, Edwards, Christensen, Prendegrast, Woods, Bunn, Kolhouse, “New anti-lung-cancer antibody cluster 12 reacts with human folate receptors present on adenocarcinoma,”  Int J Cancer-Supplement,  8:89-95, 1994.   Gatenby, Kessler, Rosenblum, Coia, Moldofsky, Hartz, Broder, “Oxygen distribution in squamous cell carcinoma metastases and its relationship to outcome of radiation therapy,”  Int J Radiat Oncol Biol Phys,  14:831-838, 1988.   Ginobbi, Geiser, Ombres, Citro, “Folic acid-polylysine carrier improves efficacy of c-myc antisense oligodeoxynucleotides on human melanoma (M14) cells,”  Anticancer Res,  17:29-35, 1997a.   Goh, Pricher, Lobie, “Growth hormone promotion of tublin polymerization stabilizes the microtubule network and protects against colchicine-induced apoptosis,”  Endocrinology,  139:4364-4372, 1998.   Goldsmith, “Receptor imaging: Competitive or complementary to antibody imaging,”  Sem Nucl Med.,  27:85-93, 1997.   Goldsmith, Macapinlac, O&#39;Brien, “Somatostatin receptor imaging in lymphoma,”  Sem Nucl Med,  25:262-271, 1995.   Gray, Conger, Elbert, Morsney, Scold, “The concentration of oxygen dissolved in tissues at the time of irradiation as a factor in radiotherapy,”  Br J Radiol,  26:638-648, 1953.   Hall, “The oxygen effect and reoxygenation,” In: E. J. Hall (ed.) Radiobiology for the radiobiologist, 3rd edition J. B. Lippincott Co., Philadelphia, Pa., 137-160, 1988.   Harada, Smith, Smith et al., “Insulin-induced egr-1 and c-fos expression in 32D cells requires insulin receptor, Shc, and mitogen-activated protein kinase, but not insulin receptor substrate-1 and phosphatidylinositol 3-kinase activation,”  J. Biol. Chem.  271(47):30222-6, 1996.   Hay, Wilson, Moselen, Palmer, Denny, “Hypoxia-selective antitumor agents. Bis(nitroimidazolyl)alkanecarboxamides: a new class of hypoxia-selective cytotoxins and hypoxic cell radiosensitizers,”  J Med. Chem.,  37:381-391, 1994.   Hermann, Patel. “Adaptive recognition by nucleic acid aptamers,”  Science,  287(5454):820-5, 2000.   Holm, Hansen, Hoier-Madsen, Sondergaard, Bzorek, “Folate receptor of human mammary adenocarcinoma,”  APMIS,  102:413-419, 1994.   Hsueh and Dolnick, “Altered folate-binding protein MRNA stability in KB cells grown in folate-deficient medium,”  Biochem Pharmacol,  45:2537-2545, 1993.   Imbert, “Discovery of podophyllotoxins,”  Biochimie,  80:207-222, 1998.   Jamar, Stoffel, Van Nerom, et al., “Clinical evaluation of Tc-99m L,L-ethylenedicysteine, a new renal tracer, in transplanted patients,”  J Nucl Med,  34:129P, 1993a.   Jamar, Van Nerom, Verbruggen, et al., “Clearance of the new tubular agent Tc-99m L,L-ethylenedicysteine: Estimation by a simplified method,”  J Nucl Med,  34:129P, 1993b.   Kabasakal. “Technetium-99m ethylene dicysteine: a new renal tubular function agent,”  Eur. J Nucl. Med.  27(3):351-7, 2000.   Kikukawa, Toyama, Katayama, et al., “Early and delayed Tc-99m ECD brain SPECT in SLE patients with CNS involvement,”  Ann Nucl Med.  14(l):25-32, 2000.   