Lonidamine As a Modulator of Alkylating Agent Activity in Vitro and in Vivo1

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[CANCER RESEARCH 51, 780-784, February 1, 1991] Lonidamine as a Modulator of Alkylating Agent Activity in Vitro and in Vivo1

Beverly A. Teicher,2 Terence S. Herman, Sylvia A. Holden, Ron Epelbaum, Shi-de Liu, and Emil Frei III

Dana-Farber Cancer Institute Â¡B.A. T., T. S. H., S. A. H., R. E., S-d. L., E. F.] and Joint Center for Radiation Therapy Â¡B.A. T., T. S. H.], Boston, Massachusetts 02115

ABSTRACT lonidamine can be reversible if the drug is removed after short time exposure (7). Ultrastructural studies by DeMartino et al. We are searching for relatively nontoxic compounds that can positively (2) indicate that the inhibition of energy metabolism in cells by modulate the efficacy of antitumor alkylating agents. Lonidamine inhibits lonidamine is a consequence of damage to the inner and outer cellular energy metabolism and could potentially increase damage by alkylating agents if cellular defenses are energy requiring. Exposure of mitochondrial membranes which leads to inhibition of respira cells to lonidamine (500 MM)for 2 h under hypoxic conditions followed tion and glycolysis and finally loss of cell viability (2). by I-li exposures to lonidamine plus alkylating agents under normally Lonidamine could be an important component of a combined oxygenated conditions in vitro significantly increased the cell kill modality regimen if repair of damage by a cytotoxic treatment achieved by m-diamminedichloroplatinum(ll) (CDDP) approximately re is an energy-dependent process. Working with Chinese hamster fold and by o-tetraplatin approximately 10-fold at 90% inhibitory con HA-1 cells in culture, Hahn et al. (8) showed that, at concen centration in MCF-7/CDDP (CDDP-resistant) cells. Carboplatin cyto- trations achievable in vivo, lonidamine inhibited the repair of toxicity, however, was little changed. In the MCF-7 parent cell line, potentially lethal damage caused by X-rays, methyl methane treatment with lonidamine increased CDDP cytotoxicity by approxi sulfonate, bleomycin, and hyperthermia. Kim et al. (9-11) mately 10-fold, D-tetraplatin by approximately 10-fold, and car hoplatin by approximately 8-fold at the 90% inhibitory concentration. For i - showed that lonidamine potentiated the effects of radiation and phenylalanine mustard (melphalan), Ar,A",jV"-triethylenethiophosphor- the effects of hyperthermia (10) in murine tumor models. Lon amide (thiotepa), and Ar,jV'-bis(2-chloroethyl)-/V-nitrosourea, little re idamine has also been shown to enhance the cytotoxicity of sistance was evident in the MCF-7/CDDP lines compared with the parent several alkylating agents (12) as well as Adriamycin in culture line. Treatment with lonidamine increased the cytotoxicity of each drug (13). by 1.5- to 3-fold in both cell lines. When exposure to lonidamine was We are searching for drugs that are relatively noncytotoxic extended to 24 h before and 12 h after drug exposure in MCF-7 normally themselves but which may positively and selectively modulate oxygenated cultures, CDDP (250 MM) cytotoxicity was increased by the cytotoxicity of alkylating agents in tumor cells. The present approximately 100-fold, but melphalan cytotoxicity was increased only studies were undertaken to examine the potential of lonidamine 2- to 3-fold over the concentration range tested. In the FSallC murine to enhance the activity of alkylating agents in vitro against fibrosarcoma tumor system, five i.p. injections of 50 mg/kg of lonidamine MCF-7 and MCF-7/CDDP3 (CDDP resistant) human breast over 36 h increased the tumor cell kill by CDDP and carboplatin approximately 2- to 3-fold over the dose range tested when the platinum carcinoma cells and against the FSallC murine fibrosarcoma complexes were given i.p. immediately after the third lonidamine injec and EMT6 murine mammary carcinoma in vivo. tion. When cyclophosphamide and thiotepa were given in the same schedule, 10-fold increases in tumor cell killing were evident on tumor MATERIALS AND METHODS excision assay over the dosage ranges. The increase in bone marrow toxicity caused by lonidamine in addition to the alkylating agents was Drugs. Lonidamine was obtained as a gift from DeSanctis Consult less than for tumor cells. Finally, in the EMT6 murine