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WO2001085221A2 - Reactifs et methodes de dosage des niveaux de $i(0)6-alkylguanine-adn alkyltransferase $i(in vivo) (agt) - Google Patents

Reactifs et methodes de dosage des niveaux de $i(0)6-alkylguanine-adn alkyltransferase $i(in vivo) (agt) Download PDF

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WO2001085221A2
WO2001085221A2 PCT/US2001/015370 US0115370W WO0185221A2 WO 2001085221 A2 WO2001085221 A2 WO 2001085221A2 US 0115370 W US0115370 W US 0115370W WO 0185221 A2 WO0185221 A2 WO 0185221A2
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tumor
purin
ylamine
agt
benzyloxy
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WO2001085221A3 (fr
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Michael R. Zalutsky
Michael Colvin
Ganesan Vaidyanathan
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Duke University
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Duke University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0491—Sugars, nucleosides, nucleotides, oligonucleotides, nucleic acids, e.g. DNA, RNA, nucleic acid aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds

Definitions

  • the invention relates to the area of cancer chemotherapeutics.
  • the invention relates to methods of assessing levels of O ⁇ -alkylguanine-DNA alkyltransferase in vivo.
  • alkylating agents such as 1, 3-bis(2-chloroethyl)-l-nitrosourea (BCNU), l-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU), 5-(dimethyltriazeno)imi- dazole-4-carboxar ⁇ ide (DTIC), and temozolomide are used in the chemotherapy of various cancers (1).
  • BCNU 1, 3-bis(2-chloroethyl)-l-nitrosourea
  • CCNU l-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea
  • DTIC 5-(dimethyltriazeno)imi- dazole-4-carboxar ⁇ ide
  • temozolomide temozolomide
  • Nitrogen mustard alkylating agents such as cyclophosphamide were thought to produce their cytotoxicity through the alkylation of the Opposition of guanine (2); however, it has been recently demonstrated that a significant part of the cytotoxicity of this class of agents also is mediated by the O 6 - alkylation of guanine residues (3).
  • the DNA repair protein O 6 -alkylguanine-DNA alkyltransferase reverts alkylator cytotoxicity by transferring the O 6 -alkyl group from the modified DNA guanine to cysteine-145 in its active site, thereby restoring a normal guanine at the site of the modified base (4).
  • AGT O 6 -alkylguanine-DNA alkyltransferase
  • AGT levels in individual tumors could greatly facilitate the process of optimizing the efficacy of chemotherapy.
  • assessing AGT levels in tumors in vivo would permit selection of patients most likely to benefit from such chemotherapy and would enable clinicians to monitor the effect of known and potential chemotherapeutic agents.
  • Patent 5,407,804 teaches an assay for MGMT in tumor biopsy samples which uses monoclonal antibodies.
  • Ciocco et al. (43) discloses O 6 -(p-hy ⁇ oxy[ 3 H]memylbenzyl)guanine (in which the radiolabel is associated with a hydroxymethylbenzyl moiety) and its use as a reagent for detecting AGT in cultured cells using autoradiography.
  • BG and "O 6 -benzylguanine” are used interchangeably in this disclosure.
  • Derivatives of O 6 -benzylguanine which inactivate AGT in vitro also have been described.
  • Moschel et al. (16) discloses substituted (non-radiolabeled) benzylguanine molecules, particularly O 6 -(p-fluorobenzyl)guanine, which inactivate AGT in vitro in human tumor cell extracts and intact tumor cells.
  • Moschel et al. suggests administering the substituted benzylguanine molecules to enhance the cytotoxic effects of O 6 -alkylating antitumor drugs.
  • Moschel et al. does not disclose use of the substituted benzylguanine molecules to assess AGT levels in vivo.
  • Chae et al. (17) discloses O 6 -benzyIguanine derivatives, including 0 6 -(p- bromobenzyl)guanine, which inactivate AGT in vitro. Administration of the benzylguanine derivatives to enhance anti-rumor chemotherapy also is disclosed. Chae et al. suggests that the pharmacokinetics of AGT-inactivating agents could be altered, enabling the agents to be directed selectively to tumor cells. Again, the derivatives are not radiolabeled, and use of the derivatives to assess AGT levels in vivo is not taught or suggested.
