Pharmacological Effects of the Ruthenium Complex NAMI-A Given Orally to CBA Mice With MCa Mammary Carcinoma

NAMI-A, imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate, is a ruthenium based compounds capable of inhibiting the growth of lung metastases of solid tumours in a number of experimental conditions.The aim of this study was to investigate the potential use of NAMI-A by the oral route to treat lung metastases of MCa mammary carcinoma in the CBA mouse. treatment of mice, carrying intramuscular tumours in advanced stage of growth, for 11 consecutive days caused a significant reduction of the weight of lung metastases over the range of doses from 150 to 600 mg/kg/day. No sign of toxicity was observed at the histological analysis in the gut epithelium or in the kidney parenchyma, and NAMI-A concentration in the kidney was more than 10-fold lower than after intraperitoneal treatments. NAMI-A is thus active against metastases also by the oral route, suggesting the use of this way to treat tumour bearing hosts for long periods.


INTRODUCTION
Metastases of solid tumours represent the most-important target for therapy in that they are the most frequent responsible for the failure of cancer therapy. Metastases are always scattered, often in more than one vital organ, and ate not erasable by conventional surgery and/or radiotherapy. On the other hand, cytotoxic cancer chemotherapy fails against solid tumour metastases in that, among all, metastases differ from the primary tumours of origin because of poor sensitivi to conventional drugs [1][2][3]. Drugs active against metastases are therefore strongly desired. These drugs should show the capacity of inhibitin metastasis growth (i.e. the growth of metastases already formed), besides metastass formation (i.e. the steps that bring turnout cells to form distant metastases) [4]. It is thus important to get the so-called antimetastasis agents rather than the mostly known antimetastatc drugs, provided that in human beings the most probable drug treatment is for already established metastases. In recent years, we accumulated a strong evidence for the effects of some ruthenium complexes on solid tumour metastases in mice bearing experimental tumours [5][6][7]. Of these complexes, the one called NAMI-A (imidazolium trans-imidazoletedimethylsulfoxidetrachlororuthenate) showed adherence to the above characteristics: it is active aainst spontaneous metastases of solid turnouts also when drug treatment occurs in mice wtth advanced turnouts, i.e. at a time at which metastases are already settled in the lungs [5,8]. Moreover, NAMI-A appears to control metastasis growth by a mechanism unrelated to direct cell cytotoxicity, typical of conventional chemotherapy agents, still showing effects similar or even superior to those of a drug of wide clinical use such as cisplatin [7,[9][10][11]. More recently, NAMI-A, though undergoing a series of molecular metabolisms in aqueous solutions, was shown capable of maintaining its antimetastatic activity in a broad range of experimental conditions, thus suggesting the possibility of handling its clinical management in a rather flexible way [12].
The aim of the present investigation was therefore that of examining the effects of NAMI-A given by the oral route to mice bearing the solid metastasising tumour MCa mammary carcinoma. The efficacy of NAMI-A by this route of administration should facilitate the treatment of tumour bearing hosts to cover a relatively long period of time, as probably required for the metastatic pathology. In fact, provided that NAMI-A has repeatedly shown efficacy against metastases wih a mechanism involving the alteration of the cancer cells with host cells/tissues [13], the reduction of metastases fill their complete removal might require a long exposure of the tumour cells to the drug. In this respect, the oral route may meet this requirement, particularly if a better compliance is also proved by the host. This study therefore examines the effects of NAMI-A on lung metastasis formation in the light of the effects of the complex on primary tumour, on gut epithelium and kidney and on i:uthenium uptake by these tissues.

MATERIALS AND METHODS
Compounds and treatment. NAMI-A was 13rep.ared according to a patented procedure (Mestroni et al., 1998). The drug was adminisfer&l orally admixed throug.hout the powered food. Animal treatment waspeforrned on days 10-21 after ttunour implantation. All the animals were pre-copditio.nedto p.owdered food for 2-weeks before the e.xperiment. Drug concentration was selected to provide 100, 150, 200, 300 and 600 mg/kg/die on the basis o-f an a.yerage daily food consumption of 5.00.1. g per mo.use, .terr.ine.d in the _pr.econditiomng period. Food consumption remained constant throughout the duration otthe experiment n either Controls or in lhe group receiving NAMI-A. Tumour line. MCa mamma, carcinoma cells (grown in CBA mice) were used for in vivo testing. CBA .mice were olbtained from a locally established breeding colony grown according to the standard procedures for inbred s(rains. The tumour lzrat p.rocedure for MCa mammary carcinoma were the same as those already described in letai[ [14]. Briefl.y, tumour cells of a single cell suspension were prep.arec[ from mincing with scissors thf primary turnout masses obtained from. donors similarly, imolanted 2-weeks before. t.u.mour..growth and lung m_etastasis, evalu.ation. Pr'.ma.ary turnout gr.owth was etermined by callper measureme,s of two orthogonal axes andthe tumour volume w.as calculate.d by the f6rmula: (:r/.6)xa xb, wherein a is fhe_shorter and b is the longer axis; the tumq, density, was .assumed to_b.e equal to one.. Lu0. g .meta.st.a.ses w_er.e counte.d .by caretully examining the surface or the lungs, immediately alter killing or the animals by.
cervicaldislocation. Lungs were dissected into the five lobes, washed with PBS an;:l ex .ained under a low 19ower microscope ecluipped with a .calibrated grid. The weight o.f e.acla metastasis.was .c.arqula.ted by. applying.the sate. e foma. ula_as, t0r primary tumours and the sum or each individual weight gave the total weight or the metastatic tumour per animal.
Histololieal analysis. Sections for light microscopy were prepared from paraffin embedd'6d kidneys and gut (from ileum) sections, which were removed, washed in water and. fixed in 10% formal.ine and processed according to the standard procedure for inclusion and followi.ng rehydration (xylene, alcohol, vater), with sections cut at 6 tm. tsections were_stained ith Cajal-Gallego mounted in Canada Balsam and were observed with a Leitz-Orthoplan microscop.e. Examinations were made on three different slides, each containing three slices for each sample. AAS of rutlienium. Ruthenium was measured in trip.licate by atomic absorption spectroscopy, using a Varian SpectrAA-300 instrumental.ion supolied with a graphite Furnace mol GT/(-96, an autosa,pler mod PSD-96, and a sp.ecffic ruthenium emission lamp (Hollow cathod 1_agap Varian P/N 56-101447-00). Rdthenium was measured in samples of 1Q 1_at 349.9 m with an ato_misingtemperature of 2,500C, .u.s.ing argon as purge gas at the flow rate of 3.0 1/min. Before dmly. analysis, a five point calitration curve _w_as performed by Ruthenium Custom-Grade Stanlard 998 txg/ml in 3.3% HC1 (Inorganic _Ventures Inc., Lakewood, NJ, USA). Statistical analy_sis. Experimental raw data were submitted to computer-assisted statistical analysis using ANOVA analysis of variance and Tukey-Kramer post-test.

