Search for new radiation potentiators

Inf. J. Radiotion

0

Oncology Biol. Phys , 1978. Vol. 4, pp. 25-35.

Pergamon Press.

Printed in the U.S.A

General Concepts SEARCH

FOR NEW RADIATION

AIBRAHAM GOLDIN, PHILIP

C. MERKER,

National Cancer

Ph.D.,* Ph.D.8

POTENTIATORSt

ISIDORE WODINSKY,

M.S. ,§

and JOHN M. VENDITTI,

Ph.D.S

Institute, National Institutes of Health, Bethesda, MD 20014, U.S.A. and Arthur D. Little, Inc., Cambridge, MA 02140, U.S.A.

The methodologies and objectives involved in the search for new radiation potentiators, as in the search for new drugs and combinations of drugs, are aimed at obtaining enhanced therapeutic effectiveness in the treatment of the tumorous host. A distinction may be made betwen radiation potentiation (with radiosensitization as a special case) and therapeutic radiation potentiation. With radiation potentiation a single parameter of response is generally employed, such as tumor cell destruction, and account is not taken of the host-tumor relationship, so that there is no measure of any enhancement of selective antitumor action. Therapeutic radiation potentiation is a form of therapeutic synergism in which treatment with a combination of radiation plus antbumor agent provides an enhanced therapeutic response which cannot be duplicated with radiation alone or drug alone. The search for new radiation potentiators may be conducted in two phases. In the initial phase, radiation potentiators may be identified employing simplified screening methodology in oitro or in uiuo, which may permit the examination of a large number of candidate materials. In the second phase of the study, investigation can ble conducted to determine whether the radiation potentiator is capable of eliciting an enhanced therapeutic response (therapeutic radiation potentiation) when employed in combination with radiation. Examples of therapeutic radiation potentiation are presented utilizing standardized experimental tumor systems. Antitumor agents, Leukemia L1210, Leukemia P388, Lewis lung tumor, B16 Melanoma, Radiation potentiation, Radiation potentiators, Ridgway osteogenic sarcoma, Screening, Therapeutic radiation potentiation, Therapeutic radiation potentiators, Therapeutic synergism.

There are a variety

However, the search for new radiation potenof definitions of synergism and tiators, as with new drugs and new combinations of potentiation which, in general, have been traceable to drugs, involves the desire to improve the therapy of historical shifts in emphasis of investigators engaged in the employment of combinations of drugs.2V6*7*‘4,‘73’9cancer and from this point of view the methodologies and objectives are similar to those which pertain to For purposes of the present discussion radiation the search for new combinations of drugs that may potentiation may be defined as a special case of exhibit enhanced therapeutic antitumor effectiveness. Goodman and Gilman’s definition6 as the combined As with single drugs and combinations of drugs, so action of radiation plus a drug such that the response is greater than that which could be achieved from the too with the combined modality of radiation plus drug sum of their individual #activities. Radiosensitization it is necessary with respect to therapeutic evaluation to take into account the triad of the host, tumor and by an agent would fall in this category since radiation-drug interrelationship. In combination radiosensitizers are capable of increasing the lethal action of ionizing radiation. Radiation potentiation chemotherapy we have employed the term “therapeutic synergism” (or “therapeutic potenand radiosensitization are applicable to the situation tiation”) to describe the situation in which the comin which a single parame:ter of response is employed bination chemotherapy results in an enhanced (e.g. tumor cell destruction). Account is not taken of the effect on the host and there is therefore no therapeutic response which can not be duplicated by the utilization of either one of the drugs definitive basis for estimation of any enhancement of alone. 2,435~“*20*2’ In like manner “therapeutic radiation selective antitumor action. TSupported by Contract -NOl-CM-53765. *Drug Research and Development Program, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, MD 20014, U.S.A.

