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When mice bearing ascitic L5178Y or L1210 lymphoblasts were treated with a combination of 1-β- d -arabinofuranosylcytosine (cytosine arabinoside) and porfiromycin, the median survival time and the number of survivors were increased. The combination is most effective when treatment is started within 24 hr after implanting the leukemie cells. The synergistic effect is noted only when the dosage level of the cytosine arabinoside component of the combination is optimal. The same synergistic effects were noted when the combination of mitomycin C and cytosine arabinoside was used.

In high performance liquid chromatography with electrochemical detection (HPLC/ECD) much attention has been paid to the performance of the applied detection system with respect to reliability and sensitivity. In general, insufficient attention has been paid to relatively easy to implant devices for improved collection and handling of detection signals. Four models of software filtering are studied and compared with hardware filtering. In the investigated chromatographic-electrochemical system, off-line parabolic filtering after on-line averaging of sixteen measurements proved to be the system of first choice, with respect to execution time, noise level, signal to noise ratio, peak height and resolution.

The bioreductive alkylating agent porfiromycin (POR) is more toxic to EMT6 cells that are hypoxic at the time of treatment than to aerobic cells. The toxicity of POR to hypoxic EMT6 cells in vitro was similar to that of mitomycin C (MC): the aerobic toxicity of POR was considerably less than that of MC. Treatment of cells in vitro with POR before and during irradiation did not sensitize either hypoxic or aerobic cells to X rays; instead, only additive cytotoxicity was produced. In contrast, treatment of solid EMT6 tumors in vivo with POR plus radiation produced supra-additive cytotoxicity, as assessed by analyses of the complete dose-response curves for the killing of tumor cells by radiation alone or by POR alone. The supra-additivity of the combination regimens appeared to reflect the preferential killing by each agent of those tumor cells which were in an environment conferring resistance to the other agent. In contrast, combinations of POR and X rays produced only additive cytotoxicities to marrow CFU-GM. Supra-additive antineoplastic effects were obtained at doses of POR which produced little hematologic or other host toxicity. The complementary cytotoxicities of radiation and POR to cells in different microenvironments in solid tumors and the absence of a similar effect in normal tissue make optimized regimens combining radiotherapy and POR unusually promising for the treatment of solid tumors.

The Kyowa Hakko Kogyo and Bristol-Myers Squibb companies reported that select mitomycin C(7) aminoethylene disulfides displayed improved pharmacological profiles compared with mitomycin C (1). Mechanisms have been advanced for these mitomycins that differ from 1. Central to many of these hypotheses is the intermediate generation of 7-N-(2-mercaptoethyl)mitomycin C (5). Thiol 5 has been neither isolated nor characterized. Two efficient methods were developed for mitomycin (porfiromycin) C(7)-substituted thiols. In the first method, the thiol was produced by a thiol-mediated disulfide exchange process using an activated mixed mitomycin disulfide. In the second route, the thiol was generated by base-mediated cleavage of a porfiromycin C(7)-substituted thiol ester. We selected four thiols, 7-N-(2-mercaptoethyl)mitomycin C (5), 7-N-(2-mercaptoethyl)porfiromycin (12), 7-N-(2-mercapto-2-methylpropyl)mitomycin C (13), and 7-N-(3-mercaptopropyl)porfiromycin (14), for study. Thiols 5 and 12-14 differed in the composition of the alkyl linker that bridged the thiol with the mitomycin (porfiromycin) C(7) amino substituent. Thiol generation was documented by HPLC and spectroscopic studies and by thiol-trapping experiments. The linker affected the structure of the thiol species and the stability of the thiol. We observed that thiols 5 and 12 existed largely as their cyclic isomers. Evidence is presented that cyclization predominantly occurred at the mitomycin C(7) position. Correspondingly, alkyl linker substitution (13) or extension of the linker to three carbons (14) led to enhanced thiol stability and the predominant formation of the free thiol species. The dominant reaction of thiols 5 and 12-14 or their isomers was dimerization, and we found no evidence that thiol formation led to mitosene production and aziridine ring-opening. These findings indicated that thiol generation was not sufficient for mitomycin ring activation. The potential pharmacological advantages of mitomycin C(7) aminoethylene disulfides compared with 1 is discussed in light of the observed thiol cyclization pathway.

One of the key issues for radiobiologists is the importance of hypoxia to the radiotherapy response. This review addresses the reasons for this and primarily focuses on one aspect, the development of bioreductive drugs that are specifically designed to target hypoxic tumour cells. Four classes of compound have been developed since this concept was first proposed: quinones, nitroaromatics, aliphatic and heteroaromatic N-oxides. All share two characteristics: (1) they require hypoxia for activation and (2) this activation is dependent on the presence of specific reductases. The most effective compounds have shown the ability to enhance the anti-tumour efficacy of agents that kill better-oxygenated cells, i.e. radiation and standard cytotoxic chemotherapy agents such as cisplatin and cyclophosphamide. Tirapazamine (TPZ) is the most widely studied of the lead compounds. After successful pre-clinical in vivo combination studies it entered clinical trial; over 20 trials have now been reported. Although TPZ has enhanced some standard regimens, the results are variable and in some combinations toxicity was enhanced. Banoxantrone (AQ4N) is another agent that is showing promise in early phase I/II clinical trials; the drug is well tolerated, is known to locate in the tumour and can be given in high doses without major toxicities. Mitomycin C (MMC), which shows some bioreductive activation in vitro, has been tested in combination trials. However, it is difficult to assign the enhancement of its effects to targeting of the hypoxic cells because of the significant level of its hypoxia-independent toxicity. More specific analogues of MMC, e.g. porfiromycin and apaziquone (EO9), have had variable success in the clinic. Other new drugs that have good pre-clinical profiles are PR 104 and NLCQ-1; data on their clinical safety/efficacy are not yet available. This paper reviews the pre-clinical data and discusses the clinical studies that have been reported.

