Antiviral properties and cytotoxic activity of platinum(II) complexes with 1,10-phenanthrolines and acyclovir or penciclovir.

Margiotta N, Bergamo A, Sava G, Padovano G, de Clercq E, Natile G.
Dipartimento Farmaco-Chimico, Universita degli Studi di Bari, via E. Orabona 4, 70125 Bari, Italy. nmargiotta@farmchim.uniba.it
J Inorg Biochem.;98(8):1385-90,

ABSTRACT

Platinum compounds containing an aromatic diimine (1,10-phenanthroline or 2,9-dimethyl-1,10-phenanthroline; phen and Me(2)phen, respectively) and antiviral guanosine-type ligands (acyclovir or penciclovir; acy and pen, respectively) have been synthesised. These compounds maintain the antiviral activity against Herpes Symplex Virus (HSV) and have greater efficacy than free acyclovir or penciclovir against Cytomegalovirus (CMV); in both cases the species with Me(2)phen are more active. The same complexes are effective against tumor cell proliferation which also results to be dependent upon the nature of the diimine ligand: all compounds containing Me(2)phen being more active than those containing phen. Although in vivo some complexes significantly reduce tumor cell proliferation, nevertheless, they do not appear to significantly affect the life time expectancy of the treated mice. The greater cytotoxicity of compounds with Me(2)phen may result from a higher reactivity towards cellular components, such as glutathione, which could cause release of the diimine, known to be highly cytotoxic.

Inhibition of hepatocellular carcinomas in vitro and hepatic metastases in vivo in mice by the histone deacetylase inhibitor HA-But.

Coradini D, Zorzet S, Rossin R, Scarlata I, Pellizzaro C, Turrin C, Bello M, Cantoni S, Speranza A, Sava G, Mazzi U, Perbellini A.
Unit of Biomolecular Determinants in Prognosis and Therapy, Experimental Department, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan.
Clin Cancer Res.;10(14):4822-30,

ABSTRACT

PURPOSE: The purpose is to evaluate the CD44-mediated cellular targeting of HA-But, a hyaluronic acid esterified with butyric acid (But) residues, to hepatocellular carcinoma cell lines in vitro and to hepatic tumor metastases in vivo. EXPERIMENTAL DESIGN: In vitro, the CD44-dependent cytotoxicity in two human hepatocellular carcinoma cell lines (HepB3 and HepG2) with high and low CD44 expression was investigated; in vivo, the effect on liver metastases originating from intrasplenic implants of Lewis lung carcinoma (LL3) or B16-F10 melanoma in mice was compared with the pharmacokinetics of organ and tissue distribution using different routes of administration. RESULTS: HepB3 and HepG2 cell lines showed different expression of CD44 (78 and 18%, respectively), which resulted in a CD44-dependent HA-But inhibitory effect as demonstrated also by the uptake analysis performed using radiolabeled HA-But ((99m)Tc-HA-But). Pharmacokinetic studies showed different rates of (99m)Tc-HA-But distribution according to the route of administration (i.v., i.p., or s.c.): very fast (a few minutes) after i.v. treatment, with substantial accumulation in the liver and spleen; relatively slow after i.p. or s.c. treatment, with marked persistence of the drug at the site of injection. The effect of s.c. and i.p. treatment with HA-But on liver metastases originating from intrasplenic implants of LL3 carcinoma or B16-F10 melanoma (both CD44-positive: 68 and 87%, respectively), resulted in 87 and 100% metastases-free animals, respectively (regardless of the route of administration), and a significant prolongation of the life expectancy compared with control groups. CONCLUSIONS: HA-But tends to concentrate in the liver and spleen and appears to be a promising new drug for the treatment of intrahepatic tumor lesions.

Cocultures of metastatic and host immune cells: selective effects of NAMI-A for tumor cells.

Bacac M, Vadori M, Sava G, Pacor S.
Department of Biomedical Sciences, University of Trieste, Via L. Giorgieri 7, 34127, Trieste, Italy.
Cancer Immunol Immunother.; Jun 25.

ABSTRACT

The effects of NAMI-A, [H(2)im][ trans-RuCl(4)(dmso-S)(Him)], a new metal-based agent for treating tumor metastases, have been investigated in vitro on splenocytes, ConA- or LPS-activated T and B lymphoblasts, and thymocytes. Splenocytes and thymocytes exposed for 1 h to 0.01-0.1-mM NAMI-A do not change their mitochondrial functionality, cell cycle distribution, protein synthesis, and CD44 expression in comparison to untreated control samples. Instead, mitochondrial functionality increased 24 h after treatment in a fraction of splenocytes. The same treatment reduced mitochondrial functionality and S phase of the cell cycle in T and B blasts (already after 1 h treatment) and reduced CD44 expression on B blasts, 24 h after treatment. On cocultures of splenocytes and metastatic cells (metGM) (1:1), NAMI-A induces a selective depolarization of mitochondrial membrane potential of metGM cells, while it stimulates splenocytes (mainly lymphocytes), as shown by the increase of the S phase, nitric oxide production, and adhesion onto metastatic cells. This, in turn, reduces the number of metastatic cells and results in the increased ratio between splenocytes and metGM in favor of diploid cells (doubling from one to two). Rosetting of leukocytes onto metastatic cells correlates with induction of CD54 expression on tumor cells after NAMI-A in vivo treatment, which in turn, might contribute to metastasis recognition by cytotoxic lymphocytes. The overall antimetastatic activity displayed by NAMI-A might therefore be the result of complex interactions with tumor cells, on which it displays selective antitumor activity, and with host immune cells through which it promotes activation of host immune defenses involved in tumor suppression.

Ruthenium anticancer drugs.

Alessio E, Mestroni G, Bergamo A, Sava G.
Department of Chemical Sciences, University of Trieste, Via L. Giorgieri 1, I-34127 Trieste, Italy. alessi@univ.trieste.it
Met Ions Biol Syst.;42:323-51, 2004.

ABSTRACT

A review paper. No abstract available.

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

Sava G, Frausin F, Cocchietto M, Vita F, Podda E, Spessotto P, Furlani A, Scarcia V, Zabucchi G.
Department of Biomedical Sciences, University of Trieste, Via L. Giorgieri 7, 34127 Trieste, Italy. g.sava@callerio.org
Eur J Cancer.; 40(9):1383-96,

ABSTRACT

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.

Electrochemical measurements confirm the preferential bonding of the antimetastatic complex [ImH][RuCl(4)(DMSO)(Im)] (NAMI-A) with proteins and the weak interaction with nucleobases.

Ravera M, Baracco S, Cassino C, Colangelo D, Bagni G, Sava G, Osella D.
Dipartimento Scienze dell'Ambiente e della Vita, Universita del Piemonte Orientale Amedeo Avogadro, Spalto Marengo 33, 15100 Alessandria, Italy.
J Inorg Biochem.; 98(6):984-90,

ABSTRACT

An electrochemical and biological study of interaction between the prototypical antimetastatic drug imidazolium trans-tetrachlorodimethylsulfoxideimidazoleruthenate (III) complex, [ImH][RuCl(4)(DMSO)(Im)] (DMSO = dimethylsulfoxide, Im = imidazole), nicknamed NAMI-A, and several biomolecules, namely DNA, bovine (BSA) and human (HSA) serum albumin, is reported. Electrochemistry offers great advantages over the existing devices based on optical techniques, since it provides rapid, simple, and low-cost information whether the interaction occurs or not. Moreover, we describe some biochemical assays to test the interaction of NAMI-A with ribonucleoprotein telomerase and protein Taq polymerase. All the data confirm the preferential interaction of NAMI-A with proteins with respect to nucleotides, especially when compared with the behaviour of the well-known alkylating drug cisplatin in the presence of the same targets.

Hyaluronic-acid butyric esters as promising antineoplastic agents in human lung carcinoma: a preclinical study.

Coradini D, Pellizzaro C, Abolafio G, Bosco M, Scarlata I, Cantoni S, Stucchi L, Zorzet S, Turrin C, Sava G, Perbellini A, Daidone MG.
Unit of Biomolecular Determinants in Prognosis and Therapy, Experimental Department, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy.
danila.coradini@istitutotumori.mi.it
Invest New Drugs.; 22(3):207-17.