RESULTS AND DISCUSSION
The effects of NAMI-A, given orally to mice bearing MCa mamm carcinoma in advanced stage of growth, on lung metastasis formation are shown in Figure 1. On 0 metastasis number, NAMI-A caused the reduction to about 55-60 A of untreated controls at any dose tested from 150 to 600 mg/kg/day; the submission of these results to the one-way analysis of variance showed a signi-ficant reduction at the doses of 150 and 600 mg/kg/day, indicating that the rather wide variation observed in the treated groups rediced the statistical significance at the other doses. Conversely, on metastasis weight NAMI-A resulted active with any dose used from 150 mg/kg/d.ay onward (Figure 1), and the reduction was around 40% of untreated controls. For comparison, the i.p. treatment at different doses and schedules showed a similar trend (i.e. marked inhibition of metastasis weight). However, the extent of metastasis reduction was generally superior being around 10-20% of untreated controls [5,8].
The effects on pdm_azy tumour growth (similarly to the i.p. treatment and independently of the dose-level used) was much less evident and statistically not significant (data not reported). Similarly, none of the doses used for the oral treatment caused host toxicity, as determined by the variation of body weight gain between the start and the end of treatment in comparison to untreated controls.  The measurement of ruthenium in the kidney of mice treated orally with NAMI-A shows that the concentration of the metal (referred as NAMI-A) increases proportionally to the daily dose used (Figure 3). Indeed, the amount ofNAMI-A found in the kidney of these animals was markedly lower than after i.p. administration. By comparison we may report the data obtained with the dose of 35 mg&g/day given daily for 6 consecutive days. In this case, the amount of NAMI-A found in the kidney was approximately 10 to 20-f01d greater than that reported in Figure 3, at the higher dose of 600 mg/kg/day for 11 consecutive days.
The kidneys of mice of Figure 1 were processed for flameless atomic absorption spectroscopy analysis of ruthenium content. Each value is the mean+S.E, obtained from at least three separate samples obtained from three different mice per group. The oral treatment with NAMI-A induces a marked modification of the appearance of tumour parenchyma, particularly at the external border which appears more regular at the interface with the surrounding tissue and with an increased density of connective tissue.
Apparently, mmour cells do not appear to be modified by the treatment with NAMI-A and their density is similar to that of the control samples. At a greater magnification it is possible to appreciate better the changes at the border of the primary mmour mass on Which severalinflammatory cells are distinguishable. These alteration are perfectly similar to those observed following i.p. treatment of this turnout [5]. A detailed examination of the cells of Figure 5 shows the presence of infiltrating leukocytes, particularly PMNs and lymphocytes (shown by arrows), already evidenced following the i.p. treatment in primary tumour masses of both i.m. and s.c. growing tumours [13]. The examination of gut epithelium and kidney parenchyma, of mice treated orally with NAMI-A, by histological staining and light microscopy analysis, showed no appreciable alteration of the architecture of the tissues examined ( Figure 6). Gut epithelium was regular and the only change consisted of an increased density of connective tissue on the basal membrane over untreated controls. Kidney tubules and glomeruli appear regular with cells well defined and no presence of cell suffering, dilation of tubules or swelling of the extracellular matrix. Figure 6. Histological appearance of kidney parenchyma (top) and gut epithelium (bottom) of control mice (left) andof mice treated Wiih NAMI-A (right). Slices were prepared from mice of the experiments of Figure 1 and represent a picture present independently of the dose of NAMi-A used and ofthe experimem chosen. The evaluation of the histological changes induced by NAMI-A was made in a single blind experiment with at least three different glasses, each mounted with three independent slices, per animal. Arrows highlight the increased thickness of collagen fibres under basal membrane.

CONCLUSION
Among the most important promising ruthenium complexes appeared in the literature [15][16], those with sulf0xide ligands still continue to show the better pharmacological properties against solid tumour metastases.
NAMI-A proved to be active against solid tumour metastases also when given by the oral route over a 10 day period to mice with advanced MCa mammary carcinoma. Although the overall effect-is slightly less pronounced than that obtained following i.p. treatment, it must be stressed that by this route of administration the kidney concentration of the compound is markedly lower and no histological toxicity is evidenced. The lack of toxicity at the gut level and on kidneys suggest the possibility to use NAMI-A by this route also for periods longer than that usedin tfi6 present study. Provided that to keep metasases under full control by non cytotoxic drugs such as NAMI-A, they should be treated for long periods, these data suggest the feasibility of this treatment and its effectiveness.