  • Mountenou et al. (21) discloses O 6 -4-[ 125 I]iodobenzylguanosine and its use as a substrate for AGT.
  • Mountenou et al. teaches that the radiolabeled benzylguanosine derivative can be used to detect AGT in tumor cells to predict whether chloroethylating chemotherapeutic agents would be effective against a particular tumor.
  • Mountenou et al. does not teach that the detection could be carried out in vivo.
  • Hotta et al. (10) teaches an assay of AGT activity in surgical samples of gliomas to predict the responsiveness of tumor cells to chloroethylnitrosoureas.
  • the assay measures transfer of radioactivity from substrate DNA containing methyl- 3 H-labeled O 6 - methylguanine to an acid-insoluble protein fraction in crude tumor extracts.
  • Hotta et al. does not teach or suggest assessing AGT levels in vivo.
  • Nesseler et al. discloses synthesis of an 18 F-labeled guanine derivative with the 18 F attached at the N 9 position as a fluoroethyl group and its use for in vivo positron emission tomography imaging of MGMT. Binding of the 18 F--V-labeled derivative was evaluated in vitro in HeLa S3 cells; the derivative bound to MGMT with only a slightly decreased affinity compared to that of ( -benzylguanine. However, despite the hope that, once dealkylated, the guanine residue would attach to the protein in a non-covalent fashion, the N'-derivative was not demonstrated to transfer the radiolabel to the protein to make it useful for AGT imaging.
  • AGT levels in mammals in vivo are provided by one or more of the embodiments described below.
  • One embodiment of the invention is a method of detecting O 6 -alkylguanine-DNA alkyltransferase (AGT) molecules in a mammal.
  • An AGT molecule in the mammal is contacted with an guanine molecule comprising at the exocyclic O 6 position a radiolabeled alkyl or benzyl moiety under conditions whereby the radiolabeled alkyl or benzyl moiety is transferred from the O ⁇ -derivatized guanine molecule to the AGT molecule to form a radiolabeled AGT molecule.
  • the radiolabeled AGT molecule is detected.
  • Another embodiment of the invention is a method of monitoring the effect of a reagent on the amount of AGT molecules in a tumor in a mammal.
  • the reagent is administered to the mammal;.
  • the amount of AGT molecules in the tumor is determined by (a) contacting AGT molecules in the tumor with O 6 -derivatized guanine molecules comprising at the exocyclic O 6 position a radiolabeled alkyl or benzyl moiety, whereby radiolabeled alkyl or benzyl moieties are transferred from the O 6 -derivatized guanine molecules to the AGT molecules to form radiolabeled AGT molecules, and (b) detecting the amount of radiolabeled AGT molecules in the tumor relative to a control in which no reagent is administered.
  • Still another embodiment of the invention is a composition useful for in vivo imaging of AGT molecules.
  • the composition comprises radiolabeled O 6 -benzyl guanine molecule and a pharmaceutically acceptable carrier.
  • the radiolabeled O 6 -benzylguanine molecule is selected from the group consisting of 6-(4-[ 18 F]fluoro-benzyloxy)-9H-purin-2- ylamine, 6-(3-[ 131 I]iodo-benzyloxy)-9H-purin-2-ylamine, 6-(3-[ 125 I]iodo-benzyloxy)-9H- purin-2-ylamine, 6-(3-[ 123 I]iodo-benzyloxy)-9H-purin-2-ylamine, 6-(3-[ 124 I]iodo- benzyloxy)-9H-purin-2-ylamine, 6-(4-[ I8 F]fluoro-3-nitrobenzyloxy)-9H-purin
  • FIG. 1 Depletion of cellular AGT activity by unlabeled 6-(4-fluoro-benzyloxy)- 9H-purin-2-ylamine ( ⁇ -4-fluorobenzylguanine ( FBG) and 6-(iodo-benzyloxy)-9H-purin- 2-ylamine (0 5 -iodobenzylguanine (IBG).
  • FBG ⁇ -4-fluorobenzylguanine
  • IBG 6-(iodo-benzyloxy)-9H-purin- 2-ylamine (0 5 -iodobenzylguanine (IBG).