N-7[2-(4-nitrophenyldithio)-ethyl] mitomycin C, (BMS-181174; previously designated as BMY25067) is a mitomycin C analog now in initial clinical trials. The experiments described in this report were performed to assess whether BMS-181174, like mitomycin C and porfiromycin, was selectively toxic to the hypoxic cells in solid tumors and might therefore prove valuable in combination with radiotherapy. In contrast to mitomycin C and porfiromycin, BMS-181174 was more toxic to aerobic EMT6 cells in vitro than to cells made acutely hypoxic. In vitro, BMS-181174 and radiation produced cytotoxicity compatible with either additive or slightly supra-additive cytotoxicity. In vivo, BMS-181174 was effective in killing cells in solid EMT6 tumors. The effects of regimens combining BMS-181174 and radiation in vivo were complex. Combinations of low doses of BMS-181174 plus a large dose of radiation were very effective in killing cells in solid tumors. However, the survival curve plateaued at high doses of BMS-181174, providing evidence for a subpopulation of tumor cells which were resistant to both BMS-181174 and radiation; this was hypothesized to be a hypoxic cell population.

Mitomycin C (1) is the prototypical bioreductive alkylating agent. Studies have shown that mitomycin C and its derivatives selectively alkylate guanine residues within di- and trinucleotide DNA sequences. This investigation sought to improve the selective DNA bonding properties of the mitomycins by coupling them with antisense oligodeoxynucleotides. Two procedures were developed that allowed the attachment of a phosphorothioate oligodeoxynucleotide containing a hexylamino spacer at the 5' terminus with a C(10)-activated mitomycin. In the first procedure, decarbamoylation of 1 (NaOCH3/ benzene) afforded 10-decarbamoylmitomycin C (10), which was treated with either dimethyl sulfate or methylthiochloroformate and base to yield 10-decarbamoylporfiromycin (11) and N(1a)-[(methylthio)-carbonyl]-10-decarbamoylmitomycin C (12), respectively. Activation of the C(10) site in 11 and 12 with 1,1'-carbonyldiimidazole or with 1,1'-thiocarbonyldiimidazole provided the N(1a)-substituted mitomycin 10-decarbamoyl-10-O-carbonylimidazoles (5, 7) and 10-decarbamoyl-10-O-thiocarbonylimidazoles (6, 8), respectively. Compounds 5-8 were reacted with glycine methyl ester hydrochloride (17) and base in both methylene chloride and aqueous buffered solutions to determine the ease and efficiency in which these C(10)-activated mitomycin derivatives coupled to amines. It was found that 5-8 all reacted with 17 in methylene chloride to give the coupled products 18-21 but that improved amine coupling yields in water were observed for the 10-decarbamoyl-10-O-thiocarbonylimidazoles 6 and 8 as compared with the 10-decarbamoyl-10-O-carbonylimidazoles 5 and 7. This finding led to the coupling of the phosphorothioate oligodeoxynucleotide, H2N(CH2)6-P(S)(OH)-GGCCCCGTG-GTGGCTCCAT (22) to 8. Compound 22 complemented a 19-base sequence in the translation initiation region of the human A-raf-1 gene. Use of excess 8 (28 equiv) with 22 gave only a 36% yield of the coupled product 23, which proved difficult to separate from 22. In the second procedure, phosphorothioate oligodexynucleotides that contained a hexylamino spacer at the 5'termini were coupled to 10-des(carbamoyloxy)-10-isothiocyanatoporfiromycin (9). Compound 9 was prepared in four steps from 11. Mesylation (methanesulfonyl chloride/pyridine) of 11 gave the C(10) mesylate 13, which was then treated with NaN3 (dimethylformamide, 90 degrees C) to give 10-des(carbamoyloxy)-10-azidoporfiromycin (14). Catalytic reduction (PtO2, H2) of 14 in pyridine afforded C(10) amine 15. Treatment of 15 with di-2-pyridyl thionocarbonate provided the desired 10-des(carbamoyloxy)-10-isothiocyanatoporfiromycin (9). Compound 9 readily coupled with 17 and base in both methylene chloride and aqueous buffered solutions to give 25. Use of the 5'hexylaminophosphorothioate oligodeoxynucleotides 32-35 in place of 17 gave the conjugated adducts 28-31, respectively, in a 12% to near-quantitative yield. The products were purified by semipreparative HPLC. Antisense agents 28-31 were designed to target a 30-base-long region from the coding region of the human FGFR1 gene. One adduct, 29, reduced the number of FGFR1 receptors in human aortic smooth cells for bFGF on the cell surface, which suggested down-regulation of FGFR1 gene expression. Further, 29 inhibited cultured human aortic smooth muscle cell proliferation and was less cytotoxic than porfiromycin (2). The biological assay data suggest that the phosphorothioate oligodexynucleotide porfiromycin conjugates may be more target selective and less toxic than either mitomycin or porfiromycin and thus be promising therapeutic agents.

