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Hypoxic cells may limit the curability of solid tumors by conventional chemotherapeutic agents and radiotherapy. Agents which are preferentially toxic to cells with low oxygen contents could therefore be useful as adjuncts to the regimens now used to treat these cancers. To date, the best agent of this type that we have tested is porfiromycin. Porfiromycin is similar to mitomycin C in its toxicity to hypoxic EMT6 cells in vitro but has much less toxicity than mitomycin C to well-oxygenated EMT6 cells. EMT6 cell sonicates reduce mitomycin C and porfiromycin to reactive electrophiles at similar rates under hypoxic conditions, a finding that correlates with cytotoxicity, whereas the rate of production of reactive species from both drugs is very slow under aerobic conditions. We also show that porfiromycin is capable of killing hypoxic radiation-resistant cells in solid EMT6 tumors. Appropriate regimens combining porfiromycin (which preferentially kills hypoxic cells) and radiation (which preferentially kills aerated cells) may therefore be especially efficacious for the treatment of solid tumors.

Mitomycin C (MC) and its structural analogs porfiromycin (PM), BMY-25282 and BL-6783 are toxic to EMT6 cells under aerobic and hypoxic conditions. The mitomycin antibiotics are hypothesized to exert cytotoxicity under hypoxic conditions by cross-linking DNA following reductive activation, while aerobic cytotoxicity may involve DNA cross-linking by these agents and/or damage due to the generation of oxygen radicals. Previous findings (Pritsos and Sartorelli, 1986) indicated that the rank order of cytotoxicity for a series of mitomycins was the same as the rank order for the rate of oxygen consumption induced by these agents. As an additional approach to explore the role of oxygen radicals in the aerobic cytotoxicity of the four agents studied, EMT6 cells were treated with the mitomycins in the presence of the superoxide dismutase inhibitor diethyldithiocarbamate (DETC). DETC, which decreased superoxide dismutase activity in EMT6 cells, increased the cytotoxicity of BMY-25282 and BL-6783 by half an order of magnitude, but did not affect the toxicity of PM or MC to these cells. DNA cross-links, a proposed cytotoxic lesion induced by BMY-25282, however, were not detectably increased in EMT6 cells exposed to this agent in the presence of DETC in spite of the large increase in cytotoxicity under these treatment conditions. No single strand breaks were detected in cells exposed to either BMY-25282 or BMY-25282 plus DETC. The findings support the concept that oxygen radicals may have a role in the aerobic cytotoxicity of some of the mitomycin antibiotics, and that the lesions responsible for cytotoxicity produced by oxygen radicals may not reside entirely at the level of DNA.

The effects of dicoumarol (DIC) on the cytotoxicity of porfiromycin (POR) were studied in vitro using EMT6 mammary tumor cells in monolayer cultures and in vivo using solid EMT6 tumors and bone marrow stem cells. In vitro, POR was more toxic to hypoxic EMT6 cells than to aerobic cells. Exposure of aerobic cultures to DIC protected against POR; in contrast, DIC sensitized hypoxic cells to POR. Treatment of mice with DIC produced a slight increase in the toxicity of POR to cells in solid tumors. The toxicity of POR to marrow stem cells (CFU-GM and CFU-MK) was not altered by DIC. Pretreatment of mice with DIC therefore produced a small improvement in the therapeutic ratio.

When the normal fermentation medium for the production of mitomycin C with Streptomyces caespitosus is supplemented with a number of primary amines, two new types of mitomycin analogs described as Type I and Type II are produced. Type I analogs are related to mitomycin C with the amine substitution at position C7 on the mitosane ring. Type II analogs also contain the same substitutions at C7 but the conformation of the mitosane ring is related to mitomycin B having an OH at positions C9a and a methyl substituted aziridine. The products obtained from the supplementation of the medium with methylamine, ethylamine, propylamine, propargylamine and 2-methylallylamine were isolated and characterized. In all cases the Type I analogs are more active in a prophage induction test and against L1210 lymphatic leukemia in mice. A number of other amines have been tested and shown to yield new products that have not yet been isolated. No secondary amines are incorporated.

The mitomycins were first obtained in 1956 from Streptomyces caespitosus by Hata and coworkers in Japan (1, 2). The isolation involved alumina chromatography of the chloroform extracts of the concentrated aqueous filtrates of the fermentation broth. Although there were many active components in the eluted fractions, only two antibiotics, mitomycins A and B were obtained in crystalline form. Later fractionations of broths of S. caespitosus yielded mitomycin C (3). By 1958, the outstanding antitumor activity of mitomycin C was a subject of great interest in spite of the fact that there existed no firm evidence as to its structure (4). In 1960, N-methylmitomycin C, called porfiromycin, was isolated from cultures of Streptoverticillatium ardus (5). In 1962 the isolation of the same four antibiotics from Streptomyces verticillatus was reported in the U.S. (6). Additionally, a fifth, but inactive member of mitomycin family, mitiromycin was obtained. The fractionation of the S. verticillatus broths to afford the crystalline antibiotics was accomplished by partition chromatography on a diatomaceous earth support.

