# Patent

## Title
Bisaminoethanethiol-targeting ligand conjugates and compositions

## Abstract

The invention provides, in a general sense, a new labeling strategy employing 99mTc chelated with ethylenedicysteine (EC). EC is conjugated with a variety of ligands and chelated to 99mTc for use as an imaging agent for tissue-specific diseases. The drug conjugates of the invention may also be used as a prognostic tool or as a tool to deliver therapeutics to specific sites within a mammalian body. Kits for use in tissue-specific disease imaging are also provided.

## Background
This is a continuation application of application Ser. No. 11/760,456, filed Jun. 8, 2007 now U.S. Pat. No. 7,615,208, which is a continuation of Ser. No. 11/627,299, filed Jan. 25, 2007, now issued as U.S. Pat. No. 7,582,281, which is a continuation application of Ser. No. 10/672,763, filed Sep. 26, 2003, now issued as 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 was 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.
[18F]FMISO has been used to diagnose head and neck tumors, myocardial infarction, inflammation, and brain ischemia (Martin 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 [18F]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 [18F]FDG was clearly demonstrated, introducing molecular imaging agents into clinical practice depends on some other factors such as easy availability and isotope cost. [18F]fluorodeoxyglucose (FDG) has been used to diagnose tumors, myocardial infarction, and neurological disease. In addition, PET radiosynthesis must be rapid because of short half-life of the positron isotopes. 18F chemistry is also complex. The 18F 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 99mTc due to low cost ($0.21/mCi vs. $50/mCi for 18F) and low energy (140 Kev vs. 571 Kev for 18F). 99mTc is easily obtained from a 99Mo generator. Due to favorable physical characteristics as well as extremely low price, 99mTc has been preferred to label radiopharmaceuticals.
Several compounds have been labeled with 99mTc using nitrogen and sulfur chelates (Blondeau et al., 1967; Davison et al., 1980). Bis-aminoethanethiol tetradentate ligands, also called 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. 99mTc-L,L-ethylenedicysteine (99mTc-EC) is a recent and successful example of N2S2 chelates. EC can be labeled with 99mTc easily and efficiently with high radiochemical purity and stability, and is excreted 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). 99mTc-EC-neomycin and 99mTc-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 N2S2 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 99mTc labeled diethylester (99mTc-L,L-ECD) is known as a brain agent. 99mTc-L,L-ethylenedicysteine (99mTc-L,L-EC) is its most polar metabolite and was discovered to be excreted rapidly and efficiently in the urine. Thus, 99mTc-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, endostatin, 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 fungi. 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-NH2 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 99mTc, 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 188Re, 186Re, 153Sm, 166Ho, 90Y, 89Sr, 67Ga, 68Ga, 111In, 153Gd, and 59Fe. 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, 186/188Re-EC-folate) and head and neck cancer (using for example, 186/88Re-EC-nitroimidazole).
Specific embodiments of the present invention include 99mTc-EC-annexin V, 99mTc-EC-colchicine, 99mTc-EC-nitroimidazole, 99mTc-EC-glutamate pentapeptide, 99mTc-EC-metronidazole, 99mTc-EC-folate, 99mTc-EC-methotrexate, 99mTc-EC-tomudex, 99mTc-EC-neomycin, 99mTc-EC-kanamycin, 99mTc-EC-aminoglycosides, (glucosamine, EC-deoxyglucose), 99mTc-EC-gentamycin, and 99mTc-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 N2S2 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 99mTc in the presence of a reducing agent to form an N2S2 chelate between the ethylenedicysteine and the 99mTc.
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 99mTc labeled ethylenedicysteine-tissue specific ligand conjugate and detecting a radioactive signal from the 99mTc 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 99mTc. 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 99mTc binding moiety. The 99mTc binding moiety is either directly attached to the tissue specific ligand or is attached to the ligand through a linker as described above. The 99mTc binding moiety is preferably an N2S2 chelate between 99mTc 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 99mTc-EC-folate.
FIG. 2. Synthetic scheme of 99mTc-EC-MTX (methotrexate).
FIG. 3. Synthetic scheme of 99mTc-EC-TDX (tomudex).
FIG. 4. Biodistribution studies for 99mTc-EC and 99mTc-EC-folate.
FIG. 5. Blocking studies for tumor/muscle and tumor/blood count ratios with 99mTc-EC-folate.
FIGS. 6A and 6B. Scintigraphic images of tumor in 99mTc-EC-folate injected group as compared to 99mTc-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 1H-NMR confirmation of the structure.
FIG. 9. Biodistribution studies (tumor/blood ratios) for 99mTc-EC-MN, [18F]FMISO and [131I]IMISO.
FIG. 10. Biodistribution studies (tumor/muscle ratios) for 99mTc-EC, [18F]FMISO and [131I]IMISO.
FIGS. 11A and 11B. Scintigraphic images of tumor in 99mTc-EC-MN (FIG. 11A) and 99mTc-EC (FIG. 11B) injected groups.
FIG. 12. Autoradiograms performed at 1 hour after injection with 99mTc-EC-MN.
FIG. 13. Illustrates stability of 99mTc-EC-NIM in dog serum samples.