ABSTRACT

New promising compounds, derived from the esterification of hyaluronic acid with butyric acid, were investigated in vitro on a non-small cell lung carcinoma cell line (NCI-H460) and an its metastatic subclone (NCI-H460M). All new compounds exerted a dose-dependent inhibitory effect on both cell lines, which expressed CD44, the specific surface receptor for hyaluronic acid, in a very high percentage of cells (90%). HE1, the most effective of these compounds, was 10-fold more effective than sodium butyrate (NaB) in inhibiting cell proliferation. Similarly to NaB, after 24 hours of treatment, HE1 affected the expression of three cell cycle-related proteins (p27(kip1), p53 and p21(waf1)) responsible for growth arrest, indicating that the presence of the hyaluronic acid backbone does not interfere with the biologic activity. Intratumoral treatment with HE1 demonstrated a marked efficacy on primary tumor growth and on lung metastases formation of the murine Lewis Lung Carcinoma model. Altogether, present findings suggest a possible clinical application of these novel butyric pro-drugs in primary and metastatic lung cancer.

Intratumoral NAMI-A Treatment Triggers Metastasis Reduction, Which Correlates to CD44 Regulation and Tumor Infiltrating Lymphocyte Recruitment.

Pacor S, Zorzet S, Cocchietto M, Bacac M, Vadori M, Turrin C, Gava B, Castellarin A, Sava G.
Department of Biomedical Sciences, University of Trieste, via L. Giorgieri 7-9, 34127 Trieste, Italy. pacorsab@univ.trieste.it
J Pharmacol Exp Ther.; 310(2):737-44, Aug 2004.

ABSTRACT

Intratumor (i.t.) injection of 35 mg/kg/day NAMI-A for six consecutive days to CBA mice bearing i.m. implants of MCa mammary carcinoma reduces primary tumor growth and particularly lung metastasis formation, causing 60% of animals to be free of macroscopically detectable metastases. The i.t. treatment allows study of the effects of NAMI-A on in vivo tumor cells exposed to millimolar concentrations for a relatively prolonged time. Under these conditions, NAMI-A reduces the number of CD44+ tumor cells and changes tumor cell phenotype to a lower aggressive behavior, as shown by scanning electron microscopy analysis. On primary tumor site, NAMI-A causes unbalance between 2n and aneuploid cells in favor of lymphocytes. Furthermore, in tumor tissue, nitric oxide production is increased and active matrix metalloproteinase 9 is decreased, and these effects are accompanied by a reduced hemoglobin concentration. These data are in agreement with the reduction of tumor invasion and metastasis and suggest the therapeutic usefulness of NAMI-A in neoadjuvant or tumor reduction treatments for preventing metastasis formation. These data further stress the usefulness of intratumor treatments as experimental preclinical model for studying in vivo the mechanism of tumor cell interactions after prolonged exposure to ruthenium-based compounds to be developed for metastasis inhibition.

Structure-dependent in vitro cytotoxicity of the isomeric complexes [Ru(L)(2)Cl(2)] (L= o-tolylazopyridine and 4-methyl-2-phenylazopyridine) in comparison to [Ru(azpy)(2)Cl(2)].

Hotze AC, Caspers SE, de Vos D, Kooijman H, Spek AL, Flamigni A, Bacac M, Sava G, Haasnoot JG, Reedijk J.
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300 RA, Leiden, The Netherlands.
J Biol Inorg Chem.; 9(3):354-64, Apr 2004.

ABSTRACT

The dichlorobis(2-phenylazopyridine)ruthenium(II) complexes, [Ru(azpy)(2)Cl(2)], are under renewed investigation due to their potential anticancer activity. The three most common isomers alpha-, beta- and gamma-[RuL(2)Cl(2)] with L= o-tolylazopyridine (tazpy) and 4-methyl-2-phenylazopyridine (mazpy) (alpha indicating the coordinating Cl, N(pyridine) and Nazo atoms in mutual cis, trans, cis positions, beta indicating the coordinating Cl, N(pyridine) and Nazo atoms in mutual cis, cis, cis positions, and gamma indicating the coordinating Cl, N(pyridine) and Nazo atoms in mutual trans, cis, cis positions) are synthesized and characterized by NMR spectroscopy. The molecular structures of gamma-[Ru(tazpy)(2)Cl(2)] and alpha-[Ru(mazpy)(2)Cl(2)] are determined by X-ray diffraction analysis. The IC(50) values of the geometrically isomeric [Ru(tazpy)(2)Cl(2)] and [Ru(mazpy)(2)Cl(2)] complexes compared with those of the parent [Ru(azpy)(2)Cl(2)] complexes are determined in a series of human tumour cell lines (MCF-7, EVSA-T, WIDR, IGROV, M19, A498 and H266). These data unambiguously show for all complexes the following trend: the alpha isomer shows a very high cytotoxicity, whereas the beta isomer is a factor 10 less cytotoxic. The gamma isomers of [Ru(tazpy)(2)Cl(2)] and [Ru(mazpy)(2)Cl(2)] display a very high cytotoxicity comparable to that of the gamma isomer of the parent compound [Ru(azpy)(2)Cl(2)] and to that of the alpha isomer. These biological data are of the utmost importance for a better understanding of the structure-activity relationships for the isomeric [RuL(2)Cl(2)] complexes.

Synthesis and chemical-pharmacological characterization of the antimetastatic NAMI-A-type Ru(III) complexes (Hdmtp)[trans-RuCl4(dmso-S)(dmtp)], (Na)[trans-RuCl4(dmso-S)(dmtp)], and [mer-RuCl3(H2O)(dmso-S)(dmtp)] (dmtp = 5,7-dimethyl[1,2,4]triazolo[1,5-a]pyrimidine).

Velders AH, Bergamo A, Alessio E, Zangrando E, Haasnoot JG, Casarsa C, Cocchietto M, Zorzet S, Sava G.
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
a.velders@chem.leidenuniv.nl
J Med Chem.;47(5):1110-21., Feb 26 2004.

ABSTRACT

Ruthenium compounds have gained large interest for their potential application as chemotherapeutic agents, and in particular the complexes of the type (X)[trans-RuCl4(dmso-S)L] (X = HL or Na, NAMI-A or NAMI, respectively, for L = imidazole) are under investigation for their antimetastatic properties. The NAMI(-A)-like compounds are prodrugs that hydrolyze in vivo, and the investigation of their hydrolytic properties is therefore important for determining the nature of the potential active species. The NAMI-A-type Ru(III) complex 1, (Hdmtp)[trans-RuCl4(dmso-S)(dmtp)] (dmtp is 5,7-dimethyl[1,2,4]triazolo[1,5-a]pyrimidine), and the corresponding sodium analogue 2, (Na)[trans-RuCl4(dmso-S)(dmtp)], were synthesized. The hydrolyses of 1 and 2 in water as well as in buffered solutions were studied, and the first hydrolysis product, [mer-RuCl3(H2O)(dmso-S)(dmtp)].H2O (3), was isolated and characterized. The molecular structures of 1 and 3 were determined by single-crystal X-ray diffraction analyses and prove the importance of the hydrogen-bonding properties of dmtp to stabilize hydrolysis products. In vitro 1 (a) is not cytotoxic on tumor cells, following challenges from 1 to 72 h and concentrations up to 100 microM, (b) inhibits matrigel invasion at 0.1 mM and MMP-9 activity with an IC50 of about 1 mM, and (c) is devoid of pronounced effects on cell distribution among cell cycle phases. In vivo compound 1, similar to NAMI-A, significantly inhibits metastasis growth in mice bearing advanced MCa mammary carcinoma tumors. In the lungs, 1 is significantly less concentrated than NAMI-A, whereas no differences between these two compounds were found in other organs such as tumor, liver, and kidney. However, 1 caused edema and necrotic areas on liver parenchyma that are more pronounced than those caused by NAMI-A. Conversely, glomerular and tubular changes on kidney are less extensive than with NAMI-A. In conclusion, 1 confirms the excellent antimetastatic properties of this class of NAMI-A-type compounds and qualifies as an interesting alternative to NAMI-A for treating human cancers.

The hydrolysis of the anti-cancer ruthenium complex NAMI-A affects its DNA binding and antimetastatic activity: an NMR evaluation.

Bacac M, Hotze AC, Schilden K, Haasnoot JG, Pacor S, Alessio E, Sava G, Reedijk J.
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300, RA Leiden, The Netherlands.
J Inorg Biochem.;98(2):402-12,Feb 2004.

