WO2010036837A1 - Coordination of a technetium imaging complex on a bioactive apoptosis targeting agent - Google Patents

Coordination of a technetium imaging complex on a bioactive apoptosis targeting agent Download PDF

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WO2010036837A1
WO2010036837A1 PCT/US2009/058283 US2009058283W WO2010036837A1 WO 2010036837 A1 WO2010036837 A1 WO 2010036837A1 US 2009058283 W US2009058283 W US 2009058283W WO 2010036837 A1 WO2010036837 A1 WO 2010036837A1
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gsao
compound
formula
kit
monodentate ligand
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PCT/US2009/058283
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French (fr)
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Raghoottama Pandurangi
Mary Dyszlewski
Michael J. Bushman
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Mallinckrodt Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F13/00—Compounds containing elements of Groups 7 or 17 of the Periodic Table
    • C07F13/005—Compounds without a metal-carbon linkage

Definitions

  • This invention relates to radiopharmaceutical compounds comprising technetium-99m and to processes for direct labeling bioactive molecules with technetium- 99m to form radiopharmaceutical compounds.
  • Nuclear medicine uses radioactive material for diagnostic and therapeutic purposes by injecting a patient with a dose of the radioactive material.
  • the radioactive material then concentrates in certain organs or biological regions of the patient.
  • Numerous radioisotopes are used in nuclear medicine including Tc-99m, In-111, Tl-201, 1-123, and Ga-67 for diagnostic gamma imaging, F-18 for diagnostic PET imaging, and Y-90, Lu-177, 1-131, and Sm- 153 for radiotherapy.
  • Chemical forms of radioactive materials naturally concentrate in a particular tissue, for example, iodide (1-131) concentrates in the thyroid.
  • Radioactive materials are often combined with a tagging or bioactive agent, which targets the radioactive material for the desired organ or biologic region of the patient.
  • radiopharmaceuticals are typically referred to as radiopharmaceuticals in the field of nuclear medicine.
  • a radiation imaging system e.g., a gamma camera
  • Irregularities in the image are often indicative of a pathology, such as cancer.
  • Higher doses of the radiopharmaceutical may be used to deliver a therapeutic dose of radiation directly to the pathologic tissue, such as cancer cells.
  • 99m Tc Technetium-99m
  • 99m Tc is the most commonly used radioisotope in nuclear medicine due to its resolution properties and relatively short half-life of 6 hours.
  • 99m Tc is produced from a technetium-99m generator comprising decaying molybdenum-99. As the molybdenum-99 nuclide decays, it produces a technetium-99m daughter nuclide. The technetium-99m is eluted from the molybdenum-99 and may then be complexed with the appropriate bioactive molecule.
  • GSAO 4-(N)-((S-glutathionylacetyl)amino) phenyl arsine oxide
  • Kits are typically used to synthesize 99m Tc radiopharmaceutical complexes.
  • the kits contain a targeting agent, and other ingredients that can promote the stability of the kit.
  • the kits can be lyophilized to enhance the shelf life of the kits and permit the kits to be stored for periods of time until they are used to synthesize a radiopharmaceutical complex.
  • the targeting agent is typically a bioconjugate molecule where one portion of the compound is the biologically active moiety and the other portion contains a chelating moiety for complexing the Tc-99m, for example, diethylenetriamine pentaascetic acid (DTPA), mercaptoacetyltriglycine (MAG3), 1,4,7,10 tetraazacyclododecane-tetraacetic acid (DOTA), N 2 O 21 N 2 S 2 , or N3S
  • DTPA diethylenetriamine pentaascetic acid
  • MAG3 mercaptoacetyltriglycine
  • DOTA 1,4,7,10 tetraazacyclododecane-tetraacetic acid
  • N 2 O 21 N 2 S 2 or N3S
  • a linking group comprised of one or more atoms is used to attach the chelating group to the biologically active moiety.
  • GSAO or a GSAO equivalent is directly labeled with 99m Tc which minimizes the synthesis steps for forming a radiopharmaceutical.
  • the 99m Tc atom directly bonds to donor atoms on the GSAO or GSAO equivalent core, providing a smaller molecule as compared to those in the art that require external chelating agents .
  • the present invention is directed to a radiopharmaceutical compound having the formula
  • D is As, Ge, Se, Sn, B, Ph, or Al
  • R 1 to R 4 are independently H, X, OH, CN, NH 2 , CO, SCN, -CH 2 NH 2 , -NHCOCH 3 , -NHCOCH 2 X or NO
  • X is a halogen
  • A is
  • R 5 is hydrogen, -OH, -COOH
  • Re is -NHCH 2 COOH or -OH; and M is a monodentate ligand.
  • the monodentate ligand is an aliphatic amine, a heterocyclic amine, a heterocyclic imine, a thiol, a thiolate, a thioether, an isocyanide, or a phosphine.
  • the monodentate ligand is methoxyisobutyl isonitrile.
  • the monodentate ligand is tris-hydroxy methyl phosphine.
  • D may be As.
  • R 1 , R 2 , R3 and R 4 may be hydrogen.
  • A is (I) and Rs is
  • A is (III) and R 5 is
  • the invention is also directed to a kit for use in preparing a radiopharmaceutical compound, the kit comprising: an amount of a compound having the formula
  • D is As, Ge, Se, Sn, B, Ph, or Al
  • R 1 to R 4 are independently H, X, OH, CN, NH 2 , CO, SCN, -CH 2 NH 2 , -NHCOCH 3 , -NHCOCH 2 X or NO, X is a halogen
  • R 5 is hydrogen, -OH, -COOH,
  • the kit may include a carbonyl reaction vial.
  • the kit may also include an acid for neutralization.
  • Another aspect of the present invention is a process for direct labeling a compound of Formula (2A) or (2B) to form a radiopharmaceutical, the process comprising reacting the compound with a solution of 99m Tc(CO) 3 (OH 2 ) 3 to form an intermediate and reacting the intermediate with a monodentate ligand to form the radiopharmaceutical of Formula (8).
