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.

Pharmacological control of lung metastases of solid tumours by a novel ruthenium complex.

Imidazolium trans-imidazoledimethylsulphoxidetetrachlororuthenate ImH[trans-RuCl4(DMSO)Im] (NAMI-A), a ruthenium compound that replaces Na+ with ImH+ in the molecule of Na[trans-RuCl4(DMSO)Im] (NAMI), was studied for the anti-metastasis effects in models of solid metastasizing tumours of the mouse. NAMI-A, given i.p. at 35 mg/kg/day for six consecutive days, a dose equimolar to that of NAMI, to mice bearing Lewis lung carcinoma and MCa mammary carcinoma, markedly reduces lung metastasis weight by 80-90%, with an effect equal or even superior to that of NAMI, depending on the experimental system adopted. Correspondingly, NAMI-A increases the content of connective tissue in the tumour matrix, around blood vessels, and in the tumour capsule, augments the percentage of tumour cells in G2/M phase and reduces the amount of CD45+ cells infiltrating the tumour parenchyma. The effects of the same doses on spleen lymphocytes correspond to an increase of CD8+ subset without any change of the distribution of cells in G0/G1, S and G2/M phases. The study shows that NAMI-A behaves similarly to NAMI on the several parameters examined in comparison experiments and therefore we suggest to credit NAMI-A with all the biological actions already described for NAMI during the last 3 years. The replacement of Na+ with ImH+ therefore, besides the better chemical stability of the molecule, confers to [trans-RuCl4(DMSO)Im]- a closer similarity with a true drug to be used in humans, and suggests this molecule for future studies of preclinical toxicology and phase I and II clinical trials.

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.

Stimulation of GALT and activation of mesenteric lymph node lymphocytes by a modified lysozyme in CBA mice with MCa mammary carcinoma.

Lysozyme (hen egg-white lysozyme) and its derivative mPEG-lyso (lysozyme coupled with polyoxyethyleneglycol) were tested in CBA mice bearing MCa mammary carcinoma for their effects on intestinal mucosal immunity (GALT) and mesenteric lymph node lymphocytes (MLNL), after oral administration. Following a cycle of administration of 100 mg/kg/day lysozyme or 350 mg/kg/day mPEG-lyso for 9 consecutive days, GALT was analyzed by using optical histology, and mesenteric lymph node lymphocytes were studied by cytofluorimetric analysis of CD3, CD4 and CD8 antigens, and of DNA and RNA content following in vitro culture with concanavalin A. Both lysozymes significantly increase the number of lymphatic nodules on gut epithelium as determined by histological analysis of sections of small bowel. mPEG-lyso, unlike native lysozyme, gives protection from the decline of the blastogenic activity of MLNL observed at early stages of tumor growth, as shown by the increased nucleic acid content of these cells. On the same cells, both lysozyme and mPEG-lyso also seem to prevent the decline of CD4+ cells observed during tumor growth in control animals. These data confirm the effects of lysozyme on GALT and show that the new lysozyme derivative mPEG-lyso has effects on host immunity greater than those of the native molecule.

Antimetastatic action and lymphocyte activation by the modified lysozyme mPEG-Lyso in mice with MCa mammary carcinoma.

The effects of Lysozyme (hen egg-white lysozyme) and of its modified derivative mPEG-Lyso, (Lysozyme coupled with monomethoxypolyethylenglycol) were tested on CBA mice bearing MCa mammary carcinoma. mPEG-Lyso, given by the oral route at a dose comparable to 100 mg/kg/day of native Lysozyme, is at least as active as Lysozyme for the activation of lymphocytes obtained from different districts along the axis GALT-spleen. These effects were evidenced by measuring the in vitro response of lymphocytes of animals treated in vivo with ConA and LPS using the SRB test, and measuring the content of nucleic acids by cytofluorimetric analysis. Lymphocytes obtained from the mesenteric lymph nodes of animals treated with mPEG-Lyso, show a response to ConA and to LPS at early stages of treatment, when tumor growth reduces the response to controls. mPEG-Lyso, was also effective on lung metastasis formation. Considering that mPEG-Lyso,, compared to the native Lysozyme, completely lost its enzymatic action on Micrococcus lysodehycticus cell walls, this data suggest that the effects of lysozyme on immunity and on tumour growth are unrelated to the production of immunoactive peptidoglycans in the gut.