Raymond P. Baumann

The antineoplastic efficacy of the prodrug Cloretazine is produced by the synergistic interaction of carbamoylating and alkylating products of its activation

Cloretazine {1,2-bis(methylsulfonyl)-1-[(2-chloroethyl)-2-(methylamino)carbonyl]hydrazine; VNP40101M; 101M} is a sulfonylhydrazine prodrug that possesses broad spectrum antitumor efficacy against transplanted murine and human tumor models and has shown activity in clinical trials against relapsed or refractory acute myeloid leukemia. Base catalyzed activation of this prodrug generates two different reactive intermediates: chloroethylating species that covalently interact with DNA at the O6-position of guanine residues that progress to a G-C interstrand cross-link, and a carbamoylating agent, methyl isocyanate. Previous findings from this laboratory have provided initial evidence that methyl isocyanate can contribute to the efficacy of Cloretazine by enhancing the cytotoxicity of the generated chloroethylating species. This action may be due in part to inhibition of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT); however, activity in cells devoid of AGT indicates that other actions are involved in the synergistic cytotoxicity. Herein we demonstrate that O6-benzylguanine can also produce synergistic cell kill with the alkylating component of Cloretazine but differs from methyl isocyanate in that the enhancement occurs in AGT-containing cells, but not in cells devoid of AGT. Methyl isocyanate generated by the decomposition of 1,2-bis(methylsulfonyl)-1-[methylaminocarbonyl]hydrazine also acts to enhance the activity of a variety of DNA cross-linking agents, while only producing additive cytotoxicity with methylating agents. Flow cytometric studies using annexin as a marker for apoptosis indicate that in Chinese hamster ovary cells and in human leukemia cells Cloretazine-induced apoptosis is primarily caused by the generated methyl isocyanate. Comet assays designed to detect DNA cross-links in intact cells indicate that the chloroethylating species generated by the activation of Cloretazine produce DNA cross-links, with the co-generated methyl isocyanate increasing the degree of cross-linking produced by the reactive chloroethylating species. These findings provide further evidence that the methyl isocyanate produced by the activation of Cloretazine can be a major contributor to the cytotoxicity produced by this antineoplastic agent.

Generation of Oxygen Deficiency in Cell Culture Using a Two- Enzyme System to Evaluate Agents Targeting Hypoxic Tumor Cells

Abstract Baumann, R. P., Penketh, P. G., Seow, H. A., Shyam, K. and Sartorelli, A. C. Generation of Oxygen Deficiency in Cell Culture Using a Two-Enzyme System to Evaluate Agents Targeting Hypoxic Tumor Cells. Radiat. Res. 170, 651–660 (2008). The poor and aberrant vascularization of solid tumors makes them susceptible to localized areas of oxygen deficiency that can be considered sites of tumor vulnerability to prodrugs that are preferentially activated to cytotoxic species under conditions of low oxygenation. To readily facilitate the selection of agents targeted to oxygen-deficient cells in solid tumors, we have developed a simple and convenient two-enzyme system to generate oxygen deficiency in cell cultures. Glucose oxidase is employed to deplete oxygen from the medium by selectively oxidizing glucose and reducing molecular oxygen to hydrogen peroxide; an excess of catalase is also used to scavenge the peroxide molecules. Rapid and sustained depletion of oxygen occurs in medium or buffer, even in the presence of oxygen at the liquid/air interface. Studies using CHO/AA8 Chinese hamster cells, EMT6 murine mammary carcinoma cells, and U251 human glioma cells indicate that this system generates an oxygen deficiency that produces activation of the hypoxia-targeted prodrug KS119. This method of generating oxygen deficiency in cell culture is inexpensive, does not require cumbersome equipment, permits longer incubation times to be used without the loss of sample volume, and should be adaptable for high-throughput screening in 96-well plates.

