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[CANCER RESEARCH 41, 5158-5161, December, 1981] 0008-5472/81 /0041 -OOOOS02.00 Effects of a-Difluoromethylornithine Alone and Combined with Adriamycin or Vindesine on L1210 Leukemia in Mice, EMT6 Solid Tumors in Mice, and Solid Tumors Induced by Injection of Hepatoma Tissue Culture Cells in Rats

Jacques Bartholeyns and Jan Koch-Weser

Centre de Recherche Merrell International, 16 rue d'Ankara, 67084 Strasbourg Cedex, France

ABSTRACT (initial body weight, about 20 g; Centre National de la Recherche Scientifique, OrlÃ©ans, France) were used for the leukemia model. The effects of a-difluoromethylornithine (DFMO; RMI 71782) Female BALB/c mice (initial body weight, 18 to 22 g; Charles River in combination with vindesine or Adriamycin were investigated Breeding Laboratories, France), were used for experiments with the in three different animal tumor models. When given in a con EMT6 tumor. Male Buffalo rats bred in our center (initial body weight, centration of 2% in drinking water to C57BL/6 x DBA/2 FI 160 to 200 g) were used for the experiments on solid tumors induced mice inoculated i.p. with L1210 leukemia cells, DFMO pro by HTC cells. The animals were housed in metal cages with free access to food and water or a solution of DFMO. Fluid intake and body weight longed the survival time 1.2-fold. Treatment with vindesine (0.1 were measured at regular intervals. Room temperature (21-23Â°), hu mg/kg/week i.p. or Adriamycin (2.5 mg/kg/week i.p.) in midity (45 to 55%), and a 12-hr light cycle (beginning at 6 a.m.) were creased the mean survival time 1.4- and 2.3-fold, respectively. kept constant throughout the investigations. DFMO with vindesine doubled survival time, while DFMO with Cells and Tumors. L1210 leukemia was propagated and maintained Adriamycin increased it 3.5-fold and yielded 30% long-term in vivo in inbred BD2Fi mice. Cells were transferred every week by i.p. survivors. The growth of solid tumors induced in Buffalo rats transplantation of 106 L1210 cells contained in 0.1 ml 0.15 M NaCI to by i.m. injection of hepatoma tissue culture cells was inhibited new acceptor mice. Mouse mammary EMT6 cells were grown as described previously 65% after 2 weeks of DFMO treatment. Similar inhibition of (12, 13) on Waymouth's medium supplemented with 15% fetal calf growth could be achieved by weekly i.p. injections of vindesine (0.2 mg/kg) or Adriamycin (2.5 mg/kg). When the same doses serum (Grand Island Biological Co., Grand Island, N. Y.). Cell cultures were trypsinized and suspended in phosphate-buffered saline (8 g/l of these drugs were administered in combination with DFMO, NaCI, 0.2 g/l KCI, 1.15 g/l Na2HPO4, 0.2 g/l KH2PO4) to a density of the growth of this hepatoma was completely arrested. Com 10" cells/ml. Animals were inoculated s.c. in the interscapular region bined treatment of BALB/c mice bearing s.c. solid EMT6 with 10s cells/mouse. Mice were sacrificed by cervical dislocation, tumors with DFMO and adriamycin or vindesine also resulted and the tumors were removed and weighed. in enhanced inhibition of tumor growth compared to single- HTC cells, derived from Morris hepatoma 7288C induced in Buffalo drug therapy. These results indicate that combination of DFMO rats, were grown in spinner culture according to Hershko and Tomkins with vindesine or Adriamycin is an effective approach to the (6). Cells were harvested from their culture medium, counted, and treatment of several animal cancers. washed twice with 0.9% NaCI solution prior to use. The tumors were induced in Buffalo rats by i.m. injection of 2 x 106 HTC cells contained in 0.5 ml 0.15 M NaCI into the leg. Animals were examined every week INTRODUCTION for tumor growth, and the diameter of their legs at the tumor level was measured with calipers in 2 perpendicular directions (breadth and Previous studies from our Centre (11, 12) have shown that width). The tumoral cross-section was considered an ellipse, and the DFMO1 (RMI 71782), a specific and nontoxic irreversible inhib following formula was used to calculate its size: itor of ODC, has antitumoral properties. It prolongs survival in mice bearing L1210 leukemia and retards the growth rate of w/4 x [(f,f2) - (c,c2)] EMT6 murine tumors in vitro and in vivo. Recent evidence suggests that combination of DFMO with MGBG (4), with where fi and (2 are the perpendicular axes of the tumoral left leg, and vitamin A analogs (5), or with cyclophosphamide (12) may c, and c2 are the perpendicular axes of the control right leg (3). produce a greater therapeutic response than does single-drug Biochemistry. ODC activity was measured according to the method therapy. This study aimed to assess the antitumoral effects of of Prakash ef al. (11 ) on freshly homogenized tumors in 9 volumes of DFMO given in combination with 2 potent antitumoral drugs, ice-cold phosphate buffer (0.1 M; pH 7.2) containing 1 ITIMdithiothreitol, vindesine (2) or Adriamycin (1 ), in 3 animal models. 0.1 HIM disodium EDTA, and 10 /IM pyridoxal phosphate. Administration of Drugs. DFMO was administered p.o. by offering a 2% solution of the compound in tap water as the sole drinking fluid. MATERIALS AND METHODS Average daily fluid intake per animal was estimated from the total fluid intake per group. Vindesine sulfate, dissolved in 0.15 M NaCI, was Animals. Male C57BL/6 x DBA/2 F, (hereafter called BD2F,) mice injected i.p. at a dose of 0.1 or 0.25 mg/kg. Adriamycin was admin istered similarly at a dose of 2.5 mg/kg. 1 The abbreviations used are: DFMO, DL-a-difluoromethylornithine; ODC, or- DFMO (RMI 71782) was synthesized in this Centre (10). Vindesine nithine decarboxylase; (EC 4.1.1.17); MGBG, methylglyoxal-bis(guanylhydrazone); HTC, hepatoma tissue culture; BCNU, 1,3-bis<2-chloroethyl)-1 -nitro- sulfate was a gift from Eli Lilly (Indianapolis, Ind.), and Adriamycin sourea. (doxorubicin hydrochloride) was purchased from Roger Bellori Labo Received May 20, 1981; accepted September 16, 1981. ratories (Neuilly, France).