ABSTRACT

The coordination of the antimetastatic agent NAMI-A, [H(2)im][trans-RuCl(4)(dmso-S)(Him)], (Him=imidazole; dmso=dimethyl sulfoxide), to the DNA model base 9-methyladenine (9-MeAde) was investigated in water. NMR spectroscopy was first applied for the study of the molecular stability and hydrolysis of NAMI-A in aqueous solution over a range of pH (3.0-7.4) and chloride ion concentrations (0-1 M) at 37.0 degrees C. In physiological conditions (phosphate buffer, pH 7.4) NAMI-A disappears from the solution in 15 min due to chloride and dmso hydrolysis, leading to uncharacterised poly-oxo Ru species. Conversely, at lower pH (3.0-6.0) and in water (pH approximately 5.5), only a partial dmso hydrolysis occurs, slowly forming the [trans-RuCl(4)(H(2)O)(Him)](-) complex. This latter species coordinates to 9-MeAde (via the N7 of 9-MeAde), forming the [trans-RuCl(4)(9-MeAde)(Him)](-) complex. NAMI-A and [trans-RuCl(4)(H(2)O)(Him)](-) give comparable intracellular ruthenium concentrations and accumulate in KB cells (human mouth carcinoma) and accumulate these at the G(2)/M phase, while poly-oxo Ru species do not, and their cell uptake is reduced to 50%. On the contrary, G(2)/M arrest and protein content in the murine metastatic cell line metGM, are not influenced by NAMI-A hydrolysis. Hydrolysed NAMI-A species apparently are easier taken up by the metGM cells, showing intracellular ruthenium concentrations one order of magnitude greater than those of intact NAMI-A. Therefore, it is proposed that the selective antimetastatic activity of NAMI-A during in vivo experiments can be attributed to its hydrolysed species.

Solution, solid state and biological characterization of ruthenium(III)-DMSO complexes with purine base derivatives.

Turel I, Pecanac M, Golobic A, Alessio E, Serli B, Bergamo A, Sava G.
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva 5, 1000, Ljubljana, Slovenia.
J Inorg Biochem.;98(2):393-401, Feb 2004.

ABSTRACT

Two new complexes of Ru(III) with purine base derivatives, [mer-RuCl(3)(acv)(DMSO-S)(C(2)H(5)OH)].C(2)H(5)OH (1) (acv=acyclovir, DMSO=dimethyl sulfoxide) and [trans-RuCl(4)(guaH)(DMSO-S)].2H(2)O (2) (guaH=protonated molecule of guanine), were prepared from the same Ru(III) precursor, [trans-RuCl(4)(DMSO-S)(2)](-), by substitution of one DMSO-S. Coordination of acv induced also replacement of one chloride by an ethanol molecule. This reactivity difference was explained by striking contrasts in the hydrogen bonding schemes of the two complexes, evidenced in their X-ray crystal structures. In 1 the guanine derivative acyclovir is coordinated to ruthenium through the N(7) atom, while in 2 the protonated guanine molecule is bound through the N(9) atom. Both complexes were also characterized by various physico-chemical methods in the solid state and in the solution. In vitro, the biological activity of 2 and of the previously described complexes [mer-RuCl(3)(acv)(DMSO-S)(CH(3)OH)].0.5CH(3)OH (3) and [mer-RuCl(3)(acv)(DMSO-S)(H(2)O)].H(2)O (4) on tumour cells appear to be very similar to that of NAMI-A (NAMI-A=[ImH][trans-RuCl(4)(DMSO-S)Im]). All compounds are only weakly active on tumour cell proliferation but show an interesting proadhesive effect that suggest possible activity on tumour malignancy.

Synthesis, characterization and biological activity of copper complexes with pyridoxal thiosemicarbazone derivatives. X-ray crystal structure of three dimeric complexes.

Ferrari MB, Bisceglie F, Pelosi G, Tarasconi P, Albertini R, Dall'Aglio PP, Pinelli S, Bergamo A, Sava G.
Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica e Chimica Fisica, Parco Area delle Scienze 17 A, Universita di Parma, I-43100, Parma, Italy
J Inorg Biochem.;98(2):301-312, Feb 2004.

ABSTRACT

A dimeric copper complex of the unsubstituted pyridoxal thiosemicarbazone (H(2)L), [{Cu(HL)(OH(2))}(2)]Cl(2).2H(2)O, previously tested on Friend murine cell lines has been recently resynthesized to evaluate its behavior on different murine and human leukemic cell lines and has been compared, in vitro and in vivo, with its monomeric counterpart [Cu(H(2)L)(OH(2))Cl]Cl. On TS/A murine adenocarcinoma cell line in vitro, both compounds significantly inhibit cell proliferation at micromolar concentrations, although the dimeric compound is more active. Despite this cytotoxicity they lack activity on TLX5 lymphoma. The unsubstituted dimeric [{Cu(HL)(OH(2))}(2)]Cl(2).2H(2)O induces apoptosis on CEM and U937 human cell lines, with IC(50) concentrations of 1.2x10(-5) and 6.7x10(-6) M, respectively, but it is inactive on K562. Moreover, it alters significantly the cell cycle of U937 and CEM lines and decreases the telomerase activity of U937.To verify if other dimeric copper complexes show relevant biological activity new complexes with N-substituted pyridoxal thiosemicarbazones have been synthesized and characterized using spectroscopic techniques. Three of them, namely [Cu(Me(2)-HL)Cl](2).6H(2)O (Me(2)-H(2)L=pyridoxal N1,N1-dimethylthiosemicarbazone) (1), [Cu(MeMe-HL)Cl](2)Cl(2).4H(2)O (MeMe-HL=pyridoxal N1,N2-dimethylthiosemicarbazone) (2), [Cu(Et-H(2)L)Cl](2)Cl(2).2H(2)O (Et-H(2)L=pyridoxal N1-ethylthiosemicarbazone) (3), were also characterized by X-ray diffractometry. These complexes are dimeric and all three present a square pyramidal coordinative geometry with the ligand showing an SNO tridentate behavior. Their biological activities have been tested in vitro on U937, CEM and K562 cell lines to ascertain their effectiveness in comparison to the corresponding unsubstituted complex [{Cu(HL)(OH(2))}(2)]Cl(2).2H(2)O. Compound 1 shows weak proliferation inhibition on all three cell lines, but it does not induce apoptosis and it does not inhibit telomerase activity, compound 2 is not effective at low concentration and is toxic at higher doses; compound 3 inhibits CEM cell growth better than complex 1 but it does not exert any other biological effect.

Reduction of in vivo lung metastases by dinuclear ruthenium complexes is coupled to inhibition of in vitro tumour invasion.

Bergamo A, Stocco G, Casarsa C, Cocchietto M, Alessio E, Serli B, Zorzet S, Sava G.
Foundation Callerio-Onlus, I-34127 Trieste, Italy. a.bergamo@callerio.org
Int J Oncol.;24(2):373-9, Feb 2004.

ABSTRACT

Mononuclear ruthenium-dmso compounds showed interesting antimetastatic properties on experimental models of solid tumours. In line with the interesting results with multinuclear platinum complexes, which proved to overcome cisplatin resistance, we thought it worthwhile to test the pharmacological properties of some dinuclear ruthenium complexes to ascertain the possible advantages due to the introduction of a second metal centre over NAMI-A and its mononuclear analogues. These compounds belong to the general formula X2[[RuCl4(dmso-S)]2(mu-L)] or [X][[RuCl4(dmso-S)](mu-L)[RuCl3(dmso-S)(dmso-O)]] where L is a nitrogen donor ligand (pyrazine; pyrimidine; 4,4'-bipyridine; 1,2-bis(4-pyridyl)ethane; 1,2-bis(4-pyridyl) ethylene; 1,3-bis(4-pyridyl)propane) and X a counterion. We focused on parameters related to metastatic ability such as gelatinase activity, detected by zymography, and invasive potential, measured by means of a transwell chamber. These activities were correlated to the ability to inhibit tumour metastases in vivo. All dinuclear complexes, except compound D8 ([NH4]2[[RuCl4(dmso-S)]2(mu-pyz]), decrease the number of tumour cells that cross a matrigel barrier, and inhibit MMP-9 gelatinolytic activity at concentrations lower than that of NAMI-A and of other mononuclear ruthenium complexes. In vivo compounds D5 (Na2[[RuCl4(dmso-S)]2(mu-ethylbipy)]) and D7 ([NH4][[RuCl4(dmso-S)](mu-pyz)[RuCl3(dmso-S) (dmso-O)]]) show anti-metastasis activity, at two dose levels, with mild or null effect on primary tumour growth; compound D8 is the weakest active. All compounds tend to accumulate in liver and kidneys, rather than in tumour and lungs. However, compound D5, the most active in vitro on invasion and gelatinases and active in vivo on metastasis, is better concentrated in the lungs than compound D8 which is less active or inactive in vitro and in vivo. Histological analysis show liver, as well as kidney toxicities that limit in vivo activity. These data thus suggest dinuclear ruthenium complexes as promising anti-invasive agents for cancer treatment.