mammary ants (Montreal, Canada), prepared in PBS, and stored at -20Â°C.CDDP carcinoma, use of lonidamine at 500 mg/kg twice daily along with CDDP, (cisplatin) and carboplatin were gifts from Dr. Donald H. Picker and carboplatin, thiotepa, and cyclophosphamide significantly increased tu Dr. Michael J. Abrams, Johnson Matthey, Inc. (West Chester, PA), mor growth delays by approximately 1.6- to 3.0-fold. The results suggest and were prepared in PBS and stored at -20Â°C. o-Tetraplatin was a that lonidamine can positively modulate antitumor alkylating agent cy gift from the Upjohn Co. (Kalamazoo, MI). Melphalan (L-phenylala- totoxicity and may be a clinically useful adjunctive therapy with these nine mustard) and CTX were purchased as pure powders from Sigma drugs. Chemical Co. (St. Louis, MO). L-PAM was dissolved in HCl-acidified ethanol and diluted with PBS just prior to use. BCNU and thiotepa were purchased from the Dana-Farber Cancer Institute pharmacy. INTRODUCTION Cell Lines. The MCF-7 cell line is a human adenocarcinoma of the breast, developed by Dr. M. Rich of the Michigan Cancer Foundation. Lonidamine, 1-[(2,4-dichlorophenyl)methyl]-1 //-indazole-3- This line is estrogen receptor positive and retains certain characteristics carboxylic acid, affects the energy metabolism of cells (1-6). In of breast adenocarcinoma. MCF-7 has been used as a model for in vitro both normal and neoplastic cells, oxygen consumption is studies of breast carcinoma (14, 15). MCF-7 human breast carcinoma strongly inhibited by this drug. Furthermore, in tumor cells, cells grow as monolayers in Dulbecco's minimal essential medium aerobic and anaerobic glycolysis are additionally affected (1,3- supplemented with antibiotics, L-glutamine, and 10% fetal bovine 6). Based on these data, mitochondria have been considered the serum. This cell line has a plating efficiency of 25 to 40%. The MCF-7/CDDP cell line was developed by a dose escalation primary intracellular targets of the drug. As data have been process as described previously (16, 17). The resistant subline was accrued, it has become evident that lonidamine is not equally screened for degree of resistance, generation times similar to those of effective in all cell types and that the mitochondrial effects of the parent line, and relative stability of resistance (up to 2 mo). Every Received 8/14/90; accepted 11/9/90. 2 mo, a vial of early passage cloned cells was used to ensure that all The costs of publication of this article were defrayed in part by the payment experiments were carried out with the same subline. The MCF-7/ of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 'The abbreviations used are: CDDP, c/s-diamminedichloroplatinum(II); L- ' This work was supported by a grant from DeSanctis Consultants. Montreal. PAM, L-phenylalanine mustard (melphalan); thiotepa, A',W,A"'-triethylenethio- Canada; National Cancer Institute Grant 1P01-CA38493; and a grant from the phosphoramide; CTX, cyclophosphamide; BCNU, A',A"-bis(2-chloroethyl)-A'-ni- Mathers Foundation. trosourea; CFU-GM, granulocyte-macrophage colony-forming unit; PBS, phos 2To whom requests for reprints should be addressed. phate-buffered saline. 780

CDDP cells are grown under the same medium and serum conditions lated as the days taken by each individual tumor to reach 500 mm3 as are the MCF-7 parent cell line and have a plating efficiency of 25 to compared with the untreated controls. Tumor volume was calculated 30%. as a hem Â¡ellipsoid.Untreated EMT6 tumors reached 500 mm3 in 12.2 Survival Studies. MCF-7 cells or MCF-7/CDDP cells in exponential Â±0.4 days. Each treatment group had seven animals, and the experi growth were treated with lonidamine (500 Â¿JM)for2 h under hypoxic ment was repeated 3 times. Days of tumor growth delay are the means conditions (18) and then exposed to various concentrations of the Â±SE for the treatment group compared with the controls. alkylating agents along with the lonidamine for l h under normally oxygenated conditions. In other experiments, MCF-7 cells in exponen tial growth were exposed to lonidamine (500 Â¿IM)under normally RESULTS oxygenated conditions for 24 h prior to exposure to the combination of lonidamine (500 IÂ¡M)and various concentrations of CDDP or L- Exposure to lonidamine (500 pM) for 2 h under hypoxic PAM for l h and followed by an