The antimetastatic drug NAMI-A potentiates the phenylephrine-induced contraction of aortic smooth muscle cells and induces a transient increase in systolic blood pressure.

The ruthenium-based drug imidazolium trans-imidazoledimethylsulphoxidetetrachlorido ruthenate (NAMI-A) is a novel antitumour drug under clinical evaluation. In this study, NAMI-A is tested on aortic rings in vitro and on the systolic blood pressure in vivo with the aim of evaluating its effects on smooth muscle cells and, more in general, on the vascular system. Pre-incubation of aortic rings with 10 µM NAMI-A for 10 min potentiates the contraction induced by phenylephrine (PE). The reduction of the B max value of [(3)H]-prazosin bound to NAMI-A-treated aortic rings and the ability of NAMI-A to displace [(3)H]-prazosin and [(3)H]-IP3 binding by 25 and 42%, respectively, suggest the involvement of α1-adrenoceptor in mediating the effects on smooth muscle cells. NAMI-A also decreases the number of maximal sites of [(3)H]-prazosin bound to kidney membrane preparation from 34 to 24 fmol/mg proteins. A single i.p. dose (105 mg/kg) or a repeated treatment for 6 consecutive days (17 mg/kg/day) in Wistar rats increases the systolic blood pressure, respectively, 1 h and 3 days after treatment, and the responsiveness of rat aortic rings to PE. Atomic absorption spectroscopy confirms the presence of ruthenium in the aortic rings excised from the treated rats. These findings suggest monitoring the cardiovascular parameters when the drug is used in humans for treating cancer patients, particularly if the drug is associated with chemicals that are potentially active at the cardiovascular level.

Influence of the binding of reduced NAMI-A to human serum albumin on the pharmacokinetics and biological activity.

NAMI-A is a ruthenium-based drug endowed with the unique property of selectively targeting solid tumour metastases. Although two clinical studies had already been completed, limited information exists on the behavior of NAMI-A after injection into the bloodstream. PK data in humans informs us of a rather low free drug concentration, of a relatively high half-life time of elimination and of a linear relationship between the administered dose and the corresponding AUC for up to toxic doses. In the present study, we examined the chemical kinetics of albumin binding with or without the presence of reducing agents, and we evaluated how these chemical aspects might influence the in vivo PK and the in vitro ability of NAMI-A to inhibit cell migration, which is a bona fide, rapid and easy way to suggest anti-metastatic properties. The experimental data support the binding of NAMI-A to serum albumin. The reaction is facilitated when the drug is in its reduced form and, in agreement with already reported data, the adduct formed with albumin maintains the biological activity of the ruthenium drug. The formation of the adduct is favored by low ratios of NAMI-A : HSA and by the reduction of the drug with ascorbic acid. The difference in in vivo PK and the faster binding to albumin of the reduced NAMI-A seem to suggest that the drug is not rapidly reduced immediately upon injection, even at low doses. Most probably, cell and protein binding prevail over the reduction of the drug. This observation supports the thesis that the reduction of the drug before injection must be considered relevant for the pharmacological activity of NAMI-A against tumour metastases.

Orally administered microencapsulated lysozyme downregulates serum AGE and reduces the severity of early-stage diabetic nephropathy.

AIM: Diabetic nephropathy is the leading cause of end-stage kidney disease in developed countries and is related to chronic hyperglycaemia. The increased production and tissue deposition of advanced glycation end products (AGE) are known to play a major role in the pathogenesis of diabetic kidney damage. This study was undertaken to determine if lysozyme (LZ), microencapsulated in orally administrable chitosan-coated alginate microspheres (MS), is effective against the early changes seen in the initial stages of diabetic nephropathy. METHODS: LZ-containing MS (MSLZ) and an equivalent dose (equidose) of nonencapsulated LZ were given as oral treatments. LZ was administered to Wistar rats for seven weeks after diabetes induction with streptozotocin. RESULTS: The results showed that microencapsulated LZ treatment significantly reduced the concentration of serum AGE in the circulation and their deposition in the kidneys. Likewise, MSLZ significantly prevented the development of microalbuminuria compared with untreated diabetic rats. Furthermore, MSLZ significantly prevented the development of glomerular and renal hypertrophy as well as overexpression of AGE receptors (RAGE). An equidose of free LZ had little or no effect whatsoever. CONCLUSION: Our study supports a relationship between serum AGE and nephropathy in diabetes, and suggests that orally administered microencapsulated LZ can exert kidney-protective activity in a diabetic animal model.

