Classification of Antineoplastic Agents by Their Selective Toxicities Toward Oxygenated and Hypoxie Tumor Cells1

[CANCER RESEARCH 41, 73-81, January 1981] 0008-5472/81/1141-OOOOS02.00 Classification of Antineoplastic Agents by their Selective Toxicities toward Oxygenated and Hypoxie Tumor Cells1

Beverly A. Teicher,2 John S. Lazo, and Alan C. Salterelli

Department ot Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine. New Haven, Connecticut 06510

ABSTRACT tic agents toward cells that are hypoxic. It has been well- established that hypoxic cells exist in solid tumors and that The cytotoxicities of a number of antineoplastic agents to these cells are relatively resistant to the cytotoxic effects of oxygenated and hypoxic EMT6 mouse mammary tumor cells Â¡n ionizing radiation. Thus, the hypoxic cell population limits the culture were examined. Based on the relative sensitivities of curability of experimental animal tumors by large doses of cells under aerobic and hypoxic conditions, drugs were placed radiation (52). Since hypoxic cells may be either noncycling or into three categories. Drugs that were preferentially toxic to slowly progressing through the cell cycle (9, 14, 54), they are cells under oxygenated conditions were classified as type 1 also presumed to be relatively resistant to cell cycle-specific agents; this group includes bleomycin, procarbazine, strepto- chemotherapy. To develop a therapeutic program designed to nigrin, actinomycin D, and vincristine. Type 2 agents were approach the cure of solid tumors, the use of agents with those preferentially toxic to cells under hypoxic conditions. cytotoxic actions directed toward each of the physiologic cel These include mitomycin C and Adriamycin. On the basis of lular components of the tumor population would appear to be other published reports, the glucose analogs, 5-thio-o-glucose required. and 2-deoxy-D-glucose, and the radiosensitizers, misonidazole In this study, representative compounds from several classes and metronidazole, can also be placed in this category. Several of anticancer drugs were tested for cytotoxic activity toward antineoplastic agents showed no major preferential toxicity to cultured EMT6 tumor cells under conditions of normal aeration cells under the conditions of oxygÃ©nation or hypoxia used in and chronic hypoxia to determine whether preferential cytotox- these experiments and were placed in a third class. This group icity toward hypoxic cells was a property of any currently used (type 3) includes 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2- chloroethyl)-3-cyclohexyl-1 -nitrosourea, c/'s-diamminedichlo- antineoplastic agents. Based on the results obtained from these studies, agents were grouped into 3 distinct classes. The roplatinum(ll), 5-fluorouracil, and methotrexate. The success possible use of these findings to fashion an approach to the of many combination chemotherapy and combined modality selection of agents to use in combination in the clinical treat treatments may be due to their ability to kill both the hypoxic ment of solid tumors is discussed. and aerobic cell populations of solid tumors. Future chemotherapeutic regimens for the treatment of solid tumors should MATERIALS AND METHODS include agents and modalities directed toward the hypoxic cell population of the tumor, as well as toward the proliferating and Drugs. Mitomycin C and bleomycin (Bleoxane) (1.6 units/ nonproliferating tumor cell compartments; a therapeutic ap mg) were the gifts of Dr. Maxwell Gordon and Dr. William T. proach to the selection of antineoplastic agents for use in Bradner, respectively, of the Bristol-Myers Company (New combination based upon physiological considerations of the York, N. Y.). Vincristine sulfate (Oncovin) was obtained from architecture of solid tumors is presented. Eli Lilly and Company (Indianapolis, Ind.). Streptonigrin and Adriamycin were the gifts of Dr. John D. Douros of the Division INTRODUCTION of Cancer Treatment of the National Cancer Institute (Bethesda, Md.); c/s-diamminedichloroplatinum(ll), BCNU,3 and CCNU Solid tumors are refractory to cytotoxic agents for several were also obtained from the Division of Cancer Treatment. reasons including: (a) many antineoplastic agents do not reach Procarbazine-HCI was obtained from Hoffman-LaRoche Inc. the poorly vascularized regions of the tumor; and (b) the cellular (Nutley, N. J.) and actinomycin D from Calbiochem-Behring populations in solid tumors are physiologically more hetero Corp. (La Jolla, Calif.). 