Efficient Synthesis of Chelators for Nuclear Imaging and Radiotherapy: Compositions and Applications

• US 20080107598A1
• Filed: 06/28/2007
• Published: 05/08/2008
• Est. Priority Date: 10/05/2006
• Status: Active Grant

• Current Assignee: Cell>Point, LLC, Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Cell>Point, LLC
• Inventors: Yu, Dongfang, Yang, David J.
• Granted Patent: US 10,925,977 B2
• Time in Patent Office: Days
• US Class Current: 424/1.73
• CPC Class Codes: A61K 49/0002 General or multifunctional contrast agents, e.g. chelated agents
• CPC Class Codes: A61K 49/06 Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
• CPC Class Codes: A61K 51/0491 Sugars, nucleosides, nucleotides, oligonucleotides, nucleic acids, e.g. DNA, RNA, nucleic acid aptamers
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/06 Antiarrhythmics
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Abstract

Novel methods of synthesis of chelator-targeting ligand conjugates, compositions comprising such conjugates, and therapeutic and diagnostic applications of such conjugates are disclosed. The compositions include chelator-targeting ligand conjugates optionally chelated to one or more metal ions. Methods of synthesizing these compositions in high purity are also presented. Also disclosed are methods of imaging, treating and diagnosing disease in a subject using these novel compositions, such as methods of imaging a tumor within a subject and methods of diagnosing myocardial ischemia.

Claims

1. A method of synthesizing a chelator-targeting ligand conjugate comprising conjugating a chelator of the following formula:

2. The method of claim 1, wherein the conjugation is carried out in an organic medium.

3. The method of claim 2, wherein the organic medium comprises a polar or a non-polar solvent.

4. The method of claim 2, wherein the organic medium comprises dimethylformamide, dimethylsulfoxide, dioxane, methanol, ethanol, hexane, methylene chloride, acetonitrile, tetrahydrofuran, or a mixture thereof.

5. The method of claim 1, further comprising at least one purification step, wherein the purification step comprises silica gel column chromatography, HPLC, or a combination thereof.

6. The method of claim 5, wherein the chelator-targeting ligand conjugate is between about 70% and about 99.9% pure.

7. The method of claim 5, further comprising chelating a metal ion to the chelator to generate a metal ion labeled-chelator-targeting ligand conjugate.

8. The method of claim 5, further comprising removing each protecting group in one or more steps.

9. The method of claim 8, wherein the chelator-targeting ligand conjugate is between about 70% and about 99.9% pure.

10. The method of claim 8, wherein the chelator-targeting ligand conjugate is between about 80% and about 99.9% pure.

11. The method of claim 8, wherein the chelator-targeting ligand conjugate is between about 90% and about 99.9% pure.

12. The method of claim 8, further comprising chelating a metal ion to the chelator to generate a metal ion labeled-chelator-targeting ligand conjugate.

13. The method of claim 12, wherein the metal ion labeled-chelator-targeting ligand conjugate is between about 70% and about 99.9% pure.

14. The method of claim 12, wherein the metal ion labeled-chelator-targeting ligand conjugate is between about 80% and about 99.9% pure.

15. The method of claim 12, wherein the metal ion labeled-chelator-targeting ligand conjugate is between about 90% and about 99.9% pure.

16. The method of claim 12, wherein the synthesis of the metal ion labeled-chelator-targeting ligand conjugate comprises:

(a) removing at least one protecting group from the chelator-targeting ligand conjugate; and

(b) chelating a metal ion to the chelator of the chelator-targeting ligand conjugate.

17. The method of claim 12, wherein the synthesis of the metal ion labeled-chelator-targeting ligand conjugate comprises:

(a) chelating a metal ion to the chelator to generate a metal ion labeled-chelator;

(b) conjugating the metal ion labeled-chelator to a targeting ligand; and

(c) removing at least one protecting group from the metal ion labeled-chelator-targeting ligand conjugate.

18. The method of claim 12, wherein the metal ion is selected from the group consisting of a technetium ion, a copper ion, an indium ion, a thallium ion, a gallium ion, an arsenic ion, a rhenium ion, a holmium ion, a yttrium ion, a samarium ion, a selenium ion, a strontium ion, a gadolinium ion, a bismuth ion, an iron ion, a manganese ion, a lutetium ion, a cobalt ion, a platinum ion, a calcium ion and a rhodium ion.