  • C ⁇ O cells transfected with pCMV-AGT were incubated with varying concentrations of IBG ( ⁇ ) or FBG (•) for 4 hours, and the AGT activity associated with the cells was determined. The results are expressed as the percentage of the AGT activity present in cell cultures that were not treated with FBG or
  • FIG. 2 Binding of [ I8 F]FBG to purified AGT as a function of unlabeled FBG concentration.
  • [ 18 F]FBG was incubated for 30 minutes at 37 °C, in the presence or absence of increasing amounts of unlabeled FBG, with 10 ⁇ g of AGT (•), or to control for nonspecific binding, 10 ⁇ g of BSA (A) in a Tris-buffer. The protein-associated activity was determined by TCA precipitation.
  • FIG. 3 Binding of [ 131 I]IBG to purified AGT as a function of unlabeled IBG concentration. The assay was performed as detailed under FIG. 2 by incubating [ 131 I]IBG with AGT (•) or BSA (A) .
  • FIG. 4. Scheme 1, preparation of 18 F-labeled compound 3 and coupling to the guanine skeleton.
  • FIG. 5. Scheme 2, preparation of compound 7 from compound 4 and commercially available 3-iodobenzyl alcohol in 60% isolated yield and converted to compound 8 by treatment with sodium hydride or potassium tert-butoxide, and SEM-C1.
  • FIG. 6. Chart 1, preparation of compound 11 from the reported compound 10.
  • AGT levels can be detected and measured in vivo using radiolabeled O 6 -derivatized guanine molecules which bind selectively to AGT.
  • Applications of this technique include the selection of patients most likely to benefit from alkylator therapy, monitoring the effect of these drugs on AGT levels during the therapeutic course, correlating therapeutic success with endogenous AGT, identifying test compounds as potential anti-cancer chemotherapeutic agents, and evaluating the clinical effectiveness of AGT-depleting agents.
  • Radiolabeled O 6 -Derivatized Guanine Molecules such as 125 1, 123 1, 12 1, 131 I, and 18 F can be used to provide a radiolabeled O 6 - derivatized guanine molecule.
  • the radiolabel must reside within an alkyl or a benzyl moiety attached to the exocyclic Opposition of the guanine, because this is the moiety that is transferred to the AGT molecule (16).
  • An O 6 -derivatized guanine molecule useful in the present invention comprises at the exocyclic O 6 position a benzyl moiety or an alkyl moiety, such as an ethyl, n-propyl, or n-butyl moiety.
  • Preferred moieties include fiuoromethyl, fluoroethyl, fluoro-n-propyl, fluoro-n-butyl, rtAo-fluoromethylbenzyl, ortbo-fluoroethylbenzyl, ortho- fluoropropylbenzyl, met ⁇ -fluoromethylbenzyl, met ⁇ -fluoroethylbenzyl, meta- fluoropropylbenzyl, j-> ⁇ r -fluoromethylbenzyl, ⁇ r ⁇ -fluoroethylbenzyl, or para- fluoropropylbenzyl.
  • the guanine molecule is an O 6 -benzylguanine molecule.
  • a variety of substituents are tolerated in the benzene ring of O 6 -benzylguamne (16).
  • FBG [6-(4-fluoro-benzyloxy)-9H-purin-2-ylamine; O 6 -4-fluorobenzylguanine] is among the purine and pyrimidine derivatives that have been shown to be AGT depletors (16-20).
  • the ability of FBG to deplete AGT in ⁇ T29 cell-free extracts and intact cells was shown to be similar to that of O 6 -benzylguanine itself (16).
  • a radiolabeled FBG such as !8 F-Iabeled FBG
  • fluorine- 18 has a 1.8 h half-life and is the most commonly used radionuclide for positron emission tomography.
  • Iodinated O 6 -benzylguanine-derivatives also are useful in methods of the present invention.
  • Iodine has a spectrum of radionuclides with different physical properties that are appropriate for external imaging. For example, I23 I 13 h) is an excellent radionuclide for use in single photon emission tomography.
  • the present invention provides methods for the preparation of 3-D3G, and the exmplified labeled compounds 6-(4-[ 18 F]fluoro-benzyloxy)-9 ⁇ -purin-2-ylamine( f 8 F]FBG) and 6-(3-[ I3i I]iodo-benzyloxy)-9H-purin-2-ylamine (3-[ 13I ⁇ jTBG), which are described in the specific examples below.