The therapeutic potential of a variety of bioreductive agents, including misonidazole, RSU-1069, NFVO, mitomycin C, porfiromycin, and SR-4233 was evaluated using Chinese hamster V79 multicell spheroids in vitro. Fluorescence-activated cell sorting techniques were used to selectively recover cells from various depths within the spheroids to measure the differential cytotoxicity in the cells near the hypoxic core of the spheroid relative to the well oxygenated peripheral cells. At the high cell density found in spheroids (as in tissues in vivo) the differential toxicity observed was typically much less than expected, based on data from single cell systems. In some cases, this was due to lack of sufficient hypoxia in the spheroids; in other cases, drug treatment itself produced reoxygenation through metabolic or toxic effects during treatment. An unexpected observation of considerable concern was rapid bioreduction of the more active agents; this sometimes occurred at rates that exceeded drug delivery, resulting in considerably less efficacy when large hypoxic fractions were present (e.g. mitomycin C, NFVO, and SR-4233). This suggests that induction of hypoxia prior to bioreductive agent therapy may not be the most productive approach. Though none of the agents showed "ideal" properties, porfiromycin was judged to give the best combination of differential toxicity, longevity in situ, and ability to reach the entire hypoxic cell subpopulation.

Mitomycin-C, a widely used antitumor antibiotic, and its analogues mitomycin-A and porfiromycin are considered for AM1–RHF type semiempirical quantum chemical treatment. The structural and energetic aspects, as well as certain molecular orbital characteristics of these structures have been compared.

Several mitomycin antibiotics were evaluated for their capacities to kill EMT6 tumor cells and to produce DNA crosslinks under conditions of oxygenation and hypoxia. The agents examined included mitomycin C, porfiromycin, and the 7-aminomethyl dithioacetal derivative of mitomycin C (BMY-43324), all of which caused greater kill of hypoxic cells than of their oxygenated counterparts; the N,N'-dimethylaminomethylene derivative of mitomycin C (BMY-25282), which was considerably more cytotoxic under oxygenated conditions than in hypoxia; and the N,N'-dimethylaminomethylene derivative of porfiromycin (BL-6783), which was equal in its toxicity to hypoxic and oxygenated cells. All of these agents produced DNA crosslinks in EMT6 cells, as measured by alkaline elution. The number of crosslinks required to produce a given amount of cell kill was similar, regardless of the mitomycin employed or the degree of oxygenation, suggesting that the crosslinking of DNA was a major lesion in the cytodestructive action of the mitomycins.

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.

Abasic sites (AP sites) are generated in DNA either directly by DNA-damaging agents or by DNA glycosylases acting during base excision repair. These sites are repaired in human cells by the HAP1 protein, which, besides its AP-endonuclease activity, also possesses a redox function. To investigate the ability of HAP1 protein to modulate cell resistance to DNA-damaging agents, CHO cells were transfected with HAP1 cDNA, resulting in stable expression of the protein in the cell nuclei. The sensitivity of the transfected cells to the toxic effect of various agents, e.g. methylmethane sulfonate, bleomycin and H 2 O 2 , was not modified. However, the transfected cells became more sensitive to killing by mitomycin C, porfiromycin, daunorubicin and aziridinyl benzoquinone, drugs that are activated by reduction. To test whether the redox function of HAP1 protein was involved in this increased cytotoxicity, we have constructed a mutated HAP1 protein endowed with normal AP-endonuclease activity but deleted for redox function. When this mutated protein was expressed in the cells, elevated AP-endonuclease activity was measured, but sensitization to the lethal effects of compounds requiring bioreduction was no longer observed. These results suggest that HAP1 protein, besides its involvement in DNA repair, is able to activate bioreduction of alkylating drugs used in cancer chemotherapy.

The solvolysis rates for the substituted C(7)-cyclohexylamino- or C(8)-cyclohexyliminomitomycins 8-19 were determined in buffered methanolic solutions (0.06 M bis-Tris.HCl, pH: 5.5) at 25 degrees C and then compared with mitomycin C (1) and porfiromycin. Kinetic studies showed that C(8)-cyclohexyliminomitomycins 8-13 underwent solvolysis 150-230 times faster than mitomycin C (1) to give C(1)-methoxymitosene products. The solvolysis rates were slightly faster than that reported for 6. The C(7)-(2'-hydroxy)cyclohexylaminomitomycins 16-19 exhibited comparable solvolysis rates with 1 and porfiromycin.