19. The method of claim 12, wherein the metal ion is ¹⁸⁷Re, ^99mTc, or ¹⁸⁸Re.

20. The method of claim 12, wherein the metal ion is a radionuclide.

21. The method of claim 20, wherein the radionuclide is selected from the group consisting of ^99mTc, ¹⁸⁸Re, ¹⁸⁶Re, ¹⁵³Sm, ¹⁶⁶Ho, ⁹⁰Y, ⁸⁹Sr, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ¹⁸³Gd, ⁵⁹Fe, ²²⁵Ac, ²¹²Bi, ²¹¹At, ⁴⁵Ti, ⁶⁰Cu, ⁶¹Cu, ⁶⁷Cu, ⁶⁴Cu and ⁶²Cu.

22. The method of claim 21, further comprising the addition of a reducing agent.

23. The method of claim 1, wherein the chelator is ethylenedicysteine (EC).

24. The method of claim 23, wherein the two thiol groups of ethylenedicysteine are protected and the two amine groups of ethylenedicysteine are protected.

25. The method of claim 12, wherein the chelator is ethylenedicysteine (EC).

26. The method of claim 1, wherein the at least one functional group of the targeting ligand comprises an atom selected from the group consisting of O, N, S and P.

27. The method of claim 26, wherein the at least one functional group of the targeting ligand is selected from the group consisting of amino, amido, thiol, hydroxyl, ether, ester, carbonyl, carboxylic acid, sulfonamido, thioether, thioester and thiocarbonyl.

28. The method of claim 1, wherein A, D, E and F are each independently —

COOH, —

NH₂, or thiol.

29. The method of claim 1, wherein any three or four of the groups A, B, C, D, E and F together form a chelate selected from the group consisting of NS₂, N₂S, S₄, N₂S₂, N₃S and NS₃.

30. The method of claim 29, wherein the chelate is N₂S₂.

31. The method of claim 29, wherein at least one of A, D, E and F is a protected thiol.

32. The method of claim 31, wherein the protected thiol is protected using a thiol protecting agent selected from a group consisting of an alkyl halide, a benzyl halide, a benzoyl halide, a sulfonyl halide, a triphenylmethyl halide, a methoxytriphenylmethyl halide and cysteine.

33. The method of claim 29, wherein at least one of A, D, E and F comprises a protected amine or at least one of B and C comprises a secondary amine.

34. The method of claim 33, wherein the protected amine is protected using an amine protecting agent selected from the group consisting of benzylchloroformate, p-nitro-chlorobenzylformate, ethylchloroformate, di-tert-butyl-dicarbonate, triphenylmethyl chloride and methoxytriphenylmethyl chloride.

35. The method of claim 1, wherein the chelator-targeting ligand conjugate further comprises a linker between the chelator and the targeting ligand.

36. The method of claim 35, wherein the linker is selected from the group consisting of a peptide, glutamic acid, aspartic acid, bromo ethylacetate, ethylene diamine, lysine and any combination of one or more of these groups.

37. The method of claim 1, wherein the chelator is conjugated to more than one targeting ligand.

38. The method of claim 1, wherein the targeting ligand is a tissue-specific ligand, an antimicrobial, an antifungal, or an imaging agent.

39. The method of claim 38, wherein the targeting ligand is a tissue-specific ligand selected from the group consisting of a drug, a DNA topoisomerase inhibitor, an antimetabolite, a disease cell cycle targeting compound, a gene expression marker, an angiogenesis targeting ligand, a tumor marker, a folate receptor targeting ligand, an apoptotic cell targeting ligand, a hypoxia targeting ligand, a DNA intercalator, a disease receptor targeting ligand, a receptor marker, a peptide, a nucleotide, an antibody, an antisense molecule, an siRNA, a glutamate pentapeptide, an agent that mimics glucose, amifostine, angiostatin, monoclonal antibody C225, monoclonal antibody CD31, monoclonal antibody CD40, capecitabine, deoxycytidine, fullerene, herceptin, human serum albumin, lactose, quinazoline, thalidomide, transferrin and trimethyl lysine.

