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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.

Purpose Previous randomized trials have shown a benefit with concurrent use of the hypoxic cell cytotoxin mitomycin C (MC) and radiation (RT) in the management of squamous cell cancer of the head and neck (SCCHN). We conducted a randomized trial comparing MC with porfiromycin (POR) in combination with RT in the management of SCCHN. Methods and materials Between 1992 and 1999, 128 patients with SCCHN were enrolled in this prospective randomized trial. Patients were stratified by management intent, and balanced with respect to stage and site of disease. They were randomized to receive MC (15 mg/M 2 ) or POR (40 mg/M 2 ) on Days 5 and 47 (or last day) of RT. Of 121 evaluable patients, 61 were randomized to MC and 60 to POR. Patients were treated with standard daily RT to a total median dose of 64 Gy over 47 days. Patients were well balanced with respect to management intent, stage, site, age, sex, hemoglobin levels, tumor grade, radiation dose, and days on treatment. Results There were no significant differences between the two arms with respect to acute hematologic or nonhematologic toxicities. As of January 2003 with a median follow-up of 6.3 years, there have been 19 local relapses (4 MC vs. 15 POR), 21 regional relapses (7 MC vs. 14 POR), 24 distant metastases (11 MC vs. 13 POR), and 66 deaths (33 MC vs. 33 POR). MC was superior to POR with respect to 5-year local relapse-free survival (91.6% vs. 72.7%, p = 0.01), local-regional relapse-free survival (82% vs. 65.3%, p = 0.05), and disease-free survival (72.8% vs. 52.9%, p = 0.026). There were no significant differences between the two arms with respect to overall survival (49.2% vs. 54.4%) or distant metastasis–free rate (79.9% vs. 75.9%). Conclusions Despite promising preclinical data, and an acceptable toxicity profile, POR was inferior to MC as an adjunct to RT in the management of SCCHN. This randomized trial emphasizes the need for randomized studies to evaluate new agents in the management of SCCHN.

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.

The isolation and identification of the major metabolites of porfiromycin formed in the presence of a rat liver preparation under aerobic conditions were performed with high‐performance liquid chromatography and electrospray ionization mass spectrometry. Porfiromycin was extensively metabolized by the rat liver preparation in an aqueous 0.1 M potassium phosphate buffer (pH 7.4) containing an NADPH generating system at 37°C. A total of eight metabolites was identified as mitosene analogs. Of these, three primary metabolites are 2‐methylamino‐7‐aminomitosene, 1,2‐ cis and 1,2‐ trans ‐1‐hydroxy‐2‐methylamino‐7‐aminomitosene, which are consistent with those previously observed in hypoxia using purified rat liver NADPH‐cytochrome c reductase. Interestingly, 2‐methylamino‐7‐aminomitosene is a reactive metabolite, which undergoes further activation at the C‐10 position by the loss of carbamic acid and then links with the 7‐amino group of the primary metabolites to yield two dimeric adducts. In addition, three phosphate adducts, 10‐decarbamoyl‐2‐methylamino‐7‐aminomitosene‐10‐phosphate, 1,2‐ cis and 1,2‐ trans ‐2‐methylamino‐7‐aminomitosene‐1‐phosphate, were also identified in the incubation system. The configurations of the diastereoisomeric metabolites were determined with 1 HNMR and phosphatase digestion. On the basis of the metabolite profile, we propose in vitro metabolic pathways for porfiromycin. The findings provide direct evidence for understanding the reactive nature and hepatic metabolism of the drug currently in phase III clinical trials. © 2000 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89:191–198, 2000

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

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.

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.

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.

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.

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.

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 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.

In an effort to develop effective combination treatments for use with radiation against solid tumors, the cytotoxic effects of the addition of mitomycin C or porfiromycin on treatment with Fluosol-DA/carbogen (95% O2/5% CO2) breathing and radiation in the FSaIIC tumor system were studied. In vitro mitomycin C and porfiromycin were both preferentially cytotoxic toward hypoxic FSaIIC cells. After in vivo exposure, however, the cytotoxicity of mitomycin C toward single cell tumor suspensions obtained from whole tumors was exponential over the dose range studied, but for porfiromycin a plateau in cell killing was observed. With Fluosol-DA/carbogen breathing and single dose radiation, addition of either mitomycin C or porfiromycin increased the tumor cell kill achieved at 5 Gy by approximately 1.2 and 1.0 logs, respectively. Less effect was seen with addition of the drugs at the 10 and 15 Gy radiation doses. In tumor growth delay experiments, the addition of either mitomycin C or porfiromycin to Fluosol-DA/carbogen breathing and radiation resulted in primarily an additive increase in tumor growth delay. The survival of Hoechst 33342 dye-selected tumor cell subpopulations indicated that Fluosol-DA/carbogen breathing increased the cytotoxicity of radiation (10 Gy) more in the bright cell subpopulation (4-fold) than in the dim cell subpopulation (2-fold) resulting in an overall 4-fold sparing of the dim subpopulation. Mitomycin C and porfiromycin were both more toxic toward the dim cell subpopulations. Addition of mitomycin C or porfiromycin to Fluosol-DA/carbogen breathing and radiation (10 Gy) resulted in a primarily additive effect of the drugs and radiation killing in both tumor cell subpopulations. Thus, with mitomycin C/Fluosol-DA/carbogen and radiation there was a 2-fold sparing of dim cells and with porfiromycin in the combined treatment a 1.6-fold sparing of the dim cell population. Our results indicate that treatment strategies directed against both oxic and hypoxic tumor subpopulations can markedly increase the tumor cell kill achieved by radiation.