5-Fluorouracil and methotrexate were geneous with respect to oxygÃ©nationand proliferation than are purchased from Sigma Chemical Company (St. Louis, Mo.). All the cellular components of hematological or particularly well- other reagents were obtained from standard chemical sources. vascularized tumors. Drugs were dissolved in acetone, ethanol, sterile distilled water, Most of the currently used chemotherapeutic agents are or sterile phosphate-buffered saline (8.0 g/l NaCI, 0.2 g/l KCI, more active against cells in exponential growth than against 1.15 g/l Na2HPO4, 0.2 g/l KH2PO4) for addition to the tissue cells in the plateau phase, in which a relatively great proportion culture system. of the cells are not actively traversing the cell cycle (16, 68). Tumor Cells and Cytotoxicity Studies. Experiments were However, little is known about the toxicity of most antineoplas- performed using EMT6 mouse mammary tumor cells in vitro. The techniques used for propagating the cells and measuring ' This research was supported in part by USPHS Grants CA-02817 and CA- their survival by colony formation have been described in detail 16359 from the National Cancer Institute and a grant from the Bristol-Myers previously (50, 82, 83). Cells were grown as monolayers in 25 Company. 2 Recipient of a postdoctoral fellowship (CA-06365) from the National Cancer Institute To whom requests for reprints should be addressed. 3 The abbreviations used are: BCNU. 1,3-bis(2-chloroethylM-nitrosourea; Received March 17. 1980; accepted September 23, 1980. CCNU. 1-<2-chloroethyl)-3-cyclohexyl-1 -nitrosourea.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. B. A. Teicher et al. sq cm Corning plastic culture flasks in Waymouth's medium oxygenated cells than to chronically hypoxic cells. The com supplemented with 15% fetal bovine serum and used for these pounds grouped as type 2 were agents which were more toxic experiments when in exponential growth. To produce hypoxia, to hypoxic cells than to cells under conditions of normal aera flasks were fitted with sterile rubber sleeve serum stoppers and tion. Type 3 agents and treatment modalities were essentially exposed to a continuously flowing 95% nitrogen/5% CO2 equitoxic to oxygenated and hypoxic cells. humidified atmosphere for 4 hr at 37Â°prior to drug treatment. Type 1 Agents. Bleomycin produces fragmentation of DMA These conditions produce a degree of hypoxia sufficient to in a reaction which is dependent upon the presence of ferrous result in radiobiological resistance (oxygen concentration 10 ions and molecular oxygen (89, 90). Reactive free radicals of ppm or less). Parallel flasks were maintained in humidified 95% oxygen may be responsible for the cleavage of DNA by bleo- air/5% COj. At this time, each of the drugs or vehicle was mycin (61, 73, 99). Sausville ef al. (90) showed that the added to the flasks by injection through the rubber stoppers reaction of bleomycin and iron(ll) with adenovirus [3H]DNA in without breaking the hypoxia. After exposure to each agent for phosphate buffer was virtually completely inhibited when the 1 hr at 37Â° under hypoxia or normal aeration, the cells were reaction mixture was equilibrated with argon. The survival of washed with 3 ml of sterile phosphate-buffered saline, sus EMT6 cells exposed to bleomycin for 1 hr under either oxy pended by treatment with 0.05% trypsin in phosphate-buffered genated or hypoxic conditions is shown in Chart 1. As reported saline for 15 min, plated in replicate dishes at 3 dilutions in by others (7, 85), the survival-dose response curve to short Waymouth's medium plus 15% fetal calf serum, and the surviv time exposure to bleomycin is bi- or multiphasic. At all concen ing fraction of cells was measured by colony formation. No trations examined, bleomycin was more toxic to oxygenated difference existed between the survival of untreated or vehicle- cells than to cells maintained in a hypoxic atmosphere for 4 hr treated cells maintained under the aerobic and hypoxic condi prior to exposure to the drug. At 150 milliunits of the antibiotic tions used; the plating efficiency for these control cultures was 65 to 80%. Cells exposed to hypoxic conditions appeared to continue traversing the cell cycle during the course of these experiments, since no decrease in the rate of [3H]thymidine I 00 incorporation into acid-insoluble material (data not shown) or BLEOMYCIN in the mitotic index was observed after 4 hr of incubation in the hypoxic atmosphere. Each drug was tested in at least 3 sepa rate experiments.