Koh, Rasey, Evans, Grierson, Lewellen, Graham, Krohn, Griffin, “Imaging of hypoxia in human tumors with [ 18 F]fluoroniisonidazole,”  Int J Radiat Oncol Biol Phys,  22:199-212, 1992.   Kranz, Patrick, Brigle, Spinella, Roy, “Conjugates of folate and anti-T-cell-receptor antibodies specifically target folate-receptor-positive tumor cells for lysis,”  Proc Natl Acad Sci,  92:9057-9061, 1995.   Krenning, Kwokkeboom, Bakker, et al., “Somatostatin receptor scintigraphy with [In-111-DTPA-D-Phe] and [I-123-Tyr]-octretide: The Rotterdam experience with more than 1000 patients,”  Eur J Nucl Med,  7:716-731, 1995.   Lambert, Bakker, Reubi, Krenning, “Somatostatin receptor imaging in vivo localization of tumors with a radiolabeled somatostatin analog,”  J. Steoid Biochem Mol Biol,  37:1079-1082, 1990.   Leamon and Low, “Cytotoxicity of momordin-folate conjugates in cultured human cells,”  J Biol Chem,  267:24966-24971, 1992.   Leamon and Low, “Delivery of macromolecules into living cells: a method that exploits folate receptor endocytosis,”  Proc Natl Acad Sci,  88:5572-5576, 1991.   Leamon, Pastan, Low, “Cytotoxicity of folate-pseudomonas exotoxin conjugates toward tumor cells,”  J Biol Chem,  268:24847-24854, 1993.   Lee and Low, “Delivery of liposomes into cultured KB cells via folate receptor-mediated endocytosis,”  J Biol Chem,  269:3198-3204, 1994.   Lennon, Martin, Cotter, “Dose-dependent induction of apoptosis in human tumor cell lines by widely diverging stimuli,”  Cell Prolif,  24:203-214, 1991.   Lu, “Antimitotic agents,” In: Foye, WO. Ed., “Cancer chemotherapeutic agents,” Washington, D.C.: American Chemical Society, 345-368, 1995.   Martin, Caldwell, Rasey, Grunbaum, Cerqueia, Krohn, Enhanced binding of the hypoxic cell marker [ 18 F]fluoromisonidazole in ischemic myocardium,”  J Nucl Med,  30:194-201, 1989.   Mathias, Hubers, Trump, Wang, Luo, Waters, Fuchs, Low, Green, “Synthesis of Tc-99m-DTPA-folate and preliminary evaluation as a folate-receptor-targeted radiopharmaceutical (Abstract),”  J Nucl Med , (Supplement); 38:87P, 1997a.   Mathias, Wang, Waters, Turek, Low, Green, “Indium-111-DTPA-folate as a radiopharmaceutical for targeting tumor-associated folate binding protein (Abstract),”  J Nucl Med , (Supplement) 38:133P, 1997b.   Mathias, Wang, Lee, Waters, Low, Green, “Tumor-selective radiopharmaceudcal targeting via receptor-mediated endocytosis of Gallium-67-deferoxamine-folate,”  J Nucl Med,  37:1003-1008, 1996.   Moller, Benecke, Flier. “Biologic activities of naturally occurring human insulin receptor mutations. Evidence that metabolic effects of insulin can be mediated by a kinase-deficient insulin receptor mutant,”  J Biol Chem.  15; 266(17):10995-1001, 1991.   Mochizuki, Inaki, Takeymoto, “Synthesis of polyglutamates containing 5-substituted uracil moieties,”  Nucleic Acids Res.,  16:121-124, 1985.   Nordsmark, Overgaard, Overgaard, “Pretreatment oxygenation predicts radiation response in advanced squamous cell carcinoma of the head and neck,”  Radiother Oncol,  41:31-39, 1996.   Offield, Jetton, Labosky, et al., “PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum,”  Development.  122(3):983-95, 1996.   