  • Radiolabeled O 6 -derivatized guanine molecules can be provided in a composition comprising a pharmaceutically acceptable carrier, such that the radiolabeled O 6 -derivatized guanine molecules can be administered to a mammal.
  • Pharmaceutically acceptable carriers are well known in the art and include physiologically compatible buffers such as Hanks' solution, Ringer's solution, dextrose, physiologically buffered saline, or water.
  • a composition of the invention can be administered by any suitable method which will provide the radiolabeled O 6 -derivatized guanine molecules to a tumor.
  • Tumors in which radiolabeled AGT molecules can be imaged include, but are not limited to, gliomas, glioblastomas, astrocytomas, medulloblastomas, Hodgkin's tumors, and tumors of the colon, breast, ovary, prostate, kidney, uterus, pancreas, lung, testis, and muscle.
  • Administration can be by any appropriate route to reach the desired tumor, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, transdermal, subcutaneous, epidural, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
  • the composition can be injected directly into a tumor or into an organ in which a tumor is located, such as a breast, prostate, uterus, liver, kidney, pancreas, lung, or muscle.
  • compositions of the invention can be administered to any mammal, including, but not limited to, dogs, cats, mice, rats, guinea pigs, horses, gorillas, chimpanzees, baboons, pigs, cows, monkeys, and humans.
  • the radiolabeled moiety attached through the exocyclic oxygen of an O 6 -derivatized guanine molecule will be transferred to an AGT molecule; the result of this transfer is a radiolabeled AGT molecule which can be detected by a scintigraphic method.
  • the dose of the radiolabeled O 6 -derivatized guanine molecule to be administered can be determined empirically, depending on the route of administration, the size of the mammal, and the type of tumor in which the level of AGT molecules is to be assessed.
  • concentrations of radiolabeled O ⁇ -derivatized guanine molecules in a composition of the invention will be in the range of no carrier added to 10 mg/kg, 10 to 25 mg/kg, 50 to 100 mg/kg, 75 to 200 mg/kg, 100 to 300 mg/kg, and 250 to 500 mg/kg. If desired, multiple administrations of a composition can be delivered.
  • PET planar imaging positron emission tomography
  • SPECT single photon emission tomography
  • PET is preferred as a detection method.
  • PET is a functional imaging modality that can probe altered biochemical pathways and in some cases can be used to assess metabolic levels quantitatively (14).
  • the number of radiolabeled AGT molecules can be quantitated. For example, the intensity of a signal generated by radiolabeled AGT molecules can be compared with the intensities of signals along a standard curve. Identifying a Test Compound as a Potential Anti-Cancer Chemot erapeuti Agent
  • test compounds can be pharmacologic agents already known in the art or can be. compounds previously unknown to have any pharmacological activity.
  • the compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
  • a test compound is administered to a mammal which bears a tumor.
  • the tumor can be either experimentally induced or naturally occurring.
  • a variety of tumor models suitable for use in this method are well known in the art, such as the murine colon 26-B carcinoma tumor model, the B16 mouse melanoma model, athymic mice bearing a D341MED human brain tumor xenograft, or nude mice injected with HT29 colon tumor cells, A172 glioblastoma cells, or human brain tumor cell lines such as SF767 and U251 MG.
  • test compound can be administered by any suitable means, including, but not limited to, oral, intravenous, intratumoral, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
  • the level of radiolabeled AGT in the tumor in response to the test compound is assessed as described above. If desired, radiolabeled AGT can be detected at multiple time points and/or after multiple administrations or concentrations of the test compound.
  • a test compound which decreases the level of radiolabeled AGT molecules in the tumor relative to the absence of the test compound identifies the test compound as a potential anti-cancer chemotherapeutic agent.
  • the test compound decreases the level of radiolabeled AGT molecules in the tumor by at least 10, 25, 50, or 75%, preferably by at least 85, 90, or 95%.
  • test compound or a known chemotherapeutic agent can be monitored by assessing the level of AGT in the tumor in response to the reagent.
  • a test compound or a known chemotherapeutic agent is administered to a mammal which bears a naturally occurring or an experimentally induced tumor.