An improved high-performance liquid chromatographic assay for the cytostatic drug mitomycin C in plasma is presented. The principal steps are precipitation of plasma proteins with acetonitrile, lyophilization of the supernatant and reversed-phase chromatography on a Hypersil ODS 5 microm column with 0.01 M NaH2PO4 buffer (pH 6.5)-methanol (70:30, v/v) in isocratic mode. At a flow-rate of 1.3 ml/min a column pressure of 180-220 bar resulted. Porfiromycin served as internal standard. UV detection was performed at 365 nm. Quantitation limit based on a coefficient of variation

A resistant subline (AH130/5A) selected from rat hepatoma AH130 cells after exposure to adriamycin (ADM) showed remarkable resistance to multiple antitumor drugs, including mitomycin C (MMC) and porfiromycin (PFM). PFM, vinblastine (VLB), and ADM accumulated in AH130/5A far less than in the parent AH130 (AH130/P) cells. AH130/5A cells showed overexpression of P-glycoprotein (PGP), an increase in glutathione S-transferase activity, and a decrease in DT-diaphorase and glutathione peroxidase activity. The resistance to MMC and VLB of AH130/5A cells was partly reversed by H-87, an inhibitor of PGP. Buthionine sulfoximine, an inhibitor of glutathione synthase, did not affect the action of MMC. tert-Butylhydroquinone induced DT-diaphorase activity, increased PFM uptake, and enhanced the growth-inhibitory action of MMC in AH130/5A cells. Dicumarol, an inhibitor of DT-diaphorase, decreased PFM uptake and reduced the growth-inhibitory action of MMC in AH130/P cells. These results indicated that the adriamycin treatment of hepatoma cells caused multifactorial multidrug resistance involving a decrease in DT-diaphorase activity.

DT-Diaphorase catalyzes a two-electron reduction of mitomycin C (MC) and porfiromycin (POR) to reactive species. Many cell lines that overexpress DT-diaphorase and are sensitive to the mitomycins are protected from the aerobic cytotoxicity of these drugs by the DT-diaphorase inhibitor dicumarol. The cytoprotective properties of this relatively non-specific inhibitor, however, vanish under hypoxic conditions. To ascertain the role of DT-diaphorase in mitomycin bioactivation and cytotoxicity in living cells, a rat liver DT-diaphorase cDNA was transfected into Chinese hamster ovary cells. MC was equitoxic to the parental cells under oxygenated and hypoxic conditions. In contrast, POR was less toxic than MC to these cells under aerobic conditions, but significantly more toxic than MC under hypoxia. Two DT-diaphorase-transfected clones displayed increases in DT-diaphorase activity of 126- and 133-fold over parental cells. The activities of other oxidoreductases implicated in mitomycin bioreduction were unchanged. MC was more toxic to both DT-diaphorase-transfected lines than to parental cells; the toxicity of MC to the transfected lines was similar in air and hypoxia. POR was also more toxic to the DT-diaphorase-elevated clones than to parental cells under oxygenated conditions. Under hypoxia, however, the toxicity of POR to the transfected clones was unchanged from that of parental cells. The findings implicate DT-diaphorase in mitomycin bioactivation in living cells, but suggest that this enzyme does not contribute to the differential toxicity of MC or POR in air and hypoxia.

Hypoxic-specific cytotoxins are a new, and as yet clinically untested, mode of treatment of solid tumors. If they can be given at high enough concentrations and sufficiently often, they should prove extremely effective both in combination with standard radiotherapy and also with certain chemotherapeutic drugs. It is likely that their optimum use with turn hypoxic cells in solid tumors from a therapeutic disadvantage to an advantage. In this report, we review the rationale for the use of hypoxia-specific cytotoxins, including both the theoretical basis for combining them with fractionated radiation and the preclinical results that have been obtained to date combining these agents with fractionated radiation. We also discuss the three major classes of bioreductive drugs, including the quinones (mitomycin C, porfiromycin, and E09), nitroaromatic compounds (including RB6145 and various deoxyribonucleic acid [DNA] targeted aromatics), and finally the N-oxides of which tirapazamine is the lead compound. We also briefly discuss new approaches to bioreductive drug development. The best ways to use these agents are also covered. These include using them in combination with radiation, in combination with chemotherapy, and in combination with agents that increase tumor hypoxia. Finally, the importance of the selection of patients for clinical trials is illustrated by showing how dramatically the number of patients in a clinical trial has to increase to obtain statistical significance for a procedure targeted towards hypoxic cells if some of the patients in the trials have well-oxygenated tumors.