  • Fig. 1 is a graphical depiction of the number of radioactive cell counts during radioimaging of cell cultures exposed to different amounts of camptothecin;
  • Fig. 2 is a graphical depiction of the biodistribution in mice organs after injection of 99m Tc(CO) 3 (GSAO) complex with and without the addition of a monodentate ligand.
  • GSAO is a compound of Formula (1)
  • D is As, Ge, Se, Sn, B, Ph, or Al
  • R 1 to R 4 are independently H, X, OH, CN, NH 2 , CO, SCN, -CH 2 NH 2 , -NHCOCH 3 , -NHCOCH 2 X or NO, and X is a halogen.
  • a "GSAO equivalent" is any compound of Formula (2A) or (2B)
  • R5 is hydrogen, -OH, -COOH
  • GSAO equivalent compounds include the GSAO molecule itself.
  • Compounds such as GSAO that contain an arsenoxide component are especially well suited for imaging apoptotic cells.
  • an arsenoxide component i.e., wherein D is As
  • glutathione is readily transportable across cell membranes
  • compounds containing a glutathione moiety are well suited for imaging apoptotic cells.
  • GSAO and GSAO equivalents may be prepared by reacting glutathione or a glutathione equivalent with N-(4-arsorylphenyl)-2-bromoacetamide (i.e., 4-(N- bromoaceryl)amino)phenylarsenoxide (BRAO)) as disclosed in US Publication No. 2005/0101524 which is incorporated herein by reference.
  • Glutathione is a compound of Formula (3)
  • a “glutathione equivalent” is a compound of Formula (4)
  • glutathione equivalent compounds include glutathione itself.
  • the 99m Tc(GSAO) complex or 99m Tc(GSAO equivalent) complex is formed by first reacting GSAO or a compound of Formula (2) with 99m Tc(CO) 3 (OH 2 ) 3 to form an intermediate.
  • 99m Tc(CO) 3 (OH 2 ) 3 solution can be prepared by adding sodium pertechnetate 99m Tc ( 99m TcO 4 Na + ) to a carbonyl reaction vial (e.g., ISOLINK vial available from Mallinckrodt Inc., St. Louis, MO), heating the solution and optionally adding acid to neutralize the solution.
  • Sodium pertechnetate 99m Tc solution may be produced as the eluate from a technetium generator (e.g., ULTRA-TECHNEKOW DTE, available from Mallinckrodt Inc., St. Louis, MO).
  • Technetium-99m used in accordance with the principles of the present invention and as produced from the technetium generator is typically in the +1 oxidation state (i.e., 99m Tc(I)).
  • a monodentate ligand can react with the complex by breaking the weak bond between the technetium-99m and GSAO or GSAO equivalent (e.g., the bond with the sulfur atom) and forming a stable bond with the technetium-99m atom to produce a bioactively stable complex.
  • the technetium-99m makes two strong covalent bonds with the GSAO or GSAO equivalent molecule and keeps an open coordination site as shown in Formula (6)
  • the technetium-99m makes two strong covalent bonds with the GSAO or GSAO equivalent molecule and keeps an open coordination site as shown in Formula (7)
  • a monodentate ligand can react with the complex by reacting in the open coordination site.
  • the 99m Tc(CO) 3 (GSAO) solution or 99m Tc(CO) 3 (GSAO equivalent) solution is prepared it is reacted with a monodentate ligand ("M") to prepare a 9 9m Tc(CO) 3 (GSAO)(M) or 99m Tc(CO) 3 (GSAO equivalent)(M) complex, which is the radiopharmaceutical compound of the invention.
  • M monodentate ligand
  • the monodentate ligand reacts directly with the 99m Tc atom either by substituting for a weaker GSAO or GSAO equivalent bond or by complexing with an open coordination site on the 99m Tc atom
  • the resulting structure is very stable and exhibits in vivo stability (See Example 3 below).
  • Suitable monodentate ligands include, but are not limited to, an aliphatic amine, a heterocyclic amine, a heterocyclic imine, a thiol, athiolate, athioether, an isocyanide, or a phosphine.
  • Methoxyisobutyl isonitrile (“MIBI”) and tris-hydroxy methyl phosphine are especially well-suited monodentate ligands for coordination with the 99m Tc (I) (CO) 3 (GSAO) or 99m Tc (I) (CO) 3 (GSAO equivalent) complex.
  • radiopharmaceutical compounds formed by the direct labeling process of the invention have the Formula (8)
  • D is As, Ge, Se, Sn, B, Ph, or Al
  • R 1 to R 4 are independently H, X, OH, CN, NH 2 , CO, SCN, -CH 2 NH 2 , -NHCOCH 3 , -NHCOCH 2 X or NO, X is a halogen;
  • R 5 is hydrogen, -OH, -COOH
  • R 6 is -NHCH 2 COOH or -OH; and M is a monodentate ligand.
  • the bioactive molecule is GSAO or when the terminal group of the GSAO equivalent molecule is -NHCH 2 COOH, the 99m Tc atom can bind to the -NHCH 2 COOH terminus to form a compound of Formula (9)
  • the 99m Tc atom can bind to the -OH terminus to form a compound of Formula (11)
  • the 99m Tc atom may bind at the other terminus and form a compound of Formula (12)
  • the 99m Tc atom may bind at the penicillamine terminus and form a compound of Formula (13)
  • a three or four component kit containing the constituent chemicals can be used to prepare 99m Tc (I) (CO) 3 (GSAO)(M) and 99m Tc (I) (CO) 3 (GSAO equivalent)(M) complexes.
  • the components of the kit are 1) a carbonyl reaction vial; 2) an optional vial of acid for neutralization, such as 1 N HCl; 3) a vial containing the GSAO or GSAO equivalent; and 4) a vial containing the monodentate ligand.
  • the contents of any vial in the kit may be lyophilized.
  • the preferred carbonyl reaction vial is a lyophilized formulation of 8.5 mg sodium tartrate, 2.85 mg Na 2 B 4 O 7 . 10H 2 O, 7.15 mg of sodium carbonate, and 4.5 mg sodium boranocarbonate.
  • a kit and a technen ' um-generator can be used to produce the complexes in the same facility in which they are administered to the patient.