RESULTS AND DISCUSSION

Classification of Antineoplastic Agents. Based upon phys iological considerations, solid tumors may be envisioned to consist of at least 3 classes of neoplastic cells. These include: (a) cells which are well oxygenated, are relatively rapidly trav ersing the cell cycle, and may correspond in drug sensitivities to logarithmically growing cells in culture; (b) non- or slowly proliferating oxygenated cells which may correspond in their susceptibilities to anticancer agents to plateau-phase cells in culture; and (c) cells in various degrees of hypoxia (108, 109). This last population may be composed of neoplastic cells with either relatively normal or prolonged cell cycle times or with cells blocked in their progression through the cell cycle. Anti neoplastic agents can be grouped into classes based upon 15 IO 25 50 75 100 ISO their cytotoxicities toward the neoplastic cell populations pres DRUG CONCENTRATION, mil/ml ent in each of these compartments (Table 1). Type 1 agents Chart 1. Survival of aerobic (â€¢)and hypoxic (O) EMT6 cells treated for 1 hr and treatment modalities were those which were more toxic to with various concentrations of bleomycin in cell culture.

Table 1 Classification of antineoplastic agents and treatment modalities based on cellular oxygÃ©nation Preferential toxicity to aerobic cells Preferential toxicity to hypoxic Minimal or no selectivity based on cel (type 1) cells (type 2) lular oxygÃ©nation (type 3) Bleomycin Mitomycin C 5-Fluorouracil Methotrexatea Procarbazine Adriamycin Streptonigrin Misonidazole, metronidazole c/s-Diamminedichloroplatinum (II) Actinomycin D 5-Thio-o-glucose. 2-deoxy-D-glucose BCNU, CCNU Vincristine High linear energy transfer radiation Ionizing radiation 3 Under the test conditions used in these experiments, hypoxic cells are still capable of DNA synthesis and of cellular replication. These agents have cytotoxic effects primarily on cells in the S phase of the cell cycle. Thus, in hypoxic cells that are blocked in their progression through the cell cycle or are cycling slowly, agents such as these that act on the S phase of the cell cycle would be expected to be relatively noncytotoxic.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. Drug Cytotoxicity and Cellular OxygÃ©nation per ml, a 9-fold difference in drug sensitivity was observed. 100-fold more sensitive to the lethal actions of actinomycin D That bleomycin showed some toxicity toward chronically hy- than are hypoxic cells. Thus, there may be a secondary oxy poxic cells may be the result of (a) residual antibiotic which gen-dependent cytotoxic mechanism of action for actinomycin exerted its cytotoxic effect after the hypoxic state was dis D at relatively high drug concentrations. rupted, (Â£>)residual oxygen in hypoxic cells, or (c) a non- oxygen-dependent mechanism of drug cytotoxicity. STREPTONIGRIN Procarbazine is rapidly oxidized in aqueous solution in the presence of oxygen to an azo derivative which is further oxi dized in vivo and in vitro to alkylating species (112). Procar COjH bazine is considerably more toxic to bacteria under aerobic conditions than under anaerobiasis (81). As shown in Chart 2, this differential cytotoxicity is also expressed with mammalian tumor cells, the drug being approximately 7 times more toxic to normally aerated EMT6 cells than to these cells maintained under conditions of chronic hypoxia during drug exposure. At a drug concentration of 1 Â¡J.M,therewas no kill of hypoxic cells, yet greater than 70% of the aerobic cells were killed. It is unclear whether the cytotoxicity of procarbazine to hypoxic cells is due to residual drug or to a non-oxygen-dependent mechanism. Streptonigrin appears to damage DNA through the obligatory intermediacy of Superoxide radical (22). As shown in Chart 3, only 20% survival of normally aerated EMT6 cells was obtained at a concentration of only 1 pw Streptonigrin; under these conditions, the survival of hypoxic EMT6 tumor cells was unaffected. At all drug concentrations examined, Streptonigrin was about 10 times more toxic to normally aerated cells than OOOOOOI OOOOOI OOOOI OOOI 001 01 to hypoxic cells. DRUG CONCENTRATION, ^M Actinomycin D binds to double-stranded DNA, permitting initiation of RNA synthesis but blocking the elongation process. Chart 3. Survival of aerobic {â€¢)and hypoxic (O) EMT6 cells treated for 1 hr with various concentrations of Streptonigrin in cell culture. The cytotoxicity of actinomycin D appears to be primarily a consequence of this interaction with DNA (36, 110). Chart 4 shows that, at concentrations less than 0.01 /Â¿M,no major difference was observed in the cytotoxicity of this antibiotic toward normally aerated and hypoxic cells; however, at drug concentrations approaching 1 /ÃŒM,oxygenatedcells are almost

