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The content of ruthenium in blood and different organs of healthy CBA mice was determined by AAS after single i.v. treatment of 200 mg kg-1 of NAMI-A, a new antimetastatic ruthenium compound. Ruthenium concentration in blood falls 5 min after i.v. treatment. In the kidney, ruthenium concentration is markedly higher than in any other analysed tissue. No ruthenium was detected in brains. Pharmacokinetic parameters for a mono- or a bi-compartment model are identifiable: t1/2 is 10.45 h vs 12.02 (t1/2 alpha 0.023 h + t1/2 beta 12 h) with Cltot of 1.60 ml*h-1 vs 1.59); Vd is 24.15 vs 27.48 ml and (model dependent) AUC is 689 vs 694 mg*L-1*h. AUC(0--infinity) calculated by noncompartmental method (linear trapezoidal rule) is 719.77 mg*L-1*h. NAMI-A is rapidly cleared from the blood compartment immediately after i.v. administration. Apparently, there is no differential accumulation of ruthenium in the lungs which might account for a selective antimetastatic effect caused by a cytotoxic concentration in this site, nor in any other specific organ examined.

A group of 4 organometallic complexes of Rhodium (I) and 1 Iridium(I) was tested for the evaluation of their anti-inflammatory activity on carrageenin paw edema in rats. All the compounds used inhibited the development of paw edema by more than 50% at different dose-levels. The activity of the pyridinalmethylimine derivative [Rh(COD)PMI]+ Cl- had better results than that of [Rh(NBD)PMI]+ Cl- and even more than the dimeric complexes tested. The higher activity of [Ir(COD)Cl]2 as compared with [Rh(COD)Cl]2 suggests that it would be of interest to examine further Iridium(I) complexes, among which [Ir(COD)PMI]+ Cl- could be a good candidate.

The antitumor effects of three rhodium(I) complexes were evaluated using two transplantable tumors of the mouse: Lewis lung carcinoma and P388 lymphocytic leukemia. The examination of the differential effects on primary tumor growth and on the formation of spontaneous pulmonary metastases, in mice bearing Lewis lung carcinoma, indicated that the complex having qualitatively the higher solubility in aqueous solutions and the higher resistance to inactivation via oxidation, displayed the more pronounced antineoplastic activity. The antileukemic effects, in mice bearing P388 lymphocytic leukemia, also seemed to depend on the chemical characteristics of the complex used, and the previously reported trend was particularly evident using an acute treatment performed 24 hr after tumor transplantation.

The antitumor action of two square planar Rhodium complexes was tested on MCa mammary carcinoma and was seen to depend upon the compound used. The complex cyclooctadiene(2-pyridinalmethylimine)-Rh(I)chloride [Rh(COD)PMI]+ Cl- confirmed the antineoplastic action already shown in the Lewis lung carcinoma model. The examination of the activity of [Rh(COD)PMI]+ Cl- on the lung metastatic tumor indicates that its antineoplastic properties do not seem simply related to a cytotoxic action. It appears more likely that modifications occurring at the primary tumor level, probably different from lethal effects directed to tumor cells, are responsible for the reduction of spontaneous lung metastasis formation observed in the treated animals. The trophic effects observed on the spleen of the treated animals seem to suggest that these compounds are endowed with properties typical of biological response modifiers.

The effects of square planar rhodium, [RhacacCOD]o and iridium, [IracacCOD]o complexes and of octahedral ruthenium, [cis-RuCl2 (DMSO)4]o complex have been examined in comparison with cis-dichlorodiammino platinum(II) (cis-PDD). The toxicity in BDF1 mice varies widely and decreasing LD50-values, ranging from 0.94 mg/kg to 1000 mg/kg, have been obtained for cis-PDD, [RhacacCOD]o, [IracacCOD]o and [cis-RuCl2(DMSO)4]o, respectively. All the tested complexes similarly inhibit the growth of subcutaneous Lewis lung carcinoma and the development of spontaneous as well as of artificial metastases, with the exception of [IracacCOD]o which is inactive on metastases. The antitumor activity of [RhacacCOD]o and [cis-RuCl2(DMSO)4]o appears interesting, since it is of the same magnitude as that of cis-PDD, considering also that they were found to be only marginally nephrotoxic.

