Efficacy of 5-FU Combined to Na[trans-RuCl4(DMSO)Im], A Novel Selective Antimetastatic Agent, on the Survival Time of Mice With P388 Leukemia, P388/DDP subline and MCa Mammary Carcinoma

The combinational treatment between the selective antimetastatic agent, sodium-trans-rutheniumtetrachloridedimethylsulfoxideimidazole, Na[trans-RuCl4(DMSO)Im], and the cytotoxic drug 5-fluorouracil (5-FU) on primary tumor growth and on the survival time of experimental tumors results in an effect significantly greater than that of each single agent used alone either with the solid metastasizing MCa mammary carcinoma of the CBA mouse or with the lymphocytic leukemia P388 and its platinum resistant P388/DDP subline. Thus the inorganic compound Na[trans-RuCl4(DMSO)Im], known for its potent and selective antimetastatic effects, positively interacts with the antitumor action of an organic anticancer agent such as 5-FU on both a solid metastasizing tumor and a tumor of lymphoproliferative type. In particular, the effects of the combinational treatment on the survival time of tumor bearing mice seem to be related to the selective antimetastatic activity of the ruthenium complex that joins the potent cytotoxicity of 5-FU for the tumor. Moreover, these data show that Na[trans-RuCl4(DMSO)Im] is almost as effective on the subline of P388 made resistant to cisplatin as it was on the parental line.


INTRODUCTION
On the wave of enthusiasm for the positive results obtained with platinum complexes , the search for novel anticancer agents has brought to the characterization of new compounds based on complexes of transition metals. Of these, ruthenium complexes attracted the interest of many groups because of some promising chemical characteristic and pharmacologic activity in different models of experimental tumors2-4. One of the last compounds that have been discovered is certainly Na[trans-RuCI4(DMSO)Im], a drug of the future endowed with a particular effectiveness against solid tumor metastasesS, 6. Considering that anticancer chemotherapy is practically always done by drugs active on primitive tumors, we thought it worthwhile testing the effects of a combination with the selective antimetastatic agent Na[trans-RuCI4(DMSO)lm] and a cytotoxic drug of a large use such as 5fluorouracil (5-FU). The purpose of the study was that of measuring the existence of a combination of effects on the primary and on the metastatic tumor, with a significant amelioration of life-time expectancy. The study was made either on a solid metastasizing tumor, the MCa mammary carcinoma of the CBA mouse 7 or with a tumor of lymphoproliferative type, the P388 lymphocytic leukemia and its platinum resistant P388/DDP subline.
.A..ni..ma!s. CBA female mice of a locally established breeding colony, originally obtained from Chester Beatty Institute of London, UK, were used together with BD2F1 female mice purchased from Charles River, Calco, Como, Italy. All animals were of 22+1 gr body weight and bout 5 to 6 weeks old. Animal studies were carried out according to the guidelines currently in force in Italy (DL 116, 27/1/1992) and in compliance to the 'Guide for the care and use of laboratory animals' DHHS Publ. No (NIH)86-23. Bethesda, MD: NIH, 1985.
Tumors. MCa mammary carcinoma was transplanted i.m. into the calf of the left hind leg of CBA mice (106 viable tumor cells per mouse). The single cell preparation was performed according to standard procedures, starting from tumor masses obtained by donors with 2-week old tumorss.

RESULTS
Effects on Mc.a. mammary carcinoma. In female CBA mice Na[trans-RuCI4(DMSO)lm] was given i.v. at 60 mg/Kg/day on days 1,5,9,13 from i.m. implantation of 106 cells of MCa mammary carcinoma; treatment with 5-FU was made i.p. on the same days and at two dose levels of 30 and 50 mg/kg/day. The dose used for Na[trans-RuCI4(DMSO)lm] is one of those already shown active on survival time and on metastasis formation in mice with solid metastasizing tumors including MCa mammary carcinoma8, whereas those chosen for 5-FU represent the doses that resulted more effective in preliminary studies of tumor inhibition. On primary tumor (Figure 1)    Effects on P388 leukemia. The effects of in vivo treatment with 5-FU alone (treatment with 10 or 20 mg/kg/day) on P388 leukemia and on its cisplatin resistant subline was generally more effective than that with Na[trans-RuCI4(DMSO)lm at the same dosages and treatment schedule ( Figure 3 and Figure 4) (p<0.05).
The combined and simultaneous treatment with 5-FU and Na[trans-RuCI4(DMSO)lm showed a prolongation of the life span of the treated animals greater than that observed with each single agent (p<0.05) and in some cases greater than the sum of the singular effects of each single agent (20 mg/kg/day 5-FU plus 20 mg/kg/day Na[trans-RuCI4(DMSO)lm on the P388 parental line). The experimental condition used was identical to that reported in Figure 3.

DISCUSSION
These data show that the inorganic compound Na[trans-RuCI4(DMSO)lm can combine its antitumor effects to those of an organic anticancer agent such as 5-FU on both a solid metastasizing tumor and a tumor of lymphoproliferative type. The choice of 5-FU for this test was based on the proposed role of ruthenium(Ill) complexes for treating colo-rectal tumors 2,e where 5-FU is the drug most often employed. Anyhow, it must also be emphasized that the aim of this paper was simply that of giving evidence that the emerging antimetastatic drug Na[trans-RuCI4(DMSO)Im], in combinational treatments, does not reduce the antitumor action of a drug of common clinical use and that with some combinations it might also increase the overall response of the tumor being treated. In fact, it is interesting to note that, when the effect of the combined treatment is lower that that expected from the sum of the effects of each individual drug, on the solid metastasizing tumor it is at least as great as that of Na[trans-RuCI4(DMSO)lm and on the P388 tumors it is equivalent to that of 5-FU; in both cases with no modification of host toxicity, expressed as % loss of body weight gain vs untreated controls. Thus Na[trans-RuCI4(DMSO)lm] has no negative effects on the antitumor activity of 5-FU and to some extent, it potentiates the effects of 5-FU. The effects of the combinational treatment on the survival time of mice with MCa mammary carcinoma should be attributed to the antimetastatic activity of the ruthenium complex. The reduction of metastases by the ruthenium complex, besides the selective conditions in which it has often been tested,o, is thus therapeutically useful even in the present experimental model, a paradigm that does not emphasize the role of distant metastases for survival in that the presence