[Gann, 68, 459～464; August, 1977]

INTERACTION OF ANTITUMOR AGENTS INCLUDING OR IN SARCOMA-180 SYSTEM

Masaaki IIGO, Fumihiko KANZAWA, Asako NAKAMURA, Akio HOSHI, and Kazuo KURETANI Pharmacology Division, National Center Research Institute*

Combination effect of antitumor agents, including doxorubicin and dauno- rubicin, was evaluated on the concept of pharmacological synergism in ascites sarcoma-180 system. In alternate administration, combinations of doxorubicin plus , thio-TEPA, , actinomycin-D, , , , , 6-, or L- show- ed synergism, but in simultaneous one, only three agents, cyclophosphamide, carboquone, and cytarabine, were synergistic. On the other hand, combination of daunorubicin plus one of 8 agents (thio-TEPA, mitomycin-C, , actinomycin-D, vinblastine, ancytabine, 6-mercaptopurine, and L-asparaginase) and 6 agents (cyclophosphamide, thio-TEPA, mitomycin-C, bleomycin, actino- mycin-D, and vinblastine) provided synergism in alternate and simultaneous administration. Combination effect of agents was affected by the schedule of drug administration for doxorubicin, but weak for daunorubicin. Toxicity of doxorubicin or daunorubicin in combination with other drugs was also affected by the schedule of administration. Combination of a larger number of agents in simultaneous administration provided antagonism compared with an alternate administration.

Combination provides a the L-1210 system, we developed a reason- clinical advantage over single-drug treat- ably quantitative method for examining the ment.5,13) As the number of new drugs in- activity and toxicity of a combination of creases yearly, the number of possible com- agents separately.7,9,10) By the extension of binations also increases. The most appropriate these studies, interaction of antitumor agents, combination of agents and treatment schedule including daunorubicin and doxorubicin, in must be selected among numerous possible activity against sarcoma-180 and lethal toxi- candidates. Up to the present, most of the city to the host animals, and of schedule of combination therapy in animal models was administration on the interaction of anti- examined with the L-1210 system14) tumor agents were examined. and the results were evaluated by the "ther- apeutic synergism" and not by pharmacol- MATERIALS AND METHODS ogical synergism. By the former method, Animals and Tumor System Female ddN antitumor activity and toxicity to the host mice weighing 20 ± 2g were used , and 0.05ml animals cannot be evaluated separately, and (1 × 107 cells) of 7-day-old ascites sarcoma-180 the method cannot be applied to other tumor was inoculated intraperitoneally to six mice in systems in which antitumor activity is eval- one group.6) Antitumor Agents As shown in Table I, 16 uated by the inhibition of tumor growth. antitumor agents were used. Cyclophosphamide In order to determine the combination effect (Endoxan; Shionogi & Co., Osaka), thio-TEPA of antitumor agents in systems other than (Tespamine; Sumitomo Chemical Industries, * Tsukiji 5-1-1 , Chuo-ku, Tokyo 104(飯 郷 正 明,官 沢 文 彦,中 村 朝 子,星 昭 夫,榑 谷 和 男) .

68(4) 1977 459 M. IIGO, ET AL.