New mitomycin C and porfiromycin analogues were prepared by treating mitomycin A and N-methylmitomycin A with a variety of amines, including aziridines, allylamines, propargylamines, chloroalkylamines, hydroxyalkylamines, glycine derivatives, aralkylamines, and heterocyclic amines. All analogues were evaluated against P-388 murine leukemia and selected ones were examined for their leukopenic properties. Certain analogues were found to be superior to mitomycin C in potency, efficacy, and therapeutic ratio in the P-388 assay. The most active substituents at the mitosane 7 position included aziridine, 2-methylaziridine, propargylamine, furfurylamine, methyl glycinate, and 3-aminopyridine. Mitomycin A and the 7-aziridino, 7-(2-methylaziridino), and 3-aminopyridine analogues were less leukopenic than mitomycin C. Certain other analogues, including propargylamino and methyl glycinate, were highly leukopenic. The three compounds tested against B-16 melanoma in mice were significantly more effective than mitomycin C in this assay. Previously established structure--activity relationships were found inadequate to account for all of the new data.

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 mechanism of uptake and efflux of porfiromycin (PFM) by HCT 116 human colon carcinoma cells or freshly obtained human RBC was investigated. The time course of uptake of radioactivity upon exposure of HCT 116 cells to [14C]PFM showed one fast and one slow phase of linear increase. The initial phase of PFM uptake was not saturable with external drug concentrations from 2 to 100 microM. PFM accumulation was temperature dependent with a temperature coefficient (Q10 24-37 degrees C) of 2.3 +/- 0.3. PFM uptake was not affected either by individual inhibitors such as 1 mM 2,4-dinitrophenol, sodium azide, iodoacetic acid, ouabain, 0.02 mM oligomycin, p-hydroxylmercuribenzoate, 0.2 mM N-ethylmaleimide, or by combinations of inhibitors. PFM uptake did not demonstrate competitive inhibition by unlabeled PFM and mitomycin C. Efflux of cellular radioactivity was not affected by the above mentioned inhibitors or by verapamil, diltiazem, or trifluoperazine. Only aliphatic alcohols accelerated the initial influx rate. The RBC, however, only exhibited the initial fast accumulation of [14C]PFM, and all the 14C accumulated by RBC was exchangeable. These data demonstrate that the uptake and the efflux of PFM in HCT 116 cells and RBC comprise a passive diffusion process.

Mitomycin C (MC) was first isolated from Streptomyces caesipitosus (1). It was demonstrated to have antibiotic and antitumor properties. Closely related antibiotics, designated mitomycin A and B and porfiromycin have also bee isolated (2). Mitomycins are colored, basic substances forming reddish-violet, plate or needle-shaped crystals. The antibiotic family was described as having unique, naturally occurring structural configurations, in that it represents the first recorded example of the formation by a microbial system of an aziridine group, the pyrrol-(1,2-a)- indole ring system, an aminobenzoquinone, and a pyrrolizine. The common mitomycin nucleus is termed Mitosane. The different compounds only vary by minor substituent groupings (3).

The mitomycins and porfiromycin (Fig. 1) constitute a complex of anticancer antibiotics which are produced by Streptomyces caespitosus (Hata et al., 1956), Streptomyces ardus sp. n (DeBoer et al., 1960) Streptomyces verticillatus (Lefemine etal., 1962) and Streptomyces michiganensis (Wolf et al., 1975). Their structures have been determined by chemical, physicochemical, and X-ray methods (Webb etal., 1962a, 1962b; Stevens etal., 1965; Tulinsky and Van den Hende, 1967; Yahashi and Matsubara, 1976). Mitomycin A (Ia) (Tulinsky and Van den Hende, 1967) and Mitomycin B (Ib) (Yahashi and Matsubara, 1976) differ stereochemically at C-9. The structure of mitiromycin (II), a biologically inactive close relative, has been reported (Morton et al., 1970). The mitomycins and the Streptomyces metabolite (S)-2,3-dicarboxyaziridine (III) (Naganawa etal., 1975) are unique among naturally occurring metabolites owing to the presence of an aziridine ring. A related ring system, an azirine ring, occurs in the Streptomyces aureus antibiotic azirinomycin (IV)Miller etal., 1971

