Development of ruthenium antitumor drugs that overcome multidrug resistance mechanisms.

Vock CA, Ang WH, Scolaro C, Phillips AD, Lagopoulos L, Juillerat-Jeanneret L, Sava G, Scopelliti R, Dyson PJ.
Institut des Sciences et Ingenierie Chimiques, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
J Med Chem. ; 50(9):2166-75; 3 May 2007; Epub 10 Apr 2007.

ABSTRACT

Organometallic ruthenium(II) complexes of the general formula [Ru(eta6-p-cymene)Cl2(L)] and [Ru(eta6-p-cymene)Cl(L)2][BPh4] with modified phenoxazine- and anthracene-based multidrug resistance (MDR) modulator ligands (L) have been synthesized, spectroscopically characterized, and evaluated in vitro for their cytotoxic and MDR reverting properties in comparison with the free ligands. For an anthracene-based ligand, coordination to a ruthenium(II) arene fragment led to significant improvement of cytotoxicity as well as Pgp inhibition activity. A similar, but weaker effect was also observed when using a benzimidazole-phenoxazine derivative as Pgp inhibitor. The most active compound in terms of both Pgp inhibition and cytotoxicity is [Ru(eta6-p-cymene)Cl2(L)], where L is an anthracene-based ligand. Studies show that it induces cell death via inhibition of DNA synthesis. Moreover, because the complex is fluorescent, its uptake in cells was studied, and relative to the free anthracene-based ligand, uptake of the complex is accelerated and accumulation of the complex in the cell nucleus is observed.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 17419606 [PubMed - in process]

Ruthenium complexes can target determinants of tumour malignancy.

Bergamo A, Sava G.
Callerio Foundation Onlus, via A Fleming 22-31, Trieste, Italy.
Dalton Trans. ; (13):1267-72; 7 Apr 2007 ; Epub 28 Feb 2007.

ABSTRACT

T Metastases are more decisive for tumour prognosis than primary lesions, because of their multiple locations, low accessibility to surgery and/or radiotherapy, and generally poor responsiveness to chemotherapy. The metastasis should therefore be the primary target for drug therapy. Among ruthenium complexes, NAMI-A is a leading compound that shows selective effects for solid tumour metastases related to a mechanism of action involving the inhibition of the processes of tumour invasiveness. NAMI-A opens an avenue to new perspectives in cancer chemotherapy. This includes novel compounds directed at targets selectively expressed by tumour metastases, thus reducing the typical side effects of the current metal-based drugs that are active via their unselective DNA interaction.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 17372640 [PubMed - in process]

The most popular Dalton Transactions article during March 2007 (778 hits) and in the top ten during April 2007.

Synthesis, characterization, and in vitro evaluation of novel ruthenium(II) eta6-arene imidazole complexes.

Vock CA, Scolaro C, Phillips AD, Scopelliti R, Sava G, Dyson PJ.
Institut des Sciences et Ingenierie Chimiques, Ecole Polytechnique Federale de Lausanne, CH-1015 Lausanne, Switzerland.
J Med Chem. ; 49(18):5552-61; 7 Sep 2006.

ABSTRACT

Ten complexes of general formula [Ru(eta6-arene)Cl2(L)], [Ru(eta6-arene)Cl(L)2][X], and [Ru(eta6-arene)(L)3][X]2 (eta6-arene = benzene, p-cymene; L = imidazole, benzimidazole, N-methylimidazole, N-butylimidazole, N-vinylimidazole, N-benzoylimidazole; X = Cl, BF4, BPh4) have been prepared and characterized by spectroscopy. The structures of five representative compounds have been established in the solid state by single-crystal X-ray diffraction. All the new compounds were assessed by the same in vitro screening assays applied to [imidazole-H][trans-RuCl4(DMSO)(imidazole)] (NAMI-A) and [Ru(eta6-arene)Cl2(1,3,5-triaza-7-phosphaadamantane)] (RAPTA) compounds. It was found that the new compounds show essentially the same order of cytotoxicity as the RAPTA compounds toward cancer cells. Several of the compounds were selective toward cancer cells in that they were less (or not) cytotoxic toward nontumorigenic cells that are used to model healthy human cells. Thus, two of the compounds, [Ru(eta6-p-cymene)Cl(vinylimid)2][Cl] (vinylimid = N-vinylimidazole) and [Ru(eta6-benzene)(mimid)3][BF4]2 (mimid = N-methylimidazole), have been selected for a more detailed in vivo evaluation.

