Effects of the ruthenium-based drug NAMI-A on the roles played by TGF-ß1 in the metastatic process.

The ruthenium-based drug NAMI-A, characterised by its selectivity against solid tumour metastases, promotes TGF-ß1-dependent fibrosis and the reduction of the release of MMPs in the primary tumour. The aim of the study was to examine the interaction of NAMI-A with TGF-ß1 in the process of metastasis formation. NAMI-A (1) affects the secretion of TGF-ß1 in metastatic MDA-MB-231 cells rather than in non-tumorigenic HBL-100 cells, (2) prevails over TGF-ß1 with regard to the invasive capacity of the treated cells, and (3) contrasts integrin-dependent migration stimulated by TGF-ß1. It, thus, appears that the effects of NAMI-A on cell invasion and migration are best summarised as an interference with TGF-ß1 and a reduction of its activity in these events. At a molecular level, the similar activity of NAMI-A and TGF-ß1 on RhoA GTPase supports its interaction with cell surface integrins while TGF-ß1 can activate it by interaction with its TGFßR receptor. The inhibition of TGF-ß1-induced migration of MDA-MB-231 cells by NAMI-A cannot simply be attributed to a modulation of the Smad2 and p38MAPK pathways. In conclusion, the effects of NAMI-A on the biological role of TGF-ß1 in cancer metastasis are insufficient to attribute the responsibility for the anti-metastatic activity of the ruthenium-based drug to this target alone.

Dermoscopy of scalp tumours: a multi-centre study conducted by the international dermoscopy society.

BACKGROUND: Little is known about the dermoscopic features of scalp tumours. Objective To determine the dermoscopic features of scalp tumours. METHODS: Retrospective analysis of dermoscopic images of histopathologically diagnosed scalp tumours from International Dermoscopy Society members. RESULTS: A total of 323 tumours of the scalp from 315 patients (mean age: 52 years; range 3-88 years) were analysed. Scalp nevi were significantly associated with young age (<30 years) and exhibited a globular or network pattern with central or perifollicular hypopigmentation. Melanoma and non-melanoma skin cancer were associated with male gender, androgenetic alopecia, age >65 years and sun damage. Atypical network and regression were predictive for thin (≤1 mm) melanomas, whereas advanced melanomas (tumour thickness > 1 mm) revealed blue white veil, unspecific patterns and irregular black blotches or dots. CONCLUSIONS: The data collected provide a new knowledge regarding the clinical and dermoscopy features of pigmented scalp tumours.

Chitosan-coated alginate micro-particles delivery of active principles through conventional pelleted food - A study in Tilapia (Oreochromis niloticus).

The search for alternatives to antibiotics in aquaculture has focused on the use of vaccines for immune-prophylaxis. The purpose of this study was to examine the feasibility and characteristics of chitosan-alginate microparticles for the oral delivery of immune-prophylactics to finfish. The microparticles, which incorporate fluorescent-labelled lysozyme, were produced by spray-drying method; their structural properties and uptake from the gastrointestinal tract of Tilapia (Oreochromis niloticus) were assessed by microscopy. The main findings show that the microparticles are able to retain their content in an acidic environment and to release it later in slightly alkaline conditions such as those found in the intestines. Moreover, both the microencapsulation procedure and the biopolymers used have no deleterious impact on the lysozyme lytic activity, which is maintained after the protein has been released from the microparticles. Administered in vivo in Tilapia by medicated food, the microparticles transit unaffected through the stomach, and reach the anterior intestines, in particular the villum sectum and the basal lamina of epithelial cells, 2 and 4 h after feeding. Overall, the evidence obtained here supports the potential of these chitosan-alginate microparticles as agents for oral immune-prophylaxis in the management of fish diseases.

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.

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.

Ruthenium-based compounds and tumour growth control (review).

Heavy metals have often been represented, as an uncertain entity related to renal and other risks of toxicity. In favour of this thought there are several lines of evidence, first of all traffic pollution, other evidence that metals such as arsenite, mercury, cadmium or even iron or radioactive heavy metals, that may be introduced into the body by accident, have been responsible of well known pathologies (for example saturnism with lead) or acute toxicity. Therefore, the biological and medical literature have debated on this subject, mainly from the toxicological point of view, rather than studying possible advantages that might come from compounds based on these metals. Exceptions are represented by studies on the role of metal ions in the biochemistry of enzymes and energy production and, although with less emphasis, on their possible use for correcting metabolic malfunctions. Ruthenium, as a metal, has received an even poorer interest and besides the use in histology, neither ruthenium ions nor ruthenium compounds have a clear place in medicine and biology. Nevertheless, since the middle seventies, many studies have been published, showing in a convincible and repetitive manner, the possible advantages of ruthenium as a base for new competitive drugs. The aim of this review is therefore that of critically examining the past and the actual work on ruthenium compounds with emphasis on their proposed role in cancer therapy.

Effects of L-(adamant-2-yl)glycyl-L-alanyl-d-isoglutamine on the antitumor action of cyclophosphamide, 5-fu, Cisplatin and dacarbazine on advanced carcinomas of the mouse.