  • 99m Tc(GSAO) or 99m Tc(GSAO equivalent) complex from the kit, 99m Tc is eluted from a technetium generator, for example, as sodium pertechnetate 99m Tc ( 99m TcO 4 -Na + ).
  • a technetium generator for example, as sodium pertechnetate 99m Tc ( 99m TcO 4 -Na + ).
  • the generator eluate is added to a carbonyl reaction vial, and the solution is typically heated at 75 - 100 C for up to 20 minutes.
  • GSAO or GSAO equivalent is then added to the 99m Tc(CO) 3 (OH 2 ) 3 SoIuUOn to prepare a solution of 99m Tc(CO) 3 (GSAO) or 99m Tc(CO) 3 (GSAO equivalent).
  • the reaction is conducted at room temperature for about 30-60 minutes. In other instances, the reaction mixture can be heated to 100°C for about 10-15 minutes.
  • a monodentate ligand is then added to the solution of 99m Tc(CO) 3 (GSAO) or 9 9m Tc(CO) 3 (GSAO equivalent) to prepare 99m Tc (I) (CO) 3 (GSAO)(M) or 99m Tc (I) (CO) 3 (GSAO equivalent)(M) which can be administered to the patient.
  • the reaction is conducted at 37°C for about 30-60 minutes.
  • the kit is useful to prepare 99m Tc (I) (CO) 3 (GSAO)(M) and 9 9m Tc (I) (CO)J(GSAO equivalent)(M) complexes for use as imaging agents for detection of apoptotic, necrotic, or any dead or dying cells in the body, for example, cells that have died after chemotherapy or radiotherapy.
  • the radiopharmaceuticals are well suited for single photon emission computed tomography (SPECT), positron emission tomography (PET), and optical imaging. Synthesized complexes can also be used to monitor GSAO when used as an angiogenesis inhibitor.
  • PDI protein disulfide isomerase
  • Example 4 illustrates the favorable biodistribution of (i.e., low non-target binding) of a compound of the invention which makes it an effective radioimaging agent, particularly for SPECT imaging.
  • This example demonstrates the affinity of GSAO towards apoptotic cells and the ability of the 99m Tc (I) (CO) 3 (GSAO)(MIBI) complex to image apoptotic cells.
  • U937 cells were plated in growth medium in 6-well tissue culture plates at a density of 2.4 x 10 5 cells per well. Freshly prepared camptothecin was added to the cells to produce final camptothecin concentrations of approximately 3.5 and 7 micromolar. Untreated cells were used as negative controls. The treated and non-treated cells were placed overnight in a humidified incubator at 37°C with a 5% CO 2 /95% ambient air atmosphere.
  • the treated and nontreated cells were incubated for approximately 16 hours and then the cells were quantitatively transferred to separate 15-mL centrifuge tubes and placed on ice. Cells were washed in ice cold phosphate buffered saline ("PBS”), resuspended in annexin binding buffer (1 mL), and placed on ice.
  • PBS ice cold phosphate buffered saline
  • the 99m Tc (I) (CO) 3 (GSAO)(MIBI) preparation was diluted to 0.1 mCi/mL in annexin binding buffer. 50 ⁇ l of the diluted solution containing 5 ⁇ Ci 9 9m Tc (I) (CO) 3 (GSAO)(MIBI) was added to each sample. Samples were swirled gently and incubated on ice for 30 minutes.
  • ice cold RPMI (Roswell Park Memorial Institute) media (10 mL) was added to each tube. Cells were mixed gently and centrifuged at 250 times gravity for 10 minutes. The supernatant was decanted. Cells were washed two more times in ice cold RPMI media (10 mL). Cells were quantitatively transferred to counting tubes using 2 x 0.5 mL aliquots of RPMI media and the cell-associated radioactivity was measured using a Cobra B5OO5 Gamma Counter.
  • Fig. 1 The results of the study are shown in Fig. 1. As can be seen from the Figure, cell counts were more than twice as high for cells exposed to camptothecin, i.e., apoptotic cells. This illustrates the affinity of 99m Tc (I) (CO) 3 (GSAO)(MIBI) towards apoptotic cells.
  • Example 3 In Vitro Stability Comparison of 99m Tc (I) (CO) 3 (GSAO) and 9 9m Tc (I) (CO) 3 (GSAO)(MIBI)
  • This example evaluates the in vitro stability effect of adding MIBI to 99m Tc(CO) 3 direct labeled GSAO. While the experiment was performed in vitro, it is useful to predict the in vivo stability Of 99m Tc(CO) 3 (GSAO) and 99m Tc (I) (CO)3(GSAO)(MIBI). Two studies were performed, a dilution study and a histidine challenge experiment.
  • Nuclear medicine agents possess two critical parameters: (1) the stability of the metal complex in dilute amounts and (2) the stability of the metal complex after exposure to biological environment. To simulate biological media, a solution of histidine was used in the experiments. Histidine forms a strong complex with the TC(CO) 3 moiety.
  • Both solution preparations also were reacted with 100 mM histidine (155 mg His/10 mL saline). 0.3 mL of the 100 mM histidine solution was added to 0.3 mL of a solution preparation, and the resulting solution was heated at 37°C for 30 minutes. Radiochemical purity was determined by HPLC as described above.
  • This example evaluates the effect of adding a monodentate ligand (MIBI) to the 99m Tc(CO) 3 direct labeled GSAO.
  • MIBI monodentate ligand
  • mice Eighteen conscious restrained female mice (strain C57BL6) were given IV injections (0.20 ml). Nine of the mice were injected with 5 ⁇ Ci of 99m Tc(CO) 3 (GSAO) (25 ⁇ Ci/ml) and nine with 5 ⁇ Ci Of 99m Tc(CO) 3 (GSAO)(MIBI) (25 ⁇ Ci/ml). Three mice of each set were euthanized at one, four and twenty-four hours after injection. The tissues collected and analyzed were the liver, heart, spleen, kidney, lung, injection site, muscle, blood and urine/feces. The tissues were analyzed for the percentage of injected dose per gram.

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Abstract

Processes for direct labeling GSAO and GSAO equivalent compounds with 99mTc(CO)3, radiopharmaceuticals produced by such methods and kits for preparing the direct-labeled radiopharmaceutical compounds.