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

Bergamo A, Messori L, Piccioli F, Cocchietto M, Sava G.
Callerio Foundation Onlus, Via A. Fleming 22-31, 34127 Trieste, Italy. a.bergamo@callerio.org
Invest New Drugs.;21(4):401-115, Nov 2003.

ABSTRACT

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.

NAMI-A inhibits the PMA-induced ODC gene expression in ECV304 cells: involvement of PKC/Raf/Mek/ERK signalling pathway.

Debidda M, Sanna B, Cossu A, Posadino AM, Tadolini B, Ventura C, Pintus G.
Department of Biomedical Sciences, Division of Biochemistry, Laboratory of Cardiovascular Research, National Institute of Biostructures and Biosystems, University of Sassari, I-07100 Sassari, Italy
Int J Oncol.;23(2):477-82, Aug 2003.

ABSTRACT

Imidazolium trans-imidazole dimethyl sulfoxide tetrachlororuthenate (NAMI-A) is a new compound active against lung metastasis of solid metastasizing tumours. While its in vivo effect has been studied, the molecular insights that underlie its action are largely unknown. Among the possible pathways responsible for malignant transformation, PKC arose as one of the most promising targets for new antineoplastic drugs. We demonstrated the capability of NAMI-A of inhibiting PMA induced-PKC activity in ECV304 in a dose-dependent fashion. Furthermore, NAMI-A through modulation of PKC activity has been proved capable of reducing the phorbol ester induced expression of ornithine decarboxilase (ODC) gene and to abrogate the activation of the Raf/MEK/ERK pathway. Taken together these results suggest that many of the in vivo outcomes of NAMI-A treatment may be the result of a direct action on PKC.

Antiangiogenic properties of selected ruthenium(III) complexes that are nitric oxide scavengers.

Morbidelli L , Donnini S, Filippi S, Messori L, Piccioli F, Orioli P, Sava G, Ziche M.
British Journal of Cancer; 88: 1484  1491, 2003.

ABSTRACT

The nitric oxide synthase (NOS) pathway has been clearly demonstrated to regulate angiogenesis. Increased levels of NO correlate with tumour growth and spreading in different experimental and human cancers. Drugs interfering with the NOS pathway may be useful in angiogenesis-dependent tumours. The aim of this study was to pharmacologically characterise certain ruthenium-based compounds, namely NAMI-A, KP1339, and RuEDTA, as potential NO scavengers to be used as antiangiogenic/antitumour agents. NAMI-A, KP1339 and RuEDTA were able to bind tightly and inactivate free NO in solution. Formation of ruthenium NO adducts was documented by electronic absorption, FT-IR spectroscopy and 1H-NMR. Pretreatment of rabbit aorta rings with NAMI-A, KP1339 or RuEDTA reduced endothelium-dependent vasorelaxation elicited by acetylcholine. This effect was reversed by 8-BrcGMP. The key steps of angiogenesis, endothelial cell proliferation and migration stimulated by vascular endothelial growth factor (VEGF) or NO donor drugs, were blocked by NAMI-A, KP1339 and RuEDTA, these compounds being devoid of any cytotoxic activity. When tested in vivo, NAMI-A inhibited angiogenesis induced by VEGF. It is likely that the antitumour properties previously observed for ruthenium-based NO scavengers, such as NAMI-A, are related to their NO-related antiangiogenic properties.

Development of a LC method for pharmaceutical quality control of the antimetastatic ruthenium complex NAMI-A.

Bouma M, Nuijen B, Jansen MT, Sava G, Picotti F, Flaibani A, Bult A, Beijnen JH.
J Pharm Biomed Anal 26; 31(2):215-28, Feb 2003.

ABSTRACT

Imidazolium trans-tetrachloro(dimethylsulfoxide)imidazoleruthenium(III) (NAMI-A) is a novel ruthenium complex with selective activity against metastases currently in Phase I clinical trials in the Netherlands. Pharmaceutical quality control of NAMI-A drug substance and lyophilized product warranted the development of an assay for determination and quantification of NAMI-A and degradation products. A high performance liquid chromatography (HPLC) method was developed, consisting of a C18 column with 0.50 mM sodium dodecylsulfate in 3% methanol at pH 2.5 (acidified using trifluoromethanesulfonic acid) as the mobile phase and UV-detection at 358 nm. The HPLC method was proven to be linear, accurate and precise. Stress testing showed that degradation products were separated from the parent compound. By combining results of nuclear magnetic resonance (NMR) and HPLC experiments, one degradation product was identified as the mono-hydroxy species of NAMI-A. HPLC analysis with off-line detection of the eluate with flameless atomic absorption spectrometry (F-AAS) showed that under most conditions, all ruthenium-containing compounds show a peak in the HPLC chromatogram and that all ruthenium applied to the column is recovered quantitatively. For completely degraded solutions of NAMI-A some ruthenium is retained on the column. Suitability of the HPLC method for the pharmaceutical quality control of NAMI-A lyophilized product was demonstrated.

Molecular structure, solution chemistry and biological properties of the novel [ImH][trans-IrCl(4)(Im)(DMSO)], (I) and of the orange form of [(DMSO)(2)H][trans-IrCl(4)(DMSO)(2)], (II), complexes.

Messori L, Marcon G, Orioli P, Fontani M, Zanello P, Bergamo A, Sava G, Mura P.
J Inorg Biochem 1; 95(1):37-46, May 2003.

ABSTRACT

The new iridium(III) complex, imidazolium[trans(DMSO,imidazole)tetrachloroiridate(III)], (I) (DMSO=dimethyl sulfoxide), and the orange form of [(DMSO)(2)H][trans(DMSO)(2)-tetrachloroiridate(III)], (II) have been prepared and characterized, both in the solid state and in solution, by X-ray diffraction and by various physicochemical techniques. Single crystal X-ray diffraction studies point out that complex (II) is isomorphous to the ruthenium(III) analogue, [(DMSO)(2)H][trans-RuCl(4)(DMSO)(2)], (III). Crystallographic data are the following: a=16.028(2) A, b=24.699(3) A, c=8.262(1) A, in space group Pbca (Z=8) for (imidazolium) [trans(DMSO,imidazole)tetrachloroiridate(III)], (I); and a=9.189(2) A, b=16.511(4) A, c=14.028(3) A, beta=100.82(2) degrees in space group P2/n (Z=4) for [(DMSO)(2)H][trans(DMSO)-(2)tetrachloroiridate(III)], (II). Visible absorption spectra show that both complexes are stable for several days, at pH 7.4, at room temperature. No significant chloride hydrolysis is observed, even at high temperature (70 degrees C), over 24 h. The extreme stability of these iridium(III) complexes within a physiological buffer was further assessed by (1)H NMR; in addition, cyclic voltammetry measurements evidenced a high stability of the oxidation state +3. Preliminary biological studies show that both complexes do not bind appreciably bovine serum albumin nor inhibit significantly the proliferation of representative human tumor cell lines, suggesting that hydrolysis of coordinated chlorides is a crucial feature for the biological properties and the antitumor activity of the parent ruthenium(III) complexes.

Distinct effects of dinuclear ruthenium(III) complexes on cell proliferation and on cell cycle regulation in human and murine tumor cell lines.

Bergamo A, Stocco G, Gava B, Cocchietto M, Alessio E, Serli B, Iengo E, Sava G.
J Pharmacol Exp Ther; 305(2):725-32, May 2003.