  • Test compounds are those which are described above, including potential alkylating agent, and can be administered at doses ranging from 1 to 10 mg/kg, 5 to 50 mg/kg, 25 to 100 mg/kg, 75 to 200 mg/kg, 100 to 300 mg/kg, and 250 to 500 mg/kg.
  • chemotherapeutic agents include, but are not limited to, alkylating agents such as chloroethylating nitrosoureas, such as l,3-bis(2-chloroethyl)-l-nitrosourea (BCNU), l-(4- ammo-2-memyl-5-pyrimidinyl)memyl-3-(2-cMoroethyl)-3-nifrosourea(A(_MJ),methyl-6- [3-(2-crdoroe l)-3-ru ⁇ oso ⁇ reido]-6-deoxy- ⁇ -D-glucopyranoside(MCl ⁇ ,CCNU,DTIC, temozolomide, and cyclophosphamide, as well as procarbazine, dacarbazine, and streptozotocin. Typical doses of such chemotherapeutic agents are well known in the art.
  • alkylating agents such as chloroethylating nitrosoure
  • the level of radiolabeled AGT in the tumor in response to the test compound or known chemotherapeutic agent is assessed as described above.
  • multiple time points can be assayed; if desired, assays can be carried out after administering multiple concentrations or multiple doses of the same concentration of the test compound or chemotherapeutic agent.
  • the level of radiolabeled AGT molecules in the tumor relative to the absence of the reagent reflects the effect of the reagent on the tumor.
  • a decrease in the level of radiolabeled AGT molecules of at least 10, 25, 50, 75, 85, 90, or 95% indicates a therapeutic effect of the reagent on the tumor.
  • FBG (unlabeled) was obtained as a gift from Dr. Robert Moschel of National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD. Sodium
  • [ I8 F]Fluoride activity was produced on the Duke University Medical Center CS-30 cyclotron via the 18 O(p,n) 18 F reaction by irradiating [ 18 O]H 2 O in a small volume (300 ⁇ l) silver target.
  • the activity was delivered to a solution of Kryptofix (10 mg in 1 ml CH 3 CN) and potassium carbonate (1 mg in 5 ⁇ l water) in a glass tube and then evaporated with argon in an oil bath at 80 °C. The dried activity was resolubilized in 50-100 ⁇ l of dry
  • HPLC High pressure liquid chromatography
  • Partisil (10 ⁇ ) (Alltech, Deerfield, IL).
  • Analytical TLC was performed on aluminum-backed sheets (Silica gel 60 F 254 ), and normal-phase column chromatography was performed using Silica gel 60, both obtained from EM Science (Gibbstown, NJ).
  • Column chromatographic fractions were collected using a Gilson model 203 micro fraction collector (Middleton, WI) or an ISCO Foxy 200 fraction collector (Lincoln, NE). Products identified by TLC.
  • an ISCO UA- 6 UN-VIS detector was placed between the column outlet and the fraction collector to identify fractions.
  • Preparative thick layer chromatography was performed using 20 x 20 cm, 1000 ⁇ plates (Whatman, Clifton, ⁇ J).
  • Radio-TLC was initially analyzed using a System 200 Imaging Scanner (BioScan, Washington, D.C.) and then cut into strips and counted using an automatic gamma counter (LKB 1282, Wallac, Finland).
  • ⁇ MR spectra ( ⁇ -300 MHZ and I3 C-75 MHZ) were obtained on a Varian Mercury 300 spectrometer.
  • Mass spectra were obtained on a Hewlett-Packard GC/MS/DS Model HP- 5988 A instrument, or on JEOL SX-102 high resolution mass spectrometer. Elemental analyses were provided by Galbraith Laboratories (Knoxville, T ⁇ ).
  • F ⁇ r ⁇ -[ I8 F]fluorobenzaldehyde was prepared following previously described procedures (26). Briefly, 50-100 mCi of [ 18 F]fluoride were resolubihzed in DMSO (50-100 ⁇ l), added to 1-2 mg of 4-formyl-(N,N,N-trimethyl)anilinium trifluoromethane sulfonate in a 5-ml Reacti ® vial. The mixture was heated in an oil bath at 150 °C for 10 minutes. The cooled reaction mixture was diluted with water and passed through an activated CI 8 solid-phase cartridge (Waters).