Bioreductive alkylating agents require reductive activation prior to exerting their cytotoxic actions. This property results in preferential toxicity to hypoxic cells. Previous data have demonstrated that mitomycin C is activated by hypoxic tumor cells and is selectively cytotoxic to these oxygen-deficient cells. The biotransformation of mitomycin C was studied in liver microsomes and nuclei and in a reconstituted, partially purified cytochrome P-450 drug-metabolizing system to provide information on these reductive processes. Both the metabolism of mitomycin C, measured by disappearance of the quinone portion of the substrate, and the formation of an alkylating metabolite(s), determined by employing 4-(p-nitrobenzyl)pyridine as a trapping agent, required anaerobic conditions and an NADPH-generating system, and were inhibited by O2 and CO in both microsomes and nuclei. A reconstituted enzyme system consisting of NADPH, NADPH-cytochrome P-450 reductase, phospholipid and cytochrome P-450 converted mitomycin C to a reactive metabolite(s) under hypoxic conditions. Omission of N2 or any component of the system decreased the metabolic activation of mitomycin C. These findings support the concept that the cytochrome P-450 system is capable of activating mitomycin C under hypoxic conditions to the alkylating metabolite(s) that is responsible for antineoplastic activity.

Under anaerobic conditions and with proper electron donors, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and xanthine oxidase (EC 1.2.3.2) similarly reductively metabolized mitomycin C. Reversed phase high performance liquid chromatography was used to separate, detect, and isolate several metabolites. Three metabolites were identified by mass spectrometry and thin layer chromatography as 1,2-cis- and trans-2,7-diamino-1-hydroxymitosene and 2,7-diaminomitosene. Three metabolites were phosphate-dependent, and two of them were identified to be 1,2-cis- and trans-2,7-diaminomitosene 1-phosphate. The amounts of the five identified metabolites generated during the reduction of mitomycin C varied with pH and nucleophile concentration. At pH 6.5, 2,7-diaminomitosene was essentially the only metabolite formed, whereas from pH 6.8 to 8.0, trans- and cis-2,7-diamino-1-hydroxymitosene increased in quantity as 2,7-diaminomitosene decreased. The disappearance of mitomycin C and the production of metabolites were enzyme and mitomycin C concentration-dependent. Substrate saturation was not reached for either enzyme up to 5 mM mitomycin C. Electron paramagnetic resonance studies demonstrated the formation of mitomycin C radical anion as an intermediate during enzymatic activation. Our results indicate that either enzyme catalyzed the initial activation of mitomycin C to a radical anion intermediate. Subsequent spontaneous reactions, including the elimination of methanol and the opening of the aziridine ring, generate one active center at C-1 which facilitates nucleophilic attack. Simultaneous generation of two reactive centers was not observed. All five primary metabolites were metabolized further by either flavoenzyme. The secondary metabolites exhibited similar changes in their absorbance spectra and were unlike the primary metabolites, suggesting that a second alkylating center other than C-1 was generated during secondary activation. We propose that secondary activation of monofunctionally bound mitomycin C is probably a main route for the bifunctional binding of mitomycin C to macromolecules and that the cytotoxic actions of mitomycin C result from multiple metabolic activations and reactions.

Cell division and DNA synthesis wereeirreversibly inhibited in HeLa cells by 0.25 μg/ml mitomycin C or 2.5 μg/ml porfiromycin. Vaccinia virus multiplied in the inhibited cells as evidenced by the uptake of [3H]thymidine into DNA and the subsequent appearance of radioactive, infectious virus. Higher levels of the drugs suppressed virus multiplication.