Solid neoplasms may contain deficient or poorly functional vascular beds, a property that leads to the formation of hypoxic tumor cells, which form a therapeutically resistant cell population within the tumor that is difficult to eradicate by ionizing irradiation and most existing chemotherapeutic agents. As an approach to the therapeutic attack of hypoxic cells, we have measured the cytotoxicity and DNA lesions produced by the bioreductive alkylating agents mitomycin C and porfiromycin, two structurally similar antibiotics, in oxygen-deficient and aerobic cells. Mitomycin C and porfiromycin were preferentially cytotoxic to hypoxic EMT6 cells in culture, with porfiromycin producing a greater differential kill of hypoxic EMT6 cells relative to their oxygenated counterparts than did mitomycin C. Chinese hamster ovary cells were more resistant to these quinone antibiotics; although in this cell line, porfiromycin was significantly more cytotoxic to hypoxic cells than to aerobic cells, and the degree of oxygenation did not affect the toxicity of mitomycin C. Alkaline elution methodology was utilized to study the formation of DNA single-strand breaks and DNA interstrand cross-links produced by mitomycin C and porfiromycin in both EMT6 and Chinese hamster ovary cells. A negligible quantity of DNA single-strand breaks and DNA interstrand cross-links were produced in hypoxic and aerobic Chinese hamster ovary cells by exposure to mitomycin C or porfiromycin, a finding consistent with the considerably lower sensitivity of this cell line to these agents. In EMT6 tumor cells, no single-strand breaks appeared to be produced by these antitumor antibiotics under both hypoxic and aerobic conditions; however, a significant number of DNA interstrand cross-links were formed in this cell line following drug treatment, with substantially more DNA interstrand cross-linking being produced under hypoxic conditions. Mitomycin C and porfiromycin caused the same amount of cross-linking under conditions of oxygen deficiency; however, mitomycin C produced considerably more DNA cross-linking than did porfiromycin in oxygenated cells. DNA interstrand cross-links were observed in hypoxic EMT6 cells throughout a 24-h period following removal of mitomycin C and porfiromycin, with a decrease in DNA interstrand cross-links observed at 24 h. An increase in DNA interstrand cross-links occurred in aerobic EMT6 cells treated with mitomycin C and porfiromycin at 6 h after drug removal, with a decrease in these lesions being observed by 24 h, suggesting that the rate of formation of the cross-links may be slower and the removal of cross-links more rapid under aerobic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Since the cure of solid tumors is limited by the presence of cells with low oxygen contents, we have approached the development of treatment regimens and of new drugs for these tumors by investigating agents which are preferentially bioactivated under hypoxia. Major emphasis has been directed at studying the mode of action of the mitomycin antibiotics, as bioreductive alkylating agents. Using primarily the EMT6 mouse mammary carcinoma as a solid tumor model, we have found that mitomycin C and porfiormycin are preferentially toxic to cells with low oxygen contents. The mitomycin analog BMY-25282 is more toxic to hypoxic cells than are mitomycin C and porfiromycin; however, unlike these antibiotics, BMY-25282 is preferentially toxic to well-oxygenated cells. With these three mitomycins, we have observed a correlation between cytotoxicity to hypoxic cells, the rate of generation of reactive products, and the redox potentials of the drugs. Investigations of the enzymes in EMT6 cells that could possibly activate mitomycin C have revealed that cytochrome P-450 and xanthine oxidase are not present in measurable quantities and therefore are not responsible for activation of mitomycin C. Activities representative of NADPH-cytochrome c reductase and DT-diaphorase are present in these neoplastic cells. Comparison of these enzymatic activities in EMT6, CHO, and V79 cells with the rate of generation of reactive products under hypoxia shows a direct correlation between these two parameters, but there is no quantitative correlation between these two parameters and the amount of cytotoxicity. Use of purified NADPH-cytochrome c reductase and inhibitors of this enzyme demonstrated that NADPH-cytochrome c reductase can activate mitomycin C, but that it is probably not the only enzyme participating in this bioactivation in EMT6 cells. The DT-diaphorase inhibitor dicoumarol was employed to show that this enzyme is not involved in the activation of mitomycin C to a cytotoxic agent. Instead, DT-diaphorase appears to metabolize mitomycin C to a nontoxic product. This property has been exploited to develop a new treatment regimen for solid tumors. Using X-rays to eliminate well oxygenated cells of a solid tumor implant of the EMT6 carcinoma, we have found that the combination of dicoumarol plus mitomycin C is more toxic to hypoxic tumor cells in vivo than mitomycin C alone. Furthermore, knowledge of the biochemical mechanism of mitomycin C activation permits a prediction of which tumors can best be treated with this combination of drugs by measuring enzymatic activities in biopsy specimens.