FIG. 14A and FIG. 14B. Illustrates breast tumor uptake of 99mTc-EC-NIM vs. 99mTc-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 99mTc-EC-NIM vs. 99mTc-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 99mTc-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 99mTc-EC-NIM after treatment with paclitaxel.
FIG. 16. Synthetic scheme of EC-GAP (pentaglutamate).
FIG. 17. Scintigraphic images of breast tumors in 99mTc-EC-GAP injected group.
FIG. 18. Scintigraphic images of breast tumors in 99mTc-EC-ANNEX V injected group at different time intervals.
FIG. 19A and FIG. 19B. Comparison of uptake difference of 99mTc-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 99mTc-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 99mTc-EC-COL.
FIG. 24. Ratios of tumor to muscle and tumor to blood as function of time for 99mTc-EC.
FIG. 25. In vivo imaging studies in breast tumor bearing rats with 99mTc-EC-COL.
FIG. 26. In vivo imaging studies in breast tumor bearing rats with 99mTc-EC.
FIG. 27. Computer outlined region of interest after injection of 99mTc-EC-COL vs. 99mTc-EC.
FIG. 28. SPECT with 99mTc-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 99mTc-EC-MN of 73 year old male patient one day after stroke at one hour post-injection.
FIG. 31. SPECT with 99mTc-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 99mTc-EC and 99mTc-EC-neomycin (100 μCi/rat, iv.) showed that the tumor could be well visualized from 0.5-4 hours postinjection.
FIG. 37B. Scintimammography with 99mTc-EC-neomycin (30 mCi, iv.) of a breast cancer patient. Images taken two hours post-injection.
FIG. 38A. 1H-NMR of EC.
FIG. 38B. 1H-NMR of neomycin.
FIG. 38C. 1H-NMR of EC-neomycin.
FIG. 39. Mass spectrometry of EC-neomycin (M+1112.55).
FIG. 40A. UV wavelength scan of EC.
FIG. 40B. UV wavelength scan of neomycin.
FIG. 40C. UV wavelength scan of EC-neomycin.
FIG. 41. Radio-TLC analysis of 99mTc-EC-neomycin.
FIG. 42. HPLC analysis of 99mTc-EC-neomycin (radioactive detector).
FIG. 43. HPLC analysis of 99mTc-EC-neomycin (UV 254 nm).
FIG. 44. HPLC analysis of 18F-FDG (radioactive detector).
FIG. 45. HPLC analysis of 18F-FDG (UV 254 nm).
FIG. 46. In vitro cellular uptake assay of a series of 99mTc-EC-drug conjugates in lung cancer cell line. 99mTc-EC-neomycin showed highest uptake in the agents tested.
FIG. 47. Effect of glucose on cellular (A549) uptake of 99mTc-EC-neomycin and 18F-FDG.
FIG. 48A and FIG. 48B. Effect of glucose on cellular (H1299) uptake of 99mTc-EC-neomycin and 18F-FDG illustrated as percent of drug uptake (FIG. 48A) and as percent of change with glucose loading (FIG. 48B).
FIG. 49. Synthetic scheme of 99mTc-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 99mTc-EC-GAP-glucosamine.
FIG. 55A, FIG. 55B, FIG. 55C. In vitro cellular uptake assay of 99mTc-EC (FIG. 56A), 99mTc-EC-deoxyglucose-GAP (FIG. 56B), and 18F-FDG (FIG. 56C) in lung cancer cell line (A549). 99mTc-EC-DG showed similar uptake compared to 18F-FDG.
FIG. 56. Tumor-to-tissue count density ratios of 99mTc-EC-GAP in breast tumor-bearing rats.
FIG. 57 In vitro cellular uptake of 18PDG with glucose loading at 2 hours post-injection in breast cancer cell line (13762).
FIG. 58. In vivo tissue uptake of 99mTc-EC-neomycin in breast tumor-bearing mice.
FIG. 59. Synthetic scheme of 99mTc-EC-deoxyglucose.
FIG. 60. Mass spectrometry of EC-deoxyglucose.
FIG. 61. 1H-NMR of EC-deoxyglucose (EC-DG).
FIG. 62. 1H-NMR of glucosamine.
FIG. 63. Radio-TLC analysis of 99mTc-EC-DG.
FIG. 64. HPLC analysis of 99mTc-EC-deoxyglucose and 99mTc-EC- (radioactive detector).
FIG. 65. HPLC analysis of 99mTc-EC-deoxyglucose and 99mTc-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 99mTc-EC-deoxyglucose, 99mTc-EC and 18F-FDG in lung cancer cell line (A549). 99mTc-EC-DG showed similar uptake compared to 18F-FDG.
FIG. 70. Effect of d- and l-glucose on breast cellular (13762 cell line) uptake of 99mTc-EC-DG.
FIG. 71. Effect of d- and l-glucose on breast cellular (13762 cell line) uptake of 18F-FDG.
FIG. 72. Effect of d- and l-glucose on lung cellular (A549 cell line) uptake of 18F-FDG.
FIG. 73. Effect of d- and l-glucose on breast cellular (A549 cell line) uptake of 99mTc-EC-DG.
FIG. 74. Effect of in vivo blood glucose level induced by glucosamine and EC-DG (1.2 mmol/kg, i.v.).
FIG. 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 99mTc-EC-deoxyglucose in breast tumor-bearing rats.
FIG. 77. In vivo biodistribution of 99mTc-EC-deoxyglucose in breast tumor-bearing rats.
FIG. 78. In vivo tissue uptake of 99mTc-EC-deoxyglucose in lung tumor-bearing mice.
FIG. 79. In vivo tissue uptake of 99mTc-EC-neomycin in lung tumor-bearing mice.
FIG. 80. In vivo tissue uptake of 18F-FDG in lung tumor-bearing mice.
FIG. 81. Planar image of breast tumor-bearing rats after administration of 99mTc-EC and 99mTc-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 99mTc-EC-DG of a patient with malignant astrocytoma.
FIG. 83A. MRI of a patient with hemorrhagic astrocytoma.
FIG. 83B. SPECT with 99mTc-EC-DG of a patient with malignant astrocytoma.
FIG. 84A. MRI of a patient with benign meningioma.
FIG. 84B. SPECT with 99mTc-EC-DG of a patient with benign meningioma showed no focal intensed uptake.
FIG. 85A. CT of a patient with TB in lung.
FIG. 85B. SPECT with 99mTc-EC-DG of a patient with TB showed no focal intensed uptake.
FIG. 86A. CT of patient with lung cancer.
FIG. 86B. Whole body images of 99mTc-EC-DG of a patient with lung cancer.
FIG. 86C. SPECT with 99mTc-EC-DG of a patient with lung cancer, the tumor showed focal intensed uptake.



DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
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 67Ga, 99mTc, 111In, 123I, 125I, 169Yb or 186Re. Due to better imaging characteristics and lower price, attempts have been made to replace the 123I, 131I, 67Ga and 111In labeled compounds with corresponding 99mTc labeled compounds when possible. Due to favorable physical characteristics as well as extremely low price ($0.21/mCi), 99mTc has been preferred to label radiopharmaceuticals. Although it has been reported that DTPA-drug conjugate could be labeled with 99mTc effectively (Mathias et al., 1997), DTPA moiety does not chelate with 99mTc as stable as with 111In. (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. 99mTc 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 thiolsulfur and two amine nitrogen atoms. (Davison et al., 1980; 1981; Verbruggen et al., 1992). 99mTc-L,L-ethylenedicysteine (99mTc-EC) is the most recent and successful example of N2S2 chelates. (Verbruggen et al., 1992; Van Nerom et al., 1993; Surma et al., 1994). EC, a new renal imaging agent, can be labeled with 99mTc easily and efficiently with high radiochemical purity and 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 99mTc-EC as a labeling agent to target ligands to specific tissue types for imaging. The advantage of conjugating the EC with tissue targeting ligands is that the specific binding properties of the tissue targeting ligand concentrates the radioactive signal over the area of interest. While it is envisioned that the use of 99mTc-EC as a labeling strategy can be effective with virtually any type of compound, some suggested preferred ligands are provided herein for illustration purposes. It is contemplated that the 99mTc-EC-drug 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'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, 99mTc-EC has not been used in conjunction with any ligands, other than as the diethylester (Kabasakal, 2000), prior to the present invention. The diethylester of EC was used as a cerebral blood flow agent (Kikukawa, et al., 2000).
Although optimal for radioimaging, the chemistry of 99mTc has not been as thoroughly studied as the chemistry of other elements and for this reason methods of radiolabeling with 99mTc are not abundant. 99mTc is normally obtained as 99mTc pertechnetate (TcO4−; 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, 99mTc 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 99mTc either to insoluble technetium dioxide or back to pertechnetate.
For the purpose of radiolabeling, it is particularly advantageous for the 99mTc 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 99mTc 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 N2S2 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 99mTc 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 (SnCl2) 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 SnCl2 per about 100 to about 300 mCi of Tc pertechnetate. The most preferred amount is about 0.1 mg SnCl2 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 99mTc 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-NH2
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 166Ho, 188Re, 186Re, 153Sm, and 89Sr. Preferred β-emitters include 90Y and 225Ac. Preferred γ-emitters include 67Ga, 68Ga, 64Cu, 62Cu and 111In. Preferred α-emitters include 211At and 212Bi. 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 99mTc. 99mTc labeled scintigraphic imaging agents according to the present invention can be prepared by the addition of an appropriate amount of 99mTc or 99mTc 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 99mTc 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.
99mTc 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 99mTc 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 99mTc-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 99mTc 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'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's time and many thousands of dollars before the effectiveness of the drug can be determined.
The 99mTc 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; Holm 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 67Ga-deferoxamine-folate and 111In-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 99mTc-EC-folate, 99mTc-EC-methotrexate (99mTc-EC-MTX), 99mTc-EC-tomudex (99mTc-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., 18F, 123I, 99mTc) may be useful for differentiating a hypoxic but metabolically active tumor from a well-oxygenated active tumor by PET or planar scintigraphy. [18F]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 [18F]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 99mTc-EC-2-nitroimidazole and 99mTc-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 99mTc-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 99mTc-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, 99mTc-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. 99mTc-EC-2-nitroimidazole and 99mTc-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. 99mTc-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 99mTc-EC-metronidazole (99mTc-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 99mTc-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 99mTc-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 99mTc-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 99mTc-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 99mTc-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 1H-NMR and fast-atom bombardment mass spectroscopy (FAB-MS).
Synthesis of Aminoethylamido Analogue of Methotrexate (MTX-NH2)
MIX (227 ma, 0.5 mmol) was dissolved in 1 ml of HCl solution (2N). The pH value was <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); 1H-NMR (D2O) δ 2.98-3.04 (d, 8H, —(CH2)2CONH(CH0)2NH2), 4.16-4.71 (m, 6H, —CH2− pteridinyl, aromatic-NCH3, NH—CH—COOH glutamate), 6.63-6.64 (d, 2H, aromatic-CO), 7.51-753 (d, 2H. aromatic-N), 8.36 (s, 1H, pteridinyl). FAB MS m/z calcd for C22H28, N10, O4(M)+ 496.515, found 496.835.
Synthesis of Aminoethylamido Analogue of Folate (Folate-NH2)
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). 1H-NMR (D2O) δ1.97-2.27 (m, 2H, —CH2 glutamate of folate), 3.05-3.40 (d, 6H, —CH2CONH(CH2)2NH2), 4.27-4.84 (m, 3H, —CH2-pteridinyl, NH—CH—COOH glutamate), 6.68-6.70 (d, 2H, aromatic-CO), 7.60-7.62 (d, 2H, aromatic-N), 8.44 (s, 1H, pteridinyl). FAB MS m/z calcd for C21H25N9, O5(M)+ 483, found 483.21.
Synthesis of Ethylenedicysteine-Folate (EC-Folate)
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-NH2 (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); 1H-NMR (D2O) δ1.98-2.28 (m, 2H, —CH2 glutamate of folate), 2.60-2.95 (m, 4H and —CH2—SH of EC). 3.24-3.34 (m, 10H, —CH2—CO, ethylenediamine of folate and ethylenediamine of EC), 4.27-4.77 (m, 5H, —CH-pteridinyl, NH—CH—COOH glutamate of folate and NH—CH—COOH of EC), 6.60-6.62 (d, 2H, aromatic-CO), 7.58-7.59 (d, 2H. aromatic-N), 8.59 (s, 1H, pteridinyl). Anal. calcd for C29H37N11S2O8Na2 (8H2O), FAB MS m/z (M)+ 777.3 (free of water). C, 37.79; H, 5.75; N, 16.72; S, 6.95. Found: m/z (M)+ 777.7 (20), 489.4 (100). C, 37.40; H, 5.42; N, 15.43; S, 7.58.
Radiolabeling of EC-Folate and EC with 99mTc
Radiosynthesis of 99mTc-EC-folate was achieved by adding required amount of 99mTc-pertechnetate into home-made kit containing the lyophilized residue of EC-folate (3 mg), SnCl2 (100 μg), Na2HPO4 (13.5 mg), ascorbic acid (0.5 mg) and NaEDTA (0.5 mg). Final pH of preparation was 7.4. 99mTc-EC was also obtained by using home-made kit containing the lyophilized residue of EC (3 mg), SnCl2 (100 μg), Na2, IPO4 (13.5 mg), ascorbic acid (0.5 mg) and NaEDTA (0.5 mg) at pH 10. Final pH of preparation was then adjusted to 7.4. Radiochemical purity was determined by TLC (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 >95% for both radiopharmaceuticals. Radio-TLC data are summarized in Table 2. Synthesis of 99mTc-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 or thiotape