Description

COORDINATION OF A TECHNETIUM IMAGING COMPLEX ON A BIOACTIVE
APOPTOSIS TARGETING AGENT
BACKGROUND OF THE INVENTION
[0001] This invention relates to radiopharmaceutical compounds comprising technetium-99m and to processes for direct labeling bioactive molecules with technetium- 99m to form radiopharmaceutical compounds.
[0002] Nuclear medicine uses radioactive material for diagnostic and therapeutic purposes by injecting a patient with a dose of the radioactive material. The radioactive material then concentrates in certain organs or biological regions of the patient. Numerous radioisotopes are used in nuclear medicine including Tc-99m, In-111, Tl-201, 1-123, and Ga-67 for diagnostic gamma imaging, F-18 for diagnostic PET imaging, and Y-90, Lu-177, 1-131, and Sm- 153 for radiotherapy. Chemical forms of radioactive materials naturally concentrate in a particular tissue, for example, iodide (1-131) concentrates in the thyroid. Radioactive materials are often combined with a tagging or bioactive agent, which targets the radioactive material for the desired organ or biologic region of the patient. These radioactive materials alone or in combination with a tagging agent are typically referred to as radiopharmaceuticals in the field of nuclear medicine. At relatively low doses of the radiopharmaceutical, a radiation imaging system (e.g., a gamma camera) may be utilized to provide an image of the organ or biological region that collects the radiopharmaceutical. Irregularities in the image are often indicative of a pathology, such as cancer. Higher doses of the radiopharmaceutical may be used to deliver a therapeutic dose of radiation directly to the pathologic tissue, such as cancer cells.
[0003] Technetium-99m ("99mTc") is the most commonly used radioisotope in nuclear medicine due to its resolution properties and relatively short half-life of 6 hours. 99mTc is produced from a technetium-99m generator comprising decaying molybdenum-99. As the molybdenum-99 nuclide decays, it produces a technetium-99m daughter nuclide. The technetium-99m is eluted from the molybdenum-99 and may then be complexed with the appropriate bioactive molecule.
[0004] 4-(N)-((S-glutathionylacetyl)amino) phenyl arsine oxide ("GSAO") is a bioactive molecule which accumulates in dead and dying cells.
[0005] Kits are typically used to synthesize 99mTc radiopharmaceutical complexes. The kits contain a targeting agent, and other ingredients that can promote the stability of the kit. The kits can be lyophilized to enhance the shelf life of the kits and permit the kits to be stored for periods of time until they are used to synthesize a radiopharmaceutical complex. The targeting agent is typically a bioconjugate molecule where one portion of the compound is the biologically active moiety and the other portion contains a chelating moiety for complexing the Tc-99m, for example, diethylenetriamine pentaascetic acid (DTPA), mercaptoacetyltriglycine (MAG3), 1,4,7,10 tetraazacyclododecane-tetraacetic acid (DOTA), N2O21N2S2, or N3S However, incorporation of an external chelating agent may interfere with the targeting capabilities of the 99mTc-radiopharmaceuticals in some circumstances, particularly when the bioactive component is small, as in the case of GSAO. In some instances, a linking group comprised of one or more atoms is used to attach the chelating group to the biologically active moiety.
SUMMARY OF THE INVENTION
[0006] According to embodiments of the present invention, GSAO or a GSAO equivalent is directly labeled with 99mTc which minimizes the synthesis steps for forming a radiopharmaceutical. The 99mTc atom directly bonds to donor atoms on the GSAO or GSAO equivalent core, providing a smaller molecule as compared to those in the art that require external chelating agents .
[0007] The present invention is directed to a radiopharmaceutical compound having the formula
Figure imgf000003_0001
wherein Z is -D=O or -D(OH)2, D is As, Ge, Se, Sn, B, Ph, or Al; R1 to R4 are independently H, X, OH, CN, NH2, CO, SCN, -CH2NH2, -NHCOCH3, -NHCOCH2X or NO; X is a halogen; A is
Figure imgf000004_0001
wherein when A is (I) or (II), R5 is hydrogen, -OH, -COOH,
Figure imgf000005_0001
Figure imgf000006_0001
,or
;and
Figure imgf000006_0002
when A is (III), Rs is
Figure imgf000006_0003
-
, or
Figure imgf000007_0002
Figure imgf000007_0001
Re is -NHCH2COOH or -OH; and M is a monodentate ligand. In some instances, the monodentate ligand is an aliphatic amine, a heterocyclic amine, a heterocyclic imine, a thiol, a thiolate, a thioether, an isocyanide, or a phosphine. In some cases, the monodentate ligand is methoxyisobutyl isonitrile. In other cases, the monodentate ligand is tris-hydroxy methyl phosphine. D may be As. Z may be -As(OH)2 or -As=O. R1, R2, R3 and R4 may be hydrogen. In some cases, A is (I) and Rs is
Figure imgf000007_0003
In other instances, A is (III) and R5 is
Figure imgf000008_0001
[0008] The invention is also directed to a kit for use in preparing a radiopharmaceutical compound, the kit comprising: an amount of a compound having the formula
Figure imgf000008_0002
or
Figure imgf000008_0003
wherein Z is -D=O or -D(OH)2, D is As, Ge, Se, Sn, B, Ph, or Al; R1 to R4 are independently H, X, OH, CN, NH2, CO, SCN, -CH2NH2, -NHCOCH3, -NHCOCH2X or NO, X is a halogen; R5 is hydrogen, -OH, -COOH,
Figure imgf000009_0001
Figure imgf000010_0001
, or
Figure imgf000010_0002
and R6 is -NHCH2COOH or -OH; and an amount of monodentate ligand. The kit may include a carbonyl reaction vial. The kit may also include an acid for neutralization.
[0009] Another aspect of the present invention is a process for direct labeling a compound of Formula (2A) or (2B) to form a radiopharmaceutical, the process comprising reacting the compound with a solution of 99mTc(CO)3(OH2)3 to form an intermediate and reacting the intermediate with a monodentate ligand to form the radiopharmaceutical of Formula (8).