5158 CANCER RESEARCH VOL. 41

RESULTS resumed growth at a rate similar to that in control rats (Chart 2). On the other hand, it was possible to slow the growth of Effect of DFMO Alone or in Combination with Vindesine or tumors by beginning p.o. administration of DFMO even 21 days Adriamycin on Survival of BD2F, Mice Inoculated with L1210 after inoculation (Chart 2). Cells. DFMO was administered p.o. as a 2% solution in drinking Vindesine (0.2 mg/kg i.p.) injected once a week into Buffalo water, resulting in a mean daily intake of 2.4 g/kg (Table 1). rats beginning 18 days after inoculation inhibited tumor growth When DFMO was given from Day 1 after inoculation of 106 by 35% to 45% (Chart 3). Combination therapy with 2% DFMO L1210 cells, the treatment prolonged the survival time of BD2Ft in drinking water from Day 4 and vindesine (0.2 mg/kg/week mice 1.2-fold (Chart 1; Table 1). The cumulative mortality curve i.p.) from Day 18, almost completely inhibited the growth of the was shifted slightly more to the right when mice were given i.p. HTC solid tumor. Adriamycin (2.5 mg/kg i.p.) injected once a injections once a week of vindesine (0.1 mg/kg; Chart 1). week from Day 18 inhibited tumor growth by 60% to 70% Therapy with a combination of DFMO and vindesine was more (Chart 3). Combination chemotherapy with DFMO resulted in a effective than either drug given alone, and the effect was more complete arrest of tumor growth and even in some reduction of than additive (Chart 1; Table 1). Vindesine (0.2 mg/kg) injected the tumor mass (Chart 3). twice a week prolonged survival as compared with weekly 0.1 - Effect of DFMO Alone or in Combination Therapy on the mg/kg doses (Table 1). When this schedule of vindesine was Growth of Solid Tumors Induced by EMT6 Cells in Mice. combined with DFMO, the mean survival increased to 18.5 BALB/c mice inoculated with 105 EMT6 cells/mouse were days (Table 1). Withdrawal of DFMO for 24 hr starting 16 hr given 2% DFMO in water as drinking fluid beginning 8 days prior to each injection of vindesine (0.1 mg/kg) did not shorten after injection of the cells. Tumors were removed 16 days after survival time significantly compared to maintaining DFMO inoculation and weighed (Table 2). Compared to animals re throughout (mean survival times, 15.6 and 16.1 days, respec ceiving no chemotherapy, mice receiving 2% DFMO during 1 tively). week (2.8 g/kg/day) had 43% less tumor mass. Vindesine Adriamycin (2.5 mg/kg) given i.p. once a week prolonged survival time 2.3-fold, but all mice had died by Day 24 (Chart 1). When Adriamycin was given in combination with DFMO l l I i I I I i (Chart 1; Table 1), the survival time of the mice was prolonged more than with either agent alone, and 30% of the mice were still alive 50 days after inoculation. Effect of DFMO Alone or in Combination with Vindesine on the Development of Solid Tumors Induced by HTC Cells in Buffalo Rats. Buffalo rats inoculated i.m. with a suspension of 2 x 106 HTC cells rapidly developed a solid tumor. Tumor growth could be followed by the increase in cross-section for up to 2 months. At this time, necrosis followed by death K> 19 20 25 0 K> 20 3O 4O 50 occurred. Tumor growth rate in control rats was compared to DAYS that in rats treated from Day 4 with 2% DFMO in the drinking Chart 1. Effect of DFMO, vindesine, and Adriamycin alone or in combination water (Chart 2). This treatment resulted in a daily DFMO intake on survival of mice bearing L1210 tumors. BD2F mice were given i.p. injections of 10e L1210 cells on Day 0. O. control mice (n = 20); â€¢,mice receiving 2% of about 1.5 g/kg by the rats and significantly slowed the DFMO in water as sole drinking fluid beginning 1 day after inoculation (n = 20); growth of the tumor so that its size on Day 21 averaged about A, mice given i.p. injections of vindesine (0.1 mg/kg/week) alone (n - 20); A, 25% of that in controls. Thereafter, the cytostatic effect of mice given i.p. injections of vindesine with 2% DFMO in drinking water (n = 20); D, mice given i.p. injections of Adriamycin (2.5 mg/kg/week) alone (n = 10); DFMO appeared to be somewhat reduced (Chart 2). In animals â€¢,mice given i.p. injections of adriamycin with 2% DFMO Â¡ndrinking water from whom DFMO was withdrawn on Day 21, the tumors (n = 10); Arrows, days of injection of cytotoxic drugs.