  • the cartridge was further washed with water (5-10 ml), and 4-[ 18 F]fluorobenzaldehyde was finally eluted with methylene chloride (1-2 ml).
  • the methylene chloride solution was dried with sodium sulfate and passed through a silica cartridge to remove any polar byproducts. Methylene chloride was removed using a rotary evaporator until a volume of about 0.1 ml was reached. Residual solution was transferred to a 1-dram vial.
  • Use of argon to evaporate methylene chloride results in substantial loss of activity. Even with a rotary evaporator, it is important that the evaporation is stopped before the mixture dries.
  • ENV Sep Pak ENV Sep Pak; Waters activated with ethanol and water.
  • the cartridge was washed with 5 ml of 0.9%) saline, and the activity was eluted with 0.25 ml portions of ethanol. Most of the activity elutes in fractions 3-5. These pooled ethanol fractions were concentrated. For in vitro assays, the activity was reconstituted in Tris-buffer.
  • THF was evaporated from a 1M solution of potassium tert-butoxide in THF (0.83 ml, 830 mmol). An equivalent amount of sodium hydride was used with similar results. To the residue was added 7 (300 mg, 0.82 mmol) followed by DMF (silylation grade;
  • the suspension was filtered through a bed of Celite, and the bed was washed thoroughly with ethyl acetate. The filtrate was concentrated to obtain an oil. This oil was applied to a bed of silica gel, and very non- polar impurities were removed by eluting with hexane. The required product and other byproducts were isolated from the silica gel bed by eluting with ethyl acetate. Ethyl acetate was removed from this solution.
  • Solvents from HPLC fractions containing [ 131 I]8 were evaporated to a small volume, transferred to a ⁇ -dram vial. The solvents were again evaporated to dryness. The residual radioactivity was treated with trifluoroacetic acid (50 ⁇ l) for 5 minutes at room temperature. Most of the trifluoroacetic acid was evaporated with an argon flow and triturated with 50 ⁇ l of ethyl acetate twice to insure its complete removal. Methanolic ammonia (50 ⁇ l) was added to the vial. The vial was vortexed, and methanol and ammonia were evaporated off under a flow of argon.
  • the radioactivity was reconstituted in methanol and injected onto a normal-phase HPLC column eluted with 0.1 % acetic acid in ethyl acetate at a flow rate of 1 ml/rnin.
  • CHO-K1 cells were maintained in ⁇ MEM (Gibco, Grand Island, NY) containing 10%) fetal bovine serum. CHO cells were transfected with a plasmid expressing human AGT (46) using FuGENE (Roche Molecular Biochemicals) according to the manufacturer's protocol for transfection of adherent cells. After 48 hours, geneticin (Gibco) was added at a concentration of 500 ⁇ g/ml. Clones were isolated from individual cell foci.
  • Inactivation of cellular AGT was measured by adding varying concentrations of FBG or IBG to cell cultures that had reached 80-90% confluence. After 4 hours of drug treatment, cell extracts were prepared. AGT activity was measured by assaying the loss of [ 3 H]-O D -methylguanine from a [ 3 H] methylated calf thymus DNA substrate as described (6). The results are expressed as the percentage of the AGT activity present in cell cultures that were not treated with FBG or IBG. For each concentration, the assay was performed in duplicate.
  • the precipitated proteins were collected on GF-C (Whatman) filters, which were washed extensively with 5% TCA. The results are expressed as the percentage of input activity retained on the filter. For each concentration, the assay was performed in triplicate. The assay was performed twice for both [ I8 F]FBG and [ I3! I]IBG.
  • IBG depletes AGT from cells to a greater degree than FBG
  • [ I8 F]FBG had a higher binding to the purified protein than [ I31 I]IBG. This may be due to differential transmembrane transport of the two compounds as a result of differences in their lipophilicity.
  • the lipophilicities of IBG and FBG were not determined per se. Intuitively, IBG is expected to be more lipophilic. Reversed-phase HPLC has been used to determine lipophilicities (45). In comparison to FBG, a higher percentage of acetonitrile was needed to elute IBG from a revered-phase column, suggesting that IBG is more lipophilic.
  • radiochemical syntheses be performed with the least number of steps possible, particularly for short half-life radionuclides such as 18 F (110 minutes).