[0010] Other objects and features will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Fig. 1 is a graphical depiction of the number of radioactive cell counts during radioimaging of cell cultures exposed to different amounts of camptothecin; and
[0012] Fig. 2 is a graphical depiction of the biodistribution in mice organs after injection of 99mTc(CO)3(GSAO) complex with and without the addition of a monodentate ligand.
DETAILED DESCRIPTION
[0013] The direct labeling method described herein may generally be used to label GSAO and any GSAO equivalent with technetium-99m. GSAO is a compound of Formula (1)
Figure imgf000011_0001
wherein Z is -D=O or -D(OH)2, D is As, Ge, Se, Sn, B, Ph, or Al; R1 to R4 are independently H, X, OH, CN, NH2, CO, SCN, -CH2NH2, -NHCOCH3, -NHCOCH2X or NO, and X is a halogen. As used herein, a "GSAO equivalent" is any compound of Formula (2A) or (2B)
or
Figure imgf000012_0001
Figure imgf000012_0002
wherein Z and R1 to R4 are as defined above; R5 is hydrogen, -OH, -COOH,
Figure imgf000012_0003
Figure imgf000013_0001
Figure imgf000014_0001
, or
Figure imgf000014_0002
and R6 is -NHCH2COOH or -OH. As can be seen from Formula (2), GSAO equivalent compounds include the GSAO molecule itself.
[0014] Compounds such as GSAO that contain an arsenoxide component (i.e., wherein D is As) are especially well suited for imaging apoptotic cells. In addition, because glutathione is readily transportable across cell membranes, compounds containing a glutathione moiety are well suited for imaging apoptotic cells.
[0015] GSAO and GSAO equivalents may be prepared by reacting glutathione or a glutathione equivalent with N-(4-arsorylphenyl)-2-bromoacetamide (i.e., 4-(N- bromoaceryl)amino)phenylarsenoxide (BRAO)) as disclosed in US Publication No. 2005/0101524 which is incorporated herein by reference. Glutathione is a compound of Formula (3)
Figure imgf000015_0001
As used herein, a "glutathione equivalent" is a compound of Formula (4)
Figure imgf000015_0002
wherein Rs and Re are as defined for Formula (2) above. As can be seen from Formula (4), glutathione equivalent compounds include glutathione itself. A GSAO equivalent of Formula (2B), such as S-Penicillamine-arsenoxide (i.e., PENAO) having the formula
Figure imgf000015_0003
can be prepared by reacting S-penicillamine with BRAO as described in WO 2008/052279 which is incorporated herein by reference.
Preparation of the 99mTc(GSAO) or 99mTc(GSAO equivalent) Complex bv the Direct Labeling Method [0016] The 99mTc(GSAO) complex or 99mTc(GSAO equivalent) complex is formed by first reacting GSAO or a compound of Formula (2) with 99mTc(CO)3(OH2)3 to form an intermediate. 99mTc(CO)3(OH2)3 solution can be prepared by adding sodium pertechnetate 99mTc (99mTcO4 Na+) to a carbonyl reaction vial (e.g., ISOLINK vial available from Mallinckrodt Inc., St. Louis, MO), heating the solution and optionally adding acid to neutralize the solution. Sodium pertechnetate 99mTc solution may be produced as the eluate from a technetium generator (e.g., ULTRA-TECHNEKOW DTE, available from Mallinckrodt Inc., St. Louis, MO). Technetium-99m used in accordance with the principles of the present invention and as produced from the technetium generator is typically in the +1 oxidation state (i.e., 99mTc(I)).
[0017] After the 99mTc(CO)3(OH2)3 solution is prepared, it is combined with the GSAO or GSAO equivalent solution to prepare 99mTc(CO)3(GSAO) intermediate or ""TC(CO)3(GSAO equivalent) intermediate. Without being bound to any particular theory, it is believed that an intermediate compound is produced with three alternative structures. Under the first structure, technetium-99m makes two strong covalent bonds and one weak covalent bond with the GSAO or GSAO equivalent molecule. For instance, when 99mTc(CO)3(OH2)3 is reacted with GSAO or a GSAO equivalent of Formula (2) wherein R6 is -NCH2COOH, a compound of Formula (5) is formed
Figure imgf000016_0001
wherein Z and R1 to R5 are as defined for Formula (2) above. In this structure, a monodentate ligand can react with the complex by breaking the weak bond between the technetium-99m and GSAO or GSAO equivalent (e.g., the bond with the sulfur atom) and forming a stable bond with the technetium-99m atom to produce a bioactively stable complex. [0018] In the second alternative structure, the technetium-99m makes two strong covalent bonds with the GSAO or GSAO equivalent molecule and keeps an open coordination site as shown in Formula (6)
Figure imgf000017_0001
wherein Z and R1 to Rs are as defined for Formula (2) above. In this arrangement, a monodentate ligand can react with the complex by reacting in the open coordination site.
[0019] In the third alternative structure, the technetium-99m makes two strong covalent bonds with the GSAO or GSAO equivalent molecule and keeps an open coordination site as shown in Formula (7)
Figure imgf000017_0002
wherein Z and R1 to R4 and R6 are as defined for Formula (2) above. In this arrangement, a monodentate ligand can react with the complex by reacting in the open coordination site.
[0020] Once the 99mTc(CO)3(GSAO) solution or 99mTc(CO)3(GSAO equivalent) solution is prepared it is reacted with a monodentate ligand ("M") to prepare a 99mTc(CO)3(GSAO)(M) or 99mTc(CO)3(GSAO equivalent)(M) complex, which is the radiopharmaceutical compound of the invention. It is believed that the monodentate ligand reacts directly with the 99mTc atom either by substituting for a weaker GSAO or GSAO equivalent bond or by complexing with an open coordination site on the 99mTc atom Surprisingly, the resulting structure is very stable and exhibits in vivo stability (See Example 3 below). Suitable monodentate ligands include, but are not limited to, an aliphatic amine, a heterocyclic amine, a heterocyclic imine, a thiol, athiolate, athioether, an isocyanide, or a phosphine. Methoxyisobutyl isonitrile ("MIBI") and tris-hydroxy methyl phosphine are especially well-suited monodentate ligands for coordination with the 99mTc(I)(CO)3(GSAO) or 99mTc(I) (CO)3(GSAO equivalent) complex.