ABSTRACT

We have examined the biological and antitumor activity of a series of dinuclear ruthenium complexes. The aim of this study was to compare the in vitro effects of these new compounds on cell proliferation, cell distribution among cell cycle phases, and the expression of some proteins involved in cell cycle regulation. Results obtained show a mild cytotoxic activity against human and murine cell lines, more evident after prolonged exposure of cell challenge. Two of the eight dinuclear complexes [namely, compounds D3 (Na(2)[(RuCl(4)(dmso-S))(2)(mu-bipy)]) and D7 ([NH(4)][(RuCl(4)(dmso-S))(mu-pyz)(RuCl(3)(dmso-S)(dmso-O))]) modify cell cycle distribution similarly to imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate (NAMI-A), whereas the others have a low or negligible effect on this parameter. If we correlate the induction of cell cycle modifications with ruthenium uptake by tumor cells and with the modulation of proteins regulating cell cycle, we may stress that the induction of G(2)-M cell cycle arrest is related to the achievement of a threshold concentration of ruthenium inside the cells, which is dependent on the cell line being used, and that only cyclin B, among cell cycle regulating proteins examined by immunoblotting assays, appears to be significantly modified. This in vitro study shows that dinuclear ruthenium complexes may have a behavior similar to that of the monomer NAMI-A. These results encourage the future experimentation of their pharmacological properties in in vivo models.

Dual Action of NAMI-A in Inhibition of Solid Tumor Metastasis: Selective Targeting of Metastatic Cells and Binding to Collagen.

Sava G, Zorzet S, Turrin C, Vita F, Soranzo M, Zabucchi G, Cocchietto M, Bergamo A, DiGiovine S, Pezzoni G, Sartor L, Garbisa S.
Clin Cancer Res; 9(5):1898-1905, May 2003.

ABSTRACT

NAMI-A is a ruthenium complex endowed with a selective effect on lung metastases of solid metastasizing tumors. The aim of this study is to provide evidence that NAMI-A's effect is based on the selective sensitivity of the metastasis cell, as compared with other tumor cells, and to show that lungs represent a privileged site for the antimetastatic effects. The transplantation of Lewis lung carcinoma cells, harvested from the primary tumor of mice treated with 35 mg/kg/day NAMI-A for six consecutive days, a dose active on metastases, shows no change in primary tumor take and growth but a significant reduction in formation of spontaneous lung metastases. Transmission electron microscopy examination of lungs and kidney shows NAMI-A to selectively bind collagen of the lung extracellular matrix and also type IV collagen of the basement membrane of kidney glomeruli. The half lifetime of NAMI-A elimination from the lungs is longer than for liver, kidney, and primary tumor. NAMI-A bound to collagen is active on tumor cells as shown in vitro by an invasion test, using a modified Boyden chamber and Matrigel, and it inhibits the matrix metallo-proteinases MMP-2 and MMP-9 at micromolar concentrations, as shown in vitro by a zimography test. These data show NAMI-A to significantly affect tumor cells with metastatic ability. Binding to collagen allows NAMI-A to exert its selective activity on metastatic cells during dissemination and particularly in the lungs. These data also stress the wide spectrum of daily doses and treatment schedules at which NAMI-A is active against metastases.

Primary tumor, lung and kidney retention and antimetastasis effect of NAMI-A following different routes of administration.

Cocchietto M., Zorzet S., Sorc A., Sava G.
Investigational New Drugs; 21: 55-62, 2003.

ABSTRACT

Imidazolium-trans-dimethylsulfoxideimidazoletetrachlororuthenate (NAMI-A) is a ruthenium compound effective on solid tumor metastases. In this study, we evaluated the effects of different routes of administration of NAMI-A on the distribution to primary tumor, lungs and kidneys in BD2F1 hybrids with Lewis lung carcinoma or in CBA inbred mice with MCa mammary carcinoma. NAMI-A concentration and the percentage of cumulative dose (%D_tot) retained in these tissues is indipendent of the animal strain and of the tumor model used. Also the presence of the tumor does not change the distribution of NAMI-A in the lungs and in the kidneys. A dose-dependent antimetastatic effect is evident with intraperitoneal (i.p.) treatments at three different doses. Treatment of tumor bearing mice with NAMI-A administrered i.p., per os or by aerosol showed a similar effect on lung metastases, although the concentration of ruthenium reached in the lungs was markedly different. On the basis of the data obtained, we can conclude that the antimetastatic effects are related to the amount of NAMI-A administered, rather than to the lung' s concentration of the compound.

Synthesis, catalytic properties and biological activity of new water soluble ruthenium cyclopentadienyl PTA complexes [(C5R5)RuCl(PTA)2] (R= H, Me; PTA= 1,3,5-triaza-7-phosphaadamantane).

Akbayeva DN, Gonsalvi L, Oberhauser W, Peruzzini M, Vizza F, Brugeller P, Romerosa A, Sava G, Bergamo A.
Chem Commun; 264-265, 2003.

ABSTRACT

The new water soluble ruthenium complexes [(C5R5)RuCl(PTA)2] (R = H, Me; PTA = 1,3,5-triaza-7- phosphaadamantane) were synthesised and characterised.
Their evaluation as regioselective catalysts for hydrogenation of unsaturated ketones in aqueous biphasic conditions and as cytotoxic agents towards the TS/A adenocarcinoma cell line is briefly presented.

Inhibition of the MEK/ERK signaling pathway by the novel antimetastatic agent NAMI-A down regulates c- myc gene expression and endothelial cell proliferation.

Gianfranco Pintus, Bruna Tadolini, Anna Maria Posadino, Bastiano Sanna, Marcella Debidda, Federico Bennardini, Gianni Sava and Carlo Ventura.
Eur.J Biochem; 269: 5861 5870, 2002.

ABSTRACT

Imidazolium trans-imidazoledimethyl sulfoxide-tetrachlo-roruthenate (NAMI-A) is a novel ruthenium-containing experimental antimetastatic agent. Compelling evidence ascribes a pivotal role to endothelial cells in the orchestration of tumor angiogenesis and metastatic growth, suggesting antiangiogenic therapy as an attractive approach for anticancer treatment. In this context, activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway has been found fundamental in transducing extracellular stimuli that modulate a number of cellular process including cell proliferation, migration and invasion. Here we show that exposure of the transformed endothelial cell line ECV304 to NAMI-A significantly inhibited DNA synthesis, as well as the expression of the proliferating cell nuclear antigene (PCNA). These responses were associated with a marked down-regulation of ERK phosphorylation in serum-cultured cells. In addition, NAMI-A markedly reduced
serum stimulated- and completely suppressed phorbol 12-myristate 13-acetate (PMA)-triggered MAPK/ERK kinase activity. NAMI-A was also able to inhibit the phosphorylation of MEK, the upstream activator of ERK, and, similar to both the protein kinase C (PKC) inhibitor GF109203X and the MAPK/ERK (MEK) inhibitor PD98059, it completely counteracted PMA-induced ERK phosphorylation. Finally,NAMI-A and PD98059 down regulated c-myc gene expression to the same extent in serum-cultured cells and dose-dependently counteracted, and ultimately abolished, the increase in c-myc gene expression elicited by PMA in serum-free cells.These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating c-myc gene expression and ECV304 proliferation.

Tumour cell uptake of the metastasis inhibitor ruthenium complex NAMI-A and its in vitro effects on KB cells.

Fabiana Frausin, Moreno Cocchietto, Alberta Bergamo, Vito Scarcia, Ariella Furlani, Gianni Sava
Cancer Chemother Pharmacol; 50: 405-411, 2002.

ABSTRACT

Purpose:The uptake of NAMI-A (imidazolium trans -imidazoledimethylsulphoxidetetrachlororuthenate) by KB cells in vitro was compared with the effects of this compound on the cell cycle phase distribution of the cells. Methods:NAMI-A uptake was determined by flameless atomic absorption spectroscopy, and the cell cycle phase distribution was determined by flow cytometry.Results:NAMI-A uptake was proportional to its concentration in the incubation medium.The use of a number of incubation conditions showed that NAMI-A uptake from MEM was independent of the presence of serum and dependent on the presence of amino acids in the incubation medium,and that NAMI- A uptake was markedly higher when the cells were incubated in PBS.The uptake increase observed in PBS did not occur when the cells were kept at 0 4° C, suggesting the presence of active transportation of NAMI-A into cells.In addition,the presence of divalent cations such as Ca²⁺ and Mg²⁺ ,appeared to facilitate NAMI-A uptake.The anionic substance transport inhibitor probenecid significantly reduced the active transportation of NAMI-A into cells.The effects of NAMI-A on cell cycle distribution were strictly dependent on its uptake by tumour cells and not on its extracellular concentration. Conclusions:These findings suggest the interaction of NAMI-A with biological components resulting in possible consequences for the distribution of the compound itself. Furthermore, NAMI-A enters tumour cells both by passive diffusion and by active transportation.