doxorubicin + claplatin (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 ±
Etoposide



methotrexate



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
Adjuvant1: Cyclophosphamide +



methotrexate + fluorouracil (CMF);



Cyclophosphamide + Doxorubicin ±



fluorouracil (AC or CAF); Tamoxifen



Metastatic: Cyclophosphamide + methotrexate +
Paclitaxel; thiotepa +



fluorouracil (CMF) or
Doxorubicin + vin-blastine;



Cyclophosphamide + duxorubicin ±
mitomycin + vinblastine;



fluorouracil (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-sensitive2
marrow transplant3


Cervix**
Claplatin
Chlorambucil, vincristine,



Ifosfamide with means
fluorouracil, Doxorubicin,



Bleomycin + ifosfamide with means +
methotrexate, altretamine



claplatin


Chorlocarcinoma
Methotrexate ± leucovorin
Methotrexate + dactinomycin +



Dactinomycin
cyclophosphamide (MAC)




Etoposide + methotrexate +




dactinomycin +




cyclophosphamide + vincristine


Colorectal*
Adjuvant colon4: Fluorouracil + levam-isole;
Hepatic metastases:



fluorouracil + leucovorin
Intrahepatic-arterial floxuridine



Metastatic: fluorouracil + leucovorin
Mitomycin


Embryonal rhabdomyosar-coma5
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's sarcoma5
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'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 +
Induction: same ± high-dose


(ALL)7
asparaginase ± daunorubicin
methotrexate ± cyterabine;



CNS prophylaxis: Intrathecal methotrexate ±
pegaspargase instead of



systemic high-dose methotrexate with
asparaginese



leutovorin ± Intrathecal cytarabine ±
Teniposide or etoposide



Intrathecal hydrocortisone
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 transplant3.


Chronic lymphocytic leukemia
Chlorambucil ± prednisone
Cladribine, cyclophosphamide,


(CLL)
Fludarabin
pentostatin, vincristine,




Doxorubicin


Chronic myeloid leukemia


(CML)10


Chronic phase
Bone marrow transplant3
Busulfan



Interferon alfa



Hydroxyures


Accelerated11
Bone marrow transplant3
Hydroxyures, busulfen


Blast crisis11
Lymphoid: Vincristine + prednisone + L-
Tretinoln†



separaginess + intrathecal methotrexate (±
Amsecrine,† azacitidine



maintenance 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 +
carboplatin + etoposide (ICE)



vincristine (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 +


(non-small cell)**
Claplatin + Vinblastine ± mitomycin
leucovorin



Claplatin + vincrisine
Carboplatin + paclitaxel


Lymphomas


Hodgkin's12
Doxorubicin + bleomycin + vinblastine +
Mechlorethamine + vincristine +



dacarbazine (ABVD)
procarbazine + prednisone



ABVD alternated with MOPP
(MOPP)



Mechlorethamine + vincristine +
Chlorambusil + vinblastine +



procarbazine (± prednisone) + doxorubicin +
procarbazine + prednisone ±



bleomycin + vinblastine (MOP[P]-ABV)
carmustine




Etoposide + vinblastine +




doxorubicin




Bone marrow transplant3


Non-Hodgkin's


Burkitt's lymphoma
Cyclophosphamide + vincristine +
Ifosfamide with means



methotrexate
Cyclophosphamide +



Cyclophosphamide + high-dose cytarabine ±
doxorubicin + vincrletine +



methotrexate with leutovorin
prednisone (CHOP)