Table I. Activity against Sarcoma-180 Ascites be combined with A as listed in Table I. The and Toxicity to Mice of Antitumor antitumor activity was evaluated by the total Agents packed cell volume ratio on, the 7th day after tumor inoculation in the sarcoma-180 system as reported previously.6) Evaluation of Drug Toxicity LD50 of the agents in normal ddN mice was calculated by daily injection for 5 days and survivors on 14th day after the first injection by the method of Litchfield-Wilcoxon.12) Doses of two compounds (A and B) in combination were one-half of LD50 of A plus one-half of LD50 of B per day for simul- taneous administration, and LD50 of A on 1st, 3rd, and 5th day and LD50 of B on 2nd and 4th day for alternate administration, where A means doxorubicin or daunorubicin and B means one of the rest of compounds. Evaluation of Drug Interaction The terms synergism, antagonism, and additive action were used according to the definition given by Wells.15) For a combination of two drugs (A and B), if equally effective doses (a and b) of A and B are combined, it holds true that: Effect of dose a = Effect of dose b Effect(1/2 dose a + 1/2 dose b) =q(simul- Effect of dose a taneous) Effect(alternate(doseand a dose b)=q(alter- Effect of dose a Osaka), mitomycin-C (Mitomycin Kyowa-S; Kyo- nate) wa Hakko Co., Tokyo), daunorubicin (Dauno- If q = 1, there is additivity in the action of the mycin; Meiji Seika Kaisha, Tokyo), bleomycin drugs combined. If q>1, there is a synergism in (Bleo; Nippon Kayaku Co., Tokyo), actinomycin- action. If q<1, there is an antagonism in action.2) D (Cosmegen; Nippon Merck-Banyu Co., Tokyo), Interaction of agents is defined as synergism for vinblastine (Exal; Shionogi & Co.), vincristine 0～20, additive action for 21～80, and antagonism (Oncovin; Shionogi & Co.), methotrexate (Takeda for 80～100 of T/C% in tumor growth. Interaction Chemical Industries, Osaka), 6-mercaptopurine of agents for toxicity is defined as synergism for (Sigma, U. S.A.), and L-asparaginase (Leunase; 5～6, additive for 2～4, and antagonism for 0～1 Kyowa Hakko Kogyo Co.) were purchased com- dead animals out of six mice. mercially. Carboquone (carbazilquinone) was kindly supplied by Sankyo Co., Tokyo, doxorubi- cin (KW-125) and 5- by Kyowa Hakko RESULTS Kogyo Co., and ancytabine (cyclocytidine) and Interaction in Antitumor Activity of cytarabine ( arabinoside) by Kohjin Co., Agents Administered Simultaneously Tokyo. ED50 (dosage showing 50% inhibition in tumor growth) and LD50 (dosage showing 50% In combination chemotherapy, the basic lethality in treated animals) of the agents in combination is a pair of drugs and the sched- normal ddN mice are also shown in Table I. ule of administration of agents calls for two Evaluation of Antitumor Activity Test com- procedures, simultaneous and sequential ad- pounds were injected intraperitoneally once daily ministration. Therefore, interaction between for 5 days, starting 24hr after tumor inoculation. The doses of two compounds (A and B) were two drugs administered simultaneously was one-half of ED50 of A plus one-half of ED50 of B first examined. As shown in Fig. 1, three per day for simultaneous administration or ED50 compounds (cyclophosphamide, carboquone, of A on 1st, 3rd, and 5th day and ED50 of B on and cytarabine) combined with doxorubicin 2nd and 4th day for alternate administration, where A means doxorubicin or daunorubicin and showed synergism, no compound showed B means one of the remaining 15 compounds to antagonism, and the rest of 12 compounds

460 Gann EFFECT OF COMBINATION OF ANTITUMOR AGENTS

Fig. 1. Effect of doxorubicin and daunorubicin in combination with other agents in simultane- ous administration

showed an additive action, when adminis- istration on the activity was marked in this tered in equally effective dosages in the sar- case. The combinations showing synergism coma 180 system. On the other hand, when in activity were more numerous in alternate combined with daunorubicin, cyclophospha- administration than in simultaneous one. On mide, thio-TEPA, mitomycin-C, bleomycin, the other hand, eight combinations containing actinomycin-D, and vinblastine showed syner- daunorubicin were synergistic, that is, thic- gism and no compound showed antagonism. TEPA, mitomycin-C, bleomycin, actinomy- The rest of 9 combinations showed an additive cin-D, vinblastine; ancytabine, 6-mercapto- action. purine, or L-asparaginase plus daunorubicin. Interaction of Agents Administered Al- Influence of the schedule of administration ternately on Antitumor Activity Influ- on the activity was weak in this case (Fig . 2). ence of the schedule of administration on Interaction in Lethal Toxicity of Anti- antitumor activity was also examined. One tumor Agents Interaction in toxicity of of the sequential administrations was con- the agents was examined. In simultaneous sidered to be the alternate administration. administration, 1 (carboquone), 5 (mitomy- In this case, two agents were considered not cin-C, vincristine, methotrexate , 5-fluoroura- to exist in tissues at the same time. Ten cil, and 6-mercaptopurine), and 8 compounds compounds (cyclophosphamide, thio-TEPA, combined with doxorubicin showed syner- carboquone, actinomycin-D, vinblastine, vin- gism, antagonism, and additive action, re- cristine, methotrexate, cytarabine, 6-mercap- spectively (Fig. 3). In combinations contain- topurine, and L-asparaginase) showed syner- ing daunorubicin, 2 (bleomycin and cytara - gism in combination with doxorubicin. The bine), 7 (thio-TEPA , carboquone, mitomy- rest of 5 compounds showed an additive cin-C, actinomycin-D , ancytabine, 5-fluoro- action. Influence of the schedule of admin- uracil, and 6-mercaptopurine) , and 5 com- 68(4) 1977 461 M. IIGO, ET AL.