Mitomycin C (MC), mitomycin A, porfiromycin, BMY-25067, and BMY-25287, antitumor antibiotics collectively termed "mitosanes", were found to have no appreciable binding affinity to various natural and synthetic DNAs, as tested by UV spectrophotometry and equilibrium dialysis. Further tests of DNA binding applied to MC including thermal melting measurements, displacement of ethidium fluorescence, and unwinding of closed circular DNA were similarly negative. In contrast, 2,7-diaminomitosene (2,7-DAM), a major end product of the reductive activation of MC, binds to the same series of DNAs by all of these criteria. In the presence of DNA its UV absorbance at the 313 nm maximum decreased and underwent a slight red shift. This effect was used for determining DNA binding constants (Kb) by the spectrophotometric titration method. At pH 6.0 the Kbs of three natural DNAs with varying GC content, as well as poly(dA-dT).poly(dA-dT), and poly(dG-dC).poly(dG-dC), were all in the range of (1.2-5.3) x 10(4) (M nucleotide)-1, with no apparent specificity of binding. Poly(dG-m5dC).poly(dG-m5dC) displayed a slightly higher Kb ((7.5-8.4) x 10(4)). Binding of other, closely related mitosenes was tested to calf thymus DNA by equilibrium dialysis. Neither the presence of a 1-OH substituent, removal of the 10-carbamoyl group, nor methylation of the 2-amino group modifies the binding affinity of the mitosenes significantly. The 1-phosphate substituent abolishes binding. The binding of 2,7-DAM to DNA increased with decreasing pH and decreasing ionic strength. It was determined that 2,7-DAM is protonated at the 2-amino group with a pKa = 7.55, and this correlated well with the observed pH dependence of the binding, indicating that the binding affinity has a strong electrostatic component. This was confirmed by the finding that the extrapolated Kb to 1 M Na+ concentration diminishes to only 10% of the value of Kb at 0.01 M Na+ concentration. Viscosity tests showed conclusively that 2,7-DAM intercalates in DNA, in a nonspecific manner. DNA binding by 2,7-DAM is shown to be a close model of the binding of the reduced activated form of MC, previously characterized indirectly [Teng, S. P., Woodson, S. A., and Crothers, D. M. (1989) Biochemistry 28, 3901-3907]. The nonspecific precovalent binding of the active form may serve in the cell to concentrate the drug at its critical target, DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Interleukin-1α (IL-1α) has potent antitumor activity either alone or combined with alkylating agents such as cisplatin and mitomycin C or porfiromycin. Cisplatin is an effective chemotherapeutic agent in the treatment of advanced squamous cell carcinoma of the head and neck (SCCHN). In the murine SCCVII/SF squamous cell carcinoma tumor model, IL-1α induced acute hemorrhagic necrosis and increased clonogenic cell kill. Objective: To determine the efficacy of cisplatin and IL-lα, singly and in combination, in the treatment of SCCHN. Methods: Syngeneic C3H/HeN mice were treated with single-dose, concurrent, intraperitoneal injections of cisplatin and interleukin-1α 14 days after subcutaneous tumor implantation and were monitored for delayed tumor regrowth. Results: Cisplatin alone, but not IL-1α induced significant delayed tumor regrowth when compared with control. The combination of IL-1α and cisplatin was even more effective in delaying tumor regrowth than cisplatin alone. Fractional tumor volume was significantly reduced in animals treated with the combination of cisplatin and IL-1α compared with those treated with IL-1α alone. Conclusions: Results of interleukin-1α and cisplatin dose-response experiments reveal that the combination of low-dose cisplatin and interleukin-1α is more effective than high-dose cisplatin alone. Our data suggest that cisplatin and IL-1α may be efficacious in the treatment of SCCHN. (Arch Otolaryngol Head Neck Surg. 1995;121:197-200)

The immediate effects of a single dose of the chemotherapeutic DNA crosslinking agent, mitomycin C (MMC), on the expression of several constitutive and drug-inducible genes were examined in a simple in vivo system, the 14 day chick embryo. We observed no effect of MMC on the steady-state mRNA expression of the constitutively expressed beta-actin, transferrin, or albumin genes. In contrast, MMC treatment significantly altered both the basal and drug-inducible mRNA expression of two glutethimide-inducible genes, 5-aminolevulinic acid (ALA) synthase and cytochrome P450 CYP2H1. The basal expression of these genes was transiently but significantly increased over a 24 hr period following a single dose of MMC. Conversely, MMC significantly suppressed the glutethimide-inducible expression of these genes when administered 1 to 24 hr prior to the inducing drug. The effects of MMC on both basal and drug-inducible ALA synthase and CYP2H1 mRNA expression were principally a result of changes in the transcription rates of these genes. In contrast, MMC treatment had little or no effect on glutethimide-induced expression of ALA synthase or CYP2H1 when administered 1 hr after the inducing drug, suggesting that a very early event in the induction process represents the target for these MMC effects. Covalent binding studies demonstrated that the effects of MMC on gene expression were closely correlated temporally with formation of [3H]-porfiromycin-DNA adducts. These results support the hypothesis that genotoxic chemicals specifically target their effects to inducible genes in vivo.

A high-performance liquid chromatographic method is described for the determination of mitomycin C (MMC) and its metabolites 2,7-diaminomitosene (2,7-DM), 1,2-cis-1-hydroxy-2,7-diaminomitosene (cis-hydro) and 1,2-trans-1-hydroxy-2,7-diaminomitosene (trans-hydro) in tumour tissue. N-la-Methylmitomycin C (porfiromycin, PM) was used as an internal standard. Two factors were critical in resolving the metabolites: pH and buffer ionic strength, where the retention times of the four components were affected in the order 2,7-DM >> cis-hydro >> trans-hydro >> MMC. The optimal isocratic conditions (flow-rate 1 ml/min) were 18 mM sodium phosphate pH 5.8-methanol (74:26) and a column temperature of 40 degrees C on a Spherisorb ODS-2 column (25 cm x 4.6 mm I.D.). Liquid-liquid extraction [twice with chloroform-propan-2-ol-ethyl acetate (2:2:1)] is described for tumour tissue. Recoveries varied depending on the component: MMC, 71.9 +/- 12.4%; PM, 85.5 +/- 27%; 2,7-DM, 51.7 +/- 5.4%; cis-hydro, 52.0 +/- 16.8%; trans-hydro, 62 +/- 8%. When applied to the analysis of a rat mammary carcinoma treated intra-tumourally with 450 micrograms of MMC five drug-related "metabolite" peaks were detected. Three of these co-chromatographed with standards of 2,7-DM, cis- and trans-hydro, and had identical absorption maxima to their respective standards, with the possible exception of trans-hydro.