A new biological response modifier, L-(adamant-2-yl)glycyl-L-alanyl-D-isoglutamine hydrochloride (AdTP), recently synthesized and characterized for antitumor, antiviral and immunomodulating properties was studied in comparison to the peptidoglycan monomer (PGM) isolated from Brevibacterium divaricatum to test the effects of their use concomitant to that of anticancer cytotoxic drugs such as cyclophosphamide, 5-fluorouracil (5-FU), cisplatin and 4-(3,3-dimethyl-1-triazeno)-5-carboxamide (dacarbazine). The experiments, performed using both Lewis lung carcinoma and MCa mammary carcinoma of CBA mouse, indicated that: a) the cytotoxic drugs, used at the maximum tolerated doses, caused different degrees of reduction of the tumors; b) the same drugs reduced lung metastases with greater efficacy when treatments were applied at the early stages of metastasis formation; c) AdTP, similarly to PGM confirmed its ability to increase some immunological parameters of lymphocytes obtained from the spleens of the treated mice; d) AdTP increased the effects of 5-FU on lung metastases but failed to show any increase of life expectancy with any treatment performed. These data indicate that AdTP, although increasing some functional responses of lymphocytes in vitro, does not improve the therapeutic activity of cyclophosphamide, cisplatin, 5-FU and dacarbazine in the experimental models presently used.

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.

Drug control of solid tumour metastases: a critical view.

Metastasis of solid tumours represent the target of election for the pharmacological treatment of cancer. Nevertheless, commonly used treatments do not represent any selective approach, provided that drugs are mostly unspecific cytotoxics. Today many strategies adopted to interfere with metastasis growth concern the interaction with biological signals of the metastatic cells or of the host. One difference should be made between anti-metastatic and anti-metastasis drugs, in that only the latter realise the goal of selectively destroying metastasis wherever they are. In this context many agents active on newly identified molecular targets are more effective in preventing metastasis formation than in inhibiting their growth. NAMI-A, an innovative ruthenium compound, seems to provide optimism for the future and, in laboratory models, it is very active on lung metastases independently of the stage of their growth. The success of NAMI-A against metastasis should stimulate laboratory studies with appropriate experimental models to predict clinical activity, since the use of experimental conditions closely similar to those of human tumours should help the identification of more active compounds.

Lysozyme stimulates lymphocyte response to ConA and IL-2 and potentiates 5-fluorouracil action on advanced carcinomas.

The oral administration of 100 mg/Kg/day of hen egg-white lysozyme (Lysozyme) for 8 consecutive days to mice bearing advanced MCa mammary carcinomas and treated with 5-fluorouracil (5-FU) increases the efficacy of 5-FU on primary tumor growth and on lung metastasis formation and particularly on the postsurgical survival time. These effects are accompanied by the correction of the reduced in vitro response to ConA of lymphocytes obtained from the spleen of the treated mice. In vitro, lysozyme is capable of inducing proliferative activity in a population of blast cells, obtained by a mixed population of mononuclear cells harvested from the spleen of healthy mice, and of evoking a marked proliferative effect to IL-2 in a condition in which, in lysozyme untreated lymphocytes, IL-2 is completely uneffective. These data stress the effects of lysozyme on host immunity following oral administration and moreover indicate the beneficial role of this peptide in conditions in which the increase of host responses can significantly contribute to the success of the treatment.

Increase of tumour infiltrating lymphocytes in mice treated with antimetastatic doses of NAMI-A.

NAMI-A is a novel antitumour agent, based on ruthenium, which has proved effectiveness against lung metastases of solid mouse tumours. The study focuses on the effects of NAMI-A on leukocyte infiltration into the primary tumour of MCa mammary carcinoma, implanted subcutaneously (s.c.) or intramuscularly (i.m.) into CBA mice. NAMI-A, given with a cycle of daily treatments for six consecutive days on advanced tumours at 35 mg/kg/day, markedly reduces lung metastasis independently of the tumour type (Lewis lung carcinoma, MCa mammary carcinoma or TS/A adenocarcinoma) being treated and of the site of tumour implantation (s.c. or i.m.). The analysis of leukocyte infiltration of the primary tumour, performed on a single cell suspension of cells isolated from a Ficoll gradient on which a raw suspension of primary tumour cells was layered, showed NAMI-A to significantly increase tumour infiltrating lymphocytes. These lymphocytes are almost all CD3+ cells with a significant increase of the CD8+ over the CD4+ subpopulation that reduces the helper/suppressor ratio from 2.8 to 2.1. These data indicated the absence of toxicity of NAMI-A for tumour infiltrating lymphocytes and suggested that this compound might even synergize in combined treatments with cancer immunotherapy.

Treatment of metastases of solid mouse tumours by NAMI-A: comparison with cisplatin, cyclophosphamide and dacarbazine.

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.

Cytofluorimetric analysis of gut-intraepithelial and mesenteric lymph node lymphocytes of tumour bearing mice fed with egg-white lysozyme.