The effects of NAMI-A, a novel ruthenium compound endowed with selective antimetastatic action, were tested on solid metastasising tumours of the mouse in comparison to cisplatin, cyclophosphamide and dacarbazine. Each compound was administered i.p. as freshly prepared solutions in isotonic saline in combination with surgical removal of primary tumour, and was used at the maximum tolerated dose. NAMI-A significantly reduced the growth of lung metastases either when given prior to surgery (early growing tumours) on TS/A adenocarcinoma or after surgical ablation of primary tumours (already established lung metasases) on Lewis lung carcinoma. The postsurgical treatment of mice bearing MCa mammary carcinoma caused a significant prolongation of the life-span of the treated animals. In the comparison experiments, dacarbazine was completely ineffective, cisplatin was as active as NAMI-A on MCa mammary carcinoma, slightly less active than NAMI-A on TS/A adenocarcinoma and inactive on Lewis lung carcinoma, and cyclophosphamide was always more active than any other treatment performed. These data stress that NAMI-A, independently of the lack of direct cell cytotoxicity, when compared to the reference drugs, has a potent therapeutic effect in mice bearing solid metastasising tumours.

The anti-metastatic ruthenium complex NaCtransRuCI4(DMSO)lm] was given i.p. at 22 and 44 mg/kg/day, on days 8-13 after tumour implantation, to mice carrying S.C. implants of MCa mammary carcinoma. The aim ofthe study was to compare the effects on lung metastasis formation with those on primary tumour cells. This investigation was based on flow cytometry analysis after propidium iodide and acridine orange staining, histology of tumour parenchyma and RT-PCR analysis for the type-IV collagenases MMP-9 and MMP-2 and their respective inhibitors TIMP-I and TIMP-2 mRNAs. NactransRuCl,(DMSO)lm] is not cytotoxic for tumour cells but has the capacity of interacting with nucleic acids, giving a general reduction of nucleic acid content as shown by a marked reduction of acridine orange staining and a tendency to a reduction of DNA polyploidy with marked reduction of 8n and 4n cell populations. Na[trans-RuCI~(DMSO)lm] also influences a proteolytic system which has the potential of degrading the basement membrane and has been related to metastatic aggressiveness: it markedly reduces, in a dose-dependent manner, MMP-2/TIMP-2 balance, but not that of MMP-9/TIMP-I. The different enzyrne/inhibitor mRNA levels between untreated and treated tumours seem to be unaffected by tumourinfiltrating lymphocytes and are paralleled by the maintenance of connective tissue around blood vessels in the tumour mass. Correspondingly, lung metastasis formation is markedly reduced, to less than 10% of that seen in conbols. o 1996 Wiley-Liss, Inc. Basic research on synthetic drugs, effective against tumour metastases, has recently highlighted the effects of a ruthenium complex, namely sodium trans-rutheniumtetrachloridedimethylsulphoxideimidazole (hereafter indicated by Na[transRuCI4(DMSO)Im]) (Sava er al., 1992a, b, 1993, 1994). The effects of this new generation ruthenium(II1) complex on solid metastasizing tumour are particularly evident on the formation of spontaneous metastases. The selectivity of Na[transRuCI4(DMSO)Im] on lung metastases is also marked on advanced metastases and accounts for a significant prolongation of the host's survival time; combined with surgical removal of primary tumour, Na[trans-RuC14(DMSO)Im] prevents the formation of metastases and inhibits the growth of those already formed (Sava et al., 1994). The histological analysis of tumour growth and of healthy host tissues such as lung and kidney epithelia, muscle and liver cells, splenocytes and bone-marrow cells, by light microscopy and by SEM, shows a lack of significant cytotoxicity (Gagliardi et al., 1994). It thus appears that the selective anti-metastatic effects do not result directly from histological modification of the primary tumour structure.