$Arthur D. Little, Inc., Cambridge, MA 02140, U.S.A. Reprint requests to: Dr. Abraham Goldin, National Cancer Institute, National Institutes of Health, Bethesda, MD 20014, U.S.A. 25

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potentiation” may be defined as a form of therapeutic synergism in which treatment with a combination of radiation plus drug is more effective therapeutically than treatment with radiation alone or drug alone. As a corollary, when therapeutic radiation potentiation occurs, it is possible that the improved therapeutic response may be achieved with a greater margin of safety for the host, with minimal destruction of vita1 tissue and reduced risk of cumulative toxicity or lethal action. In the search for new therapeutic radiation potentiators the drug which potentiates may be inactive therapeutically per se or it may be capable of exerting its own therapeutic effect. Similarly, radiation alone may be either ineffective, as in the case where the tumor is naturally radioresistant, or it may exert a therapeutic effect. Thus there are four possible combinations that conceivably could be employed in the achievement of therapeutic radiation potentiation (Table 1). The therapeutic activity of the drug alone and radiation alone in the combinations could be: drug active-radiation active; drug active-radiation inactive; drug inactive-radiation active; and drug inactive-radiation inactive. The search for new radiation potentiators may be conducted either in vitro or in viva. In relatively extensive in vitro studies much fundamental information has been obtained concerning radiation alone and the interaction of drugs and radiation. These studies have been conducted either in tissue culture employing parameters of inhibition of cell growth or cytotoxicity or in cellular preparations with measurement of action on DNA strand breakage, rates of recovery, and enzymatic or other biochemical parameters. If one were considering very extensive screening of thousands of compounds for radiation potentiation there would be merit in the identification of in vitro systems that would be suitable as initial prescreens. Alternatively it would be possible, if sufficiently simple in viva systems could be obtained, to utilize them for extensive screening. Table 1. Possible drug (radiation potentiator)-radiation combinations yield could theoretically that “therapeutic radiation potentiation” Drug alonei’

+ + -

Radiation

alone

+ + _

t(+) Drug (radiation potentiator) is therapeutically active alone. (-) Drug

(radiation potentiator) is therapeutically inactive alone.

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Or the in uiuo systems could be employed for more detailed definitive evaluation of selected compounds identified by in vitro screens as radiation potentiators. It should be reemphasized that although the in vitro systems are capable of identifying compounds that are radiation potentiators, they provide no basis for determination of the selectivity requisite for therapeutic radiation potentiation. This is so since only a single parameter of response is involved, i.e. inhibitory or cytotoxic action, without the parameter of a limiting host response. It is of interest to examine some of the in viva systems that might be employed in the search for radiation potentiators. Toxicity studies could be conducted in normal animals or in tumor-bearing animals taking into account one or more parameters of response pertaining to the host itself such as weight loss, host mortality, specific organ toxicity or other specific toxicologic, biochemical or pharmacologic responses. An attempt could be made to determine whether the drug exerts some desirable effect with respect to radiation. Does the drug protect the host against either systemic or local radiation damage? Here, too, account would not be taken of the host-tumor relation and the value of the system is limited with respect to determination of therapeutic radiation potentiation. Where a local tumor is present in the animal it is possible to test for radiation potentiation utilizing local radiation to the tumor. The effect on the local tumor could be measured without regard to the effect on the host. It could be determined, for example, whether it is possible to achieve a designated level of inhibition of local tumor with a lower dosage of radiation when the candidate potentiator is administered. If this occurred it could provide a basis for more extensive inhibition of local tumor with equal radiation dosage. Here too if the response of the host were not taken into account there would be no basis for measuring therapeutic selectivity. Where the tumor is metastatic or systemic it is still possible to determine the extent of radiation potentiation by bioassaying various tumor-infiltrated organs, following treatment with whole body radiation plus the candidate potentiator. More extensive cell kill as measured by surviving cell fraction with the combination of radiation plus drug than with radiation or drug alone would be indicative of radiation potentiation. To determine whether new radiation potentiators are capable of eliciting therapeutic radiation potentiation our program is emphasizing the use of in viuo systems that take into account the host-tumor relationship in a manner comparable to that employed in the determination of therapeutic synergism with combinations of drugs. In these systems the effect of

Search for new radiation potentiators 0 A. GOLDIN et al.