The effects of the oral administration of 100 mg/kg/day egg-white lysozyme (EWL) on the expression of CD3, CD4, CD8 and CD25 antigens of lymphocytes harvested from IEL and mesenteric lymph nodes (MLNL) were tested in mice bearing MCa mammary carcinoma. Lysozyme, after oral administration, retains its enzymatic activity along the entire small bowel and almost 10% of the administered dose is recovered 1 hr after treatment in the middle of the jejunum. Correspondingly, the number of cells expressing the test antigens in MLNL is greater than in controls after a few days of treatment and is maintained high up to the end of treatment but returns to control values after treatment withdrawal; CD4:CD8 ratio is decreased by EWL in favour of CD8 positive cells. Treatment with EWL does not modify the ratio between CD4+ and CD8+ cells vs controls in IEL nor does it change the % of CD3 positive cells or the expression of IL-2 receptor at this level. These data support the existence of the induction of an immunity communication by EWL along the axis GALT-mesenteric lymph nodes which is in agreement with the reported effects of the oral administration of EWL on tumour growth in experimental systems and on host immunity in humans.

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.

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.

Antimetastatic properties and DNA interactions of the novel class of dimeric Ru(III) compounds Na2[[trans-RuCl4(Me2SO)]2(mu-L)] (L = ditopic, non-chelating aromatic N-ligand). A preliminary investigation.

A novel class of dianionic Ru(III) dimers of formula Na2[[trans-RuCl4(Me2SO)]2(mu-L)], with L = pyrazine (pyz, 1), pyrimidine (pym, 2), 4,4'-bipyridine (bipy, 3), and 1,2-bis(4-pyridine) ethane (etbipy, 4), was developed by us with the specific aim of assessing their antitumor properties. The dimers are in fact structurally related to the antimetastatic mononuclear compound (ImH) [trans-RuCl4(Me2SO)(Im)] (NAMI-A, Im = imidazole). Preliminary results concerning the antineoplastic activity of 1-4 against the murine MCa carcinoma model, a tumor which spontaneously metastasizes in the lungs, are reported. Similarly to what is normally observed with NAMI-A, the treatment with the dimeric complexes was scarcely effective against the growth of the primary tumor. However, dimers 1, 2, and 4 reduced very effectively the number and, in particular, the weight of lung metastases (to about 5% with respect to controls); in particular, Na2[[trans-RuCl4(Me2SO)]2(mu-etbipy)] (4) was as effective as NAMI-A in reducing the spontaneous metastases at a dosage which, in terms of moles of ruthenium, is about 3.5 times lower compared to that normally used for NAMI-A. Furthermore, in vitro tests showed that dimers 1-4 are capable of forming interstrand cross-links with linearized plasmidic DNA in a time-dependent manner. All the dimeric species are more active in inducing cross-links compared to NAMI-A, and the dimer bridged by the etbipy ligand (4) is the most effective among those tested.

Effects of ruthenium complexes on experimental tumors: irrelevance of cytotoxicity for metastasis inhibition.

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.

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.

Synthetic thymic fraction 5: effects of high dose administration on circulating lymphocytes in patients.

The synthetic pentapeptide (Arg-Lys-Asp-Val-Tyr; TP-5) corresponding to the active site of the hormone thymopoietin, was given at the dose of 300 mg/m2/day (1 day), higher than the usually administered, to a group of 27 immunodepressed patients in order to determine the tolerability and the immunomodulatory activity. The examination of a series of hematological parameters including counts of differential clustering of lymphocytes by cytofluorimetric analysis was performed 24 hr and 48 hr after treatment, and repeated at different intervals up to 14 days after treatment. TP-5 caused a significant increase of circulating lymphocytes and particularly of CD3+CD4+ and CD3+CD8+ subtypes, peaking at 48 hr and maintaining the increased values up to the last examination on day 14 from treatment. A faster increase (zenith at 24 hr) was observed for CD4+ cells, in comparison with CD8+ cells (zenith at 48 hr). The number of patients that increased total lymphocytes or lymphocyte subset after treatment ranged between 52.6 (CD4+ cells) and 69.2% (NK cells), whereas about 7.7% (NK cells) to 36.9% (CD4+ cells) remained unchanged and a smaller amount of 10.5% (CD4+ and CD8+ cells) or 23.1% (NK cells) showed a decrease greater than 10% of their respective basal value. No significant relationship between responders and non-responders can be found on the basis of previous treatments, cancer type, sex or age.

Approaching tumour therapy beyond platinum drugs: status of the art and perspectives of ruthenium drug candidates.

The study of metal complexes for the treatment of cancer diseases has resulted in the identification of some unique properties of ruthenium-based compounds. Among these inorganic-based agents, two of them, namely the ruthenium(III) drugs NAMI-A and KP1019 have undertaken with some success the clinical evaluations of phase I and preliminary phase II trials in patients. Here we highlight the strategies that have led to the discovery of metal-based (NAMI-A and KP1019) and of organometallic (RM175, RAPTA-T, RDC11 and DW1/2) ruthenium-based complexes, and we report their main biological/pharmacological characteristics and expectations for further development.