Studies on the Selectivity of Antitumor Agents1 S
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Studies on the Selectivity of Antitumor Agents1 S. GARATTINI,2 A. GUAITANI, E. NANNI, AND V. PALMA Departmentof Pharmacology,Universityof Milan, Milan, Italy CONTENTS Introduction 1309 Materials and methods 1310 Results 1311 Data in control animals 1311 Drugs 1311 Effect of drugs 1312 Alkylating agents 1312 Antimetabolites (purines, pyrimidines, and others) 1313 Antibiotics 1313 Steroids 1313 Miscellaneous 1313 Increased toxicity for the various experimental groups 1314 Calculation of an index of specificity 1314 Influence of experimental conditions on the specificity index 1315 Studies on drug combinations 1315 Conclusions . 1316 INTRODUCTION emphasis was placed on obtaining drugs with the most exten sive antitumor action without regard to the fundamental ques This report summarizes more than three years of research, tion of host toxicity, i.e., to what extent does a new drug rep having as its primary objective the design and experimental resent an improvement with respect to the ratio of antitumor testing of improved procedures for the evaluation of the spec effect to the effect on normal tissues? ificity of action of antitumor agents. It was considered that One procedure which has been employed to take into account additional emphasis on host-tumor response parameters involved the effect of the drug on both the host and the tumor is the in drug specificity could help in the selection and evaluation of therapeutic index. Usually, this is taken as the ratio of the dose more effective drugs for clinical use. One of the primary prob which elicits a specified degree of host mortality (LDio) to the lems recognized by investigators is that an increase in the inhi dose which results in a requisite degree of inhibition of tumor bition of tumor growth is generally accompanied by an increase growth (14). Alternatively, the antitumor effectiveness of drugs in undesirable toxic effects, particularly for tissues such as bone has been measured at a fixed cost in host toxicity; i.e., the de marrow and intestinal mucosa which have a high rate of pro gree of tumor inhibition or survival time of the animals is meas liferation. Although the bone marrow and intestinal mucosa are ured at a specified degree of drug-dose mortality or body weight usually the most sensitive tissues, other tissues may be more loss (5). In another system, where the maximum survival time sensitive to particular drugs or become more sensitive under of the tumor-bearing animals is determined at the optimal dos special conditions. Also, the generalized toxicity for the host age, the host response is also taken into account, since the tox may increase in relation to impaired function of liver and kidney. icity of the drug limits the observation of further increases in It is a common clinical observation that, in order to inhibit survival time at doses exceeding the optimal dose (4). tumor growth, it is necessary to employ drug doses which also In the current study it was considered desirable to examine result in toxic symptoms for the host. Thus the drugs available a pattern of host effects. The antitumor activity was evaluated today are limited in antitumor selectivity since they do not "distinguish" sufficiently between tumor cells and "normal pro in relation to effects on the following parameters of the host: (a) liferating cells." The lack of specificity of antitumor agents may normal proliferating tissues (bone marrow and intestinal mu cosa); (b) compensatory proliferation (liver regeneration, adre be attributable, at least in part, to the early historic objective nal and renal hypertrophy); (c) "nonneoplastic" growth (gran in screening for new chemotherapeutic agents. Initially, the uloma resulting from the presence of a foreign body); and (d) 1This work was supported by Contract SA-43-PH-3054, Cancer biochemical indices representing various aspects of generalized Chemotherapy National Service Center, National Cancer Institute, NIH, toxicity (liver lipids and glycogen, serum protein and lipopro- Bethesda, Maryland. tein). In the current studies, the challenge to drug effectiveness " Present address: Instituto di Ricerche Farmacologiche Mario Ne was made severe by withholding treatment until the tumor was gri, Via Eritea, 62, Milano, Italy. well established. In order to compare the relative effectiveness DECEMBER 1967 (Part 2) 1309 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1967 American Association for Cancer Research. 