[CANCER RESEARCH 47, 493-495, January 15, 1987] Effect of Drug Exposure Duration and Sequencing on Hyperthermic Potentiation of Mitomycin-C and Cisplatin1
Kent ET.Wallner2 and Gloria C. Li
Department of Radiation Oncology, University of California, San Francisco, California 94143
ABSTRACT by a regulated flow of 95% air and 5% CO?. The temperature of the water bath was controlled to within +0.01°C.Fresh medium, with or The effects of drug exposure duration and of heat and drug sequencing without drug, was placed over the cells immediately prior to heating. on hyperthermic potentiation of mitomycin-C (MMC) and cisplatin All heating was for 1 h at 42°C.Cell survival after heating at 42°Cfor (DDP) were studied. Heating for l h at 42°Cwas combined with drug 1 h in drug-free medium is 95-100%. exposure times of 1, 2,4, or 8 h. For both DDP and MMC, hyperthermic Drug Exposure. DDP and MMC stock solutions were prepared by potentiation was greatest when heating was done during drug exposure. diluting freshly mixed DDP and MMC (Bristol-Myers, Syracuse, NY)4 Dose enhancement ratios for both drugs at 1% survival were highest with to a concentration of 100 ng/ml in sterile water. Multiple 1.5 nil the shortest drug exposure times and decreased as the drug exposure aliquots were stored in the dark at —20°C.Immediately prior to each time increased from 1 to 8 h. For DDP, the dose enhancement ratio experiment, a vial of drug was thawed to room temperature and diluted decreased from 1.9 with a 1-h drug exposure to 1.2 with an 8-h drug to the appropriate concentration with complete medium. Following exposure. For MMC, the dose enhancement ratio decreased from 1.8 to completion of treatment, cells were washed once with phosphate buff 1.5 as the drug exposure duration was increased from 1 to 8 h. Our ered saline, trypsinized, counted on a Coulter Counter, and plated after results suggest that thermochemotherapy in vivo is likely to be most appropriate dilutions had been made. The dishes were incubated at effective with rapid infusions of DDP or MMC. 37°Cfor8-10 days, at which time surviving colonies were stained with crystal violet and those with 50 or more cells were counted with a INTRODUCTION dissecting microscope. Whenever possible, dishes containing 50-200 colonies were used for calculation of survival. For sequencing experi Hyperthermia potentiates the action of several chemothera- ments, cells were trypsinized and plated immediately following drug peutic agents, including bleomycin (1), l,3-bis(2-chloroethyl)- exposure, except when heating was done after drug exposure. In that 1-nitrosourea (1), cyclophosphamide (2), DDP3 (3), and MMC case, cells were rinsed with phosphate buffered saline, and overlaid with (3). While the clinical efficacy of hyperthermia has yet to be fresh, drug-free medium after drug exposure and until heating was established, several investigators are proceeding to use com completed, at which time they were trypsinized and plated. All experi bined hyperthermia and chemotherapy for cancer patients. ments were performed at least twice. Error bars represent the standard error of the mean of at least three replicate experiments. There is limited information as to the optimal way to admin ister treatment with hyperthermia and chemotherapeutic agents. Chemotherapy can be given rapidly by i.v. infusion, or RESULTS it can be infused over many hours. Heating could be done The effect of drug and heat sequencing was studied for drug before, during, or after drug administration. Both the duration exposure durations of 1, 2, 4, and 8 h. Cell killing at a given of drug exposure and the sequencing of chemotherapy with drug concentration was exponential over 8 h for both MMC heating affect the interaction between drugs and hyperthermia. and DDP. All heating was done for 1 h at 42°C. The following experiments were performed to study the effect Fig. 1 shows how the interaction between hyperthermia and of drug exposure time and drug and