Ruthenium-based NAMI-A type complexes with in vivo selective metastasis reduction and in vitro invasion inhibition unrelated to cell cytotoxicity.

Bergamo A, Gava B, Alessio E, Mestroni G, Serli B, Cocchietto M, Zorzet S, Sava G
Int J Oncol ;21(6):1331-8, Dec 2002.

ABSTRACT

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.

A kinetic study of the chemical stability of the antimetastatic ruthenium complex NAMI-A.

Bouma M, Nuijen B, Jansen MT, Sava G, Flaibani A, Bult A, Beijnen JH

Department of Pharmacy and Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute, Louwesweg 6, 1066 EC, Amsterdam, The Netherlands.
Int J Pharm 6;248(1-2):239-46, Nov 2002.

ABSTRACT

NAMI-A is a novel ruthenium complex with selective activity against cancer metastases currently in Phase I clinical trials in The Netherlands. The chemical stability of this new agent was investigated utilizing a stability-indicating reversed-phase high performance liquid chromatographic assay with ultraviolet detection and ultraviolet/visible light spectrophotometry. The degradation kinetics of NAMI-A were studied as a function of pH, buffer composition, and temperature. Degradation of NAMI-A follows first-order kinetics at pH<6 and zero-order kinetics at pH >/=6. A pH-rate profile, employing rate constants extrapolated to zero buffer concentration, was constructed, demonstrating that NAMI-A is most stable in pH region 3-4. The degradation rate is not significantly affected by specific buffer components. Storage temperature strongly influences the degradation rate.

Pharmaceutical development of a parenteral lyophilized formulation of the antimetastatic ruthenium complex NAMI-A.

Bouma M, Nuijen B, Sava G, Perbellini A, Flaibani A, van Steenbergen MJ, Talsma H, Kettenes-van den Bosch JJ, Bult A, Beijnen JH
Department of Pharmacy and Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute, Louwesweg 6, 1066 EC, Amsterdam, The Netherlands.
Int J Pharm 6;248(1-2):247-59, Nov 2002.

ABSTRACT

This paper describes the development of a stable pharmaceutical dosage form for NAMI-A, a novel antimetastatic ruthenium complex, for Phase I testing. NAMI-A drug substance was characterized using several spectrometric and chromatographic techniques. In preformulation studies, it was found that NAMI-A in aqueous solution was not stable enough to allow sterilization by moist heat. The effect of several excipients on the stability of the formulation solution was investigated. None of them provided sufficient stability to allow long-term storage of an aqueous solution of NAMI-A. Therefore, a lyophilized product was
developed. Five different formulations were prepared and subjected to thermogravimetric (TG) analysis and stability studies at various conditions for 1 year. Minimal degradation during the production process is achieved with a formulation solution of pH 3-4. Of the acids tested, only hydrochloric acid (HCl 0.1 mM) both stabilized the formulation solution and was compatible with the
lyophilized product. This product was stable for at least 1 year when stored at -20 degrees C, 25 degrees C/60% relative humidity (RH) and 40 degrees C/75% RH, and was also photostable.

Photostability profiles of the experimental antimetastatic ruthenium complex NAMI-A.

Bouma M, Nuijen B, Jansen MT, Sava G, Bult A, Beijnen JH.
Slotervaart Hospital/The Netherlands Cancer Institute, Department of Pharmacy and Pharmacology, Louwesweg 6, 1066 EC, Amsterdam, The Netherlands.
J Pharm Biomed Anal 7;30(4):1287-96, Nov 2002.

ABSTRACT

NAMI-A is a novel ruthenium complex with selective activity against metastases currently in Phase I clinical trials in The Netherlands. The photostability of this new agent in solid state and in solution has been investigated utilizing a stability-indicating reversed-phase high performance liquid chromatographic (HPLC) assay and ultraviolet/visible (UV/VIS) light spectrophotometry. In solid state, NAMI-A proved to be photostable. In solution, however, the compound degraded rapidly, in a pH-independent manner in the pH range of 2-5. At alkaline pH, the degradation rate was higher than at acidic pH. The type of buffer species had little influence. NAMI-A concentration was inversely related to the photostability. Addition of photostabilizers (5% DMSO, 2% benzyl alcohol, 0.001% curcumin) marginally increased the half-life. NAMI-A's photostability in solution was influenced to the greatest extent by addition of an alcohol, with the least polar solvent system (50% propylene glycol) providing the most stable medium. Based on the presented results, it is recommended to store NAMI-A solutions in the dark.

The anti-metastatic agent imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate induces endothelial cell apoptosis by inhibiting the mitogen-activated protein kinase/extracellular signal-egulated kinase signaling pathway.

Bastiano Sanna, Marcella Debidda, Gianfranco Pintus, Bruna Tadolini, Anna M. Posadino, Federico Bennardini, Gianni Sava, Carlo Ventura
Archives of Biochemistry and Biophysics; 403: 209 218, 2002.

ABSTRACT

Imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate (NAMI-A) is a new ruthenium compound active against lung metastasis in vivo and tumor cell invasion in vitro. Since angiogenesis was recognized as a key event in the metastasizing process, the manipulation of neo-vessel formation has been developed as a new therapeutic approach. Within this context, a pivotal role for apoptosis in regulating cellular growth has been proposed. In the present study, we exposed to NAMI-A the spontaneously transformed human endothelial cell line ECV304 and assessed a number of apoptosis-related features, including the DNA degradation rate, the activation of caspase-3 protease, the expression of Hsp27, and the release of cytochrome c. Cell treatment with NAMI-A elicited a signi?cant increment in the apoptotic response, as indicated by DNA fragmentation and caspase-3 activation, two classical hallmarks of cellular suicide. Furthermore, NAMI-A was able to down-regulate Hsp27 protein expression and provoke the release of mitochondrial cytochrome c in the cytosol. Here, we analyze the involvement of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signal transduction pathway in the induction of apoptosis elicited by NAMIA.
Such a response was associated with a marked inhibition of MAPK/ERK kinase (MEK) and ERK phosphorylation with a time course and dose dependency overlapping those observed throughout NAMI-A-induced apoptosis. In addition, we report that PD98059, a selective MEK inhibitor, is able to induce apoptosis by itself in the ECV304 cell line. These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating an apoptotic event in ECV304.

In vivo Biodistribution Studies on Hyaluronan Butyrate by Means of ^99mTc Direct Labelling and YAP Camera.

R. Rossin, S. Zorzet, A. Zanella, C. Turrin, G. Sava, G. Moschini, A. Perbellini, U. Mazzi
In: Technetium, Rhenium and Other Metals in Chemistry and Nuclear Medicine (M. Nicolini, U. Mazzi Eds.), pp 689-693, SGEditoriali, Padova, 2002.

ABSTRACT

The glycosaminoglican hyaluronan is one of the main components of the extracellular matrix. The primary receptor for HA (CD44) is overexpressed on the surface of most malignant tumour cells, suggesting the use of HA for the targeted delivery of antitumour drugs such as butyrate. In this study the potential anti-tumour prodrug HA-But, directly labelled with ^99mTc, has been injected in mice intravenous, intraperitoneum and subcutaneous, and its biodistribution has been studied by means of a YAP camera. The collected scintigrafic images showed a rapid blood clearance and liver uptake after i.v. injection and a slow diffusion after i.p. and s.c. injection. These results confirm for HA-But the biodistribution of native HA and suggest its use for local therapy of solid tumours.

Analysis of the cytotoxicity of synthetic antimicrobial peptides on mouse leucocytes: implications for systemic use.

Sabrina Pacor, Anna Giangaspero, Marina Bacac, Gianni Sava, Alessandro Tossi
Journal of Antimicrobial Chemotherapy; 50: 339-348, 2002.