degree of safety (therapeutic index or ratio) with which the treatments can be employed.16 This system has been used extensively to identify combinations of drugs that are therapeutically synergistic in increasing survival time. Using the survivaltime parameter the occurrence of therapeutic synergism with a combination of known antitumor agents is illustrated in Fig. 1.22 In this study employing dose-response curves it was observed that the combination of methotrexate plus BCNU was more effective in increasing the survival time of the mice with advanced leukemia L1210 than was BCNU alone or methotrexate alone. It may be noted that in this experiment treatment was initiated with the drugs on the seventh day following subcutaneous leukemic inoculation, at a time when the disease was systemic

the therapy on the tumor and on the host is taken into account in the determination of whether enhanced therapy has occurred with a combination modality. Systems currently in use in the National Cancer Institute program for investigation of radiation potentiation and therapeutic radiation potentiation are listed in Table 2. Lymphoid leukemia L1210 is employed in the standard screening in the program of the National Cancer Institute. Following inoculation of a standard number of tumor cells such as 10’ the disease becomes systemic rapidly (2-3 days) and the animals die within a uniform period of time (8-10 days). With this system dose-response curves may be established for individual agents, combinations of agents or a combination of radiation iplus a potential potentiator. Quantitative comparisons can be made. In one experimental design a temporal separation was provided for deaths due to toxicity and deaths resulting from leukemic: growth. Dose-response curves were established for drug-toxic mortality and drug-tumor mortality. The influence of different therapies could then be compared at equal toxicity for the host. It was in this way that it was first shown that aminopterin plus delayed citrovorum factor was more effective than aminopterin alone in the treatment of mice bearing leukemia L1210.3 With an experimental design that has been used extensively the dose-survival time response is employed. Typically, with active therapeutic agents or combined modality treatment, as the dosage is increased the survival tine of the animals is increased until a maximum response is reached. As the dosage is increased further, the toxicity of the treatment becomes manifest and the survival time diminishes. It is possible then to eva.luate antitumor effectiveness

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Fig. 1. Comparison of methotrexate (MTX) and BCNU, separately and combined, in the daily treatment of advanced leukemia L1210. In this experiment the drugs were combined over a wide range of doses at 5 BCNU: MTX dosage ratios on a mg/kg basis. The combination ratio shown (BCNU : MTX = 4: 1) included the maximally effective combination treatment level [from Ref. 221.

on the basis of the optimal increases in survival time for the modalities being tested. The maximum increase in survival time at the optimal dosages provide a measure of the relative therapeutic effectiveness of the treatments. The widths of the curves measure the Table 2. Tumor systems currently

21

in use for radiation

potentiation

studies

End points studied Tumor system L1210 Leukemia P388 Leukemia B 16 Melanoma Lewis lung carcinoma Ridgway osteogenic sarcoma EMT-6 Nettesheim squamous cell carcinoma

Host

Survival time

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X

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X X X

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28

Radiation Oncology 0 Biology 0 Physics

and could be recovered from the various organs of the body including spleen, liver, blood, etc. and the untreated animals were expected There are numerous examples

to die in a few days. of such therapeutic

synergism with combinations of drugs in the leukemia L1210 system and in at least some instances the combinations have also proven to be effective in the clinic. There have not been too many studies on testing for therapeutic radiation potentiation in the leukemia L1210 system using the above methodology. In a study of Ralph Johnson (Fig. 2)8 a combination of systemic chemotherapy and electron-beam irradiation of the entire cerebra spinal axis was compared with chemotherapy alone and irradiation alone in the treatment of mice that had been inoculated intracranially with leukemia L1210. In the experiment, treatment was initiated at 4 days following i.c. inoculation in order to permit the disease to also become