Porfiromycin was reductively metabolized by NADPH cytochrome P-450 reductase and xanthine oxidase under anaerobic conditions. The production of metabolites varied with the pH and the contents of the reaction buffer. In Tris buffer, two major metabolites were produced at pH 7.5 and above, whereas one major metabolite was produced at pH 6.5. The three major metabolites were separated and isolated by HPLC. Identification by californium-252 plasma desorption mass spectrometry showed that the two major metabolites from pH 7.5 were (trans) and (cis)-forms of 7-amino-1-hydroxyl-2-methylaminomitosene and the major metabolite from pH 6.5 was 7-amino-2-methylaminomitosene. All three major metabolites showed substitutions at the C-1 position. DNA was alkylated readily by enzyme-activated porfiromycin. Digestion of porfiromycin-alkylated DNA by DNase, snake venom phosphodiesterase, and alkaline phosphatase resulted in an insoluble nuclease-resistant fraction and a soluble fraction. The nuclease-resistant fraction reflected a high content of cross-linked adducts. Upon HPLC analysis, the solubilized fraction contained two monofunctionally linked porfiromycin adducts and a possibly cross-linked dinucleotide. The major adduct was isolated by HPLC and identified by NMR, as N2-(2'-deoxyguanosyl)-7-amino-2-methylaminomitosene. The N2 position of deoxyguanosine appeared as the major monofunctional alkylating site for DNA alkylation by porfiromycin. Thus, mitomycin C and porfiromycin (which differs from mitomycin C only by the addition of a methyl group to the aziridine nitrogen) share the same enzymatic activating mechanism that leads to the formation of the same types of metabolites and the same specificity of DNA alkylation.

The prototropic properties of mitomycin and porfiromycin were studied. pK0 values for two potentially basic groups and one acidic function could be established by titration. The kinetics of the tautom- erization preceding the prototropic reaction in an alkaline medium were investigated.

High-performance liquid chromatography (HPLC) and thermospray mass spectrometry were combined for the analysis of DNA adducts formed from the interaction of the anticancer drugs mitomycin C, porfiromycin and thiotepa with calf thymus DNA. The adducts formed from reactions of mitoycin C and porfiromycin with DNA were separated from unmodified nucleosides by HPLC on a C18 column and identified by thermospray mass spectrometry. Thiotepa DNA adducts readily depurinated from DNA and were chromatographed and identified by thermospray liquid chromatography—mass spectrometry as the modified bases without the ribose moiety attached. The utility of thermospray mass spectrometry for the identification of microgram quantities of nucleoside adducts and depurinated base adducts of these anticancer drugs was demonstrated.

The effect of porfiromycin (N-methyl mitomycin) on the cell cycle in HEP-2 cells was investigated by pulse labeling with tritiated thymidine at various intervals following 4-hours incubation with the drug. Arrest of cells during the G2 phase was the predominant effect with temporary suppression of mitotic activity as well.

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.

Blood coagulation, fibrinolysis and platelet function have been studied in a female population of 404 subjects in the age strata 38, 46, 50, 54 and 60 years. There was a significant increase in fibrinogen with increasing age. The population was also subdivided according to menopause. The results indicate that the menopause has some influence on the fibrinogen concentration. Partial thromboplastin time (PTT) in silicone tubes varied significantly with age. The curve was two-humped. This is probably the result of two opposing effects on the clotting time: the fibrinogen concentration which increases with increasing age and tends to prolong the clotting times, and an unknown factor which tends to shorten the clotting times with increasing age. This non-identified factor has probably something do to with surface activation of plasma.

THE marked sensitivity in vitro1 of various metabolic pathways in T. equiperdum to actinomycin D, mitomycin C, porfiromycin, phleomycin, and pactamycin indicated that these tumour-inhibitory antibiotics may be trypanocidal in vivo. Our results show this to be so with all except pactamycin, and also that phleomycin and porfiromycin in particular cured a high percentage of infected mice at certain dosages.

Cyanide and phenethyl alcohol greatly enhance the lethal action of mitomycin C, porfiromycin, and streptonigrin on an exponentially growing culture of Escherichia coli. Dinitrophenol similarly enhances the lethal action of mitomycin C and porfiromycin, but only slightly that of streptonigrin. Phenethyl alcohol may be functioning in these experiments as an inhibitor of electron transport.

The addition of (methylene-2H2)-3-amino-5-hydroxybenzyl alcohol (8) hydrochloride to growing cultures of Streptomyces verticillatus inhibits production of the mitomycin antibiotic porfiromycin (2) by the microorganism. No incorporation of deuterium into porfiromycin occurs, and the isotopic composition of the alcohol recovered from the fermentation is unchanged. The significance of these results with regard to the immediate metabolic precursors of 3-amino-5-hydroxybenzoic acid (3) and the reduction stages involved in the established conversion of this amino acid into mitomycins are discussed.

The efficient, specific incorporation of isotope from [carboxy-13C]-3-amino-5-hydroxybenzoic acid into the C-6 methyl group of porfiromycin by Streptomyces verticillatus establishes this amino-acid as the biogenetic precursor of the methylbenzoquinone nucleus of the mitomycin antibiotics.

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.