ABSTRACT

We have analysed the toxicity of highly cationic, artificial alpha-helical antimicrobial peptides on blood cells to assess their suitability for systemic application. Flow cytometric methods, based on the uptake of propidium iodide, were used to obtain a rapid and quantitative estimate of membrane damage to resting and concanavalin A-activated mouse lymphocytes, which was further confirmed by morphological changes as observed by scanning electron microscopy. Membrane permeabilization appeared to correlate with structural characteristics, so that the peptide L-19(9/B), which contains helix-stabilizing aminoisobutyric acid (Aib) residues and is a potent antimicrobial, was also the most lytic towards both mouse lymphocytes and human erythrocytes. Reducing the propensity for helix formation in P19(9/B) resulted in a marked reduction in in vitro cytotoxicity. Changing the helical sense in D-P19(9/B) also resulted in a significant decrease in cytolytic activity towards both erythrocytes and leucocytes. A limited assessment in BALB/c mice confirmed a lower in vivo toxicity of P19(8) than L-19(9/B). In a study of the systemic antimicotic activity of P19(8) in a mouse protection model, a modest prolongation in survival of Candida albicans-infected animals after intravenous administration was observed at 5 mg/kg peptide but not at higher doses. The implications of these observations for the systemic use of this type of peptide are discussed.

A review on usnic acid, an interesting natural compound.

Moreno Cocchietto, Nicola Skert, Pier Luigi Nimis e Gianni Sava
NATURWISSENSCHAFTEN; 89:137-146, 2002.

ABSTRACT

Lichens are a world-widespread consortium of fungal and photosynthetic partners. Usnic acid is one of the most common and abundant lichen metabolites, well known as an antibiotioc, but also endowed with several other interesting properties. This review summarises the most relevuant studies on usnic acid, focusing on a number of biological activities in different flield. On the basis of the existing literature, usnic acid seems to be an exclusive lichen product. No synthetic derivatives more effective than the natural form are known. Both the (+) and (-) enantiomers of usnic acid are effective against a large variety of Gram-positive (G+) bacterial strains, including strains from clinical isolates, irrespective of their resistant phenotype. Of particular relevance is the inhibition of growth of multi-resistant strains of Streptococcus aureus, enterococci and mycobacteria. The (+)-usnic acid enantiomer appears to be selective against Streptococcus mutans without inducing perturbing side effects on the oral saprophyte flora. On the other hand, the (-)-usnic acid enantiomer is a selective natural herbicide because of its blocking action against a specific key plant enzyme. Other recognised characteristics of usnic acid are ultraviolet absorption and preserving properties. The toxicology, the in vitro anti-inflammatory effects and the mechanism of action of usnic acid need to be investigated in greater detail in order to reach clinical trials and to allow further applications. Furthermore, more research is needed to make possible intensive lichen culture, in order to produce large quantities of lichen substances for pharmaceutical, cosmetic and agricultural purposes. Some biological aspects, i.e. the possible biological roles of usnic acid, are discussed.

Inhibition of endothelial cell functions and of angiogenesis by the metastasis inhibitor NAMI-A.

Angelo Vacca, Michele Bruno, Angela Boccarelli, Mauro Coluccia, Domenico Ribatti, Alberta Bergamo, Spiridione Garbisa, Luigi Sartor, Gianni Sava
BRITISH JOURNAL OF CANCER; 86: 993-998, 2002 www.bjcancer.com

ABSTRACT

NAMI-A is a ruthenium-based compound with selective anti-metastasis activitvy in experimentaI models of solid tumours. We studied whether this activity was dependent on anti-angiogenic ability of NAMI-A. We thus investigated its in vitro effects on endothelial cell functions necessary for angiogenesis to develop, as weIl as its in vivo effects in the chick embryo chorioallantoic membrane model. Endothelial cell proliferation, chemotaxis, and secretion of the matrix-degrading enzyme rnetalloproteinase-2 were inhibited by NAMI-A in a dose-dependent manner, and without morphologic signs of cell apoptosis or necrosis. LastIy, NAMI-A displayed a dose-dependent in vivo anti-angiogenic activity in the chorioallantoic membrane model. These data suggest that the anti-angiogenic activity of NAMI-A can contribute to its anti-metastatic efficacy in mice bearing malignant solid tumours.

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

G. Sava, A.Bergamo, S. Zorzet, B. Gava, C. Casarsa, M. Cocchietto, A. Furlani, V. Scarcia, B. Serli, E. Iengo, E. Alessio, G. Mestroni
EUROPEAN JOURNAL OF CANCER; 38: 427-435, 2002 www.ejconline.com

ABSTRACT

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.

Isolation of a murine metastatic cell line and preliminary test of sensitivity to the anti-metastasis agent NAMI-A.

Sabrina Pacor¹, Marta Vadori¹, Francesca Vita², Marina Bacac², Maria Rosa Soranzo³, Giuliano Zabucchi³, Gianni Sava<sup>1,2

1</sup>Department of Biomedical Sciences, University of Trieste. via L. Giorgieri 7;
²Callerio Foundation-Onlus, via A. Fleming 22-31;
³Department of Physiology and Pathology, University of Trieste, via A. Fleming 22, 34127-Trieste, Italy

Anticancer Research; 21:2523-2530, 2001

ABSTRACT

We have isolated a new cell line (metGM) obtained from the spontaneous lung metastases of the mouse MCa mammary carcinoma. MetGM is a stable cell line which, after one year from its isolation, grows in vitro in suspension, forming cell aggregates, with cells that show irregular blabbing borders, active protein synthesis and convoluted nuclei, and which have the capacity of invading matrigel membranes on which they give rise to a network of branching colonies. The preliminary study of the effects of the anti-metastasis ruthenium complex NAMI-A on metGM showed no direct cytotoxicity, with a mild reduction of cell proliferation, independent of the concentration of the ruthenium complex and not evident before 24 hours from treatment. A 10% DNA fragmentation was also measured on metGM cells 24 hours after challenge for 1 hour with
10-5M NAMI-A, suggesting that this compound is probably capable of apoptosis in a metastasis-derived cell line. Besides these effects on a limited percent of the cell population, NAMI-A changed the shape of the metGM cells and these alterations might account for the non-cytotoxic anti-metastatic properties of this innovative ruthenium complex. Thus MetGM appears to be a novel cell line suitable for the in vitro study of compounds endowed with anti-metastatic properties and for the development of new drugs with this activity.

Sonia Zorzet¹, Alenka Sorc², Claudia Casarsa², Moreno Cocchietto², Gianni Sava<sup>1,2

1</sup>Department of Biomedical Sciences, University of Trieste, via L. Giorgieri 7,
²Fondazione Callerio Onlus, via A. Fleming 22-31, 34127 Trieste, Italy.

Metal Based Drugs; 8:1-7, 2001.

ABSTRACT

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.

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

Alberta Bergamo¹, Sonia Zorzet², Moreno Cocchietto¹, Maria Elena Carotenuto¹, Monica Magnarin¹, Gianni Sava<sup>1,2

1</sup>Fondazione Callerio Onlus via A. Fleming 22-31,
²Department of Biomedical Sciences, University of Trieste, via L. Giorgieri 7, 34127 Trieste, Italy.

Anticancer Research; 21:1893-1898, 2001.

ABSTRACT

The ruthenium(lll) 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^-4M NAMI-A in multiwell plates for 15 min to 4 hrs shows a partial reduction of cell growth, evaluated by the MTT or the sulforhodamine-B test following a further 24 hrs cultivation of the treated cells in the absence of NAMI-A, only afler 4 hrs exposure. NAMI-A causes the increase of cells in G₂-M, measured by flow cytometry following PI staining, depending on the duration of in vitro treatment, whereas, ruthenium uptake by tumour cells, determined by flameless atomic absorption spectroscopy, increases up to 2 hrs of cell incubation. The arrest of cell cycle in the pre-mitotic G₂-M phase is transient and completely reversed by 48 hrs after treatment. This study shows that the effect of NAMI-A on cell cycle of TS/A cells is not strictly related to NAMI-A uptake as is the effect on tumour cell proliferation.

Immunostimulating effects of oral lysozyme on a vaccination treatment with Vibrio anguillarum.

Pacor S, Bacac M, Vadori M, Vismara D, Manfrin A, Sava G.
Far Ter; XVIII: 51-55, 2001.