Orr, Kreisler, Kamen, “Similarity of folate receptor expression in UMSCC 38 cells to squamous cell carcinoma differentiation markers,”  J Natl Cancer Inst,  87:299-303, 1995.   Patrick, Kranz, van Dyke, Roy, “Folate receptors as potendal therapeutic targets in choroid plexus tumors of SV40 transgenic mice,”  J Neurooncol,  32:111-123, 1997.   Piper, McCaleb, Montgomery, “A synthetic approach to poly(glutamyl) conjugates of methotrexate,”  J. Med. Chem.,  26:291-294, 1983.   Popovici, Mungiu, Trandafirescu, et al., “The influence of some antibiotics on hexokinase and pyruvate-kinase activity in the rat liver and kidney,”  Arch Int Pharmacodyn Ther.  193(1):80-6, 1971.   Raderer, Becherer, Kurtaran, Angelberger, Li, Leimer, Weinlaender, Kornek, Kletter, Scheithauer, Virgolini, “Comparison of Iodine-123-vasoactive intestinal peptide receptor scintigraphy and Indium-111 CFT-102 immnunoscintigraphy,”  J. Nucl. Med.,  37:1480-1487,1996.   Raffauf, Farren, Ullyot, “Colchicine. Derivatives of trimethylcolchicinic acid,”  J. Am Chem Soc,  75:5292-5294, 1953.   Rasey, Koh, Griesohn, Grunbaum, Krohn, “Radiolabeled fluoromisonidazole as an imaging agent for tumor hypoxia,”  Int. J Radiat Oncol Biol Phys,  17:985-991, 1989.   Rasey, Nelson, Chin, Evans, Grunbaum, “Characterization of the binding of labeled fluoromisonidazole in cells in vitro,”  Radiat Res,  122:301-308, 1990.   Ratner and Clarke, “The action of formaldehyde upon cysteine,”  J. Am Chem. Soc.,  59:200-206, 1937.   Reubi, Krenning, Lamberts et al., “In vitro detection of somatostatin receptors in human tumors,”  Metabolism,  41:104-110 (suppl 2), 1992.   Rogers, Bachorik, Nunn. “Neomycin effects on glucose transport by rat small intestine,”  Digestion.  1(3):159-64, 1968.   Ross, Chaudhuri, Ratnam, “Differential regulation of folate receptor isoforms in normal and malignant tissue in vivo and in established cell lines,”  Cancer,  73:2432-2443, 1994.   Rowinsky, Cazenave, Donehower, “Taxol: a novel investigational antimicrotuble agent,”  J. Natl. Cancer Institute,  82(15):1247-1259, 1990.   Seabold, Gurll, Schurrer, Aktay, Kirchner, “Comparison of  99m Tc-Methoxyisobutyl Isonitrile and  201 T1 Scintigraphy for Detection of Residual-Thyroid Cancer After  131 I Ablative Therapy,”  J. Nucl. Med.,  40(9):1434-1440, 1999.   Shankar, Zhu, Baron et al., “Glucosamine infusion in rats mimics the beta-cell dysfunction of non-insulin-dependent diabetes mellitus,”  Metabolism.  47(5):573-7, 1998.   Stella and Mathew, “Derivatives of taxol, pharmaceutical compositions thereof and methods for preparation thereof,” U.S. Pat. No. 4,960,790, Oct. 2, 1990.   Surma, Wiewiora, Liniecki, “Usefulness of Tc-99m-N,N′-ethylene-1-dicysteine complex for dynamic kidney investigations,”  Nucl Med Comm,  15:628-635, 1994.   Tait and Smith, “Site-specific mutagenesis of annexin V: role of residues from Arg-200 to Lys-207 in phospholipid binding,”  Arch Biochem Biophys,  288:141-144, 1991.   Valk, Mathis, Prados, Gilbert, Budinger, “Hypoxia in human gliomas: Demonstration by PET with [ 18 F]fluoromisonidazole,”  J Nucl Med,  33:2133-2137, 1992.   