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (VNP40101M): II. Role of O6-alkylguanine-DNA alkyltransferase in cytotoxicity

PurposeVNP40101M (1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine) is a sulfonylhydrazine prodrug that possesses broad spectrum antitumor efficacy in murine models. VNP40101M activation generates chloroethylating species that alkylate DNA at the O6-position of guanine, and a carbamoylating agent, methyl isocyanate, which inhibits O6-alkylguanine-DNA alkyltransferase (AGT) in model systems. We determined whether expression of AGT in Chinese hamster ovary (CHO) cells decreased sensitivity to VNP40101M and explored the mechanism of VNP40101M cytotoxicity by employing analogs of VNP40101M that generate reactive intermediates with either carbamoylating or chloroethylating activity.MethodsAGT was overexpressed in CHO cells by transfection with an expression vector containing the human AGT gene. Cell lines expressing AGT were employed in clonogenic assays to determine the cytotoxicity of VNP40101M and its analogs.ResultsVNP40101M was more active against AGT-expressing CHO cells than 90CE (1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine), a chloroethylating generator devoid of carbamoylating activity. Furthermore, the greater the degree of AGT expression the more resistance to VNP40101M cytotoxicity. Combination chemotherapy experiments support the conclusions that methyl isocyanate and the chloroethylating species generated from the activation of VNP40101M function synergistically to kill cells.ConclusionsThe findings support the concept that alkylation of the O6-position of guanine residues in DNA is the predominant lesion created by VNP40101M, and that methyl isocyanate resulting from the base-catalyzed activation of VNP40101M inhibits AGT and presumably other enzymes involved in DNA repair, thereby enhancing the yield of the DNA G-C interstrand crosslinks responsible for the antitumor activity of this agent.

Bioactivation and Resistance to Mitomycin C

Publisher Summary This chapter analyzes the bioactivation and resistance of tumor cells to mitomycin C (MC). MC is a naturally occurring antibiotic that was isolated originally from the microorganism Streptomyces caspitosus . MC exerts its antitumor activity primarily by damaging DNA through both monofunctional and bifunctional alkylations (cross-links). Numerous studies have employed HPLC separation methods to identify specific MC-DNA lesions associated with MC treatment. Monoalkylations initially occur through the C1 position of MC to the N2 position of aguanine base in DNA and may proceed to form a DNA cross-link through the C10 position of MC to an adjacent DNA guanine at its N2 position. MC-induced cross-links are believed to be primarily responsible for cell death. A single cross-link per genome has been reported to be sufficient to cause the death of a bacterial cell. In this chapter, properties of MC as a prototypic bioreductive agent are described, and mechanism of the reductive activation of MC is elaborated. The chapter presents an overview of MC resistance protein A (MCRA). Mammalian MCRA functional homolog is discussed and rapid screening for DTD activity by using a microtiter assay is discussed. Methodology for the indirect determination of MC activation is also described in the chapter.

Lethality to leukemia cell lines of DNA interstrand cross-links generated by Cloretazine derived alkylating species.

Cloretazine [1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine; VNP40101M; 101M] is a relatively new prodrug with activity in elderly acute myelogenous leukemia (AML) patients. Its therapeutic action is due largely to the production of 1-(3-cytosinyl),2-(1-guanyl)ethane cross-links (G-C ethane cross-links) in DNA. The numbers of cross-links produced in three experimental leukemia lines (L1210, U937 and HL-60) were fewer than 10 per genome at their respective LC50 concentrations. Only 1 in approximately 20,000 90CE molecules produces a cross-link in the AGT (O6-alkylguanine-DNA alkyltransferase) negative L1210 and U937 cell lines and 1 in 400,000 in the AGT positive HL-60 cell line.

Quantitative relationship between guanine O6-alkyl lesions produced by Onrigin™ and tumor resistance by O6-alkylguanine-DNA alkyltransferase