5. Garatiini, A. Guaitani, E. Nonni, and V. Palma of various drugs, fractional doses of the LD5owere employed as //. Leukocyte counts. A sample of blood diluted 20 times determined following 6 days of treatment. The experimental with Türkesolution was counted under the same conditions as results obtained permit the assessment of the potential advan I/. tage of the proposed procedures and suggest various possibilities ¡m. Leukocyte differential count. Blood was spread on a for improvement of the methods of screening. glass slide and stained with May-Grünwald-Giemsa solution. For each sample, 100 leukocytes were counted and classified as MATERIALS AND METHODS granulocytes (neutrophil, basophil, and eosinophil), lymphocytes, and monocytes. Only the granulocyte and lymphocyte counts Animals are reported in the tables. Sprague-Dawley male rats having an average weight of 180 2. Partially Hepatectomized Rats (H). Two experiments, (160-200) gm were employed. The animals had free access to involving groups of 8 animals in each, were conducted for each food (Diet Alai 56 from Alai Firm, Milan) and water. During drug at 3 dose levels. Partial hepatectomy was performed em the course of the experiments, they were fasted for 12 hours ploying light ether anesthesia in accordance with the method prior to their being killed by decapitation. of Higgins and Anderson (7). Approximately 60% of the liver (left and middle lobes) was excised after ligation at the hilus. Determination of the Subacute II) A mixture of penicillin G sodium (10,000 ILJ/rat) and strep tomycin (10 mg/rat) was injected subcutaneously. The weight Animals were treated intraperitoneally once a day for six of the excised liver was determined. The weight of the hepatic days with the compound under test. The doses were spaced by tissue found 48 hours or 6 days after operation was also de a factor of 1.2. The mortality was calculated on the basis of the termined. In animals killed after 48 hours the following deter number of deaths which had occurred by the seventh day. The minations were made: LDso was determined graphically by plotting the percent mor 2a. Liver regeneration was calculated according to the tality against dose on probit paper. method of Drabkin (2).:l 2b. Number of hepatic mitoses. Samples of liver were fixed Doses in Susa solution for 18 hours, dehydrated, embedded in paraffin, For each compound three concentrations representing i; I, cut in slices 2 n thick, and stained with hematoxylin. The num and is of the subacute LD50 (after 6 days of treatment) were ber of mitoses was determined per 1000 hepatic cells. The cells employed. For some compounds, as indicated in the tables, the in mitosis were classified as in prophase, metaphase, anaphase, doses were chosen arbitrarily. The treatments were adminis and telophase. In the tables, only the total number of cells in tered i.p. once a day for 6 days. The controls received either mitosis is reported. saline or the vehicle employed to dissolve the compound under 2c. Liver lipids. See \h. test. 2d. Liver glycogen was evaluated in accordance with the method of Kemp and Kits Van Heijningen (8). Tests and Methods In animals killed at 7 days after operation the following de 1. Intact rats (N). Two experiments, each involving groups of terminations were made: 6 animals, were carried out for each drug at 3 dose levels. Fol 2e. Body weight. See la. lowing the termination of the treatments (Day 7), the animals 2f. Spleen weight. See li. were killed and the following determinations were made. 2g. Hypophysis fresh weight. ¡a.Body weight. Difference between the weight at the be 2h. Adrenal fresh weight. ginning and at the termination of the experiment. 2i. Intestinal weight. See \f. Ib. Spleen weight. Spleens were removed immediately after 21. Intestinal DNA. See \g. death and their weight determined. They were cut into small 2m. Liver lipids. See \h. fragments on a glass slide and dried at 60°Cfor 2 days at which 2n. Erythrocyte counts. See I/'. time constant weight was observed. 2o. Leukocyte counts. See I/. Ic. Liver weight. Same as !/>., but the dry weight was de 2p. Leukocyte differential count. See 1m. termined only for the caudate lobe of the liver. 2r. Liver regeneration. See la. Id. Hypophysis fresh weight. 2s. Agar-induced granuloma. In the same animals employed le. Adrenal fresh weight. for the evaluation of liver regeneration, at the time of operation If. Intestinal weight. Small intestine was carefully isolated an agar cylinder (diameter, 7 mm; length, 7 mm) was also im from the mesentery and washed out with saline, perfused under planted subcutaneously in the intrascapular area (1). When the constant pressure. It was then compressed with filter paper and animals were killed, the granuloma was excised, opened, and weighed. the agar removed. The weight of the granuloma was determined ¡g.Intestinal DNA was determined in the last portion (5 cm) immediately before and after it was dried. of the small intestine. Chemical analysis was performed ac cording to the method of Schmidt and Thannhauser (12). "The formula used was: LR = (•--X 1001 - 52.8. LR is the % re- ¡h.Liver lipids. Total lipids were determined in pooled liver in accordance with the method of Sperry (15). //'. Erythrocyte counts. A sample of blood from the carotid generation with respect to the excised liver; A is the weight of the liver found after the sacrifice of the animal.