The first example of C-3 alkoxylation in mitomycins has been achieved. 3 alpha-iso-Propoxy-10-O-decarbamoylmitomycin D (4) and 3 alpha-iso-propoxymitomycin D (5) were derived from mitomycin D (3) under decarbamoylation conditions with iso-propoxide. Under similar conditions 3 alpha-iso-propoxy-10-O-decarbamoylporfiromycin (8) and 3 alpha-methoxy-10-O-decarbamoylmitomycin B (11) were also derived from porfiromycin (6) and mitomycin B (9), respectively. The mechanism of generation of these novel analogs was based on the premise that the key intermediate of hydroquinone iminium salt (14) was led through the iminium salt (13), followed by alkoxide addition and oxidation.

Mitomycin G (2) was derived from porfiromycin (10b) in 3 steps via the methanesulfonate (14b) in an overall yield of 39%. On the basis of the established method for the introduction of an exomethylene group in mitomycins with a 9a-methoxy group, the preparation of biologically more important 1a-demethylmitomycin G (5) from mitomycin C (1) was accomplished by the use of a protective acetyl group on the aziridine in an overall yield of 57%. 1a-Demethylmitomycin K (6) was obtained from 5 in a yield of 42%. In a preliminary evaluation of their antitumor activity, compound 5 showed superior activity against sarcoma 180 (sc-ip) to its 1a-methyl congener, i.e., mitomycin G (2).

We have directly demonstrated the involvement of human NADPH: cytochrome c (P-450) reductase in the aerobic/hypoxic differential toxicity of mitomycin C and porfiromycin in living cells by varying only this enzyme in a transfected cell line. In the same manner, we have implicated rat DT-diaphorase in the aerobic and hypoxic activation of mitomycin C, but found only a minor role for this enzyme in the aerobic activation of porfiromycin. DT-Diaphorase does not cause the production of an aerobic/hypoxic differential toxicity by mitomycin C, but rather activates this agent through an oxygen insensitive pathway. The evidence suggests that DT-diaphorase activates mitomycin C more effectively than porfiromycin, with porfiromycin being preferentially activated through a one-electron reductive pathway. The therapeutic potential of mitomycin antibiotics in the treatment of cancer can be envisioned to be enhanced for those tumors containing elevated levels of the bioreductive enzymes. However, cytogenetic heterogeneity within the tumor cell population and the various environmental factors which impact on bioreductive enzyme function, including pH and oxygen tension, may subvert this approach. Moreover, if high tumor levels of a drug activating enzyme reflect high levels in the normal tissues of the patient, normal tissue damage may also be enhanced with possibly no improvement in the therapeutic ratio. Approaches utilizing gene therapy, whereby a specific bioreductive catalyst is introduced into the tumor cell population via a targeting vehicle to activate a particular prodrug, may be more effective in that not only will the prodrug of choice be specifically activated in the tumor, but the source of the catalyst, be it bacterial, rodent, or human, will not be important. In fact, in the case of DT-diaphorase and mitomycin C, the rat form of the enzyme could be advantageous because it is more effective in activating mitomycin C than is the human form of this enzyme. Assuming targeted gene delivery to malignant cells, a non-host enzyme which is more effective at activating mitomycin C than the analogous host enzyme might also result in less drug activation in normal tissue and, hence, less normal tissue toxicity.

15 human tumour cell lines (lung, breast and colon) have been evaluated for their sensitivity to the quinone based anti-cancer drugs Mitomycin C, Porfiromycin, and E09 (3-hydroxymethyl-5-aziridinyl-l-methyl-2-(IH-indole-4,7-dione)prop-β-en-α-ol). Sensitivity has been compared with the intra-cellular levels of DT-diaphorase, an enzyme thought to be important in the reductive activation of these quinones. No correlation exists between levels of DT-diaphorase and sensitivity to Mitomycin C or Porfiromycin. However, for E09 those cell lines showing highest levels of DT-diaphorase activity tend to be the most sensitive.

Streptonigrin (SN, CAS no. 3930-19-6) is an aminoquinone antitumor antibiotic isolated from cultures of Streptomyces flocculus. This compound is a member of a group of antitumor agents which possess the aminoquinone moiety and that includes also mitomycin C, porfiromycin, actinomycin, rifamycin and geldanamycin. Because of the potential use of SN in clinical chemotherapy, the study of its genotoxicity has considerable practical significance.SN inhibits the synthesis of DNA and RNA, causes DNA strand breaks after reduction with NADH, induces unscheduled DNA synthesis and DNA adducts and inhibits topoisomerase II. At the chromosome level, this antibiotic causes chromosome damage and increases the frequency of sister-chromatid exchanges.SN cleaves DNA in cell-free systems by a mechanism that involves complexing with metal ions and autoxidation of the quinone moiety to semiquinone in the presence of NADH with production of oxygen-derived reactive species. Recent evidence strongly suggests that the clastogenic action of this compound is partially mediated by free radicals. The present review aims at summarizing past and current knowledge concerning the genotoxic effects of SN.