ABSTRACT

The aim of the present work has been of evaluating the effects of an oral treatment with 100 mg/kg hen egg-white lysozyme (HEL) for 6 days followed by 3x105 cells/day Vibrio anguillarum serotype ol (VA-o1), heat inactivated, for 3 days, on host immunity, in CBA mice. The oral treatment of HEL increases lymphocytes (+47%) in Peyer's patches as compared to untreated controls. A small subset of these lymphocytes is increased from 11% to 38% in the group treated with HEL; these cells are CD4+ (66%), CD54+ (96%) and have significantly increased the protein content. VA-o1 is devoid of such effects, whereas HEL+VA-o1 shows the same effects as HEL alone. Repetition of this schedule, after 3-weeks, causes a statistically significant increase in CD3+ cells into mesenteric lymph nodes. These results suggest the possibility of using the combination HEL+VA-o1 to add aspecific immune responses to the vaccination response with a VA-o1.

Pharmacological effects of the ruthenium complex NAMI-A given orally to CBA mice with MCa mammary carcinoma.

Zorzet S, Sorc A, Casarsa C, Cocchietto M, Sava G.
Metal Based Drugs, 8: 1-7, 2001.

ABSTRACT

NAMI-A imidazolium trans-imdazoledimethylsulfoxidetrachlororuthenate 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 600mg/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.

Effects of NAMI-A and some related ruthenium complexes on cell viability after short exposure of tumor cells.

Bergamo A, Zorzet S, Gava B, Sorc A, Alessio E, Iengo E, Sava G.
Anti-Cancer Drugs, 11: 667-672, 2000.

ABSTRACT

A series of three ruthenium complexes, i.e. trans-dichlorote-trakisdimethyl-sulfoxide ruthenium(ll) (trans-Ru), imidazolium trans-imidazoletetra-chlororuthenate (ICR) and sodium trans-tetramethylensulfoxideisoquinoline-tetrachlororuthenate (TEQU), were studied in vitro in comparison to NAMI-A, a potent ruthenium-based antimetastasis agent. In vitro challenge of TS/A adenocarcinoma or KB oral carcinoma tumor cells with 10(-4) M concentration for 1 h evidenced the lack of cytotoxicity of NAMI-A, ICR and trans-Ru, the accumulation of cells in the G2/M pre-mitotic cell phase by NAMI-A and the attachment of tumor cells to the plastic substrate was significantly greater for NAMI-A than for ICR. These data stress that in vitro cytotoxicity is not necessary for in vivo activity of ruthenium antitumor complexes: NAMI-A, ICR and trans-Ru, are in fact known to be active against murine tumors in the mouse system. Rather, TEQU, the compound free of in vivo activity, was the only one to reduce cell growth of in vitro cultured cells. In conclusion, the data on the effects of NAMI-A on in vitro cultured cells show that the increase of cell adhesion properties and the transient cell cycle arrest in the G2/M phase are much more relevant than the effects on cell properties relevant to cell growth (i.e. on CD44, CD54 or CD71 antigens) for determining in vivo antimetastasis activity.

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

Sava G, Bergamo A.
Int J Oncol; 17: 353-365, 2000.

ABSTRACT

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.

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

Magnarin M, Bergamo A, Carotenuto ME, Zorzet S, Sava G.
Anticancer Res; 20: 2939-2944, 2000.

ABSTRACT

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.

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.

Zorzet S, Bergamo A, Cocchietto M, Sorc A, Gava B, Alessio E, Iengo E, Sava G.
J Pharmacol Exp Ther; 295: 927-933, 2000.

ABSTRACT

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.

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

Cocchietto M, Sava G.
Pharmacol Toxicol; 87: 193-197, 2000.

ABSTRACT

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.

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

Sava G, Cocchietto M.
In Vivo; 14: 741-744, 2000.

ABSTRACT

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.

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

Sava G, Capozzi I, Clerici K, Gagliardi R, Alessio E, Mestroni G.
Clinical Experimental Metastasis; 16: 371-379, 1998.

ABSTRACT

Imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate 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 metastasising 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 corresponds 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.

In vitro cell cycle arrest, in vivo action on solid metastasizing tumors, and host toxicity of the antimetastatic drug NAMI-A and cisplatin.

Bergamo A, Gagliardi R, Scarcia V, Furlani A, Alessio E, Mestroni G, Sava G.
Journal of Pharmacology and Experimental Therapeutics; 289: 559-564, 1999.

ABSTRACT

The effects of NAMI-A (imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate) are compared with cisplatin on tumour cells cultured in vitro at doses of 1 to 100 mM and on tumour metastases in vivo at maximum tolerated doses. Using mouse tumours that metastasise to the lungs, NAMI-A given i.p. for 6 consecutive days at 35 mg/kg/day, was effective independently of the tumour line being treated and of the stage of metastasis growth. Conversely, cisplatin (2 mg/kg/day for 6 days) was as effective as NAMI-A on MCa mammary carcinoma and TS/A adenocarcinoma and less effective than NAMI-A on Lewis lung carcinoma. Cisplatin reduced body weight gain and spleen weight during treatment and was much more toxic than NAMI-A on liver sinusoids, kidney tubules, and lung epithelium. In vitro NAMI-A caused a transient cell cycle arrest of tumour cells in the premitotic G₂/M phase, whereas cisplatin caused a progressive dose-dependent disruption of cell cycle phases. Correspondingly NAMI-A did not modify cell growth, whereas cisplatin caused a dose-dependent reduction of cell proliferation, as determined by sulphorhodamine B test. Thus, NAMI-A, unlike cisplatin is a potent agent for the treatment of solid tumour metastases as well as when these tumour lesions are in an advanced stage of growth. NAMI-A is endowed with a mechanism of action unrelated to direct tumour cell cytotoxicity, and such mechanism of action is responsible for a reduced host toxicity.

Drug control of solid tumour metastases: a critical view.

Sava G, Bergamo A.
Anticancer Research; 19: 1117-1124, 1999.

ABSTRACT

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 metastases whereever 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 shpould 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.

Paracrine effects of IL-4 transfection on TS/A adenocarcinoma cells mediate reduced in vivo growth.

Pacor S, Gagliardi R, Spessotto P, Zabucchi G, Sava G.
Pathology and Oncology Research; 5: 110-116, 1999.

ABSTRACT

The in vitro / in vivo growth capacity and phenotype of TS/A and the IL4-transfected TS/A-IL4 cell lines were studied by cell cycle analysis, expression of ICAM-1/CD54, ransferrin receptor/CD71 and E-cadherin and by histology of the primary tumors. TS/A-IL4, unlike the TS/A line, shows in vitro a marked increase in the fibroblastoid cell type and a decreased E-cadherin expression. Administration of conditioned medium containing IL4 obtained from the TS/A-IL4 cell line, stimulates CD54 expression in the TS/A CELL LINE. TS/A-IL4 tumors grow more slowly in vivo and are ultimately rejected. These processes are accompanied by a marked increase in collagen and extracellular matrix proteins and increased recruitment and degranulation of mast cells. The paracrine effect of IL4, released by the transfected tumor cells, might be responsible for the reduced in vivo growth of the TS/A cell line in the presence of TS/A-IL4 cells.

In vitro down regulation of ICAM-1 and E-cadherin and in vivo reduction of lung metastases of TS/A adenocarcinoma by a lysozyme derivative.

Pacor S, Gagliardi R, Di Daniel E, Vadori M, Sava G.
International Journal of Molecular Medicine; 4: 369-375, 1999.

ABSTRACT

The aim of the present investigation was to examine the effects of the lysozyme derivative mPEG-lyso (hen egg-white lysozyme coupled with polyoxyethylenglycol), on TS/A adenocarcinoma cell line in vivo and in vitro. mPEG-lyso reduces the number of ICAM-1⁺ and E-cadherin⁺ cells of TS/A adenocarcinoma cell line in vitro, and causes a marked decrease of spontaneous lung metastases in vivo. In both cases, mPEG-lyso reduces the number of tumour cells in synthesis and pre-mitotic phases. In connection with the reduction of cells expressing adhesion molecules, mPEG-lyso reduces the number of infiltrating leukocytes in the primary tumour in vivo and reduces the binding capacity of splenocytes to tumour cells in vitro. These data stress, for the first time, that the in vivo control of mPEG-lyso on lung metastasis formation of solid metastasising tumours may be due to a combination of effects on tumour cells in addition to those on host’s immune system.