[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, Orléans, 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 (21-23°), 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(guanyl- hydrazone); 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 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1981 American Association for Cancer Research. Combination Therapy with DFMO, Vindesine, Adriamycin 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.