O(6)-Alkylguanine-DNA alkyltransferase (AGT) mediates tumor resistance to alkylating agents that generate guanine O(6)-chloroethyl (Onrigin™ and carmustine) and O(6)-methyl (temozolomide) lesions; however, the relative efficiency of AGT protection against these lesions and the degree of resistance to these agents that a given number of AGT molecules produces are unclear. Measured from differential cytotoxicity in AGT-ablated and AGT-intact HL-60 cells containing 17,000 AGT molecules/cell, AGT produced 12- and 24-fold resistance to chloroethylating (90CE) and methylating (KS90) analogs of Onrigin™, respectively. For 50% growth inhibition, KS90 and 90CE generated 5,600 O(6)-methylguanines/cell and ∼300 O(6)-chloroethylguanines/cell, respectively. AGT repaired O(6)-methylguanines until the AGT pool was exhausted, while its repair of O(6)-chloroethylguanines was incomplete due to progression of the lesions to AGT-irreparable interstrand DNA cross-links. Thus, the smaller number of O(6)-chloroethylguanine lesions needed for cytotoxicity accounted for the marked degree of resistance (12-fold) to 90CE produced by AGT. Transfection of human or murine AGT into AGT deficient transplantable tumor cells (i.e., EMT6, M109 and U251) generated transfectants expressing AGT ranging from 4,000 to 700,000 molecules/cell. In vitro growth inhibition assays using these transfectants treated with 90CE revealed that AGT caused a concentration dependent resistance up to a level of ∼10,000 AGT molecules/cell. This finding was corroborated by in vivo studies where expression of 4,000 and 10,000 murine AGT molecules/cell rendered EMT6 tumors partially and completely resistant to Onrigin™, respectively. These studies imply that the antitumor activity of Onrigin™ stems from guanine O(6)-chloroethylation and define the threshold concentration of AGT that negates its antineoplastic activity.

4-nitrobenzyloxycarbonyl derivatives of O(6)-benzylguanine as hypoxia-activated prodrug inhibitors of O(6)-alkylguanine-DNA alkyltransferase (AGT), which produces resistance to agents targeting the O-6 position of DNA guanine.

A series of 4-nitrobenzyloxycarbonyl prodrug derivatives of O(6)-benzylguanine (O(6)-BG), conceived as prodrugs of O(6)-BG, an inhibitor of the resistance protein O(6)-alkylguanine-DNA alkyltransferase (AGT), were synthesized and evaluated for their ability to undergo bioreductive activation by reductase enzymes under oxygen deficiency. Three agents of this class, 4-nitrobenzyl (6-(benzyloxy)-9H-purin-2-yl)carbamate (1) and its monomethyl (2) and gem-dimethyl analogues (3), were tested for activation by reductase enzyme systems under oxygen deficient conditions. Compound 3, the most water-soluble of these agents, gave the highest yield of O(6)-BG following reduction of the nitro group trigger. Compound 3 was also evaluated for its ability to sensitize 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (laromustine)-resistant DU145 human prostate carcinoma cells, which express high levels of AGT, to the cytotoxic effects of this agent under normoxic and oxygen deficient conditions. While 3 had little or no effect on laromustine cytotoxicity under aerobic conditions, significant enhancement occurred under oxygen deficiency, providing evidence for the preferential release of the AGT inhibitor O(6)-BG under hypoxia.

KS900: A hypoxia-directed, reductively activated methylating antitumor prodrug that selectively ablates O6-alkylguanine-DNA alkyltransferase in neoplastic cells

To most effectively treat cancer it may be necessary to preferentially destroy tumor tissue while sparing normal tissues. One strategy to accomplish this is to selectively cripple the involved tumor resistance mechanisms, thereby allowing the affected anticancer drugs to gain therapeutic efficacy. Such an approach is exemplified by our design and synthesis of the intracellular hypoxic cell activated methylating agent, 1,2-bis(methylsulfonyl)-1-methyl-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS900) that targets the O-6 position of guanine in DNA. KS900 is markedly more cytotoxic in clonogenic experiments under conditions of oxygen deficiency than the non-intracellularly activated agents KS90, and 90M, when tested in O(6)-alkylguanine-DNA alkyltransferase (AGT) non-expressing cells (EMT6 mouse mammary carcinoma, CHO/AA8 hamster ovary, and U251 human glioma), and than temozolomide when tested in AGT expressing cells (DU145 human prostate carcinoma). Furthermore, KS900 more efficiently ablates AGT in HL-60 human leukemia and DU145 cells than the spontaneous globally activated methylating agent KS90, with an IC(50) value over 9-fold lower than KS90. Finally, KS900 under oxygen-deficient conditions selectively sensitizes DU145 cells to the chloroethylating agent, onrigin, through the ablation of the resistance protein AGT. Thus, under hypoxia, KS900 is more cytotoxic at substantially lower concentrations than methylating agents such as temozolomide that are not preferentially activated in neoplastic cells by intracellular reductase catalysts. The necessity for intracellular activation of KS900 permits substantially greater cytotoxic activity against cells containing the resistance protein O(6)-alkylguanine-DNA alkyltransferase (AGT) than agents such as temozolomide. Furthermore, the hypoxia-directed intracellular activation of KS900 allows it to preferentially ablate AGT pools under the oxygen-deficient conditions that are present in malignant tissue.