40. The method of claim 39, wherein the targeting ligand is a drug.

41. The method of claim 40, wherein the drug is an anti-cancer agent.

42. The method of claim 41, wherein the anti-cancer agent is selected from the group consisting of tamoxifen, topotecan, LHRH, podophyllotoxin, colchicine, endostatin, tomudex, thiotepa, cyclosphosphamide, busulfan, improsulfan, piposulfan, benzodepa, carboquone, meturedepa, uredepa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, bryostatin, callystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, a sarcodictyin, spongistatin, chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, calicheamicin, dynemicin, clodronate, an esperamicin, neocarzinostatin chromophore, an aclacinomysin, actinomycin, authramycin, azaserine, a bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, a chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, epirubicin, esorubicin, idarubicin, marcellomycin, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil (5-FU), denopterin, methotrexate, pteropterin, trimetrexate, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformithine, elliptinium acetate, an epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, a maytansinoid, mitoguazone, mopidanmol, nitraerine, pentostatin;

phenamet;

pirarubicin;

losoxantrone;

podophyllinic acid;

2-ethylhydrazide;

procarbazine, PSK polysaccharide complex, razoxane, rhizoxin, sizofuran, spirogermanium, tenuazonic acid, triaziquone, 2,2′

,2″

-trichlorotriethylamine, a trichothecene, urethan, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (“

Ara-C”

), cyclophosphamide, doxetaxel, chlorambucil, 6-thioguanine, mercaptopurine, methotrexate, cisplatin, oxaliplatin, carboplatin, vinblastine, platinum, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan, RFS 2000, difluoromethylomithine (DMFO), retinoic acid and capecitabine.

43. The method of claim 40, wherein the drug is a cardiovascular drug.

44. The method of claim 43, wherein the cardiovascular drug is selected from the group consisting of an antihyperlipoproteinemic agent, an antiarteriosclerotic agent, an antithrombotic agent, a fibrinolytic agent, an antiplatelet agent, a blood coagulant, a thrombolytic agent, an antiarrythmic agent, an antihypertensive agent, a vasopressor, an anti-angiotension II agent, an afterload-preload reduction agent, a diuretic and an inotropic agent.

45. The method of claim 39, wherein the targeting ligand is a DNA topoisomerase inhibitor selected from the group consisting of a fluoroquinolone antibiotic, irinotecan, topotecan, etoposide, teniposide, lurtotecan, exatecan and rubitecan.

46. The method of claim 39, wherein the targeting ligand is an antimetabolite selected from the group consisting of azathioprine, a mercaptopurine, a pyrimidine, a sulfanilamide drug, methotrexate, tetrahydrofolate, folic acid, pemetrexed, raltitrexed, thioguanine, fludarabine, pentostatin, cladribine, fluorouracil, floxuridine and gemcitabine.

47. The method of claim 39, wherein the targeting ligand is a disease cell cycle targeting ligand selected from the group consisting of adenosine, FIAU, FIRU, IVFRU, GCV, PCV, FGCV, FPCV, FHPG, FHBG and guanine.

48. The method of claim 39, wherein the targeting ligand is a gene expression marker that is an epidermal growth factor receptor ligand.

49. The method of claim 39, wherein the targeting ligand is an angiogenesis targeting ligand selected from the group consisting of a COX-2 inhibitor, anti-EGF receptor, herceptin, angiostatin and thalidomide.

50. The method of claim 49, wherein the COX-2 inhibitor is celecoxib, rofecoxib, or etoricoxib.

51. The method of claim 39, wherein the targeting ligand is a tumor marker selected from the group consisting of PSA, ER, PR, CA-125, CA-199, CEA, AFP, an interferon, BRCA1, HER-2/neu, cytoxan, p53 and endostatin.

52. The method of claim 39, wherein the targeting ligand is a folate receptor targeting ligand selected from the group consisting of folate, methotrexate and tomudex.

53. The method of claim 39, wherein the targeting ligand is an apoptotic cell targeting ligand selected from the group consisting of a TRAIL monoclonal antibody, a substrate of caspase-3 and a Bcl family member.

54. The method of claim 39, wherein the targeting ligand is a hypoxia targeting ligand.

55. The method of claim 54, wherein the hypoxia targeting ligand is a tumor hypoxia targeting ligand, a cardiac ischemia marker, a cardiac viability tissue marker, a congestive heart failure marker, or a rest/stress cardiac tissue marker.

56. The method of claim 55, wherein the hypoxia targeting ligand is a tumor hypoxia targeting ligand selected from the group consisting of annexin V, colchicine, a nitroimidazole, mitomycin, metronidazole, 99mTc-HL91 and Cu-ATSM.