Fig. 2. Effect of doxorubicin and daunorubicin in combination with other agents in alternate administration

Fig. 3. Toxicity of doxorubicin and daunorubicin in combination with other agents in simul- taneous administration

462 Gann EFFECT OF COMBINATION OF ANTITUMOR AGENTS

Fig. 4. Toxicity of doxorubicin and daunorubicin in combination with other agents in alternate administration

pounds showed synergism, antagonism, and quone and daunorubicin plus bleomycin in additive action, respectively (Fig. 3). Com- simultaneous administration and the com- binations containing L-asparaginase could not binations of doxorubicin plus carboquone be tested because the agent was not lethal and daunorubicin plus vinblastine in alternate to mice. Therefore, its LD50 could not be administration provided synergism in both determined. activity and toxicity. In alternate administration of these agents, 1 (carboquone), 1 (cytarabine), and 12 com- DISCUSSION pounds combined with doxorubicin showed In the studies on experimental combination synergism, antagonism, and additive action, chemotherapy, almost all experiments have respectively. In combinations containing been discussed on the concept of therapeutic daunorubicin, 2 (cyclophosphamide and vin- synergism using L-1210 leukemia system.3,8) blastine), 2 (mitomycin-C and cytarabine), However, therapeutic synergism is not a and 10 compounds provided synergism, antag- pharmacological synergism and indicates a onism, and additiv eaction, respectively (Fig. situation in which a combination treatment 4). For therapeusis, the combination that results in an improved therapy as described provided synergism in activity and antag- by Venditti et al.14) Therapeutic synergism is onism in toxicity is the best. Such best com- affected by both toxicity and activity of binations were daunorubicin plus thio-TEPA, agents and, therefore, toxicity cannot be mitomycin-C, or actinomycin-D in simultane- evaluated separately from antitumor activity . ous administration, and doxorubicin plus On the other hand, pharmacological syner- cytarabine and daunorubicin plus mitomycin- gism by the administration of two agents C in alternate administration. Worst com- means an unexpected enhancement of the bination that provided antagonism in activity effect in either activity or toxicity over the and synergism in toxicity was not found. predetermined effect by the administration The combinations of doxorubicin plus carbo- of a single agent. The expected effects in the

68(4) 1977 463 M. IIGO, ET AL. present experiments were 50% inhibition in doxorubicin in combination with other drugs tumor growth and 50% lethality in treated is affected by the schedule of drug adminis- animals so that both the activity and toxicity tration, and alternate administration is ad- can be determined separately. Furthermore, vantageous over simultaneous administration the cause of therapeutic synergism is clarified in the activity but reverse is true in toxicity. by this analysis of the effect as enhancement in activity or decrease in toxicity, or both. (ReceivedFebruary 26, 1977) With these results, doses of drugs in combina- tion therapy can be selected. For example, REFERENCES when toxicity of drugs increases in combina- 1) Ansari, B. M., Tompson, E. N., Whiftaker, tion (synergism), the dose must be diminish- J. A., Br. J. Haematol., 31, 269～277 (1975). 2) Ariens, E. J., "Toxicological Problems of ed, and when there is no increase (antago- Drug Combinations," p. 137 (1972). Ex- nism), the usual doses for therapy can be cerpta Medica, Amsterdam. administered in combination. Doxorubicin 3) Carter, S. K., Cancer Chemother. Rep., Part was clinically active against solid tumors in 2, 4, 3～11 (1974). combination with cyclophosphamide, vincris- 4) Carter, S. K., Cancer Treat. Rep., 3, 141～ 174 (1976). tine, methotrexate, or 5-fluorouracil.4,11) The 5) Frei, E., III, Cancer Res., 32, 2593～2607 reason for these combination effects was con- (1972). sidered to be the enhancement in activity 6) Hoshi, A., Kuretani, K., Farumashia, 9, 464 of cyclophosphamide and both enhancement ～468 (1973). 7) Hoshi, A., Kanzawa, F., Iigo, M., Kuretani, in activity and decease in toxicity of the rest K., Gann, 66, 539～546 (1975). of 3 compounds in the present results by 8) Hoshino, A., Igaku-no-Ayumi, 85, 121～128 alternate administration. On the other hand, (1973). daunorubicin was clinically active against 9) Iigo, M., Nakamura, A., Hoshi, A., Kure- leukemia in combination with cytarabine or tani, K., Gann, 67, 641～644 (1976). 10) Kanzawa, F., Hoshi, A., Kuretani, K., Gann, vincristine,1) but such a result does not agree 65, 55～60 (1974). with the results from the present experiment. 11) Krakoff, I. H., Clin. Bull., 5, 120～123 This clinical advantage in a combination (1975). over single agent was previously rationalized 12) Litchfield, J. T., Wilcoxon, F., J. Pharmacol. Exp. Ther., 96, 99～133 (1949). in the L-1210 system.7) It suggests that the 13) Ota, K., Nippon Iji Shinpo, No. 2442, 43～51 combination effect in one tumor system is (1971). different from that in another system. In 14) Venditti, J. M., Humphreys, S. R., Mantel, this sense, combination effect of antitumor N., Goldin, A., J. Natl. Cancer Inst., 17, 631 agents must be examined in several tumor ～767 (1956). 15) Wells, J. A., "Pharmacology in Medicine," systems. Drug interaction is also affected by pp. 1～20 (1958). McGraw-Hill Book Co., the schedule of administration. Activity of New York.

464 Gann