Publication Types:
Research Support, Non-U.S. Gov't
PMID: 16942028 [PubMed - indexed for MEDLINE]

The role of cisplatin and NAMI-A plasma-protein interactions in relation to combination therapy.

Khalaila I, Bergamo A, Bussy F, Sava G, Dyson PJ.
Institut des Sciences et Ingenierie Chimiques, Ecole Polytechnique Federale de Lausanne, CH-1015 Lausanne, Switzerland.
Int J Oncol.; 29(1):261-8; Jul 2006.

ABSTRACT

The aim of the study is to evaluate the differences of protein binding of NAMI-A, a new ruthenium drug endowed with selective antimetastatic properties, and of cisplatin and to ascertain the possibility to use two drugs based on heavy metals in combination to treat solid tumour metastases. For this purpose, we have developed a technique that allows the proteins, to which metal drugs bind, to be identified from real protein mixtures. Following incubation with the drugs, the bands containing platinum and/or ruthenium are separated by native PAGE, SDS-PAGE and 2D gel electrophoresis, and identified using laser ablation inductively coupled plasma mass spectrometry. Both drugs interact with essentially the same proteins which, characterised by proteomics, are human serum albumin precursor, macroglobulin alpha2 and human serotransferrin precursor. The interactions of NAMI-A are largely reversible whereas cisplatin forms stronger interactions that are less reversible. These data correlate well with the MCa mammary carcinoma model on which full doses of NAMI-A combined with cisplatin show additive effects as compared to each treatment taken alone, independently of whether NAMI-A precedes or follows cisplatin. Furthermore, the implication from this study is that the significantly lower toxicity of NAMI-A, compared to cisplatin, could be a consequence of differences in the mode of binding to plasma proteins, involving weaker interactions compared to cisplatin.
Publication Types:
Comparative Study
Research Support, Non-U.S. Gov't
PMID: 16773208 [PubMed - in process].

Metal-based antitumour drugs in the post genomic era.

Dyson PJ, Sava G.
Institut des Sciences et Ingenierie Chimiques, Ecole Polytechnique Federale de Lausanne, CH-1015 Lausanne, Switzerland. paul.dyson@epfl.ch
Dalton Trans. ;(16):1929-33.; 28 Apr 2006; Epub 28 Mar 2006.

ABSTRACT

The discovery of new metal-based antitumour drugs, whether cisplatin derivatives or those based on other metals, has been largely based on cell viability assays (IC(50) values) and compounds that bind to DNA. This approach has been applied for more than 30 years during which time very few new drugs have entered clinical use. In this article we discuss what the future holds for metal-based drugs, in particular anti-metastasis drugs, in these enlightened times of the post genomic era.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 16609762 [PubMed - in process].

A novel retinoic/butyric hyaluronan ester for the treatment of acute promyelocytic leukemia: preliminary preclinical results.

Coradini D, Pellizzaro C, Scarlata I, Zorzet S, Garrovo C, Abolafio G, Speranza A, Fedeli M, Cantoni S, Sava G, Daidone MG, Perbellini A.
Unit of Tumor Biology and Experimental Therapy, Experimental Department, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy. danila.coradini@istitutotumori.mi.it
Leukemia; 20(5):785-92; May 2006.

ABSTRACT

All-trans retinoic acid (ATRA) represents the therapy of choice for patients with acute promyelocytic leukemia (APL). However, patients often relapse due to ATRA-resistance. The molecular basis of APL alterations indicates that addition of a histone deacetylase inhibitor to ATRA may restore the sensitivity to retinoids. We explored the in vitro and in vivo effects of a novel retinoic/butyric hyaluronan ester (HBR) on a retinoic acid (RA)-sensitive human myeloid cell line, NB4, and on its RA-resistant subclone, NB4.007/6. In vitro, HBR induced growth arrest and terminal differentiation in RA-sensitive NB4 cells (as confirmed by an increased expression of CD11 family members and nitroblue tetrazolium assay), whereas it inhibited the growth of RA-resistant cells by apoptosis, paralleled by an increase in the levels of caspase 3 and 7. In vivo, HBR treatment of NB4-inoculated severe combined immunodeficient mice resulted in a statistically significant increase in survival time (P<0.0001), comparable to that induced by a maximum tolerated dose of RA alone. Also on P388-inoculated mice, HBR was active in contrast to RA that was completely ineffective. Present findings suggest that, owing to the simultaneous presence of RA and an histone deacetylases inhibitor, HBR might be useful in controlling the proliferation of RA-resistant cells and the differentiation of RA-sensitive cells.