additional 18 h of exposure to conditions followed by an additional 1-h exposure under nor lonidamine (500 ^M). Following treatment, cells were washed 3 times with 0.9% PBS solution and suspended by treatment with 0.25% mally oxygenated conditions did not alter the survival of ex ponentially growing MCF-7 or MCF-7/CDDP cells compared trypsin. The cells were plated in duplicate at 3 dilutions for colony formation. After 2 wk the colonies were visualized by staining with with untreated controls; however, this exposure to lonidamine crystal violet, and colonies of 50 cells or greater were counted. The increased the sensitivity of both cell lines to several alkylating results were expressed as the surviving fraction of treated cells compared agents (Fig. 1). The MCF-7/CDDP cell line is about 5-fold with vehicle-treated control cells. resistant to CDDP at the 90% inhibitory concentration com Tumor Lines. The FSall fibrosarcoma (19, 20) adapted for growth pared with the MCF-7 parent cell line. Exposure of the MCF- in culture (FSallC) (20) was carried in male C3H/FeJ mice (The Jackson Laboratory, Bar Harbor, ME). For the experiments, 2x10' 7/CDDP cells to lonidamine prior to and during CDDP expo sure increased the sensitivity ofthat cell line to CDDP by about tumor cells prepared from a brei of several stock tumors were implanted 5- to 7-fold, so that the MCF-7/CDDP cells became as sensitive i.m. into the legs of 8- to 10-wk-old male C3H/FeJ mice. to CDDP as were the MCF-7 parent cells. A marked increase The EMT6 murine mammary carcinoma is an in vivo-in vitro tumor system (21-24). The EMT6 tumor was carried in BALB/c mice (Ta- in CDDP cytotoxicity was observed in the MCF-7 parent cells conic Farms, Germantown, NY). For the experiments, 2x10' tumor upon prior and concomitant exposure to lonidamine. The in cells prepared from a brei of several stock tumors were implanted i.m. crease in cytotoxicity was more than 10-fold with 100 Â¿Â¿molof into the legs of BALB/c mice 8 to 10 wk old. CDDP and more than 100-fold with 500 Â¿/molofCDDP. The Tumor Excision Assay. For each experiment, two tumors were im cytotoxicity of carboplatin was increased only slightly in the planted per mouse, and there were two animals at each dosage level; MCF-7/CDDP cells by the addition of lonidamine prior to and therefore, four tumors were pooled at each point. When the tumors during carboplatin exposure. In the MCF-7 parent cells, on the were approximately 100 mm3 in volume (about 1 wk after tumor cell other hand, there was a larger increase in carboplatin cytotox implantation), five lonidamine doses (50 mg/kg) were administered icity by prior and concomitant exposure to lonidamine which every 7 to 10 h over 36 h. The alkylating agents were administered as reached a maximum of about 10-fold between 250 and 500 single doses by i.p. injection (0.2 ml) along with the third lonidamine ÃÃmolofcarboplatin. As occurred with CDDP, exposure to (50 mg/kg) injection. Mice were sacrificed 24 h after alkylating agent lonidamine prior to and during exposure to D-tetraplatin in treatment to allow for full expression of drug cytotoxicity and repair of creased the sensitivity of the MCF-7/CDDP cells to the drug potentially lethal damage and then soaked in 95% ethanol. The tumors were excised, and a single cell suspension was prepared as described so that they were as sensitive to D-tetraplatin as were the MCF- previously (25). The untreated tumor cell suspensions had a plating 7 parent cells. In the MCF-7 parent cell line, the increase in efficiency of 8 to 12%. The results are expressed as the surviving the cytotoxicity of D-tetraplatin by prior and concomitant ex fraction Â±SE of cells from treated groups compared with untreated posure to lonidamine was greater than 10-fold at 10 to 50 Â¿IM controls from three independent experiments. D-tetraplatin and less at higher concentrations of the platinum Bone Marrow Toxicity. Bone marrow was taken from the same drug. animals used for the tumor excision assay. A pool of marrow from the femurs of two animals was obtained by gently flushing the marrow CARBOPLATIN D-TETRAPLATIN through a 23-gauge needle, and CFU-GM assay was carried out as described previously (25). Colonies of at least 50 cells were scored on an Acculile colony counter (Fisher Scientific, Springfield, NJ). The results from