i.20 r PROCARBAZINE

IO 100 1000 lO.OOO OOOOOI OOOOI OOOI 001 O.I 1.0 DRUG CONCENTRATION, pM DRUG CONCENTRATION,

Chart 2. Survival of aerobic (â€¢)and hypoxic (O) EMT6 cells treated for 1 hr Chart 4. Survival of aerobic (â€¢)and hypoxic (O) EMT6 cells treated for 1 hr with various concentrations of procarbazine in cell culture. with various concentrations of actinomycin D in cell culture.

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Although the cytotoxicity of vincristine is attributed to its taneous rearrangement of the reduced molecule can then ability to interrupt cell division in metaphase (113), other effects theoretically lead to the production of a highly reactive quinone may also contribute to cell death (23). In the range of vincristine methide intermediate capable of alkylating cellular molecules concentrations tested (i.e., 0.01 to 50 /IM), the drug showed (46, 47, 60, 92). Schwartz (91) demonstrated that liver con minimal cytotoxicity to hypoxic cells, while toxicity to oxygen tains an enzymatic system capable of metabolizing mitomycin ated cells was very clearly a dose-related process (Chart 5). C under anaerobic conditions; this enzyme system is found in This finding cannot be attributed to a decrease in mitoses in both the microsomal and nuclear fractions (51). The bioacti- the hypoxic cells, since the mitotic index was 5.79 Â±0.89% vation of mitomycin C to an alkylating species can occur in for aerobic cells and 6.59 Â± 1.69% for cells after 4 hr of neoplastic cells in a reaction requiring anaerobiasis and an hypoxia. NADPH-generating system (50, 52). In agreement with these Agents classified as type 1 are primarily drugs that require findings, mitomycin C was considerably more toxic to hypoxic molecular oxygen or oxidative biotransformation to exert a cells than to oxygenated cells over a wide range of concentra cytotoxic effect and includes the treatment modality ionizing tions (Chart 6). This difference in sensitivity reached a maxi radiation (52). Additionally, some drugs may be accumulated mum of about 10-fold at relatively high concentrations of drug; or retained intracellularly by energy-requiring processes that in addition, however, at a concentration range of 0.001 to 0.1 depend upon oxidative metabolism; therefore, intracellular con /Â¿Mmitomycin C, little or no measurable cytotoxic activity oc centrations of these drugs may be different in oxygenated and curred in oxygenated cells, while 50 to 90% of the hypoxic hypoxic cells. In this test system, streptonigrin showed the cells were unable to replicate. largest differential kill of aerobic cells, being greater than 1000 The action of Adriamycin may involve activation via metabolic times more toxic to oxygenated cells at 50% survival and reduction, either by one- or 2-electron transfer (29, 30, 62). approximately 10,000 times more toxic to aerobic cells at 20% Anaerobic incubation of Adriamycin with liver microsomes in survival. Procarbazine required greater than 5000 times more the presence of NADPH results in the appearance of an elec drug to achieve a 50% kill of hypoxic cells than to kill 50% of tron spin resonance signal attributed to the semiquinone free aerobic cells. The degree of differential kill achieved by bleo- radical (1-3, 40, 88). Although the direct reaction of the mycin, actinomycin D, and vincristine was similar, ranging anthracycline radical with tissue constituents has not been between 50 to 100 times more toxic to oxygenated EMT6 cells reported, Bachur et al. (3) have observed widespread appar than to their hypoxic counterparts at 50% survival levels. ently covalent binding of Adriamycin to tissue proteins. An Type 2 Agents. Early studies established that DNA is the explanation for this alkylating ability which involves 2-electron principal target for the expression of the antineoplastic activity reduction of the quinone nucleus of Adriamycin followed by the of mitomycin C (46, 47). The term bioreductive alkylating agent elimination of the daunosamine sugar moiety with subsequent has been used by this laboratory to describe the class of drugs formation of a quinone methide alkylating species has been which require reductive transformation to exhibit alkylating proposed by Moore (71 ). As shown in Chart 7, Adriamycin was activity (57, 58), and mitomycin C can be considered to be a more toxic to hypoxic cells at all of the concentrations of the naturally occurring bioreductive alkylating agent. Enzymatic reduction of this agent to the hydroquinone results in the loss of methanol to give an aziridinomitosene derivative (24). Spon-

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OOI O.I I.O IO OOOI O.OI O.l I.O IOO DRUG CONCENTRATION, Â¿iM DRUG CONCENTRATION,/ Chart 5. Survival of aerobic (â€¢)and hypoxic (O) EMT6 cells treated for 1 hr Chart 6. Survival of aerobic (â€¢)and hypoxic (O) EMT6 cells treated for 1 hr with various concentrations of vincristine in cell culture. with various concentrations of mitomycin C in cell culture.