Inhibition of B16 melanoma metastases with the ruthenium complex imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate and down-regulation of tumor cell invasion.

The antimetastatic ruthenium complex imidazolium trans-imidazoledimethylsulfoxide-tetrachlorouthenate (NAMI-A) is tested in the B16 melanoma model in vitro and in vivo. Treatment of B6D2F1 mice carrying intra-footpad B16 melanoma with 35 mg/kg/day NAMI-A for 6 days reduces metastasis weight independently of whether NAMI-A is given before or after surgical removal of the primary tumor. Metastasis reduction is unrelated to NAMI-A concentration, which is 10-fold lower than on primary site (1 versus 0.1 mM), and is correlated to the reduction of plasma gelatinolitic activity and to the decrease of cells expressing CD44, CD54, and integrin-beta(3) adhesion molecules. Metastatic cells also show the reduction of the S-phase cells with accumulation in the G(0)/G(1) phase. In vitro, on the highly metastatic B16F10 cell line, NAMI-A reduces cell Matrigel invasion and its ability to cross a layer of endothelial cells after short exposure (1 h) to 1 to 100 microM concentrations. In these conditions, NAMI-A reduces the gelatinase activity of tumor cells, and it also increases cell adhesion to poly-L-lysine and, in particular, to fibronectin, and this effect is associated to the increase of F-actin condensation. This work shows the selective effectiveness of NAMI-A on the metastatic melanoma and suggests that metastasis inhibition is due to the negative modulation of tumor cell invasion processes, a mechanism in which the reduction of the gelatinolitic activity of tumor cells plays a crucial role.

Vaccination trials of sea bass (Dicentrarchus labrax) against pasteurellosis using oral, intraperitoneal and immersion methods.

Photobacterium damsela subsp. piscicida (Phdp) is the aetiological agent of fish pasteurellosis, causing heavy losses in intensive mariculture plants. The present work compares the protective efficacy of five different vaccine formulation: oral, intraperitoneal, immersion, bivalent immersion (Vibrio anguillarum) and immersion associated with immunostimulants. Each of these vaccine formulations containing whole cells of Phdp formalin inactivated (FKC), was administered to 100 sea bass weighing approximately 2 g; 100 non-vaccinated sea bass were used as controls. Protection against pasteurellosis was tested for 40 days after vaccination by intraperitoneal challenge: each fish was inoculated with Phdp cells at a concentration of 2.75 x 10(4) cfu/ml. Mortality was recorded over the following 14 days, vaccine protection was evaluated using a relative percentage survival (RPS) index. The intraperitoneal formulation gave excellent protection (RPS 82.4%). The most effective immersion form was that followed by simple immersion (RPS 23.1%) followed by the group vaccinated with bivalent vaccine (RPS 18.7%). Protection conferred orally (RPS 28.6%) is of interest for practical purposes.

Free exchange across cells, and echistatin-sensitive membrane target for the metastasis inhibitor NAMI-A (imidazolium trans-imidazole dimethyl sulfoxide tetrachlororuthenate) on KB tumor cells.