Intrathecal methotrexate or cytarabine


Difuse large-cell lymphoma
Cyclophosphamide + doxorubicin +
Dexamethasone sometimes



vincristine + prednisone (CHOP)
substituted for prednisone




Other combination regimens,




which may include methotrexate,




etoposide, cytarabine,




bleomycin, procarbazine,




ifosfamide and mitoxantrone




Bone marrow transplant3


Follicular lymphoma
Cyclophosphamide or chlorambusil
Same ± vincristine and




prednisone, ± etoposide




Interferon alfa, cladribine,




fludarabin




Bone marrow transplant3




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's lymphoma


Mysloma*
Melphelan (or cyclophosphamide) +
Interferon alfa



prednisons
Bone marrow transplant3



Melphalan ∀ carmustine +
High-dose dexamethasons



cyclophosphamide + prednisons +



vincristine



Dexamethasone + doxorubicin +



vincristine (VAD)



Vincristine + carmustine + doxorubicin +



prednisons (VBAP)


Neuroblestoma*
Doxorubicin + cyclophosphamide +
Carboplatin, etoposide



claplatin + teniposide or etoposide
Bone marrow transplant3



doxorubicin + cyclophosphamide



Claplatin + cyclophosphamide


Osteogenic sarcoma5
Doxorubicin + claplatin ∀ etopside ∀
Ifosfamide with means,



ifosfamide
etoposide, carboplatin, high-




dose methotrexate with




leucovorin




Cyclophosphamide +




etoposide


Ovary
Claplatin (or carboplatin) + paclitaxel
Ifosfamide with means,



Claplatin (or carboplatin) +
paclitaxel, tamoxifen,



cyclophosphamide (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


Retinoblestoma5*
Doxorubicin + cyclophosphamide ∀
Carboplatin, etoposide,



claplatin ∀ etoposide ∀ vincristina
Ifosfamide with means


Sarcomas, soft tissue, adult*
Doxorubicin ∀ decarbazine ∀
Mitornyeln + doxorubicin +



cyclophosphamide ∀ Ifosfamide with
claplatin



means
Vincristina, etoposide


Testicular
Claplatin + etoposide ∀ bleomycin
Vinblestine (or etoposide) +



(PEB)
Ifosfamide with means +




claplatin (VIP)




Bone marrow transplant3


Wilms' tumor5
Dectinomycln + vincriatine ∀
Ifosfamide with means,



doxorubicin ∀ cyclophosphamide
etoposide, carboplatin










*Chemotherapy has only moderate activity.


**Chemotherapy has only minor activity.


1Tamoxifen 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.


2Megastrol and other hormonal agents may be effective in some patients with


tamoxifen fails.


3After high-dose chemotherapy (Medical Letter, 34: 79, 1982).


4For rectal cancer, postoperative adjuvant treatment with fluoroutacil plus


radiation, preceded and followed by treatment with fluorouracil alone.


5Drugs have major activity only when combined with surgical resection,


radiotherapy or both.


6The 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.


7High-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).


8Patients with a poor prognosis initially or those who relapse after remission.


9Some 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).


10Allogeheic 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 <15% patients in blast crisis. Disease-free survival after bone marrow


transplantations adversely influenced by age >50 years, duration of disease >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 >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.


11If 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.


12Limited-stage Hodgkin'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 %




Dactinomyclin
dectinomycin %





cyclophosphamide (MAC)





Etoposide % methotrexate %





dactinomycin %





cyclophosphamide %





vincrlatine



Colorectal*
Adjuvant colon4: Fluoroutacil %
Hepatic metastases:




lavamleole; fluoroutacil % leucovarin
Intrahepactic-arterial




Metastatic: Fluoroutacil % leucvarin
floxuridine





Mitomyclin



Embryonal
Vincriatine % dectinomycin ∀
Same % doxorubicin



rhebdomyosarcoma6
cyclophosphamide




Vincristine % Ifosfamide with means %




etoposide



Endometrial**
Megastrol or another progeetin
Fluoroutacil, tamoxifen,




Doxorubicin % claplatin ∀
altretamine




cyclophosphamide







Cancer
Drugs of Choice
Some alternatives







Esophageal*
Claplatin % Fluoroutacil
Doxorubicin, methotrexete,



Ewing's sarcoma5
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'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 %
Industion: same ∀ high-dose



Acute lymphocytic leukemia
asparaginase ∀ daunorubieln
methotrexete ∀ cyterabine;



(ALL)7
CNS prophylaxia; Intrathecal
pegaspargase instead of




methotrexete ∀ systemic high-dose
aspareginese




methotrexete with leucovorin ∀
Teniposide or etoposide




Intrethecal cytarabine ∀ Intrathecal
High-dose cytarabine




hydrocortisone




Maintenance: methotrexete
Maintenance: same %




∀mercaptopurine
periodic vincristine %




Bone marrow transplant3
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 transplant3



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.


1Available in the USA only for investigational use.


2Megestrol and other hormonal agents may be effective in some pateients when tamoxifen fails.


3After high-dose chemotherapy (Medical Letter, 34: 78, 1992).


4For rectal cancer, postoperative adjuvant treatment with fluoroutacil plus radiation, preceded and followed by


treatment with fluoroutacil alone.


5Drugs have major activity only when combined with surgical resection, radiotherapy or both.


6The 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).


7High-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).