76 CANCER RESEARCH VOL. 41

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I.O 10 100 1000 10,000 DRUG CONCENTRATION, 0.0001 0.001 0.01 O.I 1.0 Chart 8. Survival of aerobic (â€¢)and hypoxic (O) EMT6 cells treated for 1 hr DRUG CONCENTRATION, Â¿iM with various concentrations of 5-fluorouracil in cell culture. Chart 7. Survival of aerobic (â€¢)and hypoxic (O) EMT6 cells treated for 1 hr with various concentrations of Adriamycin in cell culture. of glucose for survival; aerobic cells are more resistant to depletion of the glucose supply or to inhibition of glycolysis antibiotic tested. At high drug levels, the differential kill of (96). Since hypoxic cells derive their energy primarily by an hypoxic cells was 10 times greater than the kill of oxygenated aerobic glycolysis, they are particularly vulnerable to either a cells. It appears that, under hypoxic conditions, Adriamycin diminished availability of glucose or an inhibition of glycolysis. may act as a bioreductive alkylating agent, while under condi 5-Thio-D-glucose and 2-deoxy-o-glucose, potent inhibitors of tions of normal aeration, an oxygen-dependent mechanism of glycolysis, are preferentially cytotoxic to hypoxic cells (95- cytotoxicity may be operative. In contrast to these findings, 98). Glucose analogs may be of limited clinical utility, however, Smith et al. (94) reported that hypoxic Chinese hamster ovary because of the relatively large quantities required to produce cells were more resistant to Adriamycin than their oxygenated lethality by creating a shortage of glucose in hypoxic tumor counterparts, and Harris and Schrieve (41), using an EMT6 cells. cell line, found no difference in the toxicity of Adriamycin to Type 3 Agents. The mechanism by which 5-fluorouracil kills oxygenated and hypoxic cells. The latter authors indicate that neoplastic cells is believed to require conversion to 5-fluoro- the sensitivity of their line of EMT6 to antineoplastic agents 2'-deoxyuridylic acid, which forms a stable ternary complex differed from that of others. The results of these studies, with the cofactor, 5,10-methylenetetrahydrofolate and the en however, indicate that all tumor cells may not be capable of zyme thymidylate synthetase (42, 56, 64), leading to a state of "thymineless death" (21, 44, 87). As shown in Chart 8, no carrying out the reductive reactions necessary to activate Adriamycin under hypoxic conditions. major difference was observed in the cytotoxicity of 5-fluo Except for glucose analogs, all agents classified as type 2 rouracil to oxygenated and hypoxic cells. The aerobic and seem to use mechanisms that involve enzymatic reduction of hypoxic cells used in this study incorporate [3H]thymidine into a functional group on the drug molecule to express their acid-insoluble material at equal rates, suggesting that cells cytotoxic activity. The nitroheterocyclic radiosensitizers, ex under both of these conditions are actively traversing the cell emplified by metronidazole and misonidazole, are selectively cycle during the course of the experiment. This presumably toxic to hypoxic cells in the absence of irradiation (27, 33-35, explains the sensitivity of oxygenated and hypoxic cells to this 67, 102, 107). This selective toxicity corresponds to the pref antimetabolite. erential formation of relatively large amounts of N-hydroxy and Methotrexate is a potent inhibitor of dihydrofolate reducÃase amine metabolites formed by nitro group reduction by hypoxic (1 2); interference with this enzyme activity leads to a decrease cells (8, 74, 103, 107). To attain cytotoxicity by the nitroimi- in the rate of synthesis of cellular DMA, RNA, and protein (10, dazoles requires prolonged contact times of several hr and 11). As shown in Chart 9, methotrexate was similar to 5- relatively high drug concentrations (e.g., 1 to 5 rriM misonida fluorouracil, in that little difference was observed in the degree zole) (32, 70, 115, 11 6); consequently, the selective action of of cytotoxicity exhibited toward EMT6 cells which was depend these agents on hypoxic cells may not be exploitable in the ent upon their state of oxygÃ©nation. treatment of human cancer (1 5, 26). The neutral platinum complexes interact with DNA to impair A prominent metabolic difference between aerobic and hy its function as a template for further DNA replication (80). cis- poxic cells is their degree of dependence upon the metabolism Diamminedichloroplatinum(ll) has been shown to cross-link

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METHOTREXATE 100 i.oo - BCNU

O.I IO IO IOO 001 0.1 1.0 IO 100 1000 DRUG CONCENTRATION, MM DRUG CONCENTRATION, MM Chart 1 1. Survival of aerobic (â€¢)and hypoxic (O) EMT6 cells treated for 1 hr Chart 9. Survival of aerobic (â€¢)and hypoxic (O) EMT6 cells treated for 1 hr with various concentrations of BCNU in cell culture. with various concentrations of methotrexate in cell culture.