Structure of the 99mTc(I) (CO)3(GSAO)(M) or 99mTc(I) (CO)3(GSAO equivalent)(M) Complex [0021] The radiopharmaceutical compounds formed by the direct labeling process of the invention have the Formula (8)
Figure imgf000018_0001
wherein Z is -D=O or -D(OH)2, D is As, Ge, Se, Sn, B, Ph, or Al; R1 to R4 are independently H, X, OH, CN, NH2, CO, SCN, -CH2NH2, -NHCOCH3, -NHCOCH2X or NO, X is a halogen;
A is
wherein when A is (I) or (II), R5 is hydrogen, -OH, -COOH,
Figure imgf000020_0001
Figure imgf000021_0001
,or and
Figure imgf000021_0002
when A is (III), Rs is
Figure imgf000021_0003
or
Figure imgf000022_0003
Figure imgf000022_0001
R6 is -NHCH2COOH or -OH; and M is a monodentate ligand.
[0022] When the bioactive molecule is GSAO or when the terminal group of the GSAO equivalent molecule is -NHCH2COOH, the 99mTc atom can bind to the -NHCH2COOH terminus to form a compound of Formula (9)
Figure imgf000022_0002
wherein Z and R1 to Rs are as defined for Formula (8) above when A is (I). [0023] In some structures the 99mTc atom may bind at the other terminus and form a compound of Formula (10)
Figure imgf000023_0001
wherein Z and R1 to Rs are as defined for Formula (8) above when A is (in).
[0024] When the terminal group of the GSAO equivalent molecule is -OH, the 99mTc atom can bind to the -OH terminus to form a compound of Formula (11)
Figure imgf000023_0002
wherein Z and R1 to Rs are as defined for Formula 7 above when A is (II).
[0025] In some structures the 99mTc atom may bind at the other terminus and form a compound of Formula (12)
Figure imgf000023_0003
wherein Z and R1 to Rs are as defined for Formula (8) above when A is (III).
[0026] In some structures, the 99mTc atom may bind at the penicillamine terminus and form a compound of Formula (13)
Figure imgf000024_0001
wherein Z, M and R1 to Rt are as defined for Formula (8) above.
Kits for Preparation of 99mTc(I)(CO)3(GSAO)(M) or 99mTc(I)(CO)3(GSAO equivalent)(M) Complexes
[0027] A three or four component kit containing the constituent chemicals can be used to prepare 99mTc(I)(CO)3(GSAO)(M) and 99mTc(I)(CO)3(GSAO equivalent)(M) complexes. The components of the kit are 1) a carbonyl reaction vial; 2) an optional vial of acid for neutralization, such as 1 N HCl; 3) a vial containing the GSAO or GSAO equivalent; and 4) a vial containing the monodentate ligand. The contents of any vial in the kit may be lyophilized. The preferred carbonyl reaction vial is a lyophilized formulation of 8.5 mg sodium tartrate, 2.85 mg Na2B4O7 .10H2O, 7.15 mg of sodium carbonate, and 4.5 mg sodium boranocarbonate. A kit and a technen'um-generator can be used to produce the complexes in the same facility in which they are administered to the patient.
[0028] To prepare a 99mTc(GSAO) or 99mTc(GSAO equivalent) complex from the kit, 99mTc is eluted from a technetium generator, for example, as sodium pertechnetate 99mTc (99mTcO4-Na+). To prepare the 99mTc(CO)3(OH2)3 intermediate solution, the generator eluate is added to a carbonyl reaction vial, and the solution is typically heated at 75 - 100 C for up to 20 minutes. GSAO or GSAO equivalent is then added to the 99mTc(CO)3(OH2)3 SoIuUOn to prepare a solution of99mTc(CO)3(GSAO) or 99mTc(CO)3(GSAO equivalent). In some embodiments, the reaction is conducted at room temperature for about 30-60 minutes. In other instances, the reaction mixture can be heated to 100°C for about 10-15 minutes. A monodentate ligand is then added to the solution of 99mTc(CO)3(GSAO) or 99mTc(CO)3(GSAO equivalent) to prepare 99mTc(I)(CO)3(GSAO)(M) or 99mTc(I)(CO)3(GSAO equivalent)(M) which can be administered to the patient. In some embodiments, the reaction is conducted at 37°C for about 30-60 minutes.
[0029] The kit is useful to prepare 99mTc(I)(CO)3(GSAO)(M) and 99mTc(I)(CO)J(GSAO equivalent)(M) complexes for use as imaging agents for detection of apoptotic, necrotic, or any dead or dying cells in the body, for example, cells that have died after chemotherapy or radiotherapy. The radiopharmaceuticals are well suited for single photon emission computed tomography (SPECT), positron emission tomography (PET), and optical imaging. Synthesized complexes can also be used to monitor GSAO when used as an angiogenesis inhibitor. Without being bound by any particular theory, it is believed that the -D(OH)2 or -D=O moiety of the radiopharmaceutical compound of Formula (8) reacts with proteins containing closely spaced dithiols , such as protein disulfide isomerase (PDI), which are more readily accessible in dead and dying cells compared to normal cells. Example 4 illustrates the favorable biodistribution of (i.e., low non-target binding) of a compound of the invention which makes it an effective radioimaging agent, particularly for SPECT imaging.
[0030] Having described the invention in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims.
EXAMPLES
[0031] The following non-limiting examples are provided to further illustrate the present invention.
Example 1; Preparation of 99mTc(I)(CO)3 (GSAO)(MI BI)
[0032] 99mTc(CO)3(OH2)3 was prepared as follows. 99mTcO4- solution was eluted from a technetium generator. One mL (70 mCi) 99mTcO4 ' was added to an IsoLink vial and the solution was heated in a boiling water bath for 10 minutes. 120 μL of IN HCl was then added. Reverse phase HPLC (C-18 column with a 0.05 M TEAP; pH = 2.0/methanol gradient) demonstrated greater than 95% radiochemical purity (retention time = 4 min). [0033] Next, 99mTc(CO)3(GSAO) was prepared. In a separate vial, 125 μL of the freshly prepared 99mTc(CO)3(OH2)3 solution was added to 500 μL (ca. 25 mg) GSAO solution, and the reaction was allowed to stand at room temperature for 30 minutes. Radiochemical purity based on the HPLC analysis described above was ca. 94% (retention time = 18 min).