Table 1 Effect of combination therapies using DFMO and vindesine or Adriamycin on BD2F, mice inoculated with 10e LÃ¬210 cells

in dexoftreated (survival group/survival in fluid in wtgainafter of con take(g/kg)2.42.83.02.7Dailytake(ml/mouse)2.62.433.22.83.03.13.5Av.8days(g/ ChemotherapyNoneDFMO time(days)8.5 trolgroup)1.001.211.361.691.902.182.36>3.4740-daysurvivors0/67b0/390/240/100/260/100/103/10DailyDFMOmouse)4.94.23.63.83.02.53.02.5 Â±0.2a10.3 fromDay(2%) in drinking water Â±0.311.6 1Vindesine onDays (0.1 mg/kg/ wk i.p.) Â±0.414.4 8Vindesine1 and onDays (0.2 mg/kg twice a wk) Â±0.716.1 15DFMO1.3, 8, 11, and mg/kg)on(2%) + vindesine (0.1 Â±0.718.5 5DFMODays 1, 8 and 1 mg/kg)on(2%) + vindesine (0.2 Â±1.020.1 5AdriamycinDays 1, 3, 8, 11, and 1 2,11. (2.5 mg/kg) on Days Â±2.6>29.5Therapeutic 35DFMO19, 27, and mg/kg)(2%) + Adriamycin (2.5 on Days 2, 1 1, 19, 27, and 35Survival Mean Â±S.E. ' Number of surviving animals/total number of animals used.

DECEMBER 1981 5159

(0.2 mg/kg) or Adriamycin (2.5 mg/kg) injected i.p. on Days cells, only the animals receiving Adriamycin showed a reduced 7 and 14 inhibited tumor growth by about 30%. When DFMO weight increase when compared with controls (Table 2). was given in combination with vindesine or with Adriamycin at the above doses, the reduction in tumor weight reached 60% DISCUSSION (Table 2). Tumor ODC activity was reduced after treatment with DFMO, mostly when DFMO was combined in therapy with Antiproliferative effects of DFMO have been described for vindesine (Table 2). several cell types in culture (5, 7, 9, 11, 12, 14) and against Toxicity. The only apparent side effect of DFMO treatment L1210 leukemia (11) and EMT6 s.c. tumors (12) in mice. The was diarrhea in rats. The daily fluid intake by Buffalo rats was growth-inhibiting effects of DFMO did not always equal the reduced by 25% when DFMO was given in drinking water, but antitumoral effects achieved with selected cytotoxic agents, there was no change in weight gain compared to controls. but the action of DFMO was free of obvious cytotoxicity and Combination therapy did not add any obvious toxicity. The other untoward effects. This pointed to the possible benefits of daily fluid intake and the average weight increase of the mice using DFMO in combination chemotherapy (5, 12). receiving DFMO (2% in drinking water) were similar to un Our results in 3 animal models show that, in combination treated animals, and no additive toxicity was observed when with DFMO, Adriamycin and vindesine give better therapeutic DFMO was given in combination with Adriamycin or vindesine. results than when used alone. This was particularly clear in the The average daily intake of fluid by leukemia-bearing BD2F, case of L1210 leukemia where combination therapy with Ad mice was slightly increased in animals treated with vindesine riamycin and DFMO resulted in a marked increase in survival or Adriamycin (Table 1). The apparent low weight gain mea sured in mice treated with vindesine or Adriamycin and DFMO (Table 1) probably reflects a delay in appearance of ascites, which contributes significantly to the total body weight, rather 15 than a toxic effect. For BALB/c mice inoculated with EMT6