three experiments, in which each group was measured at three cell concentrations in duplicate, were averaged. The results are expressed as the surviving fraction of treated groups compared with untreated controls. Tumor Growth Delay. For the experiments, 2 x IO6 EMT6 tumor cells prepared from a brei of several stock tumors were implanted i.m. into the legs of BALB/c mice 8 to 10 wk old. When the tumors were approximately 100 mm3 in volume (Day 7 after tumor cell implanta tion), treatment was initiated. Lonidamine (50 mg/kg) was adminis tered i.p. daily on Days 7 to 12 just prior to the alkylating agent or twice daily on Days 7 to 12 just prior to and 4 h post-alkylating agent administration. Each alkylating agent was administered i.p. alone or in combination with lonidamine on the daily or twice daily schedule. The alkylating agent treatment schedules were the following: CDDP (3.3 100 250 500 100 250 500 100 250 500 mg/kg) on Days 7, 9, and 11; carboplatin (25 mg/kg) on Days 7 to 12; Drug Concentration, ^M CTX (100 mg/kg) on Days 7, 9, and 11; and thiotepa (5 mg/kg) on Fig. I. Survival of the MCF-7 human breast carcinoma parent cell line (â€¢,O) Days 7 to 12. These are standard doses and schedules for these drugs. and CDDP-resistant MCF-7/CDDP subline (â€¢.D) to various alkylating agents The progress of each tumor was measured 3 times weekly until the in the presence (O, Q) or absence (â€¢,â€¢)oflonidamine (500 ^M). Points, mean of tumor reached a volume of 500 mm3. Tumor growth delay was calcu three independent experiments; bars, SE. 781

In these cell culture experiments, small increases in the 1.0 cytotoxicity of L-PAM, thiotepa, and BCNU were observed 0.1 when MCF-7 or MCF-7/CDDP cells were exposed to lonidamine prior to and during exposure to each of the drugs (Fig. 0.01 1). With L-PAM, addition of lonidamine to treatment with the 0.001 alkylating agent increased the cytotoxicity of the drug 2- to 3- ! 0.0001 fold in both cell lines. With thiotepa, the increase in cytotoxicity I CDDP CARBOPLATIN with the addition of lonidamine prior to and during thiotepa 0.00001 treatment was 4- to 5-fold. Finally, with BCNU, there was dose I 5 10 20 30 100 300 500 1.0 modification of the drug toxicity in the presence of lonidamine prior to and during drug treatment, so that the increase in 0.1 BCNU cytotoxicity at low drug concentrations was about 2- 0.01 fold, and at high drug concentrations, the increase was about 5-fold. 0.001

Even under prolonged exposure times, lonidamine showed 0.0001 only limited direct cytotoxicity toward MCF-7 parent cells THIOTEPA producing a surviving fraction of 0.75 after 42-h exposure to 0.00001 100 300 500 10 20 30 500 M"iol of the drug (Fig. 2). When normally oxygenated MCF-7 cells were exposed simultaneously to lonidamine (500 Drug Dose, mg/kg Fig. 3. Survival of FSalIC tumor cells (â€¢.â€¢)andbone marrow CFU-GM (O, MM)and various concentrations of CDDP for l h followed by D) from animals treated in vivo with lonidamine (5 x 50 mg/kg) over 36 h alone continued exposure to lonidamine (500 MM)for 12 h, a dose or with a single dose of an alkylating agent administered with the third dose of modification of the CDDP toxicity resulted, increasing from lonidamine (â€¢,D)or with a single dose of the alkylating agent alone (â€¢,O). about 2.5-fold at 50 MMCDDP to about 50-fold at 250 MM Points, mean of three independent experiments; bars, SE. CDDP. When exposure to lonidamine (500 MM)was maintained for 24 h prior to, during CDDP exposure, and 12 h post-CDDP given at 7- to 10 h intervals over 36 h with the alkylating agent treatment, an additional increase in CDDP cytotoxicity was being administered as a single dose i.p. immediately following observed so that 50- to 100-fold more cells were killed by the the third lonidamine injection. Lonidamine, on this treatment combination treatment at 100 to 250 MMCDDP than by CDDP schedule, resulted in about a 50% killing of the tumor cells and exposure alone. A similar study was conducted with L-PAM, no toxicity to the bone marrow CFU-GM. Each of the four and much lesser effects were seen with the addition of lonida alkylating agents produced log-linear increasing tumor cell mine to L-PAM treatment. With lonidamine (500 MM)exposure killing with increasing dose of the drug. The addition of loni for 24 h prior to, during, and for 12 