The activity of three different classes of bioreductive drug, i.e., heterocyclic nitro compounds, N -oxides and quinones are compared. The major characteristics of RB-6145, tirapazamine and E09 are summarized and future directions for development of new bioreductive drugs are outlined. The concept of potentiating bioreductive drug activity by increasing tumor hypoxia is described and illustrated in particular by the use of photodynamic therapy (PDT) in combination with RSU-1069. Examples of how the therapeutic effectiveness of this approach can be studied by the use of 3'P magnetic resonance spectroscopy is described. The effects of manipulation of nitric oxide (NO) levels in tumors by the use of modifiers of NO-synthase activity is illustrated by studies with the inhibitor nitro- L -arginine in experimental tumors. Associated changes in tumor physiology indicate promise for potential applications in therapy. Finally, changes in expression of reductase enzyme levels are considered in the context of the heterogenous nature of the tumor microenvironment.

Tirapazamine is the second clinical anticancer drug (after porfiromycin) that functions primarily as a hypoxia-selective cytotoxin. Hypoxic cells in tumours are relatively resistant to radiotherapy and to some forms of chemotherapy and are also biologically aggressive, thus representing an important target population in oncology. Tirapazamine undergoes metabolism by reductases to form a transient oxidising radical that can be efficiently scavenged by molecular oxygen in normal tissues to re-form the parent compound. In the absence of oxygen, the oxidising radical abstracts a proton from DNA to form DNA radicals, largely at C4' on the ribose ring. Tirapazamine can also oxidise such DNA radicals to cytotoxic DNA strand breaks. It therefore shows substantial selective cytotoxicity for anoxic cells in culture (typically approximately 100-fold more potent than under oxic conditions) and for the hypoxic subfraction of cells in tumours. Preclinical studies showed enhanced activity of combinations of tirapazamine with radiation (to kill oxygenated cells) and with conventional cytotoxics, especially cisplatin (probably through inhibition of repair of cisplatin DNA cross-links in hypoxic cells). Phase II and III clinical studies of tirapazamine and cisplatin in malignant melanoma and non-small cell lung cancer suggest that the combination is more active than cisplatin alone and preliminary results with advanced squamous cell carcinomas of the head and neck indicate that tirapazamine may enhance the activity of cisplatin with fractionated radiotherapy.

Mitomycin C (MMC) is a bioreductive alkylating agent that is capable of generating oxygen radicals. Porfiromycin (PM) is an analog to MMC that generates oxygen radicals at a significantly lower level than the parent compound. Under aerobic conditions, the toxicity of MMC to EMT6 cells is 2.5-fold that of PM, whereas hypoxically the two are equitoxic. In the present studies, the protective effect of PZ-51 in combination with NAC was assessed against the dose-dependent toxicity of either MMC or PM under both aerobic and hypoxic conditions. Aerobically, the PZ-51 and NAC combination inhibited the toxicity of MMC at concentrations of between 0.25 and 2 microM but had no effect on PM toxicity. Under hypoxic conditions, the PZ-51 and NAC combination had no effect on either MMC or PM toxicity. These findings support a role for oxygen radical generation in the aerobic toxicity of MMC at clinically relevant doses.

Mitomycin C is being used as an adjunct to ionizing radiation in the treatment of some solid tumors. A rationale for this is that radioresistant hypoxic cells in solid tumors will have enhanced sensitivity to this bioreductively activated drug, compared to aerobic cells. The role of oxygen concentration and enzymatic drug reduction in bioreductive drug activation have been investigated. Techniques are reviewed for the in vitro determination of the oxygen concentration dependency of bioreductive drug activation. One of these techniques, an open cell suspension system using Chinese hamster ovary cells, is described. Results are shown that indicate that the oxygen concentration dependency of toxicity of mitomycin C and one of its analogues profiromycin, though qualitatively complementing the oxygen dependency of ionizing radiation toxicity, are not quantitatively optimal. Using a mitomycin C resistant human cell strain (3437T) from a cancer prone family, a possible role for DT-diaphorase, an oxygen insensitive 2-electron transfer enzyme, is suggested. A correlation between a low level of DT-diaphorase in 3437T cells and mitomycin C resistance under aerobic exposure conditions is seen. Under hypoxic exposure conditions this resistance is lost, suggesting 1-electron transfer enzymes control hypoxic cell bioreductive activation. An activation role for DT-diaphorase in mitomycin C toxicity in the treatment of solid tumors is contrasted to a potential detoxification role for the enzyme with other xenobiotics in the cancer prone family phenotype.