  • a prerequisite for insertion of 18 F onto a benzene ring by nucleophilic substitution is that the ring contains a suitable leaving group, such as -NO 2 or a quaternary ammonium triflate, that is positioned ortho or para to a strongly electron withdrawing group such as NO 2 (22, 23).
  • a suitable leaving group such as -NO 2 or a quaternary ammonium triflate
  • NO 2 22, 23
  • an FBG precursor such as the one with an -NO 2 or a quaternary ammonium group in the place of fluorine, from which [ 18 F]FBG may be produced in a single step.
  • Unlabeled FBG was originally prepared by the treatment of 2-amino-6-chloropurine (ACP) at 100-130 °C for over 24 hours with an excess of the sodium salt of 4-fluorobenzyl alcohol in 4-fluorobenzyl alcohol as solvent (16). These conditions are not adaptable to 18 F labeling. Even if one started with Curie-quantities of [ 18 F]3 and microgram amounts of ACP, the concentration of 3 would be substantially sub-stoichiometric. In addition, the long reaction time is not suitable with I8 F. We initially attempted this chemistry by conducting the reaction in a solvent such as THF or DME without success.
  • ACP 2-amino-6-chloropurine
  • radioiodmated compounds One of the most commonly used techniques for the preparation of radioiodmated compounds is the radioiododestannylation of the corresponding tin precursor. Initially, we envisaged the preparation of 11 (Chart I, FIG. 6) from the reported compound 10 (17). It may be possible to prepare 12 or its radioiodmated analogue by the halodestannylation of 11; however, attempts to convert 10 to 11 by treatment with hexamethylditin and bis- triphenylphosphine palladium dichloride in dioxane were unsuccessful. This was probably due to the insolubility of 10 in dioxane.
  • Guanine chemistry is often challenging due to the amphoteric nature of guanine derivatives (32). Chemical manipulations can be made more facile if the starting compound can be converted to a non-polar derivative by the introduction of a protecting group. For example, this strategy has simplified the preparation of polar guanidines (33). Guanine derivatives modified with different moieties at the N'-position have been reported (34-36). These derivatives have sufficient non-polar nature to permit purification by silica gel chromatography. Although some of these N 3 -substiruents (34, 36) may be potentially used as protecting groups, the reported work was not done with that objective.
  • Radioiodination of 9 to [ I31 I]8 was performed easily by sonication for 30 seconds with 131 I and a mixture of acetic acid and hydrogen peroxide. The radiochemical yield was more than 90%.
  • Tetrabutylammonium fluoride (TBAF) and TFA are among reagents that have been used for the removal of the SEM group.
  • TFA Tetrabutylammonium fluoride
  • TMSE-ACP ⁇ -(trimethylsilylethyl) ACP
  • TMSE-ACP was treated with TBAF in DMF or DMSO at room temperature and at 60°C.
  • TMSE-ACP remained intact at room temperature for at least one hour. Analysis of the reaction mixtures left at room temperature for 20 hours and those heated at 60°C for 5 minutes by HPLC showed several by-product peaks. On the other hand, a single product peak corresponding to ACP was observed when TMSE-ACP was treated with TFA for 5 minutes at room temperature. Treatment of [ 131 I]8 with TFA at room temperature for 5 minutes gave 55-60% radiochemical yield of [ 131 I]7; 15-20%> of unreacted [ 131 I]8 was recovered. Conducting the reaction for a longer time did not improve the radiochemical yield.
  • Li B.F. Methods of assaying DNA-repairing enzymes and their alkylated derivatives. U.S. Patent 5,879,899, 1999.

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Abstract

On peut utiliser des molécules de guanine dérivées O6- marquées pour constituer des molécules d'AGT marquées que l'on peut détecter in vivo. Le dosage In vivo des niveaux d'AGT s'avère utile pour optimiser l'efficacité de la chimiothérapie. En outre, au moyen du dosage des niveaux d'AGT dans des tumeurs in vivo, on peut sélectionner les patients les plus susceptibles de bénéficier d'une telle chimiothérapie et les cliniciens peuvent surveiller l'effet des agents chimiothérapeutiques connus et potentiels. Notamment, on a développé des méthodes destinées à la synthèse efficace de [18F]FBG et [131I]IBG, qui se lient tous deux spécifiquement à l'AGT purifiée. On peut utiliser ces deux analogues de benzylguanine O6 marqués pour doser des niveaux d'AGT in vivo.