h after L-PAM treatment, damine prior to, during, and following treatment with CDDP there were increases of 2- to 3-fold in cell killing compared with resulted in a 2- to 3-fold increase in tumor cell killing and a L-PAM alone. smaller increase in bone marrow CFU-GM killing. A very Using the FSalIC fibrosarcoma in vivo-in vitro tumor system, similar effect was observed with carboplatin in vivo where, in the responses of tumors in vivo was examined to increasing the presence of lonidamine treatment, there was a 2- to 2.5- single doses of alkylating agents in the presence or absence of fold increase in the killing of FSalIC tumor cells and a smaller lonidamine (Fig. 3). Tumor cell kill was quantified by colony increase in the killing of bone marrow CFU-GM. formation, and the survival of bone marrow from the same The addition of lonidamine to treatment with cyclophospha- animals was measured by the CFU-GM assay. The treatment mide in vivo resulted in 10-fold additional tumor cell killing in schedule for lonidamine was five i.p. injections of 50 mg/kg the normal therapeutic range of CTX doses (100 to 300 mg/ kg) and a much smaller increase in the killing of bone marrow CFU-GM of 0- to 3-fold in that same dosage range (Fig. 3). LONIDAMINE(500uM) LONIDAMINE(500pM) There was a similar 10-fold increase in the killing of tumor + + CDDP L-PAM cells by thiotepa with the addition of lonidamine to treatment with that drug. The 10-fold increase in tumor cell killing by thiotepa persisted over the entire dosage range (10 to 30 mg/ kg) examined. The killing of bone marrow CFU-GM by thi otepa with the addition of lonidamine increased to a lesser 0.01 extent than did the tumor cell killing, so that there was equal or greater tumor cell than bone marrow CFU-GM killing by thiotepa over the dosage range examined. 0.001 The EMT6 mouse mammary carcinoma was used for tumor growth delay studies (Table 1). Lonidamine was administered 0.0001 either daily or twice daily for 5 days at a dose of 50 mg/kg. On the daily schedule, lonidamine was administered i.p. immedi 0.00001 ately prior to the alkylating agent, which was also administered 100 250 500 100 250 500 i.p. On the twice daily schedule, the first dose of lonidamine Drug Concentration, uM was administered i.p. immediately prior to the alkylating agent, Fig. 2. Survival of MCF-7 human breastcarcinoma parent cell line exposedto and the second dose was administered i.p. 4 h later. Lonidamine lonidamine for 42 h (O), CDDP or L-PAM for l h (â€¢),lonidamine(500 /JM)and on either treatment schedule had only a small effect on tumor an alkylating agent for l h followed by an additional 12 h to lonidamine (500 Â¿IM)(G),and an alkylating agent for l h followed by lonidamine (500 /JM)for an growth. Each of the alkylating agents was administered on a additional 12 h (â€¢).Points,mean of three independent experiments; bars, SE. multiple dose schedule over the 5-day period of lonidamine 782

Table 1 Growth delay of the EMT6 mouse mammary carcinoma produced by conditions resulted in significant increases in the cytotoxicity lonidamine and various alkylating agents of CDDP, carboplatin, and D-tetraplatin in MCF-7 human The EMT6 tumor was grown subcutaneously in BALB/c mice. Treatment was administered on Day 7 post-tumor cell implantation when the tumors were 50 to breast carcinoma cells. The presence of lonidamine in these 100 mm3 in volume. Tumor growth delay is the difference in days for treated experiments essentially overcame the CDDP resistance of the tumors to reach 500 mm3 compared with untreated controls. MCF-7/CDDP cells, but the degree of enhancement of cyto +alkylating toxicity of CDDP, carboplatin, and D-tetraplatin by lonidamine agent agent (5 X 50 in the MCF-7/CDDP cells was much less than in the MCF-7 groupLonidamine"CDDPTreatment alone6.5 mg/kg)1.0 (10 X 50mg/kg)2.0 parent cell line. In cell culture, much lesser effects were seen Â±0.4*6.8 Â±0.511.0 with L-PAM, thiotepa, and BCNU. In vivo, however, a some (3 x 3.3 mg/kg)' Â±1.5 Â±1.1 Â±1.3 what different pattern of enhancement in alkylating agent ac Carboplatin (5 x 25 mg/kg) 4.9 Â±0.7 6.4 Â±1.2 8.2 Â±1.621.3 CTX(3x 100 mg/kg) 9.7 Â±1.3 11.2Â±1.37.2 Â±2.0 tivity was observed. Lonidamine, administered prior to, during, Thiotepa (5x5 mg/kg)Alkylating5.7 Â±1.1LonidamineÂ±1.0Lonidamine16.5 Â±1.8 and after alkylating agent treatment in the tumor cell survival Â°Lonidamine (5 x 50 mg/kg) was administered i.p. daily immediately before assay using the FSallC fibrosarcoma, produced essentially ad the antitumor alkylating agent on Days 7 to 12. Lonidamine (10 x 50 mg/kg) ditive tumor cell killing with both CDDP and carboplatin. was administered i.p. twice daily immediately before and 4 h after the antitumor alkylating agent on Days 7 to 12. However, greater than additive enhancement in (calculated by * Mean Â±SE. 