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systemic. Irradiation alone, over a series of dosages was essentially ineffective in increasing the survival time of the animals. Cyclophosphamide, which is not capable of crossing the blood brain barrier, at its optimal dose produced only a 4 day increase in survival time. However, the combined modality of cyclophosphamide plus irradiation resulted in a median survival time of greater than 60 days and there were 7 out of 10 long term survivors. In this study the survival time patterns were essentially identical whether the drug was administered 4 hr prior or 4 hr subsequent to irradiation indicating that although therapeutic radiation potentiation was observed, the cyclophosphamide did not sensitize the radiation dosage. In one of the controls, therapy and irradiation of the pelvis did not achieve prolonged survival suggesting that the effect of the irradiation, where a therapeutic response was observed, was direct. Thus, this type of system can lend itself well to the study of therapeutic radiation potentiation. In a more generalized model the leukemic cells could be inoculated i.p. or S.C. and whole body irradiation plus systemic chemotherapy applied in the treatment of early or advanced systemic disease. The leukemia P388 system is now in general usage as a prescreen in the program at the National Cancer Institute. It is similar to the leukemia L1210 system in that the disease becomes systemic and the animals die relatively early in a uniform period of time. The utilization of this system for combination chemotherapy is illustrated in Table 3.26The combination of adriamycin plus cyclophosphamide was more effective than adriamycin alone or cyclophosphamide alone in increasing the survival time of the animals. Wodinsky et al.23s24investigated the combination of -y-radiation and chemotherapy against lymphocytic P388 leukemia in Go. Ten clinically useful agents, methotrexate, nitrogen mustard, alanine mustard, 5Table

3. Combination cyclophosphamide plus adriamycin. leukemia P388 Cyclophosphamide mg/kg/inj.

(CP)

1 0 1 50 1 100 1 200 1 300 J

%ILS I 81 1 18 1 14: 1 1.50 I 01

Fig. 2. (A) Median and individual survival times for animals receiving no treatment, irradiation only, or cyclophosphamide (CTX) only. 0 = day of death of individual mice. (B) Median and individual survival times for animals receiving combined systemic cyclophosphamide and central nervous system irradiation. RAD = radiation; 0 = day of death of individual mice [from Ref. 83.

68 1 145 268 I 209

*lo6 cells in 0.1 ml i.p. [from Ref. 261. CP-Day 5 only 1 x day i.p. AD-Day 5 only 1 x day i.p.

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with y-radiation Table 4. Activity of selected agents administered alone and in combination lymphocytic leukemia [from Ref. 241

to mice bearing

i.p. P388

Optimal % ILS (combination)

Drug Methotrexate Mechlorethamine Prednisone Alanine mustard S-FU Cyclophosphamide Vinblastine Cytosine arabinoside Vincristine Procarbazine cis-Diamminedichloroplatinum

Optimal % ILS (y-radiation alone)

Optimal % ILS (drug alone)

Drug given 4 hr before y-radiation

Drug given just prior to y-radiation

Drug given 4 hr after y-radiation

45 29 41 49 50 22 45 23 40 49 50

79 66 0 95 55 277 59 27 63 9 100

100 104 33 127 80 409 91 73 90 59 104

108 166 33 131 80 322 127 82 90 81 114

129 183 24 113 90 368 118 95 113 59 114

fluorouracil, cyclophosphamide, vinblastine, cytosine arabinoside, vincristine, procarbazine and cis-diamminedichloroplatinum were more effective in combination with whole body y-radiation in the treatment

of i.p. inoculated lymphocytic leukemia P388 than drug alone or y-irradiation alone (Table 4). The timing of the y-radiation and drug administration was not critical. Therapeutic radiation potentiation was observed in most instances whether the drug was given 4 hr before, simultaneously, or 4 hr after the y-radiation. With prednisone plus -y-radiation no therapeutic potentiation was observed against the i.p. P388. In a second experiment where the leukemic cells were inoculated i.v., therapeutic radiation potentiation was nitrogen mustard, obtained with methotrexate, cyclophosphamide, vinblastine, cytosine arabinoside and procarbazine, but not with Sfluorouracil. An additional parameter for determination of therapeutic synergism involves the total eradication of tumor cells as reflected in the incidence of longterm survivors or “cures”. In one example a priming dose therapy of BCNU was employed in the treatment of advanced leukemia L1210 in order to drastically reduce the number of leukemic cells. When this single treatment with BCNU was followed by daily treatment with cytosine arabinoside there was an increase in the percentage of survivors (80% survivors) over that observed with BCNU alone (20% survivors) or ara-C alone (0% survivors).‘V’8 The percentage “cure”’ parameter of response can also be employed for determination of therapeutic radiation potentiation. An investigation has been conducted on the potentiation of -y-radiation by adriamycin and daunorubicin. Mice inoculated i.p. with leukemia P388 were treated i.p. with adriamycin t2-Methyl-5-nitroimidazole-l-ethanol