heat sequencing on hyper DDP changes as the duration of DDP exposure is increased thermic potentiation of MMC and DDP. from 1 to 8 h. Each data point represents survival when heating for 1 h at 42°Cis done at that time point in relation to DDP MATERIALS AND METHODS exposure. Survival after exposure to DDP without heating is shown on the left-hand side of each graph. As the exposure Cell Culture. All experiments were performed with Chinese hamster fibroblasts, HA-1 cells. The cells were grown in Eagle's Minimal interval was increased, the concentration of DDP was reduced to yield isosurvival for each exposure time at 37°C.Hyper- Essential Medium (GIBCO, Santa Clara, CA) supplemented with 15% fetal calf serum and gentamycin (25 /¿g/ml).Cell cultures were kept at thermic potentiation of DDP is greatest if heating is done 37°Cina humidified incubator with a mixture of 95% air and 5% CO2 during DDP exposure. With DDP exposure times longer than and routinely checked for mycoplasma contamination. All experiments 1 h, there was a tendency for greater hyperthermic potentiation were performed with plateau phase cells, prepared by plating 4 x IO4 if heating was done during the first hour of drug exposure. As cells in 35-mm Petri dishes on day 0. The cultures were fed daily the duration of DDP exposure increases, hyperthermic poten starting on day 3 and used for experiments on days 8-10, when the cell tiation of DDP decreases. Survival curves shown in Fig. 2 were density had stabilized at 1.0-1.2 x IO6 cells/cm2. Plating efficiencies done with increasing DDP exposure times and with heating were between 65 and 85%. done only during the first hour of drug exposure. The curves Heating. Heating of cell monolayers was done in a specially designed with and without 1-h heating at 42°Capproach each other as water bath at 42*C. The pH of the medium was maintained at 7.2-7'.4 the drug exposure time increases, indicating a gradual decrease in hyperthermic potentiation of DDP. Received 8/5/86; revised 9/26/86; accepted 10/3/86. The costs of publication of this article were defrayed in part by the payment The interaction between hyperthermia and MMC when heat of page charges. This article must therefore be hereby marked advertisement in ing is done at various times during MMC exposure durations accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in part by Grants CA-31397 and CA-09215 from of 1, 2, 4, and 8 h is shown in Fig. 3. The concentration of the National Cancer Institute, Department of Health, Education, and Welfare. MMC was decreased as the exposure duration was increased, 2To whom requests for reprints should be addressed, at Dept. of Radiation to give isosurvival levels at 37°Cfor each exposure time. Sur Oncology, Long Hospital, Room L-75, University of California, San Francisco, CA 94143. vival after exposure to MMC without heating is shown along 3The abbreviations used are: DDP, cisplatin [ru-diamminedichloroplat- inum(II)]; MMC, mitomycin-C; DER, dose enhancement ratio. 4 Kindly supplied by Dr. James H. Keller. 493
Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research. HEAT AND DRUG SEQUENCING
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s HEAT BEFORE HEAT AFTER DRUG DRUG
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HOURS HOURS HOURS HOURS Fig. 1. Effect of increasing DDP exposure time (1, 2, 4, and 8 h) and Fig. 3. Effect of increasing MMC exposure times (1, 2, 4, and 8 h) and sequencing of hyperthermia with DDP. The concentration of DDP was decreased sequencing of hyperthermia with MMC. The concentration of MMC was de as the drug exposure time was increased. Data points represent the midpoint of creased as the drug exposure time was increased. Data points represent the each 1-h heat exposure at 42'C. Survival after exposure to DDP without heating midpoint of each 1-h heating at 42'C. Survival after exposure to MMC without is shown on the left side of each graph. heating is shown on the left side of each graph.