Antineoplastic agents considered to be type 3 were those in cis-DIAMMINEDICHLOROPLATINUM (H) which the ratio of drug concentrations required to achieve IOO - either a 50% or 20% survival of oxygenated and hypoxic cells ranged between 0.5 and 5. Drugs in this category include alkylating agents, such as the 2-haloethylnitrosoureas and the neutral platinum compounds, and since in the methodology used in this study both oxygenated and hypoxic cells appear to be actively traversing the cell cycle, the S-phase inhibitors 5-fluorouracil and methotrexate fall into this group (45). It is essential to recognize, however, that long-term chronically hypoxic cells which are either noncycling or are slowly moving through the cycle would not be expected to be sensitive to these agents. High linear energy transfer radiation also has no oxygen requirement for cytotoxic activity and may be placed in this class. Penetration. A property of major importance for hypoxic cell directed chemotherapeutic agents is the ability of the drugs to penetrate to poorly vascularized regions of the tumor to reach target hypoxic cells in therapeutically useful concentrations. Furthermore, the intracellular concentrations of the cytotoxic agents which can be achieved may differ for oxygenated and hypoxic cells. Although molecular oxygen penetrates only a oooooi ooooi 0.001 0.01 O.I 10 IOO short distance through tumor tissue, largely because of its DRUG CONCENTRATION,/iM rapid metabolic utilization, some dyes and some drugs can Chart 10. Survival of aerobic (â€¢)andhypoxic (O) EMT6 cells treated for 1 hr with various concentrations of c/s-diamminedichloroplatinum(ll) in cell culture. diffuse into the tumor mass over much greater distances (108, 109). Although the nitroimidazole radiosensitizers and the glu DMA in mammalian cells (79). As might be expected for an cose analogs are both type 2 agents, since they are not agent which can covalently bind to critical molecules in cells exceedingly potent cytotoxic agents for hypoxic cells, large regardless of their physiological state, no difference in the doses would appear to be required to achieve effective drug cytotoxicity of c/s-diamminedichloroplatinum(ll) toward oxy concentrations in the tumor. Adriamycin is exceedingly effec genated and hypoxic cells was observed (Chart 10). tive against hypoxic cells, but this agent has been reported to The 2-haloethylnitrosoureas, BCNU and CCNU, are chemi have poor tumor penetrating properties (101). In contrast, cally reactive compounds that decompose nonenzymatically at mitomycin C, also a potent type 2 agent, has been shown to relatively rapid rates under physiological conditions (28, 43, have the ability to penetrate to hypoxic regions of a rodent 69, 78). Furthermore, BCNU and related 2-haloethylnitrosou solid tumor (84). Therefore, we believe that at present mito reas covalently cross-link DMA (48, 49, 93). As shown in Chart mycin C is the most promising agent with which to attack the 11, BCNU has little selective cytotoxicity that is based upon hypoxic cell compartment of solid tumors currently available the cellular state of oxygÃ©nation. A similar result was obtained for clinical use. with CCNU. Evidence for the Clinical Utility of the Concept. A variety of

