A simple and inexpensive method to control oxygen concentrations within physiological and neoplastic ranges.

Traditional methods for regulating oxygen concentration ([O2]) in in vitro experiments over the range found in normal and tumor tissues require the use of expensive equipment to generate controlled gas atmospheres or the purchase of a range of gas cylinders with certified O2 percentages. Here we describe a simple and inexpensive enzymatic method for generating low, precise steady-state [O2] levels that are stable for several hours. This method is particularly applicable to the in vitro study of some classes of hypoxia-targeted antitumor prodrugs and bioreductively activated agents.

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (VNP40101M): I. Direct inhibition of O6-alkylguanine-DNA alkyltransferase (AGT) by electrophilic species generated by decomposition.

PURPOSE: To investigate the interaction of the electrophilic species generated by the decomposition of the antineoplastic prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (VNP40101M) on the ability of O(6)-alkylguanine-DNA alkyltransferase (AGT) to repair alkylated O(6)-chloroethylguanine and/or N(1),O(6)-ethanoguanine DNA lesions. MATERIALS AND METHODS: The contributions of inhibitory electrophilic species generated from VNP40101M towards AGT was assessed using analogues that selectively generated either the chloroethylating or the carbamoylating components of VNP40101M. The activity of AGT was determined from the inhibition of crosslink formation from O(6)-chloroethylguanine and/or N(1),O(6)-ethanoguanine lesions. The half-lives of sulfonylhydrazine derivatives and isocyanates were measured using an acidification assay which gives a change in absorbance proportional to the release or consumption of small quantities of protons. RESULTS: Both of the reactive components produced by VNP40101M directly inactivated cloned human AGT; the carbamoylating moiety (IC(50) about 13 micro M) was approximately seven- to eight-fold more potent than the alkylating component(s) (IC(50) about 100 micro M). These inhibitory actions were moderated by the addition of naked T5 bacteriophage DNA. Thus, AGT bound to DNA was markedly more resistant than free AGT to these electrophilic species. DNA also blocked the spontaneous loss of AGT activity which occurred upon incubation of this protein under mild conditions. CONCLUSIONS: The reaction of AGT with the methyl isocyanate generated from the decomposition of VNP40101M increased the net number of crosslinks generated by VNP40101M compared to a sulfonylhydrazine prodrug that formed the equivalent alkylating species in the absence of the cogeneration of methyl isocyanate. These actions may be of significance to the antineoplastic activity of VNP40101M.

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

PURPOSE: VNP40101M (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 O(6)-position of guanine, and a carbamoylating agent, methyl isocyanate, which inhibits O(6)-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. METHODS: AGT 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. RESULTS: VNP40101M 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. CONCLUSIONS: The findings support the concept that alkylation of the O(6)-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.

Inhibition of DNA cross-linking by mitomycin C by peroxidase-mediated oxidation of mitomycin C hydroquinone.

Mitomycin C requires reductive activation to cross-link DNA and express anticancer activity. Reduction of mitomycin C (40 microm) by sodium borohydride (200 microm) in 20 mm Tris-HCl, 1 mm EDTA at 37 degrees C, pH 7.4, gives a 50-60% yield of the reactive intermediate mitomycin C hydroquinone. The hydroquinone decays with first order kinetics or pseudo first order kinetics with a t(12) of approximately 15 s under these conditions. The cross-linking of T7 DNA in this system followed matching kinetics, with the conversion of mitomycin C hydroquinone to leuco-aziridinomitosene appearing to be the rate-determining step. Several peroxidases were found to oxidize mitomycin C hydroquinone to mitomycin C and to block DNA cross-linking to various degrees. Concentrations of the various peroxidases that largely blocked DNA cross-linking, regenerated 10-70% mitomycin C from the reduced material. Thus, significant quantities of products other than mitomycin C were produced by the peroxidase-mediated oxidation of mitomycin C hydroquinone or products derived therefrom. Variations in the sensitivity of cells to mitomycin C have been attributed to differing levels of activating enzymes, export pumps, and DNA repair. Mitomycin C hydroquinone-oxidizing enzymes give rise to a new mechanism by which oxic/hypoxic toxicity differentials and resistance can occur.