The duration of cell proadhesive effects induced by imidazolium trans-imidazole dimethyl sulfoxide tetrachlororuthenate (NAMI-A), a compound endowed with in vivo antimetastatic properties, was tested in vitro on the human epithelial tumor cell line KB. The intensity of proadhesive effects continues to increase up to 48 to 72 h after NAMI-A withdrawal and declines only after 96 h. The proadhesive effect on cells seeded on fibronectin is greater than on plastic, since it already reaches its maximum after 24 h. This effect suggests a role for integrin activation, which is further stressed by the inhibitory activity of the disintegrin molecule echistatin. The intensity and duration of NAMI-A's proadhesive effects are correlated to cell exposure time and to the rapid release of NAMI-A metabolites in the culture medium in the first 5 min after drug withdrawal. These metabolites are probably neutral species with ruthenium-bound bioligands to allow for the rapid exchange between cells and extracellular medium. These data suggest a long-lasting effect of NAMI-A in biological systems, even at very low concentrations, and stress the low and reversible effects on kidney, where it naturally concentrates. These data on proadhesive effects are, further, relevant for in vivo antimetastatic effects, as this adhesion is associated to cell motility and invasion, which in turn are related to tumor malignancy and metastasis.

Actin-dependent tumour cell adhesion after short-term exposure to the antimetastasis ruthenium complex NAMI-A.

Imidazolium trans-imidazoledimethylsulphoxidetrachlororuthenate (NAMI-A) was tested in vitro on the pro-adhesive properties, evaluated as resistance to trypsin treatment, which is a bona fide measure of adhesion strength, of KB and HeLa carcinoma cell lines and on human polymorphonuclear neutrophils (HPMN). NAMI-A increased the pro-adhesive activity of KB cells at 0.001 mM concentration, after few minutes incubation and this effect was not influenced by the vehicle used for cell challenge, neither did it depend on NAMI-A concentration or on temperature. The same effect occurred on HeLa cells at 0.01 mM NAMI-A. This effect, detected at concentrations up to 100 times lower than those necessary to block cells at the G(2)-M premitotic phase of cell cycle, or to inhibit matrix metalloproteinase release or cell invasion, was not related to ruthenium uptake by tumour cells. HeLa cells and healthy HPMN, following short exposure to 0.1 mM NAMI-A, assumed a different shape, with the extrusion of filopodia (HeLa) and of large lamellopodia (HPMN), which increased their interactions with the substrate. This effect was attributed to stabilisation, altered turnover and sensitivity to cytochalasin D of actin filaments. Provided that adhesion is associated with cell motility and invasion, these data suggest that NAMI-A may exert antimetastatic properties at concentrations lower than those observed in the lungs at the end of a conventional intraperitoneal treatment in vivo.

Biological role of adduct formation of the ruthenium(III) complex NAMI-A with serum albumin and serum transferrin.

NAMI-A is an innovative ruthenium(III) complex with a very encouraging preclinical profile of metastasis inhibition, which is undergoing initial phases of clinical trials. To assess the pharmacological relevance of the drug fraction associated to plasma proteins, adducts of NAMI-A with either serum albumin or serum transferrin were prepared and their biological effects tested in vitro and in vivo. Specifically, adducts of NAMI-A with either serum albumin or serum transferrin, prepared and characterized at a ruthenium-to-protein molar ratio of 4:1, were evaluated in vitro on the KB human tumor cell line and in vivo on the MCa mammary carcinoma tumor. The effects of NAMI-A/protein adducts on cell viability and on cell cycle progression were found to be far smaller than those produced by free NAMI-A. GFAAS measurements point out that the amount of ruthenium that gets into cells is drastically reduced when NAMI-A is presented in its protein-bound form. In vivo use of NAMI-A adducts with albumin and transferrin resulted markedly less effective on lung metastasis reduction, than free NAMI-A. Overall, the present results suggest that binding to plasma proteins causes a drastic decrease of NAMI-A bioavailability and a subsequent reduction of its biological activity, implying that association to plasma proteins essentially represents a mechanism of drug inactivation.

Influence of chemical stability on the activity of the antimetastasis ruthenium compound NAMI-A.

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.

Tumour cell uptake G2-M accumulation and cytotoxicity of NAMI-A on TS/A adenocarcinoma cells.