1 1 1 10 Commi Control

i VINQ+ DFMO

10 20 3O 40 10 20 30 40

DAYS DAYS IO 20 30 Chart 3. Effect of DFMO. vindesine. and Adriamycin alone or Â¡ncombination on in vivo HTC cell tumor growth in rats. Buffalo rats were given i.m. injections of DAYS 2 x 10e HTC cells on Day 0. O. control rats (n = 10); â€¢,rats receiving 2% Chart 2. Effect of DFMO on the growth of HTC cell solid tumors in rats. DFMO in drinking water beginning 4 days after inoculation (n â€”10); A, rats given Buffalo rats were given i.m. injections of 2 x 10e HTC cells in one leg on Day 0. i.p. injections of vindesine (0.2 mg/kg/week) alone (n = 10); A, rats given i.p. â€¢.control rats (n - 12 up to Day 21 and n = 6 thereafter; A, rats receiving 2% injections of vindesine in combination with 2% DFMO in drinking water from Day DFMO in drinking water from Day 21 (n = 6); A, rats receiving 2% DFMO in 4 (n = 10); Ã›, rats given i.p. injections of Adriamycin (2.5 mg/kg/week) alone drinking water from Day 4 (n = 12 up to Day 21 and n = 6 thereafter); O, rats (n = 4); â€¢rats given i.p. injections of Adriamycin in combination with 2% DFMO given 2% DFMO in drinking water from Day 4 to Day 21 and then tap water in drinking water from Day 19 (n = 4); Arrows, days of injection of cytotoxic (n - 6). Points, means; bars, S.E. drugs. Points, means; oars, S.E.

Table 2 Combination therapies of EMT6 tumors in mice using DFMO and vindesine or Adriamycin Mice were inoculated s.c. with 106 EMT6 cells. Tumor wt 16 % of inhibi- Tumoral ODC Daily fluid in- Av. wt gain days after im- tion of tumor activity (nmol/g/ take (ml/ in 16 days Chemotherapy plantation growth hr) mouse) (g/mouse) None 1.12 Â±0.1 r 3.55 Â±0.21 3.7 2.1 DFMO (2%) Â¡ndrinking water 0.69 Â±0.11 43 1.21 Â±0.13 3.5 2.1 Vindesine (0.2 mg/kg i.p.) on 0.77 Â±0.14 31 1.14 Â±0.24 4.0 2.3 Days 7 and 14 DFMO (2%) from Day 8 + 0.50 Â±0.07 56 0.20 Â±0.02 3.9 2.0 vindesine (0.2 mg/kg) on Days 7 and 14 Adriamycin (2.5 mg/kg i.p.) on 0.77 Â±0.10 31 2.96 Â±0.31 3.5 1.0 Days 7 and 14 DFMO (2%) from Day 8 + 0.45 Â±0.06 60 1.25 Â±0.18 4.3 1.0 Adriamycin (2.5 mg/kg) on Days 7 and 14 None; control without EMT6 4.0 1.9 tumor (normal animal) ' Mean Â±S.E. of 15 animals.