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. Drug Cytotoxicity and Cellular OxygÃ©nation clinical trials have used mitomycin C or Adriamycin, the 2 each of the cell types present in the tumor, including cycling agents which appear to be the most efficacious against the and nonclycing populations of oxygenated and hypoxic com hypoxic cell component of solid tumors, in admixture with partments. Selection of combinations of drugs (or other treat drugs capable of attacking cells in the oxygenated compart ment modalities) based upon these concepts should include: ments of solid tumors. These studies encourage the utilization (a) a bioreductive alkylating agent designed to attack the of combinations of drugs specifically designed to attack cellular hypoxic cell compartment by exploitation of the capacity of compartments based upon the physiological status of the neo- these cells to accomplish reductive reactions; mitomycin C plastic cells. They include combination chemotherapy with would appear to be the most efficacious agent of this class mitomycin C, particularly mitomycin C in admixture with 5- presently available for clinical use. To maximize the differential fluorouracil, an agent capable of attacking well-oxygenated toxicity of this agent to hypoxic cells, it should be given in tumor cells in cycle, in the treatment of advanced carcinoma of relatively low doses over a relatively long period. In addition, it the stomach, pancreas, and colon. This combination (often should be administered prior to the component(s) of the drug including additional agents) has produced significant increases combination used to kill oxygenated cells, to minimize the loss in both the percentage and duration of responses as compared of effectiveness of this agent through reoxygenation of the to either drug alone (17-19, 25, 39, 55, 63, 77). Mitomycin C hypoxic cell compartment after kill of aerobic cells, a process in combination with bleomycin, an agent whose cytotoxicity is that is capable of occurring relatively rapidly (52); (b) an agent directed primarily against oxygenated cells, or in admixture such as bleomycin or a nitrosourea would seem to be a rea with bleomycin and vincristine, also a type 1 agent, appears to sonable addition to such therapy to specifically attack any represent a very significant advance in the chemotherapy of nonproliferating oxygenated cells present in the tumor (i.e., advanced squamous cell cervical cancer (4, 5, 66). Radiation plateau phase-like cells); and (c) X-irradiation and/or an agent therapy, a type 1 modality, plus chemotherapy consisting of 5- or mixture of agents with specificity for actively proliferating fluorouracil and mitomycin C has been reported to result in no aerated cells. The drug(s) selected to attack these cellular evidence of disease in 3 patients presenting with surgically components of the malignant tumor obviously must be capable inoperable epidermoid carcinoma of the anus (72). Mitomycin of achieving biochemical lesions which lead to cell death. C in combination chemotherapy or alone has also been re ported to produce objective response in the treatment of liver REFERENCES (53, 65), breast (114), and lung cancer (86). 