The mitomycin antibiotics, because of their preferential toxicities for hypoxic cells, have significant potential as adjuncts to ionizing radiation in the treatment of solid tumors. To gain information on the mechanism by which these agents exert their cytotoxicities to hypoxic and aerobic cells, the effects of MC, POR and several of their analogs were studied in EMT6 mammary carcinoma cells. The rate of uptake of POR by these cells was directly correlated with the cytotoxicity produced by this agent under both hypoxia and aeration. At equivalent concentrations, uptake of POR into hypoxic cells was more rapid than into aerobic cells. Hypoxic cells also accumulated the antibiotic in concentrations well in excess of that present in the extracellular medium, presumably as a result of reductive activation and covalent binding of POR to cellular structures. Such activation and binding occur to a much lesser degree in aerated cells, resulting in the rapid efflux of POR from these cells when the antibiotic is removed from the extracellular environment. To gain information on the reaction of POR with DNA, mono- and bis-adducts formed in EMT6 cells exposed to this agent were measured. Three major adducts were formed. Two were mono-adducts consisting of deoxyguanosine linked at its N2-position to the C-1 of POR and of 10-decarbamoyl POR. The third was a bis-adduct in which POR was cross-linked to two deoxyguanosines at their N2-positions. More adducts were formed in hypoxia than in air, and more bis-adducts were present in hypoxic cells. Simultaneous exposure of cells to both POR and DIC reduced the total adduct level and a new unknown adduct was formed, primarily under hypoxia. Several mitomycins were evaluated for their capacity to kill EMT6 cells and to produce DNA cross-links in both hypoxia and aeration. The number of cross-links required to produce a given amount of cell kill was similar, regardless of the mitomycin employed or the degree of oxygenation. The findings support the concept that DNA is a critical target in the action of the mitomycins and that cross-linking of the DNA creates an important lesion for cytodestruction.

The new mitomycin 7-N-[2-((2-(γ- l -glutamylamino)ethyl)dithio)ethyl]mitmycin C (KW 2149) (I) proved to be active against a wide variety of experimental tumours. In order to perform pharmacokinetic studies with the new drug in Phase I sessions, a fast and reliable method has been developed based on the data of previous assays for mitomycin C. XAD-2 was preferred for isolation of I from blood plasma. The recovery of I was 50% whereas that of mitomycin C was 85%. Optimal separation was obtained on octadecyl silica columns with methanol—water (45:55, v/v) as mobile phase, while ultraviolet absorbance detection was performed at 375 nm. The assay enabled determination of I in a plasma concentration range of 20–1000 ng/ml using porfiromycin as internal standard.

The cellular pharmacology of the mitomycin bioreductive alkylating agents is complex. This reflects in part the chemical characteristics of these quinones, which have multiple sites of reactivity and the capacity to produce a large number of different lesions of biological importance. Moreover, at least six different enzymes are capable of activating these compounds; the nature of the active species and the resultant biological lesions can vary with the activating enzyme. The relative activities of these reductases vary in different cell lines and can be modulated by pH and oxygenation. The effects of a quinone bioreductive alkylating agent therefore depend upon both the cell line and the microenvironment. DNA damage appears to be critical to the cytotoxic effects of these compounds. Both monoadducts and bis-adducts (forming interstrand and intrastrand cross-links) have been identified in DNA from drug-treated cells. The pattern of adduct formation varies with the compound and the environment. Alkaline elution studies suggest a correlation between DNA cross-linking and cytotoxicity, both in air and in hypoxia. The rate of production of oxygen radicals and the importance of radical reactions in producing cytotoxic damage vary for different quinones and for different environments. While the potency of the bioreductive quinones varies with their redox potential, the direction and magnitude of the oxic/hypoxic differential cannot yet be predicted from the structures.

Stable acquired resistance to etoposide (VP-16) or teniposide (VM-26) in HCT116 human colon carcinoma cells and A549 human lung adenocarcinoma cells, was previously obtained by weekly 1-h exposures to either drug (B. H. Long, Natl. Cancer Inst. Monogr., 4: 123-127, 1987). The purpose of this study was to identify possible mechanisms of resistance present in these cells by using human mdr1 and topoisomerase II DNA probes, antibodies to these gene products, and P4 phage unknotting assay for topoisomerase II activities. HCT116(VP)35 cells were 9-, 7-, and 6-fold resistant to VP-16, VM-26, and Adriamycin, respectively, and showed no cross-resistance to colchicine and actinomycin D. These cells had no differences in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels but displayed decreased levels of topoisomerase II mRNA and enzyme activity without any alteration of drug sensitivity displayed by the enzyme. HCT116(VM)34 cells were 5-, 7-, and 21-fold resistant to VP-16, VM-26, and Adriamycin; were cross-resistant to colchicine (7-fold) and actinomycin D (18-fold); and possessed a 9-fold increase in mdr1 mRNA and increased P-glycoprotein without evidence of mdr1 gene amplification. No alterations in topoisomerase II gene or mRNA levels, enzyme activity, or drug sensitivity were observed. A549(VP)28 and A549(VM)28 cells were 8-fold resistant to VP-16 and VM-26 and 3-fold resistant to Adriamycin. Both lines were not cross-resistant to colchicine or actinomycin D but were hypersensitive to cis-platinum. No alterations in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels, but lower topoisomerase II mRNA levels and decreased enzyme activities, were observed. Of the four acquired resistant cell lines, resistance is likely related to elevated mdr1 expression in one line and to decreased topoisomerase II expression in the other three lines.