PCT/US2001/015370 2000-05-12 2001-05-14 Reactifs et methodes de dosage des niveaux de $i(0)6-alkylguanine-adn alkyltransferase $i(in vivo) (agt) Ceased WO2001085221A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004031405A1 (fr) * 2002-10-03 2004-04-15 Ecole Polytechnique Federale De Lausanne (Epfl) Substrats de o6-alkylguanine-adn alkyltransferase
WO2005085470A1 (fr) * 2004-03-02 2005-09-15 Epfl Ecole Polytechnique Federale De Lausanne Substrats specifiques de la o6-alkylguanine-adn alkyltransferase
WO2006052976A3 (fr) * 2004-11-09 2006-08-17 Schering Corp Methodes de traitement
WO2010034931A1 (fr) 2008-09-23 2010-04-01 Cis-Bio International Nouveaux substrats d'o6-alkylguanine-adn alkyltransferase et ses mutants
WO2011028507A3 (fr) * 2009-08-24 2011-07-14 Duke University Compositions, procédés et trousses pour déterminer une alkyl transférase

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
E. MOUNETOU ET AL.: "Synthesis of three no-carrier-added o6-4-[125i] iodobenzylguanosine derivates, new reagents for the assay of o6-alkyguanine-dna alkyltransferase activity." J. LABEL.COMP. AND RADIOPH., vol. XXXVI, no. 12, 1995, pages 1215-1225, XP001073774 *
GINA M. CIOCCO ET AL.: "Specific labeling of 06-alkylguanine-DNA Alkyltransferase by reaction with O6-(p-hydroxy[3HÜmethylbenzyl)guanine" CANCER RESEARCH, vol. 55, 15 September 1995 (1995-09-15), pages 4085-4091, XP002200702 MD US cited in the application *
VAIDYANATHAN G ET AL: "Radiolabeled guanine derivatives for the in vivo mapping of O(6)- alkylguanine -DNA alkyltransferase: 6-(4-Ä(18)FÜFluoro-benzyloxy)- 9H-purin-2-ylamine and 6-(3-Ä(131)IÜIodo-benzyloxy)-9H-purin-2-yl amine." BIOCONJUGATE CHEMISTRY, (2000 NOV-DEC) 11 (6) 868-75. , XP002200703 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004031405A1 (fr) * 2002-10-03 2004-04-15 Ecole Polytechnique Federale De Lausanne (Epfl) Substrats de o6-alkylguanine-adn alkyltransferase
US7799524B2 (en) 2002-10-03 2010-09-21 Ecole Polytechnique Ferdeale de Lausanne Substrates for O6-alkylguanina-DNA alkyltransferase
WO2005085470A1 (fr) * 2004-03-02 2005-09-15 Epfl Ecole Polytechnique Federale De Lausanne Substrats specifiques de la o6-alkylguanine-adn alkyltransferase
JP2007526282A (ja) * 2004-03-02 2007-09-13 ウペエフエル・エコル・ポリテクニック・フェデラル・ドゥ・ローザンヌ Ｏ６−アルキルグアニンｄｎａアルキルトランスフェラーゼ特異的基質
US8163479B2 (en) 2004-03-02 2012-04-24 Ecole Polytechnique Federale De Lausanne Specific substrates for O6-alkylguanine-DNA alkyltransferase
WO2006052976A3 (fr) * 2004-11-09 2006-08-17 Schering Corp Methodes de traitement
JP2008519584A (ja) * 2004-11-09 2008-06-12 シェーリング コーポレイション 患者のｍｇｍｔレベルに基づいて癌を処置するための、テモゾロミドの改良された投薬養生法
WO2010034931A1 (fr) 2008-09-23 2010-04-01 Cis-Bio International Nouveaux substrats d'o6-alkylguanine-adn alkyltransferase et ses mutants
WO2011028507A3 (fr) * 2009-08-24 2011-07-14 Duke University Compositions, procédés et trousses pour déterminer une alkyl transférase
US20120270812A1 (en) * 2009-08-24 2012-10-25 Duke University Compositions, methods, and kits for determining an alkyl transferase

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