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino)carbonylhydrazine (101M): a novel sulfonylhydrazine prodrug with broad-spectrum antineoplastic activity.

Our laboratory has synthesized and evaluated the anticancer activity of a number of sulfonylhydrazine DNA modifying agents. As a class, these compounds possess broad spectrum antitumor activity, demonstrating significant activity against a variety of experimental murine tumors, including the P388 and L1210 leukemias, B16 melanoma, M109 lung carcinoma, and M5076 reticulum cell sarcoma, as well as against the human LX-1 lung carcinoma xenograft. The current report describes the activity of a more recently synthesized member of this class, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino)carbonylhydrazine (101M). 101M was active in mice against the i.p. implanted L1210 leukemia over a wide range of doses and produced long-term survivors when administered as a single i.p. bolus of 10, 20, 40, 60, or 80 mg/kg, demonstrating a wider margin of safety than the nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Curative therapy was achieved with doses of 101M that did not produce depression of the bone marrow. 101M was also highly effective against the L1210 leukemia when administered by the oral route. The ability of 101M to penetrate the blood-brain barrier and eradicate leukemia cells in the brain was remarkable (>6 log kill). This agent was also curative against L1210 variants resistant to cyclophosphamide, BCNU, or melphalan. Mice implanted with the murine C26 colon carcinoma were also cured by two injections of 10 or 20 mg/kg of 101M. Administration of 101M by two different well-tolerated regimens caused complete regression of established human glioblastoma U251 xenografts in 100% of treated mice, and significant responses were also obtained with 101M against advanced murine M109 lung carcinomas in mice. The broad spectrum of anticancer activity of the sulfonylhydrazine prodrug 101M coupled with the wide range of therapeutic safety exhibited by this agent, makes 101M particularly attractive for further development and clinical evaluation.

Comparison of DNA lesions produced by tumor-inhibitory 1,2-bis(sulfonyl)hydrazines and chloroethylnitrosoureas.

1,2-Bis(sulfonyl)hydrazine derivatives, designed to generate several of the electrophilic species classically believed to be responsible for the alkylating (chloroethylating) and/or carbamoylating activities of the chloroethylnitrosoureas (CNUs), were compared with respect to the cross-linking and nicking of T7 DNA to that caused by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and 1-(2-chloroethyl)-3-(4-trans-methylcyclohexyl)-1-nitrosourea (MeCCNU). In the case of BCNU, a large proportion of T7 DNA strand nicking was found to be due to the generation of 2-chloroethylamine, produced from the hydrolysis of 2-chloroethylisocyanate, in turn formed during the decomposition of the parental nitrosourea. 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (compound 1) gave a greater yield of DNA cross-links than the CNUs. Compound 1, as well as its derivatives that were incapable of generating 2-chloroethylisocyanate, did not produce detectable levels of strand nicking, indicating that N7-alkylation of guanine did not occur to a significant extent with these agents. Since compound 1 and its derivatives are believed to generate chloronium and chloroethyldiazonium ions, it would appear that these species could not be significantly involved in the N7-alkylation of guanine caused by the CNUs. The relatively low level of N7-alkylation of guanine residues and the relatively high yield of cross-links generated by some of the 1,2-bis(sulfonyl)-1-(2-chloroethyl)hydrazine derivatives implies that they are more exclusive O6-guanine chloroethylating agents than the CNUs. O6-Guanine chloroethylation is believed to be the therapeutically relevant event produced by the CNUs; therefore, compound 1 derivatives represent promising new cancer chemotherapeutic agents, since they appear to generate lower quantities of therapeutically unimportant, yet carcinogenic lesions, and more of the therapeutically relevant O6-guanine chloroethylation than the CNUs.

Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA.

The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from cross-linking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism.

Hypoxia-selective nitrobenzyloxycarbonyl derivatives of 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazines.

Some 4- and 2-(nitrobenzyloxycarbonyl)-1, 2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazines (4, 6, and 7) were synthesized and evaluated for their ability to exert preferential toxicity to hypoxic EMT6 mammary carcinoma cells using a colony-forming assay. Of these, the 4,5-dimethoxy-2-nitro analogue 6 (50 microM, 1-h exposure) caused greater than 3 logs of kill of hypoxic cells, with relatively minor toxicity to corresponding aerobic cells. The ability of 4-nitro (4) and 4,5-dimethoxy-2-nitro (6) analogues to reach and kill hypoxic cells of solid tumors was also demonstrated using intradermally implanted EMT6 solid tumors in mice. In addition, a possible source of toxicity to normal tissue, i. e., the activation of the 4-nitrobenzyl derivative 4 by glutathione S-transferase-catalyzed thiolysis, was essentially eliminated by replacing one of the benzylic methylene protons by a methyl group. The 4-nitro (4) and 4,5-dimethoxy-2-nitro (6) analogues also appear to be reduced more easily under acidic conditions (pH 6.0) than under neutral conditions, as measured by differential pulse polarography. Since the pH in hypoxic regions is often lower than that in adjacent aerobic regions, this property should aid in the cytotoxic action of these agents against hypoxic cells of solid tumors.

Spectrophotometric assay for processes involving changes in hydrogen ion concentration in aqueous solution.

A strategy for the design of simple colorimetric assays to follow any process that involves the net generation or consumption of hydrogen ions in aqueous solutions over the pH range of 3-10 is described. This procedure relies upon the measurement of the change in absorption when a weakly or moderately buffered solution of a pH indicator is subjected to a small change in pH. Buffers and indicators are chosen with closely matching pKa values. The versatility of this type of assay technique is illustrated using three examples.

Antitumor 2-(aminocarbonyl)-1,2-bis(methylsulfonyl)-1-(2-chloroethyl)- hydrazines.

Several 2-(aminocarbonyl)-1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydr azi nes were synthesized and primarily evaluated for antitumor activity against the murine L1210 leukemia. All of the compounds tested were capable of producing "cures" of mice bearing this tumor. One of the most active agents of this class, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)- 2(-)[[2-chloroethyl)-amino]carbonyl]hydrazine, was further evaluated against a spectrum of transplanted murine and human solid tumors. Pronounced activity was found against all of the tumors including the murine B16F10 melanoma, M109 lung carcinoma, M5076 reticulum cell sarcoma, and the human LX-1 lung carcinoma. The activities observed compared favorably with those of the established antitumor drugs, cyclophosphamide, mitomycin C, and the nitrosoureas, evaluated concomitantly.

Studies on the mechanism of decomposition and structural factors affecting the aqueous stability of 1,2-bis(sulfonyl)-1-alkylhydrazines.

1,2-Bis(sulfonyl)-1-alkylhydrazines are highly active experimental antineoplastic agents which decompose with first-order kinetics in neutral aqueous solutions. These agents generate approximately 2 mol of the corresponding sulfinate, 1 mol of nitrogen, and 1 mol of the appropriate alcohol, produced as a consequence of the alkylation of water. Increasing the leaving-group ability of the sulfonyl moiety on N-1 shortens the half-life, while the converse happens with N-2 substitutions. Linear Hammett relationships are found for both types of substitutions. The predictable kinetics of decomposition makes these agents potential candidates for use in regional chemotherapy, where compounds with tunable short half-lives may offer some advantage. Prodrugs of extremely short-lived derivatives of this class may also have utility as targeted alkylating agents.

Synthesis and evaluation of 1-acyl-1,2-bis(methylsulfonyl)-2-(2- chloroethyl)hydrazines as antineoplastic agents.