(NSC-69587).

or daunorubicin 4 hr prior to or 4 hr following whole body y-radiation with ?Zo. Maximum therapeutic radiation potentiation was observed, as indicated by the number of long term survivors, when adriamycin or daunorubicin were administered 4 hr prior to the y-radiation. Pretreatment with adriamycin (Fig. 3) yielded 8 out of 10 long term (60 day) survivors whereas post treatment with adriamycin gave only 3 out of 10 long term survivors, the latter being equal to that observed with the same dose of adriamycin alone. With daunorubicin administered 4 hr prior to y-irradiation (Fig. 4) there were 40% long term survivors whereas no long term survivors were observed with y-radiation alone or daunorubicin alone or daunorubicin administered after the y-radiation. In the above study comparisons were made at optimal dosages yielding maximum percentage survival. It is of interest to review a preliminary study with the radiation sensitizer metronidazolet in relation to the potential application to therapy. In an experiment with radiation plus metronidazole in the P388 system the combination was not more effective in increasing the survival time than radiation alone (Fig. 5). There were no long term survivors with any of the therapeutic regimens employed. The metronidazole by itself was completely ineffective. Thus in this experiment there was no evidence for therapeutic radiation potentiation. Although in the experiment illustrated above there was no therapeutic radiation potentiation by metronidazole, it may be noted that the highest dose of whole body radiation employed was 600r which is in the area of the highest tolerated dose. In another experiment whole body radiation was increased to 4000r and the radiosensitization by metronidazole

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Fig. 3. Survival curves for the optimal increases in lifespan of P-388 tumor-bearing mice treated with Control; ----, -y-radiation (600R and 400R -y-radiation and adriamycin as single agents or combined. -, adriamycin 10 mg/kg 4 hr prior to 600R y-radiation; .....+, combined); -m--, adriamycin 20 mg/kg; -.e--, adriamycin 10 mg/kg just prior to 400R y-radiation; -A-, adriamycin 20 mg/kg 4 hr after 200R y-radiation.

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Fig. 4. Survival curves for the optimal increases in lifespan of P-388 tumor-bearing mice treated y-radiation and daunorubicin as single agents or combined. -, Control; ---, y-radiation (600R and combined); -m---, daunorubicin 2.5 mglkg; -. .-, daunorubicin 5 mg/kg 4 hr prior to 600 R y-radiation; daunorubicin 2.5 mg/kg just prior to 600 R y-radiation; -A-, daunorubicin 2.5 mg/kg 4 hr after y-radiation. was measured by transplantation bioassay (Fig. 6). A linear relationship was observed between the number of cells required to yield 50% takes (TDso) on transplantation into recipient mice and the dose of radiaWhen metronidazole was adtion employed.

with 4OOR ...e.., 600 R

ministered in conjunction with the radiation a linear relationship was also obtained between the TDso and the dose of radiation, but with an increased slope. At the radiation dose of 2000r and 4OOOr the addition of metronidazole resulted in a marked increase in the

Search for new radiation potentiators ?? A.

GOLDIN

et al.

31

Dose, rad

Fig. 5. Effect of Vo-y-radiation and metronidazole (Met. NSC-69587) on the average lifespan of CDF, female mice bearing P-388 lymphocytic leukemia. Metronidazole administered 30 min prior to whole-body y-irradiation, 24 hr after tumor implantation. -, Radiation alone; -.-, + MET 250 mg/kg; -. .-, + MET 500 mg/kg; -----, + MET 100 mg/kg; --, + MET 2000 mg/kg. 108

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Fig. 6. Number of P-388 leukemia cells required to produce Radiation alone; --, tumors in 50% of the mice (TI&). -, + MET 500 mg/kg; ----, + MET 1000 mg/kg.