The influence of chemical stability on the antimetastatic ruthenium(III) compound imidazolium trans-imidazoletetrachlorodimethylsulphoxideruthenium(III) (NAMI-A) in aqueous solution was studied both in vitro and in vivo. The loss of dimethyl-sulphoxide (DMSO) ligand from the compound was tested by using a NAMI-A solution acidified with HCl at pH 3.0 and aged for 0, 4, 8 and 24 h prior to intraperitoneal (i.p.) injection into CBA mice bearing advanced MCa mammary carcinoma. The activity of NAMI-A on lung metastases showed no change even after the loss of DMSO ligand from up to 50% of the molecules. The reduction of NAMI-A did not modify the number of KB cells blocked in the S+G2M phases, independent of whether the reduction occurred outside the cells or after loading the cells with the compound prior to treatment with the reductants (ascorbic acid, glutathione or cysteine). In vivo, the complete reduction of NAMI-A with equivalent amounts of ascorbic acid, glutathione or cysteine prior to administration to mice bearing advanced MCa mammary carcinoma was more active than NAMI-A alone. The data show that NAMI-A, although undergoing a series of chemical modifications, maintains its antimetastatic activity in a broad range of experimental conditions.

The effects of the new molecule ImH[trans-RuCl4(DMSO)Im] (NAMI-A), administered orally or intraperitoneally to adjuvant-arthritic rats or orally to mice bearing s.c. or i.m. implants of MCa mammary carcinoma, were studied. NAMI-A was not able to modify the progression of chronic inflammation in the complete Freund-adjuvant injected animals. Histology indicated a significant worsening of the inflammatory process, characterised by an increased infiltration of inflammatory cells, as well as by a remarkable deposition of connective tissue fibres around the blood vessels and alveolar walls. NAMI-A had no effect on primary i.m. implanted MCa mammary carcinoma growth and its lung metastasis formation, but significantly interfered with the cell cycle of primary tumor cells following bolus oral administration. On the contrary, NAMI-A caused a significant inhibition of lung metastasis accompanied by a dramatic deposition of connective tissue fibres around the primary tumor mass, when given as medicated food to mice implanted s.c. with MCa tumor. These data indicated that NAMI-A is well absorbed after oral administration although there is no connection between lung concentration and the antimetastatic activity. Conversely, the marked deposition of connective tissues in NAMI-A treated animals is in agreement with the reported effects of the compound on extracellular matrix and tumor blood vessels.

A series of 18 ruthenium(III) complexes, structurally related to the selective antimetastatic drug Na[trans-RuCl4(DMSO)Im], and characterized by the presence of sulfoxide and nitrogen-donor ligands were tested on TLX5 lymphoma and some of them on MCa mammary carcinoma to evaluate the dependence of the degree of cytotoxicity and of antimetastatic activity on the chemical properties. In vitro cytotoxicity is present only at high concentrations (> 10(-4) M), depends upon lipophilicity and is markedly affected by the presence of 5% serum or plasma samples in the culture medium. The comparison of the effects on in vitro cytotoxicity with in vivo antitumor and antimetastatic action points out that these compounds reduce metastasis formation by a mechanism unrelated to a direct tumor cell cytotoxicity. If on one hand Na[trans-RuCl4(TMSO)Iq], the compound that shows the most potent in vitro cytotoxic effects, is the least effective against metastases, on the other Na[trans-RuCl4(DMSO)Im], the compound that better reduces metastasis formation, is rather devoid of cytotoxic effects on tumor cells kept in vitro. In particular, Na[trans-RuCl4(DMSO)Im] seems to distinguish between artificially induced metastases and spontaneous metastases and reduces only the former by a cytotoxic mechanism. Out of all the tested compounds, with the exception of Na[trans-RuCl4(DMSO)Ox], Na[trans-RuCl4(DMSO)Im] is confirmed to be the most selective antimetastatic agent of the group.