Trypanosoma equiperdum , a member of the brucei-evansi group of African trypanosomes, is sensitive to the following tumor-inhibitory antibiotics: actinomycin D, mitomycin C, porfiromycin, phleomycin, and pactamycin. Only pactamycin inhibited trypanosomal carbohydrate metabolism in vitro , an action that might have contributed to the ability of this antibiotic to depress protein synthesis and various pathways of nucleic acid metabolism. The inhibitory effects of the other antibiotics were restricted to trypanosomal nucleic acid metabolism. Actinomycin D, 1.6 × 10 −8 M, inhibited the incorporation of 3 H-adenine and 3 H-uracil into RNA by approximately 50%, but had no effect at this concentration upon the incorporation of derivatives of these isotopes into DNA. Higher concentrations had no effect upon the incorporation of 3 H-adenine and 3 H-thymidine into DNA, but did inhibit the incorporation of derivatives of 3 H-uracil into DNA. The distribution of radioactivity in the nucleotide components of the acid-soluble fractions suggested that the predominant action of actinomycin D was to inhibit RNA polymerase. Mitomycin C and its methyl congener, porfiromycin, 2.2 × 10 −4 M and 5.7 × 10 −4 M, respectively, inhibited the incorporation of derivatives of 3 H-adenine and 3 H-uracil into RNA and DNA by approximately 50%. At 7.5 × 10 −6 M and 1.1 × 10 −5 M, mitomycin C and porfiromycin inhibited the incorporation of 3 H-thymidine into DNA by approximately 50%, and at 7.5 × 10 −5 M and 7.2 × 10 −5 M, the evolution of 14 CO 2 derived from orotic acid- 14 COOH. The ability of the mitosane antibiotics, at relatively low concentrations, to inhibit the incorporation of thymidine into DNA suggested that interference with DNA replication was a prominent feature of this group. The data also indicated that at higher concentrations, mitomycin C and porfiromycin inhibited RNA polymerase—whether by direct action or as a consequence of their effects upon DNA synthesis remains unsettled. Phleomycin, 1.3 × 10 −4 M, inhibited the incorporation of 3 H-adenine and 3 H-uracil into RNA by approximately 50%. At this concentration, phleomycin inhibited the incorporation of derivatives of 3 H-uracil into DNA to a greater extent than the derivatives of 3 H-adenine. At 3.2 × 10 −6 M, phleomycin inhibited the incorporation of 3 H-thymidine into DNA by approximately 50%. The action of phleomycin seemed to be qualitatively similar to that of the mitosane antibiotics, except for its inability to inhibit orotidylic acid decarboxylase.

The kinetics of solvolysis of the antibiotic porfiromycin are quantified in the pharmaceutically useful pH ranges in acetate, phosphate, and borate buffers. The devised ultraviolet spectrophotometric assays of solution degraded antibiotic are correlated with the plate-disk assay against S. aureus and the paper chromatographic assay against S. lutea . General acid catalyzed solvolysis is indicated as well as specific acidbase. In acetate buffers, the primary degradative mechanism is the hydrolysis of the fused ring aziridine group, whereas mild alkali substitutes a hydroxyl for the quinoid ring amine. In the intermediate pH ranges characterized by phosphate buffers reaction at both sites is implied and maximum stability at pH 8 at 30° can be predicted. Conditions for isolation of solvolytic products and their characterizations are given.

The mass spectra of the four known mitomycin antibiotics, mitomycin A, B, C, porfiromycin, and four related compounds are reported. Characteristic fragmentation patterns for this group of antibiotics are discussed. The nature of major fragment ions from the aziridine ring has been established via deuterium labeling.

Induction experiments with the antibiotics mitomycin A, mitomycin B, and porfiromycin demonstrated that they are capable of inducing λ-development in Escherichia coli K12(λ), with viable phage yields 52–75% of that obtained by mitomycin C induction. Determination of the localization of [ 14 C]porfiromycin in cell extracts of E. coli K12(λ) showed 25% of the 14 C in the ribosomal fraction. 60–80% of the [ 14 C]porfiromycin bound in cell extracts is apparently attached to protein. Lysogenic induction of E. coli K12(λ) with [ 14 C]porfiromycin leads to the formation of λ particles which contain 14 C.

Solid tumors have inadequate vascular networks; thus, significantly less blood flows through these tumor vascular beds than through those of the tissue of origin, and as neoplastic masses enlarge, the blood supply to these tumors further decreases [l-3]. This phenomenon is believed to be due in part to a slower growth rate for endothelial cells of blood vessels than for malignant cells; such a differential results in an inability to maintain an adequate vascular supply [4]. In addition, as tumor masses enlarge, changes in interstitial pressure are possible which can cause compression of tumor capillary beds and regional changes in tumor blood flow. The end result of these oxygen deficit producing effects is the formation of chronic and acute hypoxia in the neoplastic cell population (Fig. l), which may have significant impact on the curability of malignant lesions. There is evidence that hypoxic cell populations exist in both animal [5] and human (see ref. 6 for appropriate references) tumors. Characterization of hypoxic cell populations in transplanted animal tumors has shown that most tumors contain between 5 and 30% of the malignant cell population in an oxygen-deficient state [5]. Although tumor enlargement increases the quantity of hypoxic cells, it is clear that micrometastases also contain oxygendeficient cell populations. Oxygen-deficient malignant cells are significantly more resistant to ionizing radiation than their aerobic counterparts (see for example refs 7 and 8) and, in addition, hypoxia may limit the efficacy of chemotherapy. This latter phenomenon is due to the fact that hypoxic cell populations may be composed of cells which are either blocked or are slowly progressing through the cell cycle [g-11]. Thus, cell cycle active agents may have a relatively low efficacy against hypoxic tumor cell populations. Chemotherapeutic agents may also have difficulty in reaching hypoxic regions, since they must diffuse into the