'CDDP (3 x 3.3 mg/kg) was administered i.p. on Days 7, 9, and 11; carbo- product of the surviving fractions) tumor cell killing was prob platin (5 x 25 mg/kg) was administered i.p. on Days 7 to 12; CTX (3 x 100 mg/ ably obtained with CTX and thiotepa. With each of these four kg) was administered i.p. on Days 7, 9, and 11; and thiotepa (5x5 mg/kg) was drugs, in general, the addition of lonidamine to the alkylating administered i.p. on Days 7 to 12. treatment resulted in a greater increase in the killing of tumor cells than in the killing of bone marrow CFU-GM, indicating administration. There was no change in the tumor growth delay that there would be an increase in therapeutic index if bone produced by CDDP with once daily lonidamine but, with twice marrow toxicity was dose limiting. daily lonidamine, there was a 1.7-fold increase in tumor growth In the EMT6 mouse mammary carcinoma growth delay delay from about 6.5 days to about 11.0 days. The tumor growth studies, lonidamine on two different schedules was added to delay produced by carboplatin was only slightly increased by standard doses of each of the four alkylating agents without daily lonidamine treatment. However, twice daily lonidamine any evidence of an increase in the toxicity of the combination along with carboplatin resulted in a 1.7-fold increase in tumor treatment compared with the alkylating agent alone. For growth delay from about 4.9 days to about 8.2 days. There was CDDP, carboplatin, CTX, and thiotepa, significant increases no significant increase in the tumor growth delay produced by in tumor growth delay were observed only with the twice daily CTX with the addition of daily lonidamine, but, again, twice schedule of lonidamine. As in the tumor cell survival assay, daily lonidamine resulted in a 2.2-fold increase in tumor growth lesser increases were seen with the platinum drugs, CDDP and delay compared with CTX alone. The tumor growth delay carboplatin, than with the mustard-type alkylating drugs, CTX produced by cyclophosphamide was about 9.7 days, and that of and thiotepa. the combination treatment was about 21.3 days. A similar These results indicate that lonidamine has the potential to pattern persisted with thiotepa treatment in that there was a increase the efficacy of antineoplastic alkylating agents without small increase in tumor growth delay with the addition of daily a reduction in the dosage of the alkylating agents and that a lonidamine but a larger increase of about 3-fold with daily greater potentiation of the effect of the alkylating agents may thiotepa and twice daily lonidamine. occur in the tumor compared with the bone marrow. Although in cell culture lonidamine potentiated the cytotoxicity of plati DISCUSSION num drugs to a greater degree than it potentiated the cytotox icity of mustard-type drugs, in vivo, larger enhancement in both Treatments or agents that are relatively nontoxic but can the FSallC fibrosarcoma and the EMT6 mammary carcinoma modulate the cytotoxicity of bifunctional alkylating drugs in was observed with the mustard-type drugs than with the plati tumors could be very useful in combination chemotherapeutic num drugs. Clinical protocols utilizing lonidamine with CTX regimens for the treatment of clinical neoplastic diseases. Po are being considered. tential modulators of alkylating agent cytotoxicity include the following: (a) the topoisomerase II inhibitors such as etoposide, REFERENCES VM-26, and novobiocin; (b) the nitroimidazole radiosensitizers such as misonidazole and etanidazole; (c) agents or treatments 1. DeMartino, C., Battelli, T., Paggi, M. G., Nista, A., Marcante, M. 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Beverly A. Teicher, Terence S. Herman, Sylvia A. Holden, et al.

Cancer Res 1991;51:780-784.

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