57. The method of claim 55, wherein the hypoxia targeting ligand is a cardiac ischemia marker selected from the group consisting of interleukin-6, tumor necrosis factor alpha, matrix metalloproteinase 9, myeloperoxidase, an intercellular adhesion molecule, a vascular adhesion molecule, soluble CD40 ligand, placenta growth factor, high sensitivity C-reactive protein, ischemia modified albumin, a free fatty acid, choline and adenosine.

58. The method of claim 55, wherein the hypoxia targeting ligand is a cardiac viability tissue marker selected from the group consisting of phospholipase C, myosin light-chain phosphatase, nitric oxide, prostacyclin, endothelin, thromboxane, L-arginine and L-citrulline.

59. The method of claim 55, wherein the hypoxia targeting ligand is a congestive heart failure marker selected from the group consisting of interleukin-1, cardiotrophin-1, insulin-like growth factor, epidermal growth factor, tyrosine kinase receptor, angiotensin II and metronidazole.

60. The method of claim 55, wherein the hypoxia targeting ligand is a rest/stress cardiac tissue marker selected from the group consisting of a mitogen-activated protein kinase, cyclic adenosine monophosphate, phospholipase C, phosphatidylinositol bisphosphate, isositol trisphosphate, diacylglycerol, a tyrosine kinase and metronidazole.

61. The method of claim 39, wherein the targeting ligand is a DNA intercalator selected from the group consisting of 7-aminoactinomycin, ethidium, proflavin, daunomycin, doxorubicin and thalidomide.

62. The method of claim 39, wherein the targeting ligand is a peptide selected from the group consisting of neuropeptide Y, calcitonin gene-related peptide, substance P and vasoactive intestinal peptide.

63. The method of claim 39, wherein the targeting ligand is a nucleotide selected from the group consisting of adenine, thymine, guanine, cytosine and uracil.

64. The method of claim 39, wherein the targeting ligand is an antibody that binds to a troponin, tropomyosin, a sarcolemmal, a collagen, a matrix metalloproteinase, or a tissue inhibitor of a matrix metalloproteinase.

65. The method of claim 39, wherein the targeting ligand is glutamate pentapeptide.

66. The method of claim 39, wherein the targeting ligand is an agent that mimics glucose.

67. The method of claim 66, wherein the agent that mimics glucose is selected from the group consisting of deoxyglucose, glucosamine, tetraacetylated glucosamine, neomycin, kanamycin, gentamycin, paromycin, amikacin, tobramycin, netilmicin, ribostamycin, sisomicin, micromicin, lividomycin, dibekacin, isepamicin, astromicin and aminoglycoside.

68. The method of claim 39, wherein the targeting ligand is a disease receptor targeting ligand selected from the group consisting of an estrogen, an androgen, luteinizing hormone, luteinizing hormone releasing hormone (LHRH), transferrin, a progestin, tetraacetate mannose, α

-β

-tyrosine, tyrosine, a tyrosine derivative, estrone, tamoxifen, or α

-methyltyrosine.

69. A composition comprising a chelator-targeting ligand conjugate synthesized by the method of claim 1.

70. A composition comprising a metal ion labeled-chelator-targeting ligand conjugate synthesized by the method of claim 7.

71. A composition comprising a chelator-targeting ligand conjugate synthesized by the method of claim 8.

72. The composition of claim 71, further defined as a metal ion labeled-chelator-targeting ligand conjugate synthesized by the method of claim 12.

73. The composition of claim 72, wherein:

(a) the metal ion labeled-chelator-targeting ligand conjugate comprises ethylenedicysteine chelated to a metal ion selected from the group consisting of ^99mTc, ⁶⁸Ga, ¹⁸⁸Re and ¹⁸⁷Re;

(b) the targeting ligand comprises a ligand selected from the group consisting of glucosamine, deoxyglucose, metronidazole, annexin V, guanine and LHRH; and

(c) the chelator and the targeting ligand are conjugated via an amide bond or an ester bond.

74. A kit for preparing an imaging agent, a therapeutic agent, or a radio/therapeutic agent, comprising one or more sealed containers and a predetermined quantity of a composition comprising a chelator-targeting ligand conjugate prepared by a method comprising the method of claim 8 in one or more sealed containers.