Van Nerom, Bormans, Bauwens, Vandecruys, De Roo, Verbruggen, “Comparative evaluation of Tc-99m L,L-ethylenedicysteine and Tc-99m MAG3 in volunteers,”  Eur J Nucl Med,  16:417, 1990.   Van Nerom, Bormans, De Roo, et al., “First experience in healthy volunteers with Tc-99m-L,L-ethylenedicysteine, a new renal imaging agent,”  Eur J Nucl Med,  20:738-746, 1993.   Verbruggen, Nosco, Van Nerom et al., “Tc-99m-L,L-ethylenedicysteine: A renal imaging agent. I. Labelling and evaluation in animals,”  J Nucl Med,  33:551-557, 1992.   Verbruggen, Nosco, Van Nerom, Bormans, Adriacns, De Roo, “Evaluation of Tc-99m-L,L-ethylenedicysteine as a potential alternative to Tc-99m MAG3,”  Eur J Nucl Med,  16:429, 1990.   Villevalois-Cam, Tahiri, Chauvet, et al., “Insulin-induced redistribution of the insulin-like growth factor II/mannose 6-phosphate receptor in intact rat liver,”  J Cell Biochem.  77(2):310-22, 2000   Virgolini, Raderer, Kurtaran, “Vasoactive intestinal peptide (VIP) receptor imaging in the localization of intestinal adenocarcinomas and endocrine tumors,”  N Eng J Med,  331:1116-1121, 1994.   Wang, Lee, Mathias, Green, Low, “Synthesis, purification, and tumor cell uptake of Ga-67 deferoxamine-folate, a potential radiopharmaceutical for tumor imaging,”  Bioconjugate Chem,  7:56-62, 1996.   Wang, Luo, Lantrip, Waters, Mathias, Green, Fuchs, Low, “Design and synthesis of [ 111 In]DTPA-folate for use as a tumor-targeted radiopharmaceutical,”  Bioconjugate Chem,  8:673-679, 1997.   Wang, Wang, Ichijo, Giannakakou, Foster, Fojo, Wimalasena, “Microtubule-interfering agents activate c-Jun N-terminal kinasae/stress-activated protein kinase through both Ras and apoptosis signal-regulating kinase pathways; ”  J. Biol. Chem.,  273:4928-4936, 1998.   Weitman, Frazier, Kamen, “The folate receptor in central nervous system malignancies of childhood,”  J Neuro - Oncology,  21:107-112, 1994.   Weitman, Lark, Coney et al., “Distribution of folate GP38 in normal and malignant cell lines and tissues,”  Cancer Res,  52:3396-3400, 1992a   Weitman, Weinberg, Coney, Zurawski, Jennings, Kamen, “Cellular localization of the folate receptor: potential role in drug toxicity and folate homeostasis,”  Cancer Res,  52:6708-6711, 1992b.   Wester, Herz, Weber, Heiss, Schlnidtke, Schwaiger, Stocklin, “Synthesis and radiopharmacology of —O(2-[ 18 F]fluoroethyl)-L-Tyrosine for tumor imaging,”  J. Nucl. Med.,  40:205-212, 1999.   Westerhof, Jansen, Emmerik, Kathmann, Rijksen, Jackman, Schornagel, “Membrane transport of natural folates and antifolate compounds in murine L1210 leukemia cells: Role of carrier- and receptor-mediated transport systems,”  Cancer Res,  51:5507-5513, 1991.   Yang, Wallace, Cherif, Li, Gretzer, Kim, Podoloff, “Development of F-18-labeled fluoroerythronitroimidazole as a PET agent for imaging tumor hypoxia,”  Radiology,  194:795-800, 1995.   Yoshino, Takeda, Sugimoto, et al., “Differential effects of troglitazone and D-chiroinositol on glucosamine-induced insulin resistance in vivo in rats,”  Metabolism.  48(11):1418-23, 1999.