[0034] The 99mTc(I)(CO)3(GSAO)(MIBI) solution was then prepared as follows. 20 mM MIBI solution (500 μL) was added to the 99mTc(CO)3(GSAO) solution (500 μL). The solution was heated at 37°C for 30 minutes. The radiochemical purity of the final reaction was determined to be about 85% by HPLC using the HPLC conditions described above (retention time = 21 min). For biological work, the 99mTc(I)(CO)3(GSAO)(MIBI) is HPLC purified in order to achieve radiochemical purity of greater than 90%.
Example 2: Ability of the 99mTc(I)(CO)3(GSAO)(MIBI) Complex to Bind and Image Apoptotic Cells
[0035] This example demonstrates the affinity of GSAO towards apoptotic cells and the ability of the 99mTc(I)(CO)3(GSAO)(MIBI) complex to image apoptotic cells.
1. Camptothecin Treatment of U937 Cells
[0036] U937 cells were plated in growth medium in 6-well tissue culture plates at a density of 2.4 x 105 cells per well. Freshly prepared camptothecin was added to the cells to produce final camptothecin concentrations of approximately 3.5 and 7 micromolar. Untreated cells were used as negative controls. The treated and non-treated cells were placed overnight in a humidified incubator at 37°C with a 5% CO2/95% ambient air atmosphere.
2. Harvesting Treated and Non-Treated U937 Cells
[0037] The treated and nontreated cells were incubated for approximately 16 hours and then the cells were quantitatively transferred to separate 15-mL centrifuge tubes and placed on ice. Cells were washed in ice cold phosphate buffered saline ("PBS"), resuspended in annexin binding buffer (1 mL), and placed on ice.
3. Incubation of Cells with 99mTc(I)(CO)3(GSAO)(MIBI)
[0038] The 99mTc(I)(CO)3(GSAO)(MIBI) preparation was diluted to 0.1 mCi/mL in annexin binding buffer. 50 μl of the diluted solution containing 5 μCi 99mTc(I)(CO)3(GSAO)(MIBI) was added to each sample. Samples were swirled gently and incubated on ice for 30 minutes.
[0039] After the 30 minute incubation, ice cold RPMI (Roswell Park Memorial Institute) media (10 mL) was added to each tube. Cells were mixed gently and centrifuged at 250 times gravity for 10 minutes. The supernatant was decanted. Cells were washed two more times in ice cold RPMI media (10 mL). Cells were quantitatively transferred to counting tubes using 2 x 0.5 mL aliquots of RPMI media and the cell-associated radioactivity was measured using a Cobra B5OO5 Gamma Counter.
4. Results
[0040] The results of the study are shown in Fig. 1. As can be seen from the Figure, cell counts were more than twice as high for cells exposed to camptothecin, i.e., apoptotic cells. This illustrates the affinity of 99mTc(I)(CO)3(GSAO)(MIBI) towards apoptotic cells. Example 3: In Vitro Stability Comparison of 99mTc(I)(CO)3(GSAO) and 99mTc(I)(CO)3(GSAO)(MIBI)
[0041] This example evaluates the in vitro stability effect of adding MIBI to 99mTc(CO)3 direct labeled GSAO. While the experiment was performed in vitro, it is useful to predict the in vivo stability Of99mTc(CO)3(GSAO) and 99mTc(I)(CO)3(GSAO)(MIBI). Two studies were performed, a dilution study and a histidine challenge experiment.
[0042] Nuclear medicine agents possess two critical parameters: (1) the stability of the metal complex in dilute amounts and (2) the stability of the metal complex after exposure to biological environment. To simulate biological media, a solution of histidine was used in the experiments. Histidine forms a strong complex with the TC(CO)3 moiety.
[0043] 99mTc(CO)3(GSAO) complex and "111TC^CO)3(GSAO)(MIBI) complex were prepared as described in Example 1. Radiochemical purity (RCP) was determined by reverse phase HPLC (C-18 column with a 0.05 M TEAP; pH = 2.0/methanol gradient).
[0044] Both solutions were diluted to 25 μCi/mL with normal saline (0.9% NaCl) and heated at 37°C for 4 hours. Radiochemical purity was determined by HPLC as described in the preceding paragraph.
[0045] Both solution preparations also were reacted with 100 mM histidine (155 mg His/10 mL saline). 0.3 mL of the 100 mM histidine solution was added to 0.3 mL of a solution preparation, and the resulting solution was heated at 37°C for 30 minutes. Radiochemical purity was determined by HPLC as described above.
[0046] The radiochemical purity results for the solutions are shown below.
Table 1: Comparison of Radiochemical Purity (RCP) of 99mTc(CO)3(GSAO) Complex and "111TC5^CO)3(GSAO)(MIBI) Complex Before and After Dilution and Before and After Ex osure to Histidine
Figure imgf000028_0001
[0047] As can be seen from Table 1, the complex containing MIBI was resistant to potential loss of radioactivity caused by dilution and exposure to histidine. Example 4: Bioassav Comparison of 99mTc (CO)3(GSAO) and 99mTc(I)(CO)3(GSAO)(MIBI)
[0048] This example evaluates the effect of adding a monodentate ligand (MIBI) to the 99mTc(CO)3 direct labeled GSAO.
[0049] Eighteen conscious restrained female mice (strain C57BL6) were given IV injections (0.20 ml). Nine of the mice were injected with 5 μCi of 99mTc(CO)3(GSAO) (25 μCi/ml) and nine with 5 μCi Of99mTc(CO)3(GSAO)(MIBI) (25 μCi/ml). Three mice of each set were euthanized at one, four and twenty-four hours after injection. The tissues collected and analyzed were the liver, heart, spleen, kidney, lung, injection site, muscle, blood and urine/feces. The tissues were analyzed for the percentage of injected dose per gram.