TD~o, indicative of the extent of radiation sensitization to cell kill. But it should be noted that at the maximum tolerated dose of 600r there was little to no difference in TD~o. Thus it was necessary to employ doses of y-radiation that were very much higher than the tolerated whole body dose before any substantial

radiation potentiation of tumor cell kill could be observed. The lack of radiation potentiation by metronidazole at a low dose of -y-radiation (5OOr) and its occurrence at a high dose of y-radiation (4OOOr)is also illustrated when the data are presented as percentage of survivors at the series of cell inoculum levels employed in the bioassay (Figs. 7 and 8). Thus, from the point of view of therapy it is important to employ experimental models such as this one to determine whether on treatment with radiation plus a radiosensitizer, definitive antitumor potentiation can be demonstrated at radiation dosages that can be tolerated by the host. B16 melanoma implanted intramuscularly in the gastrocnemius muscle of BDFl mice is also being employed as a model for combined y-radiation and chemotherapy. Following inoculation, the tumor becomes palpable after 6 days and increases in weight exponentially until day 13. The tumor is lethal for the host and the range of death is narrow when it is inoculated i.m. The metastatic rate of the tumor to lung, liver and spleen as determined by bioassay is somewhat unpredictable. Nevertheless all of the mice bearing i.m. tumors eventually do die of metastasis. In this system -y-radiation is administered to the local tumor site with the objective that it may provide local control and that chemotherapy may permit the eradication of metastatic disease. Parameters of response include tumor inhibition and the survival time of the animals.

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cell inoculum

Log

Fig. 7. Relation between inoculum of P-388 lymphocytic leukemia cells and percentage of survivors as bioassayed in recipient CDF, mice: Effect of radiation and metronidazole. -, Control; - - -- -, 500 RAD; ---, 500 RAD + MET 500 mg/kg; -.-, 500 RAD + MET 1000 mg/kg.

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Fig. 8. Relation between inoculum of P-388 lymphocytic leukemia cells and percentage of survivors bioassaved inrecinient CDF, mice: Effect of radiation and metronidazole. -,Control;----,4OOORAD;---, 4000 RAD + MET 500 mg/kg; - . -, 4000 RAD + MET 1000 mg/kg.

With the B16 melanoma mode1 investigations have been conducted of combined modality treatment of Sfluorouracil and -y-radiation on 3 different schedules, and cyclophosphamide and radiation on 6 different schedules and in no instance was any of the combined modality treatments significantly superior to -y-radiation alone.*’ An experiment is illustrated in Fig. 925in which cyclophosphamide was administered on days 1-S following tumor inoculation and y-radiation on days 8-12. Progressive inhibition of tumor

as

growth was observed with increasing dosage of yradiation. Treatment with y-radiation alone at a total dose of 1200r resulted at day 22 in total inhibition of tumor. The results were essentially similar with daily dosages of 3.1,6.3 or 12.5 mg/kg cyclophosphamide in combination with y-radiation. However with daily dosage of 25 mg/kg of cyclophosphamide plus yradiation total inhibition of local tumor was achieved at a lower dosage (200r total dose y-radiation). Cyclophosphamide alone yielded only partial inhibition of

Search for new radiation potentiators 0 A.

Day22 0

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potentiation utilizing the parameters of survival time and “cures”. Another tumor system of interest is the Ridgway Osteogenic Sarcoma. When growing as an i.m. tumor in AKD FI mice it does not metastasize. Relatively large tumors (2.0 cm X 1.0 cm) regress following localized treatment with @Co y-irradiation at a dose of 800rlday given for 5 successive days. With the Ridgway osteogenic sarcoma tumor model, treatment with actinomycin D, melphalan or adriamycin in combination with 6oCo y-irradiation in the dose range of 600r per day x 5 to 400r per day x 5 was more effective in increasing survival time than treatment with drug alone or radiation alone at comparable doses. This model is suitable for clinical staging and it is possible to determine the influence of therapy on extent and duration of remission and the course of relapse~10,12,‘3.15 In one illustrative experiment, tumor regression and regrowth following combined treatment with adriamycin and @Co radiation was followed (Fig. 10). In this experiment radiation was administered to large tumors (about 2 g) for 5 days 100,000