8Patients with a poor prognosis initially or those who relapse after remission


9Some 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).


10Allogenaic 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 <15% patients in blast crisis. Disease-free survival


after bone marrow transplantation is adversely influenced by age >50 years, duration of disease >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 >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 99mTc

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 99mTc-EC-folate except EC-MTX and EC-TDX were used. Synthesis of 99mTc-EC-MTX and 99mTc-EC-TDX is shown in FIG. 2 and FIG. 3.
Stability Assay of 99mTc-EC-Folate, 99mTc-EC-MTX and 99mTc-EC-TDX
Stability of 99mTc-EC-Folate, 99mTc-EC-MTX and 99mTc-EC-TDX was tested in serum samples. Briefly, 740 KBq of 1 mg 99mTc-EC-Folate, 99mTc-EC-MIX and 99mTc-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 (106 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 99mTc-EC-folate or 99mTc-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 99mTc-EC-folate was co-administrated (i.v.) 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 99mTc-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 99mTc-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 99mTc-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 99mTc-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 99mTc was achieved with high (>95%) radiochemical purity. 99mTc-EC-folate was found to be stable at 20 min. 1, 2 and 4 hours in dog serum samples.
Biodistribution of 99mTc-EC-Folate
Biodistribution studies showed that tumor/blood count density ratios at 20 min-4 h gradually increased for 99mTc-EC-folate, whereas these values decreased for 99mTc-EC in the same time period (FIG. 4). % ID/g uptake values, tumor/blood and tumor/muscle ratios for 99mTc-EC-folate and 99mTc-EC were given in Tables 3 and 4, respectively.







TABLE 3







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









% of injected 99mTc-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 99mTc-EC-folate injected group. Contrary, there was no apparent tumor uptake in 99mTc-EC injected group (FIG. 6). Both radiotracer showed evident kidney uptake in all images. Autoradiograms performed at 1 h after injection of 99mTc-EC-folate clearly demonstrated tumor activity.
Example 2
Tumor Hypoxia Targeting
Synthesis of 2-(2-methyl-5-nitro-1H imidazolyl)ethylamine (amino analogue of metronidazole, MN-NH2)
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-NH2. The structure was confirmed by 1H-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%). 1H-NMR (D20) δ 2.93 (s, 6H, nitroimidazole-CH3), 2.60-2.95 (m, 4H and —CH2—SH of EC), 3.30-3.66 (m, 8H, ethylenediamine of EC and nitromidazole-CH2—CH2—NH2), 3.70-3.99 (t, 2H, NH—CH—CO of EC), 5.05 (t, 4H, metronidazole-CH2—CH2—NH2) (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-1H-imidazolyl)propylamine (amino analogue of nitroimidazole, NIM-NH2)
To a stirred mixture containing 2-nitloimidazole (1 g, 8.34 mmol) and Cs2, CO3 (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. 1H-NMR (CDCl3) 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 MgSO4 and evaporated to dryness to afford azido analogue (0.6 g, 75%, TLC: hexane:ethyl acetate; 1:1, Rf=0.42). 1H-NMR (CDCl3) 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 MgSO4 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. 1H-NMR (D2O) 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-NH2 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 1H-NMR (D2O) (FIG. 8B).
Radiolabeling of EC-MN and EC-NIM with 99mTc
Radiosynthesis of 99mTc-EC-MN and 99mTc-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), SnCl2, (100 μg), Na2HPO4 (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 >96% for both radiotracers.
Synthesis of [18F]FMISO and [131I]IMISO
18F was produced by the cyclotron using proton irradiation of enriched 18O-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 [18F]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 Na131 (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 99mTc-EC-MN and 99mTc-EC-NIM
Stability of labeled 99mTc-EC-MN and 99mTc-EC-NIM were tested in serum samples. Briefly, 740 KBq of 1 mg 99mTc-EC-MN and 99mTc-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 99mTc-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 (106 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 99mTc-EC-MN or 99mTc-EC (n=3/time point). The injected mass of 99mTc-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 [18F]FMISO and [131I]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 99mTc-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 99mTc-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 pO2 Measurements
To confirm tumor hypoxia, intratumoral pO2 measurements were performed using the Eppendorf computerized histographic system. Twenty to twenty-five pO2 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 pO2 histogram between 0 and 100 mmHg with a class width of 2.5 mm.
Results
Radiosynthesis and Stability of 99mTc-EC-MN and 99mTc-EC-NIM
Radiosynthesis of EC-MN and EC-NIM with 99mTc were achieved with high (>95%) radiochemical purity Radiochemical yield was 100%. 99mTc-EC-MN and 99mTc-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 99mTc-EC-MN and 99mTc-EC in the tumor-bearing rats is shown in Tables 4 and 5. Due to high affinity for ionic 99mTc, there was no significant and consistent thyroid uptake, suggesting the in vivo stability of 99mTc-EC-MN (Table 5).







TABLE 4







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









% of injected 99mTc-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<0.01) with folic acid co-administrations (FIG. 5).