The ruthenium(III) complex imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate (NAMI-A) was tested on TS/A adenocarcinoma cells to evaluate the relationship between cell uptake, cell cycle arrest and cytotoxicity. The in vitro challenge of TS/A cells with 10(-4) M NAMI-A for 15 minutes to 4 hours showed a partial reduction of cell growth only after 4 hour exposure. In the same experimental conditions NAMI-A caused the increase of cells in G2-M cell cycle phase directly proportional on the length of treatment, and the ruthenium uptake by tumour cells, measured by flameless atomic absorption spectroscopy, that increases up to 2 hours of treatment and then reaches a plateau. The arrest of cell cycle in the pre-mitotic G2-M phase was transient and completely reversed by 48 hours after treatment. This study showed that the effect of NAMI-A on the cell cycle of TS/A cells is not strictly related to NAMI-A uptake as is the effect on tumour cell proliferation.

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.

Blood concentration and toxicity of the antimetastasis agent NAMI-A following repeated intravenous treatment in mice.

NAMI-A is a new generation antitumour ruthenium-based agent and characterised by strong efficacy against lung metastases of experimental solid tumours in mice. The effects of intravenous administration of 15, 35 and 50 mg/kg/day of NAMI-A for 5 consecutive days on blood concentration and host toxicity were tested on Swiss CD1 male and female mice. The blood concentration of NAMI-A, both after the first injection and at the end of the 5-day treatment fell rapidly and 5 min. after the last injection it was always below 10% of the administered dose. Kinetic parameters, calculated at the end of the 5-day treatment cycle according to a mono-compartment model (fitting with R2=0.9), indicate a t 1/2 of about 18 hr. Toxicity i) was observed only at the highest dose used (50 mg/kg/day), ii) was greater in females than in males, iii) in mice which survived treatment was completely reversed within 3-weeks of the end of the treatment. Haematological examinations, clinical chemistry data and histopathologic studies were consistent in terms of the effect on host lymphoid tissues, consisting in spleen and lymph node depletion and in a general increase of circulating leukocytes. Data on ruthenium organ retention confirm lack of brain penetration and a relatively high lung concentration which might account for the remarkable effect on lung metastases.

Lack of In vitro cytotoxicity, associated to increased G(2)-M cell fraction and inhibition of matrigel invasion, may predict In vivo-selective antimetastasis activity of ruthenium complexes.

The ruthenium complexes trans-dichlorotetrakisdimethylsulfoxide ruthenium(II) (trans-Ru), imidazolium trans-imidazoletetrachlororuthenate (ICR), sodium trans-tetramethylensulfoxideisoquinolinetetrachlororuthenate (TEQU), and imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate (NAMI-A) are tested in vitro by short exposure of MCF-7, LoVo, KB, and TS/A tumor cells to 10(-4) M concentration, and in vivo on Lewis lung carcinoma by a daily i.p. treatment for 6 consecutive days using equitoxic and maximum tolerated doses. NAMI-A 1) inhibited tumor cell invasion of matrigel, 2) induced a transient accumulation of cells in the G(2)-M phase, 3) did not modify in vitro cell growth, and 4) markedly reduced lung metastasis formation. TEQU showed significant cytotoxicity in vitro and was not antimetastatic in vivo. ICR and trans-Ru did not modify cell cycle distribution of in vitro tumor cells nor did they inhibit matrigel invasion; ICR was also devoid of antimetastasis effects in vivo. Ruthenium uptake by tumor cells did account for in vitro cytotoxicity but not for other in vitro actions or for in vivo antimetastasis activity. The contemporary absence of cytotoxicity, associated to inhibition of matrigel crossing and to transient block in the premitotic G(2)-M phase, appears to be prerequisites for a ruthenium compound to show in vivo-selective antimetastasis effect. The validation of this model for other classes of compounds will allow an understanding of the combined weight of the above-mentioned phenomena for tumor metastasis growth and control.

Fate of the antimetastatic ruthenium complex ImH [trans-RuCl4(DMSO)Im] after acute i.v. treatment in mice.

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.

Blood levels of ruthenium following repeated treatments with the antimetastatic compound NAMI-A in healthy beagle dogs.