75. The kit of claim 74, wherein the chelator-targeting ligand conjugate is between about 70% and about 99.9% pure.

76. The kit of claim 74, wherein the chelator-targeting ligand conjugate is between about 80% and about 99.9% pure.

77. The kit of claim 74, wherein the chelator-targeting ligand conjugate is between about 90% and about 99.9% pure.

78. The kit of claim 74, wherein the chelator-targeting ligand conjugate comprises ethylenedicysteine.

79. The kit of claim 74, further comprising a metal ion.

80. The kit of claim 79, wherein the metal ion is a radionuclide.

81. A method of imaging a site, diagnosing a disease, or treating a disease within a subject comprising:

(a) obtaining a metal ion-labeled-chelator targeting ligand conjugate; and

(b) administering to the subject a pharmaceutically or diagnostically effective amount of a metal ion labeled-chelator-targeting ligand conjugate,

wherein the metal ion labeled-chelator-targeting ligand conjugate is prepared by a method comprising the method set forth in claim 12, wherein the site is imaged, the disease is diagnosed, or the disease is treated.

82. The method of claim 81, wherein the metal ion labeled-chelator-targeting ligand conjugate is between about 70% and about 99.9% pure.

83. The method of claim 81, wherein the metal ion labeled-chelator-targeting ligand conjugate is between about 80% and about 99.9% pure.

84. The method of claim 81, wherein the metal ion labeled-chelator conjugate is between about 90% and about 99.9% pure.

85. The method of claim 81, wherein the metal ion labeled-chelator conjugate comprises ethylenedicysteine.

86. The method of claim 81, wherein the metal ion is a radionuclide.

87. The method of claim 81, wherein the subject is a human.

88. The method of claim 81, wherein the method is further defined as a method of treating a subject with cancer.

89. The method of claim 88, wherein the cancer is breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, a esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, or leukemia.

90. The method of claim 81, wherein the method is further defined as a method for performing dual radio/chemotherapy.

91. The method of claim 90, further comprising administering one or more secondary forms of therapy of a hyperproliferative disease.

92. The method of claim 81, further defined as a method of imaging a site within a subject comprising detecting a signal from the metal ion labeled-chelator-targeting ligand conjugate that is localized at the site.

93. The method of claim 92, wherein the signal is detected using a technique selected from the group consisting of PET, PET/CT, CT, SPECT, SPECT/CT, MRI, optical imaging and ultrasound.

94. The method of claim 81, wherein the site to be imaged is a tumor or the heart.

95. The method of claim 81, further defined as a method of imaging, diagnosing, or treating a subject with a cardiovascular disease.

96. The method of claim 95, further comprising performing one or more additional diagnostic or imaging procedures to evaluate the subject for a cardiovascular disease.

97. The method of claim 95, wherein the cardiovascular disease is a myocardial infarction, congestive heart failure, cardiomyopathy, valvular heart disease, an arrhythmia, congenital heart disease, angina pectoris, noncardiac circulatory congestion, systolic heart failure, heart failure with normal systolic function, or right-sided heart failure.

98. The method of claim 97, wherein the cardiovascular disease is a myocardial infarction, myocardial ischemia, or angina pectoris and the method further comprises imaging the heart of the subject.

99. The method of claim 81, wherein the metal ion labeled-chelator-targeting ligand conjugate is ⁹⁹Tc-EC-glucosamine, ¹⁸⁸Re-EC-glucosamine, or ¹⁸⁷Re-EC-glucosamine.

100. A method of synthesizing a protected chelator comprising:

(a) obtaining a chelator of the following formula;

101. The method of claim 100, wherein the method is carried out in an organic medium.

102. The method of claim 100, wherein the protected chelator is protected ethylenedicysteine.

103. The method of claim 100, further comprising a purification step, a chelation step comprising chelation of a metal ion, the removal of at least one protecting group, or any combination of these steps.

104. The method of claim 103, wherein the protected chelator is about 70% to about 99.9% pure.

105. The method of claim 103, wherein the protected chelator is about 80% to about 99.9% pure.

106. The method of claim 103, wherein the protected chelator is about 90% to about 99.9% pure.

107. The method of claim 100, wherein when A and D are each —

NH₂, neither B nor C is a secondary or a tertiary amine.

108. A chelator-targeting ligand conjugate of the following formula:

109. The composition of claim 108, wherein the chelator-targeting ligand conjugate is between about 80% and about 99.9% pure.

110. The composition of claim 108, wherein the chelator-targeting ligand conjugate is between about 90% and about 99.9% pure.

111. The composition of claim 108, further defined as a metal ion labeled-chelator-targeting ligand conjugate.

112. The composition of claim 111, further defined as ^99mTc-EC-glucosamine, ¹⁸⁶Re-EC-glucosamine, or ¹⁸⁷Re-EC-glucosamine.