TABLE 5







Biodistribution of 99mTc-EC-metronidazole conjugate


in breast tumor bearing rats1













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







1Each 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 99mTc-EC-MN, [18F]FMISO and [131I]IMISO, whereas these values did not alter for 99mTc-EC in the same time period (FIG. 9 and FIG. 10). [18F]FMISO showed the highest tumor-to-blood uptake ratio than those with [131I]IMISO and 99mTc-EC-MN at 30 min, 2 and 4 hrs post-injection. Tumor/blood and tumor/muscle ratios for 99mTc-EC-MN and [131I]IMISO at 2 and 4 hrs postinjection were not significantly different (p<0.05).
Scintigraphic Imaging and Autoradiographic Studies
Scintigraphic images obtained at different time points showed visualization of tumor in 99mTc-EC-MN and 99mTc-EC-NIM groups. Contrary, there was no apparent tumor uptake in 99mTc-EC injected group (FIG. 11). Autoradiograms performed at 1 hr after injection of 99mTc-EC-MN clearly demonstrated tumor activity (FIG. 12). Compare to 99mTc-EC-NM, 99mTc-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 99mTc-EC-NIM group compared to 99mTc-EC (FIG. 14A). Data obtained from percent of injected dose of 99mTc-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, 99mTc-EC-NIM could be used to assess tumor response to paclitaxel treatment.
Polarographic Oxygen Microelectrode pO2 Measurements
Intratumoral pO2 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 99mTc-EC-GAP
Radiolabeling of EC-GAP with 99mTc was achieved using the same procedure described previously. The radiochemical purity was 100%. Stability of labeled 99mTc-EC-GAP was tested in serum samples. Briefly, 740 KBq of 1 mg 99mTc-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 99mTc-EC-GAP
99mTc-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 99mTc-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 99mTc-EC-ANNEX
Radiolabeling of EC-ANNEX with 99mTc was achieved using the same procedure described in EC-GAP. The radiochemical purity was 100%. Stability of labeled 99mTc-EC-ANNEX was tested in serum samples. Briefly, 740 KBq of 1 mg 99mTc-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 99mTc-EC-ANNEX
99mTc-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 99mTc-EC-ANNEX group (FIGS. 18-20). The images indicated that highly apoptotic cells have more uptake of 99mTc-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-NH2)
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-NH2. The structure was confirmed by 1H-NMR and mass spectroscopy (FAB-MS). 1H-NMR (CDCl3) 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-NH2 (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%). 1H-NMR (D2O) 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, —OCH3), 3.84 (S, 3H, —OCH3), 3.53 (S, 3H, —OCH3), 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 99mTc
Radiosynthesis of 99mTc-EC-COL was achieved by adding required amount of 99mTc-pertechnetate into home-made kit containing the lyophilized residue of EC-COL (5 mg), SnCl2 (100 μg), Na2HPO4 (13.5 mg), ascorbic acid (0.5 mg) and NaEDTA (0.5 mg). Final pH of preparation was 7.4. 99mTc-EC was also obtained by using home-made kit containing the lyophilized residue of EC (5 mg), SnCl2 (100 μg), Na2HPO4 (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 99mTc-EC-COL
Stability of labeled 99mTc-EC-COL was tested in serum samples. Briefly, 740 KBq of 5 mg 99mTc-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 99mTc-EC-COL or 99mTc-EC (n=3/time point). The injected mass of 99mTc-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 99mTc-EC-COL and 99mTc-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 99mTc-EC-COL
Radiosynthesis of EC-COL with 99mTc was achieved with high (>95%) radiochemical purity (FIG. 21). 99mTc-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 99mTc-EC-COL and 99mTc-EC in breast-tumor-bearing rats are shown in Tables 4 and 6. Tumor uptake value (% ID/g) of 99mTc-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 99mTc-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 99mTc-EC-COL group (FIG. 23). Conversely, tumor-to-blood and tumor-to-muscle values showed time-dependent decrease with 99mTc-EC when compared to 99mTc-EC-COL group in the same time period (FIG. 24).







TABLE 6







Biodistribution of 99mTc-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†















99mTc-EC-folate
0
1 (>95%)



99mTc-EC-
0
1 (>95%)



Free 99mTc
1
1



Reduced 99mTc
0
0







*Acetone



†Ammonium Acetate (1M in water):Methanol (4:1)






Gamma Scintigraphic Imaging of 99mTc-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 99mTc-EC-COL group (FIG. 25), whereas, less tumor uptake in the 99mTc-EC group was observed (FIG. 26). Computer outlined region of interest (ROI) showed that tumor/background ratios in 99mTc-EC-COL group were significantly higher than 99mTc-EC group (FIG. 27).
Tumor Glycolysis Targeting
Example 6
Development of 99mTc-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 1H-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 1H-NMR (FIGS. 38A-B), mass spectrometry (FIGS. 39A-B) and elemental analysis (Galbraith Laboratories, Inc. Knoxville, Tenn.). Elemental analysis C39H75N10S4O19.15H2O (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 99mTc
Radiosynthesis of 99mTc-EC and 99mTc-EC-neomycin were achieved by adding required amount of 99mTc-pertechnetate into home-made kit containing the lyophilized residue of EC or EC-neomycin (10 mg), SnCl2 (100 μg), Na2HPO4 (13.5 mg) and ascorbic acid (0.5 mg). NaEDTA (0.5 mg) in 0.1 ml of water was then added. Final pH 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 >95% for both radiotracers.
Stability Assay of 99mTc-EC and 99mTc-EC-neomycin
Stability of labeled 99mTc-EC and 99mTc-EC-neomycin were tested in dog serum samples. Briefly, 740 KBq of 1 mg 99mTc-EC and 99mTc-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 99mTc-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 (106 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 99mTc-EC or 99mTc-EC-neomycin (n=3/time point). The injected mass of 99mTc-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 99mTc-EC (Table 4) and free technetium (Table 9), tumor-to tissue ratios increased as a function of time in 99mTc-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 99mTc-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 99mTc-EC-neomycin (FIG. 37B).