5160 CANCER RESEARCH VOL. 41

time and in a 30% cure rate. The curative action could not have ODC was observed in EMT6 tumors from mice receiving vin been predicted from the effects of either drug given alone. The desine together with DFMO (Table 2). It has been observed hepatoma tumor model in Buffalo rats was particularly suitable that depletion of polyamine levels in several transformed cells for evaluating combination therapy since the effects of cyto- resulted in the arrest of the cells in the S phase (14). Sunkara static drugs could be monitored for 2 months. DFMO alone ef al. (14) proposed that sequential administration of DFMO markedly slowed the growth of this tumor. Combination of and of an S-phase-specific drug should result in a synergistic DFMO with vindesine or Adriamycin resulted essentially in antiproliferative effect. DFMO did indeed potentiate the cyto complete arrest of tumor growth. Tumor polyamines and ODC toxic effects of cytosine arabinoside on HeLa cells, and this activity were not measured at the end of this experiment since, was prevented by addition of putrescine to the medium (14). in the HTC model, ODC activity is induced only between Days In conclusion, the use of DFMO could prove useful in pre 5 and 12 following inoculation. venting tumor regrowth in the interval between courses of The antitumoral action of DFMO confirmed in the present cytotoxic chemotherapy, immunotherapy, radiotherapy, or study is probably due to inhibition of ODC (5,9,11,12). after surgical excision of the tumor. Moreover, combination of Increases in ODC activity and polyamine synthesis are invari DFMO with certain cytotoxic drugs may be more effective than ably associated with rapid cell proliferation and a high rate of single-drug therapy without increasing toxicity. Much additional tumor growth (8, 15). DFMO is a potent and selective inhibitor work will be required to delineate the optimal combinations and of ODC and decrease tumoral putrescine and spermidine con treatment schedules for specific human tumors. centrations (11,12). Both the decrease in tumoral polyamine concentration and the inhibition of tumor growth by DFMO ACKNOWLEDGMENTS were reversed by treatment with an inhibitor of both S-adeno- syl-L-methionine decarboxylase and diamine oxidase, 1,1'- The authors thank Dr. P. Mamont for culture of HTC and EMT6 cells, Drs. J. Fozard and N. Seiler for their helpful comments, and M. Nagy and M. Nussli for [(methylethanediylidene)dinitrilo]bis(3-aminoguanidine) (12). technical assistance. All this suggests that DFMO influences tumor growth by deple tion of intracellular putrescine and spermidine. REFERENCES We can only speculate how DFMO interacts with antitumoral 1. Avery, T. L.. and Roberts. D. W. Adriamycin and cyclophosphamide in drugs. Antitumoral synergism has been observed when DFMO combination of L1210 leukemia. Cancer Res., 37: 678-683. 1977. was used in combination with cytotoxic drugs of different 2. Barnett. C. J., Cullinan, G. J., Gerson, K . Hoying, R. C , Jones. W. E., mechanisms of action: alkylating agents [BCNU; (7)], cyclo- Newlon, W. M., Poore, G. A., Robison, R. L.. Sweeney, M. J.. and Tood, G. C. Structure-activity relationships of dimeric catharanthus alkaloids. 1. De- phosphamide (12), intercalating agents or free radical-gener acetylvinblastine amide (vindesine) sulfate. J. Mod Chem., 21: 88-96, ating agents (Adriamycin), agents interacting with microtubules 1979. 3. Bartholeyns, J., and Zenebergh, A. in vitro and in vivo antitumor effect of (vindesine), or even retinoids (5). We have confirmed the ad dimerized ribonuclease A. Eur. J. Cancer, 75; 85-91, 1979. ditive antitumoral effects of DFMO and MGBG on L1210 leu 4. Burchenal, J., Lokys, L., Smith, R., Cartmell, S., and Warrell, R. Potentiation kemia described by Burchenal ef a/. (4), but by using 2% of methylglyoxal-bis-guanylhydrazone by o-difluoromethylornithine, stilbam- idine and pentamidine in mouse leukemia. Proc. Am. Assoc. Cancer Res., DFMO in water and MGBG (20 mg/kg) twice weekly; additive 22. 230, 1981. antitumoral effects were also observed for the HTC hepatoma 5. Chapman, S. Antitumor effect of vitamin A and inhibitors of ornithine decar in rats, while antagonism of MGBG and DFMO occurred in boxylase in cultured neuroblastoma and glioma cells. Life Sci., 26: 1359- murine EMT6 tumors.2 It seems likely that at least part of the 1366. 1980. 6. Hershko, A., and Tomkins, G. M. Studies on the Degradation of Tyrosine effectiveness of DFMO in combination therapy results from a amino-transferase in Hepatoma cells in culture. J. Biol. Chem.. 246: 710- relatively general mechanism rather than from a specific inter 714, 1971. 7. Hung, D. T., Deen, D. F., Seidenfeld, J., and Marlon, L. J. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1981 American Association for Cancer Research. Effects of α-Difluoromethylornithine Alone and Combined with Adriamycin or Vindesine on L1210 Leukemia in Mice, EMT6 Solid Tumors in Mice, and Solid Tumors Induced by Injection of Hepatoma Tissue Culture Cells in Rats

Jacques Bartholeyns and Jan Koch-Weser

Cancer Res 1981;41:5158-5161.

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