1600

roentgens

Fig. 9. Combined modality studies of the effect of yradiation and cytoxan on B-16 melanoma growth and y-Radiation tumor-bearing host lifespan [from Ref. 251.-, alone days 8-12; ----, y-radiation days 8-12 cytoxan y-radiation days 8-12 cytoxan 3.1 mg/kg days l-5; -.--, y-radiation days 8-12 cytoxan 6.3 mg/kg days l-5; ----, 12.5 mg/kg days l-5; .**.e., y-radiation days 8-12 cytoxan 25 mg.(kg days l-5. 10,000

tumor growth. No differences were observed in the survival time responses between -y-radiation and cyclophosphamide plus y-radiation. The maximum increase in survival time with y-radiation alone (1200r) was 221% and this was not exceeded by the combination therapy. Cyclophosphamide alone was ineffective in increasing survival time. In a second experilmen? where treatment with cyclophosphamide was withheld until days 8-12 and -y-radiation to days 15-19 the extent of tumor inhibition and increase in survival time were diminished as compared with treatment initiated earlier in the course of the disease, undoubtedly as the result of the greater tumor challenge represented by advanced tumor. No definitive differences were observed between the combination therapy and treatment with y-radiation alone. The i.m. inoculated B16 melanoma, particularly where the tumor is more advanced, would appear to be suitable for investigations of radiation and chemotherapy for assessment of both radiation potentiation with respect to local tumor and therapeutic radiation

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Fig. 10. Ridgway osteogenic sarcoma growth and regression following combined treatment with adriamycin (NSC123127) and ‘?Zo-radiation.

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starting on the 39th day and this was followed 3 days later by i.p. treatment for 5 days with adriamycin. It was observed that for adriamycin plus radiation (600r) regression was more extensive and relapse occurred later. In another experiment the percentage survival of ROS tumor-bearing mice following combined therapy with 6oCo y-radiation (600x) plus adriamycin was higher (100%) as compared with radiation alone (40%) or adriamycin alone (0%). The Lewis lung tumor is of particular interest since it will grow at a local site of inoculation and also metastasize preferentially to the lungs.’ Experiments showing the effect of localized ‘?Zo-radiation treatment on Lewis lung carcinoma growing intramuscularly in BDFl mice is summarized in Figs. 11 and 12. Well established 10 day old tumors failed to regress following localized radiation (fractional doses of 8OOror 400r given once daily for 5 days), although there was some transient inhibition of tumor growth. The survival time of tumor bearing mice was not prolonged following the delayed localized radiation treatment given alone. BCNU administered alone as a single i.p. treatment on day 18 (32 or 16 mg/kg) had no effect on tumor growth and did not increase the survival time of the animals. Combination treatment with y-radiation did not result in any appreciable additional inhibition of tumor growth. In dose-response studies, no definite increases in survival time were obtained with drug or radiation individually and only a moderate increase (20-25%) was observed with

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in BDF, mice.

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Days post tumor implantation

Fig. 12. Effect of BCNU (NSC-409962) and localized ‘?Toradiation treatment of Lewis lung carcinoma growing intramuscularly in BDFI mice.

the combination modality. Although the effects in these studies were only minimal it is indicated that the Lewis lung tumor model may be useful for further studies, particularly since the tumor is relatively resistant to localized radiation. It should be pointed out that in the above experiments therapy was initiated at a time when there was not only an enlarged tumor at the site of implantation but also there was extensive metastatic disease, particularly in the lungs. Animals with advanced Lewis lung disease are currently being utilized for investigation of combined modalities involving thoracic or whole body radiation and systemic therapy with or without surgical extirpation of local tumor. In summary it is evident that the overall methodology employed in the search for new antitumor agents and new combinations of drugs that may be effective in the treatment of cancer is also applicable to the search for radiation potentiators. One may consider the search as a two-step process. The initial step involves the identification of radiation potentiators or sensitizers utilizing simplified in vitro or in vim methodology which may permit more extensive screening. The second step encompasses determination of the usefulness of the potentiator as a therapeutic tool and involves utilization of methodology which permits the identification of therapeutic radiation potentiation. Where therapeutic radiation potentiation has been demonstrated for a

Search for new radiation potentiators 0 A.

new drug clinic the terest for ring the

in a tumor system that has relevance for the new radiation potentiator becomes of infurther development for clinical use. Baruncovering of prohibitive toxicologic or

GOLDIN

et al.

35

pharmacologic characteristics or insurmountable problems in development or formulation, the radiation potentiator should receive clinical trial.

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