The antitumor and antimetastatic effects of a ruthenium(III) complex, Na[trans-RuCl4(DMSO)Im], have been examined in mice bearing MCa mammary carcinoma. Na[trans-RuCl4(DMSO)Im] is capable of reducing primary tumor growth and of prolonging the survival time with different schedules of administration. However, better effects were obtained (a) with treatments started soon after tumor implantation, (b) with daily administration rather than with treatments at 4-day intervals and (c) using relatively low daily doses. The prolongation of survival time in tumor-bearing hosts, greater than that obtained with cisplatin, cannot be simply related to the effect on primary tumor growth, always less than that of cisplatin. Rather, emphasis is placed on the reduction of lung metastasis formation, obtained either by i.v. injection of tumor cells (lung colonization or spontaneously from i.m. tumor implants, which is significantly greater than the reduction of primary tumor growth. The antimetastatic effect depends on the treatment schedule and is greater with low doses given daily than with high doses administrated with drug-free intervals. Metastasis reduction involved mainly large metastatic nodules, reducing the average weight of each metastasis by 8-fold for spontaneous metastases and by 5-fold for lung colonies. The antimetastatic action of Na[trans-RuCl4(DMSO)Im] thus indicates the possibility that related analogs can represent a new generation of antitumor compounds capable of selectively interacting with metastasis formation of solid tumors.

Octahedral ruthenium(III) and ruthenium(II) complexes show antineoplastic properties on a number of experimental tumors. Tetraammine-, pentaammine-, heterocycle-, and dimethylsulfoxide-coordinated ruthenium complexes have shown high affinity for nitrogen donor ligands in vitro and as a result exhibit various degrees of biological activities including antitumor action in vivo. The chemical behavior of ruthenium(III) complexes indicates the possibility of opening a window of selective toxicity, in practice lacking in the chemotherapeutic approach to neoplastic diseases. Ruthenium ions may accumulate in tumor tissues via a mechanism mediated by transferrin transport. Moreover, binding of ruthenium to DNA is several times higher in its reduced ruthenium(II) form and the reduction from ruthenium(III) prodrugs to the more toxic ruthenium(II) compounds is particularly efficient in tumor hypoxic environments. Correspondingly, solid tumors appear to be more susceptible than those of the lymphoproliferative type. In particular, tumors of the colorectal region and lung tumors (primary or metastatic), which are generally associated with a bad prognosis, have given interesting responses in experimental models, indicating these tumors as preferential targets for the development of ruthenium anticancer drugs.

Several rhodium(I) and iridium(I) complexes displayed different degrees of antitumour activity when tested in mice bearing Ehrlich ascites carcinoma. Rhodium (I) and iridium (I) acetylacetonate derivatives caused a high percentage of cures. The rhodium (I) dimers were particularly interesting, since (bis(cycloocta-1,5-diene)micromicron' dichlorodirhodium(I) [RhCODCl]2) was highly effective, whereas its analogues, bis(bicyclo[2,2,1]hepta-2,5-diene)micromicron'-dichlorodirhodium(I) [RhNBDCl]2) and bis(1,5-hexadiene)micromicron' dichlorodirhodium(I) [RhEDCl]2) were virtually inactive. The absence of significant inhibition of DNA, RNA and protein syntheses in tumour cells found for [RhCODCl]2 at therapeutically active dosages, indicates that this substance has a different mechanism of action from that of cis-dichlorodiammine Pt(II) (cis-PDD). The amount of rhodium found in tumour cells after administration of active [RhCODCl]2 was higher than that for [RhEDCl]2, while the rhodium concentration in the ascitic fluid was much higher for [RhEDCl]2. A mechanism based on the chemical properties of the complexes is tentatively proposed for explaining these findings and selective toxicity for tumour cells.

The antitumour effects of some ruthenium(II) complexes were tested in mice bearing the solid metastasizing tumour, Lewis lung carcinoma. The toxicity of trans-RuCl2(DMSO)4 is 10-fold higher than that of its (itcis)-isomer. Qualitatively the antitumour activity of these two complexes is comparable, although trans-isomer is more potent. Both exhibit only marginal effects on primary tumour growth while significantly lowering lung metastasis formation. The effects of trans-RuCl2(DMSO)4 on primary tumour depend on the inoculum size, being more pronounced with low tumour inocula; significant antitumour effects can be achieved also by replacing Cl with I in the molecule. trans-RuCl2(DMSO)4 markedly reduces the number of lung metastases and particularly their volume; the reduction is comparable to that obtained with equitoxic treatments of cisplatin. Treatment with trans-RuCl2(DMSO)4 for 10 consecutive days, after surgical amputation of primary tumour, significantly prolongs the survival time of the treated mice; cisplatin, at equitoxic doses, is less effective. These data show that dimethylsulphoxide ruthenium(II) complexes possess a significant antitumour and antimetastatic activity in the Lewis lung system, also exhibiting an interesting therapeutic potential when combined with surgical amputation of the primary tumour.