[0050] The results are shown in Fig. 2. As depicted in the graph, addition of a monodentate ligand (in this instance MIBI) to GSAO directly labeled with ""1TC resulted in a significant decrease in tissue uptake and a corresponding improved clearance of the radiolabeled compound from the various organs, tissues, and blood. Blood clearance is significant in that it results in improved images. This improved pharmacokinetics allows for the feasibility of using the direct labeled GSAO as an imaging agent.
[0051] When introducing elements of the present invention or the preferred embodiments(s) thereof, the articles "a", "an", "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.
[0052] In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.
[0053] As various changes could be made in the above compositions and processes without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

Claims

What is claimed is:
1. A radiopharmaceutical compound having the formula
Figure imgf000030_0001
wherein Z is -D=O or -D(OH)2, D is As, Ge, Se, Sn, B, Ph, or Al; R1 to R4 are independently H, X, OH, CN, NH2, CO, SCN, -CH2NH2, -NHCOCH3, -NHCOCH2X or NO, X is a halogen; A is
Figure imgf000030_0002
Figure imgf000031_0001
or
Figure imgf000031_0002
wherein when A is (I) or GI), R5 is hydrogen, -OH, -COOH,
H
Figure imgf000031_0003
Figure imgf000032_0001
Figure imgf000032_0002
,or and
Figure imgf000032_0003
when A is (III), R? is
Figure imgf000033_0001
or
Figure imgf000033_0002
R6 is -NHCH2COOH or -OH; and M is a monodentate ligand.
2. The compound of claim 1 wherein A is (I) and Rs is
Figure imgf000034_0001
3. The compound of claim 1 wherein A is (III) and Rs is
Figure imgf000034_0002
4. The compound of any one of claims 1-3 wherein D is As.
5. The compound of any one of claims 1-3 wherein Z is -As(OH)2.
6. The compound of any one of claims 1-5 wherein R1, R2, R2 and R4 are hydrogen.
7. The compound of any one of claims 1-6 wherein the monodentate ligand is an aliphatic amine, a heterocyclic amine, a heterocyclic imine, a thiol, a thiolate, a thioether, an isocyanide, or a phosphine.
8. The compound of any one of claims 1-6 wherein the monodentate ligand is methoxyisobutyl isonitrile.
9. The compound of any one of claims 1-6 wherein the monodentate ligand is tris- hydroxy methyl phosphine.
10. A kit for use in preparing a radiopharmaceutical compound, the kit comprising: an amount of a compound having the formula
Figure imgf000035_0001
or
Figure imgf000035_0002
wherein Z is -D=O or -D(OH)2, D is As, Ge, Se, Sn, B, Ph, or Al; R1 to R4 are independently H, X, OH, CN, NH2, CO, SCN, -CH2NH2, -NHCOCH3, -NHCOCH2X or NO, X is a halogen; R5 is hydrogen, -OH, -COOH,
Figure imgf000035_0003
Figure imgf000036_0001
Figure imgf000037_0001
, or
Figure imgf000037_0002
and R6 is -NHCH2COOH or -OH; and an amount of monodentate ligand.
11. The kit of claim 10 further comprising a carbonyl reaction vial.
12. The kit of claim 10 or 11 wherein R5 is
Figure imgf000037_0003
13. The kit of any one of claims 10-12 wherein R6 is -NHCH2COOH.
14. The kit of any one of claims 10-13 wherein D is As.
15. The kit of any one of claims 10-13 wherein Z is -As(OH)2.
16. The kit of any one of claims 10-15 wherein R1, R2, R3 and R4 are hydrogen.
17. The kit of any one of claims 10-16 wherein the monodentate ligand is an aliphatic amine, a heterocyclic amine, a heterocyclic imine, a thiol, a thiolate, a thioether, an isocyanide, or a phosphine.
18. The kit of any one of claims 10-16 wherein the monodentate ligand is methoxyisobutyl isonitrile.
19. The kit of any one of claims 10-16 wherein the monodentate ligand is tris- hydroxy methyl phosphine.
20. A process for direct labeling a compound of Formula (2A) or (2B) to form a radiopharmaceutical, the process comprising reacting the compound with a solution of 99mTc(CO)3(OH2)3 to form an intermediate and reacting the intermediate with a monodentate ligand to form the radiopharmaceutical, wherein the compound has the formula
Figure imgf000038_0001
Figure imgf000039_0001
wherein Z is -D=O or -D(OH)2, D is As, Ge, Se, Sn, B, Ph, or Al; R1 to R4 are independently H, X, OH, CN, NH2, CO, SCN, -CH2NH2, -NHCOCH3, -NHCOCH2X or NO, X is halogen; R5 is hydrogen, -OH, -COOH,
Figure imgf000039_0002
Figure imgf000040_0001
Figure imgf000040_0002
,or
Figure imgf000040_0003
R6 is -NHCH2COOH or -OH; and the radiopharmaceutical is a compound of claim 1.
21. The process of claim 20 wherein R5 is
Figure imgf000041_0001
22. The process of claim 20 or 21 wherein R6 is -NHCH2COOH.
23. A process as set forth in claim 34 wherein the intermediate has either the Formula
Figure imgf000041_0002
the Formula
or
Figure imgf000041_0003
the Formula
Figure imgf000042_0001
24. The process of any one of claims 20-23 wherein D is As.
25. The process of any one of claims 20-23 wherein Z is -As(OH)2 or -As=O.
26. The process of any one of claims 20-25 wherein R1, R2, R3 and R4 are hydrogen.
27. The process of any one of claims 20-26 wherein the monodentate ligand is an aliphatic amine, a heterocyclic amine, a heterocyclic imine, a thiol, a thiolate, a thioether, an isocyanide, or a phosphine.
28. The process of any one of claims 20-26 wherein the monodentate ligand is methoxyisobutyl isonitrile.
29. The process of any one of claims 20-26 wherein the monodentate ligand is tris- hydroxy methyl phosphine.
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Citations (1)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6344178B1 (en) * 1997-04-25 2002-02-05 Mallinckrodt Inc. Method for the preparation of facial metal tricarbonyl compounds and their use in the labelling of biologically active substrates

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