A series of 1-acyl-1,2-bis(methylsulfonyl)-2-(2-chloroethyl)hydrazines, conceived as more potent analogs of 1-(2-chloroethyl)-1,2,2-tris(methylsulfonyl)hydrazine, were synthesized and evaluated for antineoplastic activity against the L1210 leukemia in mice. Of these, 1-acetyl-1,2-bis-(methylsulfonyl)-2-(2-chloroethyl)hydrazine produced "cures" of mice bearing the L1210 leukemia at dosage levels that were considerably less than those at which the tris(sulfonyl) analog produced its antineoplastic effects. This compound was also found to have pronounced activity against the P388 leukemia and against several solid tumors, including the B16F10 melanoma, the M5076 reticulum cell sarcoma, and the M109 lung carcinoma. Furthermore, the acyl derivatives were in general considerably more resistant to hydrolysis in aqueous media and more prone to protease- and thiol-mediated activation than the tris(sulfonyl) analog. The former property is important to formulation, while the latter properties may result in some degree of drug targeting and enhancement of the therapeutic indices of these agents.

The effects of the methylating agent 1,2-bis(methylsulfonyl)-1-methylhydrazine on morphology, DNA content and mitochondrial function of Trypanosoma brucei subspecies.

Repeated exposure of trypanosomes in vitro or in vivo to low concentrations of the methylating agent 1,2-bis(methylsulfonyl)-1-methylhydrazine induces a series of moderately synchronous morphological and biochemical changes. Cell division halts and the long-slender bloodstream forms transform to short-stumpy forms via larger intermediate-stage cells which contain approximately double the normal G2 content of DNA. In common with naturally occurring short-stumpy trypanosomes, drug-induced short-stumpy forms do not infect rodents and when transferred to Cunningham's medium, transform to and replicate as procyclics. Furthermore, these short-stumpy forms exhibit alpha-ketoglutarate supported motility and oxygen consumption, acquire the ability to reduce nitroblue tetrazolium (NADH diaphorase positivity) and appear to be in the G1 or G0 stage of the cell cycle based upon DNA content.

Synthesis and evaluation of 1,2,2-tris(sulfonyl)hydrazines as antineoplastic and trypanocidal agents.

Several 1,2,2-tris(sulfonyl)hydrazines, conceived as prodrugs of 1,2-bis(sulfonyl)hydrazines, were synthesized and evaluated for antineoplastic and trypanocidal activities in mice. 1-Methyl-1,2,2-tris(methylsulfonyl)hydrazine emerged as an extremely efficacious antitrypanosomal agent, whereas 1-(2-chloroethyl)-1,2,2-tris(methylsulfonyl)hydrazine was inactive. In contrast, 1-(2-chloroethyl)-1,2,2-tris(methylsulfonyl)hydrazine displayed potent antineoplastic activity, producing several 60-day "cures" of mice bearing leukemia L1210, leukemia P388, or Sarcoma 180. Furthermore, the fact that the tris(sulfonyl) derivatives will not generate isocyanates, which contribute to the host toxicity of nitrosoureas like 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), makes them agents of significant promise in trypanosomal and cancer chemotherapy.

Methylating agents as trypanocides.

Methylating agents, such as streptozotocin, procarbazine, N-methyl-N-nitrosourea, dimethyl sulfate, 1,2-dimethylhydrazine, and a series of 1,2-bis(sulfonyl)-1-methylhydrazines synthesized in this laboratory, were evaluated and shown to be therapeutically active against murine models of African trypanosomiasis. At high dose levels, methylating agents halted trypanosome proliferation and transformed cells into bizarre forms containing multiple nuclei and kinetoplasts. These cells disappeared from the bloodstream of mice bearing these organisms in 48-72 h. When administered at repetitive low doses, methylating agents induced the entire population of trypanosomes to differentiate into biochemically distinct short-stumpy forms in a synchronous manner. These results suggest that methylating agents may be used as biochemical tools in the study of trypanosome differentiation.