TABLE 8







Biodistribution of 99mTc-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 99mTc 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 99mTc-EC-Drug Conjugates

To evaluate the cellular uptake of 99mTc-EC-drug conjugates, each well containing 80,000 cells (A549 lung cancer cell line) was added with 2 μCi of 99mTc-EC-neomycin and 18F-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. 99mTc-EC-neomycin showed highest uptake among those agents tested in human lung cancer cell line (FIG. 46).
Effect of Glucose on Cellular Uptake of 99mTc-EC-Neomycin and 18F-FDG
Neomycin is known to influence glucose absorption (Rogers et al., 1968; Fanciulli et al., 1994). Previous experiments have shown that 99mTc-EC-neomycin has higher uptake than 18F-FDG in human lung cancer cell line (A549). To determine if uptake of 99mTc-EC-neomycin is mediated via glucose-related mechanism, glucose (0.1 mg-2.0 mg) was added to each well containing either 50,000 (breast) cells or 80,000 cells (lung) along with 2 μCi of 99mTc-EC-neomycin and 18F-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 99mTc-EC-neomycin in two lung cancer cell lines and one breast cell line was observed. Similar results were observed in 18F-FDG groups. 99mTc-EC (control) showed no uptake. The findings suggest that the cellular uptake of 99mTc-EC-neomycin may be mediated via glucose-related mechanism (FIGS. 47, 48A and 48B).
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 99mTc-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 99mTc-EC-DG and 18F-FDG were added to wells containing 80,000 cells each. 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. The uptake of 99mTc-EC-DG was comparable to FDG (FIG. 69).
Effect of d- and l-Glucose on Cellular Uptake of 99mTc-EC-Deoxyglucose and 18F-FDG
To evaluate if the uptake of 99mTc-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 99mTc-EC-deoxyglucose and 18F-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 99mTc-EC-deoxyglucose and 18F-FDG by d-glucose in breast and lung cancer cells was observed. However, there was no influence on both agents by l-glucose (FIGS. 70-73). The findings suggest that the cellular uptake of 99mTc-EC-deoxyglucose is mediated via d-glucose mechanism.
Effect of EC-Deoxyglucose Loading on Blood Glucose Level in Normal Rats
Previous experiments have shown that cellular uptake of 99mTc-EC-deoxyglucose is similar to FDG. For instance, the hexokinase assay (glucose phosphorylation) was positive. The uptake of 99mTc-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 99mTc-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 (106 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 (106 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 99mTc-EC or 99mTc-EC-DG (n=3/time point). The injected mass of 99mTc-EC-DG was 1 mg per rat. At 0.5, 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 99mTc-EC (Table 4) and free technetium (Table 9), tumor-to tissue ratios increased as a function of time in 99mTc-EC-DG group (FIGS. 76-80).
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 99mTc-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. 99mTc-EC-DG was able to differentiate benign versus malignant tumors. For instance, malignant astrocytoma showed high uptake (FIGS. 82A, 82B, 83A and 83B). 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.
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## Claims

1. A bis-aminoethanethiol (BAT)-targeting ligand conjugate, wherein the targeting ligand is an agent that mimics glucose; a folate receptor targeting ligand; or a tumor hypoxia targeting ligand that is annexin V, colchicine, nitroimidazole, mitomycin, or metronidazole.

2. The conjugate of claim 1, wherein the conjugate is further defined as an ethylenedicysteine-targeting ligand conjugate.

3. The conjugate of claim 1, wherein the targeting ligand is an agent that mimics glucose.

4. The conjugate of claim 3, wherein the agent that mimics glucose is glucosamine, deoxyglucose, neomycin, kanamycin, gentamicin, paromycin, amikacin, tobramycin, netilmicin, ribostamycin, sisomicin, micromicin, lividomycin, dibekacin, isepamicin, astromicin, or an aminoglycoside.

5. The conjugate of claim 4, wherein the agent that mimics glucose is glucosamine.

6. The conjugate of claim 5, wherein the conjugate is further defined as EC-glucosamine.

7. The conjugate of claim 4, wherein the agent that mimics glucose is deoxyglucose.

8. The conjugate of claim 7, wherein the conjugate is further defined as EC-deoxyglucose.

9. The conjugate of claim 1, wherein the targeting ligand is a folate receptor targeting ligand.

10. The conjugate of claim 1, wherein the targeting ligand is a tumor hypoxia targeting ligand that is annexin V, colchicine, nitroimidazole, mitomycin, or metronidazole.

11. The conjugate of claim 1, wherein the conjugate further comprises a linker between said BAT and said targeting ligand.

12. The conjugate of claim 11, wherein the linker is a water soluble peptide, glutamic acid, polyglutamic acid, aspartic acid, polyaspartic acid, bromoethylacetate, ethylenediamine or lysine.

13. The conjugate of claim 1, wherein the bis-aminoethanethiol (BAT)-targeting ligand conjugate is labeled with a metal ion.

14. The conjugate of claim 13, wherein the metal ion is a radionuclide.

15. The conjugate of claim 14, wherein the radionuclide is 99mTc, 188Re, 186Re, 183Sm, 166Ho, 90Y, 89Sr, 67Ga, 68Ga, 111In, 153Gd, 59Fe, 255Ac, 212Bi, 211At, 62Cu, or 64Cu.

16. The conjugate of claim 1, wherein the BAT comprises two carboxylic acid moieties.

17. The conjugate of claim 16, wherein a targeting ligand is conjugated to one of the carboxylic acid moieties of the BAT.

18. The conjugate of claim 16, wherein a first targeting ligand is conjugated to one of the carboxylic acid moieties of the BAT and a second targeting ligand is conjugated to another carboxylic acid moiety of the BAT.