DDP.1 HR DDP«2 HRS the left side of each graph. Hyperthermic potentiation of MMC is greatest with the shorter drug exposure durations and when 10 ' 10"' heating is done during drug exposure. For longer drug exposure times, hyperthermic potentiation tends to be greater when heating is done during the last hour of drug exposure. Complete Õ survival curves were generated using increasing MMC exposure times with heating done during the last hour of drug exposure (Fig. 4). Dose enhancement ratios were calculated as the dose of drug 10-' 42°C x 1 HR * needed to yield a 1% surviving fraction without heating, divided 37°C «1 HR by the dose needed to give a 1% survival level with heating (Fig. 5). For both MMC and DDP, the DERs are greatest with drug IO"4 10"4 0.5 1.0 1.6 exposure intervals of 1 h. The DERs decrease rapidly as the UG/ML DDP UG/ML DDP exposure time increases from 1 to 4 h. For DDP, the DER decreased from 1.9 with a 1-h heat exposure to 1.2 with an 8-h
DOP. 8 HRS drug exposure. For MMC, the DER decreased from 1.8 to 1.5 as the drug exposure duration was increased from 1 to 8 h. The g10" loss of hyperthermic potentiation with increasing exposure time is more marked for DDP than for MMC. o £ DISCUSSION Previous investigators have studied the effect of sequencing on hyperthermic potentiation of chemotherapeutic agents. Cy- 42°C x 1 HR 37°C x 3 HRS clophosphamide(2,4), l,3-bis(2-chloroethyl)-l-nitrosourea(l), cisplatin (3), and mitomycin-C (3) all showed maximal poten tiation when heating and drug exposure were done simultane
0.1 0.2 0.3 0.4 0.5 Fig. 2. Survival curves for increasing duration of exposure to DDP, with or without 1-h heating at 42'C during the first hour of drug exposure. UG/ML DDP UG/ML DDP 494
Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research. HEAT AND DRUG SEQUENCING greater when 42°Cheating was done during the first hour of
MMC x 2 MRS drug exposure for DDP, versus the last hour of drug exposure for MMC. We do not know the reason for this difference, but it points to the fact that drug and heat sequencing can affect various drugs differently. Plateau phase cells were used for these experiments so that the cell number would not change appreciably during the 10- 12 h that the experiments were performed. With exponentially growing cells, changes in the cell number during the experiment can affect the cytotoxicity of MMC and DDP. Dose enhancement ratios were highest with the shortest drug 37°Cx1 HR< 42°CX1 HR exposure times. The length of drug exposure in vivo will be determined primarily by the method of administration and by 10"• 20 4.0 60 1.0 2.0 3.0 the serum half-life of the drug. The serum half-lives of both
UG/Ml. MMC UG/ML MMC MMC and DDP are approximately l h (5, 6), so that with an i.v. bolus injection, exposure intervals will be approximately 1-
MMC x 8 MRS 2 h. In practice, however, cisplatin is usually infused over several hours to allow for adequate hydration. Also, there has been some interest in using longer infusion times for both drugs, in the hope of decreasing normal tissue toxicity (7, 8). If the drugs are infused over longer time periods, our results suggest that ocI heating should be given at the start of the infusion period for DDP, and at the end of the infusion period for MMC. The concentrations of MMC (6) and DDP (9) used in our experi 37°C ments are achievable in serum following i.v. administration in 3 i«-.»- 37°Cx 7 HRS+\ »SHBS 42°CX1 HR humans. While in vitro studies of the effect of sequencing between hyperthermia and chemotherapeutic agents is relatively simple, io- 0.6 1.0 1.6 0 0.2 0.4 0.6 the relationships will surely be more complicated in vivo. Re UG/ML MMC UG/ML MMC sponse of tumor cells in vivo will be influenced by such factors Fig. 4. Survival curves for increasing duration of exposure to MMC, with or as drug metabolism, tumor vasculature, thermotolerance, and without 1-h heating at 42°Cduring the last hour of drug exposure. drug-resistant cell populations. Additionally, these factors may vary from one patient to the next, and from one drug to the next. More information from in vitro and in vivo studies would 2.0 be valuable prior to extensive clinical testing, as suboptimal sequencing may jeopardize clinical trials. 1.8 Œ ui 1.6 REFERENCES
Q 1.4 1. Hahn, G. M. Potential for therapy of drugs and hyperthermia. Cancer Res., 39: 2264-2268, 1979.
1.2 2. Urano, M., Kim, M. S., Kahn, J., Kenton, L. A., and Li, M. L. Effect of thermochemotherapy (combined cyclophosphamide and hyperthermia) given at various temperatures with or without glucose administration on a murine 1.0 fibrosarcoma. Cancer Res., 45: 4162-4166, 1985. 3. Corry, P. M., Jabboury, K., Armour, E. P., and Kong, J. S. Human cancer DURATION OF DRUG EXPOSURE (HOURS) treatment with ultrasound. IEEE Trans. Sonics Ultra., 31:444-456, 1984. Fig. 5. Dose enhancement ratios for increasing drug exposure times, calculated 4. Dalli. O., and Mella, O. Effect of timing and sequence of hyperthermia and from data of Figs. 2 and 4. cyclophosphamide on a neurogenic rat tumor (BT
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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research. Effect of Drug Exposure Duration and Sequencing on Hyperthermic Potentiation of Mitomycin-C and Cisplatin
Kent E. Wallner and Gloria C. Li
Cancer Res 1987;47:493-495.
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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research.