Distinct mechanisms of cell-kill by triapine and its terminally dimethylated derivative Dp44mT due to a loss or gain of activity of their copper(II) complexes

Triapine, currently being evaluated as an antitumor agent in phase II clinical trials, and its terminally dimethylated derivative Dp44mT share the α-pyridyl thiosemicarbazone backbone that functions as ligands for transition metal ions. Yet, Dp44mT is approximately 100-fold more potent than triapine in cytotoxicity assays. The aims of this study were to elucidate the mechanisms underlying their potency disparity and to determine their kinetics of cell-kill in culture to aid in the formulation of their clinical dosing schedules. The addition of Cu(2+) inactivated triapine in a 1:1 stoichiometric fashion, while it potentiated the cytotoxicity of Dp44mT. Clonogenic assays after finite-time drug-exposure revealed that triapine produced cell-kill in two phases, one completed within 20 min that caused limited cell-kill, and the other occurring after 16 h of exposure that produced extensive cell-kill. The ribonucleotide reductase inhibitor triapine at 0.4 μM caused immediate complete arrest of DNA synthesis, whereas Dp44mT at this concentration did not appreciably inhibit DNA synthesis. The inhibition of DNA synthesis by triapine was reversible upon its removal from the medium. Cell death after 16 h exposure to triapine paralleled the appearance of phospho-(γ)H2AX, a marker of DNA double-strand breaks induced by collapse of DNA replication forks after prolonged replication arrest. In contrast to triapine, Dp44mT produced robust cell-kill within 1h in a concentration-dependent manner. The short-term action of both agents was prevented by thiols, indicative of the involvement of reactive oxygen species. The time dependency in the production of cell-kill by triapine should be considered in treatment regimens.

Reductive activation of the prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) selectively occurs in oxygen-deficient cells and overcomes O6-alkylguanine-DNA alkyltransferase mediated KS119 tumor cell resistance

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) is a prodrug of the 1,2-bis(sulfonyl)hydrazine class of antineoplastic agents designed to exploit the oxygen-deficient regions of cancerous tissue. Thus, under reductive conditions in hypoxic cells this agent decomposes to produce the reactive intermediate 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which in turn generates products that alkylate the O(6)-position of guanine in DNA. Comparison of the cytotoxicity of KS119 in cultured cells lacking O(6)-alkylguanine-DNA alkyltransferase (AGT) to an agent such as Onrigin, which through base catalyzed activation produces the same critical DNA G-C cross-link lesions by the generation of 90CE, indicates that KS119 is substantially more potent than Onrigin under conditions of oxygen deficiency, despite being incompletely activated. In cell lines expressing relatively large amounts of AGT, the design of the prodrug KS119, which requires intracellular activation by reductase enzymes to produce a cytotoxic effect, results in an ability to overcome resistance derived from the expression of AGT. This appears to derive from the ability of a small portion of the chloroethylating species produced by the activation of KS119 to slip through the cellular protection afforded by AGT to generate the few DNA G-C cross-links that are required for tumor cell lethality. The findings also demonstrate that activation of KS119 under oxygen-deficient conditions is ubiquitous, occurring in all of the cell lines tested thus far, suggesting that the enzymes required for reductive activation of this agent are widely distributed in many different tumor types.