The antineoplastic activity of an organometallic complex of ruthenium(II), [cis-RuCl2(DMSO)4]o, has been examined in comparison with that of cis-PDD, using three metastasizing tumors of the mouse: Lewis lung carcinoma, B16 melanoma and MCa mammary carcinoma. [cis-RuCl2(DMSO)4]o significantly reduces primary tumor growth in all the tumors tested, and its activity is similarly pronounced at three different dosages in mice bearing Lewis lung carcinoma. On the contrary, the survival time of animals having i.v. or i.m. tumor implants are only moderately increased, and also in the case of combined treatments with surgery. The antineoplastic activity of cis-PDD appears to be less pronounced than that of [cis-RuCl2(DMSO)4]o, and is limited to mice bearing B16 melanoma, which, among the three tumors used, appears to be naturally more responsive to cis-PDD and [cis-RuCl2(DMSO)4]o. The use of [cis-RuCl2(DMSO)4]o appears advantageous over that of cis-PDD since, unlike cis-PDD, its antineoplastic effects have been obtained at dosages with reduced host toxicity, indicated by the absence of significant hematological toxicity and toxicity for normal proliferating tissues.

A series of analogues of NAMI-A, a reference compound active on solid tumor metastases, were synthesized (NAMI-A type complexes). They share the same chemical structure of NAMI-A, and differ from it in the nature of the coordinated nitrogen ligand, such as pyrazole, thiazole and pyrazine, which are less basic than imidazole. This modification confers to the new NAMI-A type complexes a better stability in aqueous solution compared to the parent compound, a very important characteristic for a class of compounds that, with NAMI-A, is currently completing a phase I clinical trial at the Netherlands Cancer Institute of Amsterdam. Cytotoxicity and the effects on cell cycle and invasion were investigated on TS/A, B16-F10 and MCF-7 tumor cell lines, while the inhibition of lung metastases was determined on the mouse experimental tumors Lewis lung carcinoma and MCa mammary carcinoma. The new complexes show a pharmacological activity very similar to that of the parental compound NAMI-A: in vitro they are devoid of meaningful cytotoxicity against tumor cells, and in vivo they inhibit metastasis formation and growth approximately to the same extent as NAMI-A. Thus the new NAMI-A type complexes retain the same potent characteristic of NAMI-A to selectively interact with solid tumor metastases. However, compared to NAMI-A they do not stop cell cycle progression at G2-M level and are more active in preventing the spontaneous invasion of Matrigel by tumor cells exposed for 1 h to 10(-4) M concentration. Globally, these complexes take advantage of the knowledge on NAMI-A and appear particularly interesting for future clinical handling and applications.

NAMI-A (imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate, ImH[trans-RuCl4(DMSO)Im]) is a new ruthenium compound active against lung metastasis of solid metastasizing tumors. We have tested this compound in mice with Lewis lung carcinoma or MCa mammary carcinoma in order to compare the effects on primary tumor and lung metastases with possible alterations of cell cycle distribution of tumor cells. We have also investigated whether there were unequal tissue accumulations of the compound itself at different dose levels ranging from 17.5 to 70 mg/kg/day given for six consecutive days. NAMI-A caused a reduction of metastasis weight larger than that of metastasis number; we explain this finding as the capacity of NAMI-A to selectively interfere with the growth of metastases already settled in the lungs. However, this specificity is not simply related to a larger concentration of NAMI-A in the lungs than in other tissues. Following i.p. treatment, NAMI-A rapidly disappeared from the peritoneal cavity; its low blood concentration may be caused by rapid renal clearance. These data provide further evidence for a selective anti-metastasis effect of the ruthenium complex NAMI-A. The reduction of lung metastasis is followed by a significant prolongation of the host's life-time expectancy, indicating a therapeutic benefit of NAMI-A on lung metastases from solid tumors.