NAMI-A is a new generation ruthenium compound which is entering phase-I clinical trials anti-metastatic agent. This study analyses the effects of the i.v. injection of NAMI-A to healthy Beagle dogs at increasing doses from 0.4 (low) 4 (mid) and 8 (high) mg/kg/day, given for 5 consecutive days. Only mild signs of toxicity, consisting of emesis and mucoid faeces, from which animals completely recovered, occurred during treatment at the high dose. Decay of ruthenium concentration from the whole blood, 24 hr after 5-days treatment, was lower than that observed after 1-day treatment. T1/2 was about 20-23 hr, or slightly longer when the animals were hydrated with tap water prior to treatment; Cltot was 21-22 ml*hr-1, decreasing to 13 ml*hr-1 after hydration and increasing to 34 ml*hr-1 with the high dose. AUC was proportional to the dose used. Thus NAMI-A is well tolerated by healthy dogs with blood levels comparable to those obtained in mice treated with an about 10-times higher daily dose.

Reduction of lung metastasis by ImH[trans-RuCl4(DMSO)Im]: mechanism of the selective action investigated on mouse tumors.

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.

Modification of cell cycle and viability of TLX5 lymphoma in vitro by sulfoxide-ruthenium compounds and cisplatin detected by flow cytometry.

The effects of Na[trans-RuCl4(DMSO)Im] (NAMI), Na[trans-RuCl4(TMSO) Ind] (TIND) and Na[trans-RuCl4(TMSO)Iq] TEQU) were tested in vitro on TLX5 lymphoma cells in comparison to cisplatin by means of the sulforhodamine-B test SRB) for protein content determination, by acridine orange and propidium iodide staining and by means of the bromodeoxyuridine test, for cell cycle modifications. After 1 h drug exposure with metal-based drugs, TLX5 lymphoma cells require a further 72 h in vitro cultivation to show alteration of cell cycle. Ruthenium compounds show a different pattern of effects: TEQU causes the same dose-dependent cytotoxicity and DNA fragmentation shown by cisplatin, TIND reduces absorbance with the SRB test and slightly increases S and G2M populations with a time-dependent drug exposure of tumour cells, and NAMI is virtually devoid of any detectable effect. By in vivo bioassay of in vitro treated tumour cells, TIND and TEQU are effective independently of the time of drug exposure of tumour cells, this effect being confirmed by the same cell uptake of ruthenium after 1 or 4 h treatment, determined by atomic absorption spectroscopy. These data stress the lack of the involvement of direct cytotoxic effects in the potent anti-metastatic action of NAMI.

Comparison of the effects of the antimetastatic compound ImH[trans-RuCl4(DMSO)Im] (NAMI-A) on the arthritic rat and on MCa mammary carcinoma in mice.

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.

Down-regulation of tumour gelatinase/inhibitor balance and preservation of tumour endothelium by an anti-metastatic ruthenium complex.

The anti-metastatic ruthenium complex Na[trans-RuCl4(DMSO)Im] 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 of the 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-1 and TIMP-2 mRNAs. Na[trans-RuCl4(DMSO)Im] 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-RuCl4(DMSO)Im] 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-1. The different enzyme/inhibitor mRNA levels between untreated and treated tumours seem to be unaffected by tumour-infiltrating 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 controls.

Treatment of residual metastases with Na[trans-RuCl4 (DMSO)lm] and ruthenium uptake by tumor cells.

Treatment of MCa mammary carcinoma metastases by i.p. administration of a total dose of 450 mg/kg Na[trans-RuCl4(DMSO)lm], after successful surgical removal of primary tumor mass, causes a significant prolongation of the host's life-time expectancy. This effect, related to lung metastasis inhibition, seems not attributable to a direct inhibition of tumor cells since antimetastatic effects can be achieved also when drug treatment occurs before tumor cell injection into the host. Also, the activity of Na[trans-RuCl4(DMSO)lm] seems independent of its concentration in tumor cells. Rather it must be stressed that the fate of this compound in the blood, following i.v. administration, is fast and only a very low percent of the total dose reaches the tumor target in the lungs. These data emphasize the possibility that Na[trans-RuCl4(DMSO)lm] increases the resistance of the host against metastasis formation, possibly by the already shown mechanism of potentiation of the extracellular matrix and reduction of blood stream invasion by tumor cells.