1. Bachur, N. R. Anthracycline antibiotic pharmacology and metabolism. Adriamycin, a type 2 agent, is synergistic with several alkyl- Cancer Treat. Rep.. 63. 817-820. 1979. ating agents; thus, in clinical trials in advanced breast cancer, 2. Bachur, N. R., Gordon. S. L.. and Gee, M. V. Anthracycline antibiotic augmentation of microsomal electron transport and free radical formation. Adriamycin in combination with alkylating agents has resulted Mol. Pharmacol.. 13: 901-910, 1977. in a high percentage of responses to treatment (59, 76, 104, 3. Bachur, N. R., Gordon, S. L., and Gee, M. V. A general mechanism for 111). Adriamycin in combination chemotherapy has also been microsomal activation of quinone anticancer agents to free radicals. Cancer Res., 38. 1745-1750, 1978. effective in the treatment of advanced lung (20), bronchogenic 4. Baker, L. H.. Opipari. M. I., and Izbicki, R. M. Phase II study of mitomycin (105), and testicular carcinoma (31). The treatment of ad C, vincristine, and bleomycin in advanced squamous cell carcinoma of the vanced epidermoid carcinoma of the cervix with this agent in uterine cervix. Cancer (Phila.). 38. 2222-2224, 1976. 5. Baker, L. H., Opipari, M. I.. Wilson. H., Bottomley, R., and Coliman, C. A. combination with bleomycin gave poor results at the primary Mitomycin C, vincristine, and bleomycin therapy for advanced cervical lesion; however, a satisfactory response rate was observed in cancer. Obstet. Gynecol., 5ÃŽ.146-150, 1978. 6. Barranco. S. C., and Novak, J. K. Survival responses of dividing and metastatic growths (37). Response rates achieved with Adria nondividing mammalian cells after treatment with hydroxyurea. arabinosyl- mycin therapy appear to be of relatively short duration, perhaps cytosine. or Adriamycin. Cancer Res.. 34: 1616-1618. 1974. due to inadequate penetration of the drug to the hypoxic tumor 7. Barranco, S. C., Novak, J. K.. and Humphrey, R. M. Response of mam malian cells following treatment with bleomycin and 1,3-bis(2-chloroethyl>- cells most distal from the tumor blood supply. 1-nitrosourea during plateau phase. Cancer Res., 33. 691-694, 1973. Attack of Nonproliferating Cells. The nonproliferating cel 8. Sasaga, S. H.. Dunlop, J. R., Searle, A. J. F., and Willson. R. L. 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Beverly A. Teicher, John S. Lazo and Alan C. Sartorelli

Cancer Res 1981;41:73-81.

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