Vaccination trial of sea-bass (Dicentrarchus labrax) against pasteurellosis using oral, intraperitoneal and bath administration.

Paolini A, Ridolfi V, Zezza D, Cocchietto M, Musa M, Pavone A, Conte A, Giorgetti G.
Istituto Zooprofilattico Sperimentale dell’Abruzzo & del Molise “G. Caporale” (IZSA&M), Teramo, Italy.
Veterinaria Italiana; 41(2), 7-14, Apr-Jun 2005.

ABSTRACT

Photobacterium damsela subsp. piscicida (Phdp) is the aetiological agent of fish Pasteurellosis, causing heavy losses in intensive mariculture plants. The present work compares the protective efficiency of five different vaccine forms: oral, intraperitoneal, immersion, bivalent immersion (Vibrio anguillarum) and immersion with immunostimulant. Each of these vaccine forms, containing whole cells of Phdp inactivated with formalin (FKC) was administered to 100 sea-bass weighing approximately 2 g; 100 non vaccinated sea-bass were used as control. (The fish were tested for protection against pasteurellosis 40 days after vaccination with an intraperitoneal challenge. Each fish was inoculated with Phdp cells at concentration of 2.75 x 104 CFU/ml ). Mortality was registered in the following 14 days. Vaccine protection was evaluated using a Relative Percentage Survival (RPS) index. The intraperitoneal formulation gave an excellent protection (RPS 82.4%). Immersion form produced the best results (RPS 26.4%) with the immunostimulant, followed by the simple immersion (RPS 23.1%) to end with the group vaccinated with the bivalent vaccine (RPS 18.7%). The protection conferred by the oral form (RPS 28.6%) is interesting for its practicality.

Lysozyme-containing chitosan-coated alginate microspheres for oral immunisation.

Zorzin L, Cocchietto M, Voinovich D, Marcuzzi A, Filipovic Grcic, Mulloni C, Crembiale G, Casarsa C, Bulla R, Sava G.
Callerio Foundation Onlus, Institutes of Biological Researches, Trieste, Italy.
J. Drug Del. Sci. Tech; 16(6), 413-420, 2005.

ABSTRACT

A set of ten different chitosan-coated alginate microspheres (MS1-10), containing hen-egg white lysozyme (LZ) as immunostimulant, was developed for the oral delivery of antigens. MS10, the formulation chosen for in vivo studies, charged with heat-inactivated Vibrio anguillarum (VA), presents mean dry and swollen diameter , zeta potential value, LZ and VA encapsulation efficiency of 2.7 ± 1.1 µm, 3.2 ± 1.2 µm, 2.1 ± 0.6 mV, 45.5% and 65.0%, respectively. The immunomodulating properties of the system were preliminarily tested on CBA mice model. A 6 consecutive-day oral immunization with microencapsulated VA induces a significant humoral immune response. The presence of LZ in the system contributes to increase the immune response against co-encapsulated VA vs. VA loaded MSs (IgM and IgG) or non encapsulated VA (IgM). The microencapsulation seems to improve the VA uptake by Payer’s patches (PP) vs. free particulate. These results provide useful insights into the suitability of this system for oral immunization with microencapsulated antigens.

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

Gava B, Zorzet S, Spessotto P, Cocchietto M, Sava G..
Department of Biomedical Sciences, University of Trieste, Italy. g.sava@callerio.org.
J Pharmacol Exp Ther. ;317(1):284-91;Apr 2006; Epub Dec 20 2005.

ABSTRACT

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

Influence of Hydrogen-Bonding Substituents on the Cytotoxicity of RAPTA Compounds.

Claudine Scolaro, Tilmann J. Geldbach, Sébastien Rochat, Antoine Dorcier, Christian Gossens, Alberta Bergamo, Moreno Cocchietto, Ivano Tavernelli, Gianni Sava, Ursula Rothlisberger and Paul J. Dyson.

Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL),
CH-1015 Lausanne, Switzerland, Callerio Foundation Onlus, Via A. Fleming 22-31, 34127, Trieste, Italy,
and Dipartimento di Scienze Biomediche, UniVersita` di Trieste, Via L. Giorgieri 7-9, 34127, Trieste, Italy.
Received October 14, 2005
Organometallics; 25, 756-765; 2006.

ABSTRACT

A new series of organometallic ruthenium(II)-arene compounds of the type RuCl2(è6-arene)(phosphine)
(phosphine ) 1,3,5-triaza-7-phosphaadamantane, PTA, and 3,7-diacetly-1,3,7-triaza-5-phosphabicyclo-
[3.3.1]nonane, DAPTA) with different potential hydrogen-bonding functionalities on the arene ligand
have been prepared and studied for their antitumor activity. Cell viability studies using the TS/A mouse
adenocarcinoma cancer cell line and the nontumorigenic HBL-100 human mammary cell line, combined
with uptake determinations, are compared to the nonfunctionalized analogues, previously shown to be
active on solid metastasizing tumors. The reactivity of the functionalized RAPTA compounds with a
14-mer oligonucleotide (established by mass spectrometry) has been rationalized by DFT calculations,
which indicate that environmental factors are important.
PMID: 16368900 [PubMed - indexed for MEDLINE].

Most-Cited Articles in the last two month (Hot Papers - September 2007)PDF
Third of the Organometallic Most-Cited Articles in 2006 .

In vitro and in vivo evaluation of ruthenium(II)-arene PTA complexes.

Scolaro C, Bergamo A, Brescacin L, Delfino R, Cocchietto M, Laurenczy G, Geldbach TJ, Sava G, Dyson PJ.
Institut des Sciences et Ingenierie Chimiques, Ecole Polytechnique Federale de Lausanne (EPFL), Switzerland.
J. Med. Chem.; 48(12):4161-71, Jun 16 2005.

ABSTRACT

The antitumor activity of the organometallic ruthenium(II)-arene complexes, RuCl(2)(eta(6)-arene)(PTA), (arene = p-cymene, toluene, benzene, benzo-15-crown-5, 1-ethylbenzene-2,3-dimethylimidazolium tetrafluoroborate, ethyl benzoate, hexamethylbenzene; PTA = 1,3,5-triaza-7-phosphaadamantane), abbreviated RAPTA, has been evaluated. In vitro biological experiments demonstrate that these compounds are active toward the TS/A mouse adenocarcinoma cancer cell line whereas cytotoxicity on the HBL-100 human mammary (nontumor) cell line was not observed at concentrations up to 0.3 mM, which indicates selectivity of these ruthenium(II)-arene complexes to cancer cells. Analogues of the RAPTA compounds, in which the PTA ligand is methylated, have also been prepared, and these prove to be cytotoxic toward both cell lines. RAPTA-C and the benzene analogue RAPTA-B were selected for in vivo experiments to evaluate their anticancer and antimetastatic activity. The results show that these complexes can reduce the growth of lung metastases in CBA mice bearing the MCa mammary carcinoma in the absence of a corresponding action at the site of primary tumor growth. Pharmacokinetic studies of RAPTA-C indicate that ruthenium is rapidly lost from the organs and the bloodstream.

Second of the  Journal of Medicinal chemistry Most-Cited Articles in the last two month (Hot Papers - September 2007)

Platinum(II) complexes with antitumoral/antiviral aromatic heterocycles: effect of glutathione upon in vitro cell growth inhibition.

Papadia P, Margiotta N, Bergamo A, Sava G, Natile G.
Dipartimento Farmaco-Chimico, Università degli Studi di Bari, Via E. Orabona 4, 70125 Bari, Italy.
J. Med. Chem.; 48(9):3364-71, May 5 2005.

ABSTRACT

The compounds [Pt(Me(2)phen)(acy)(2)](NO(3))(2) (1), [Pt(Me(2)phen)(pen)(2)](NO(3))(2), [Pt(phen)(acy)(2)](NO(3))(2) (2), and [Pt(phen)(pen)(2)](NO(3))(2), containing the bidentate 1,10-phenanthroline (phen) or 2,9-dimethyl-1,10-phenanthroline (Me(2)phen, neocuproine) and the antiviral agents acyclovir (acy) or penciclovir (pen), show different in vitro toxicity, the Me(2)phen complexes being appreciably more toxic than the phen complexes. To explain the different behavior, we investigated the reaction of complexes 1 and 2 with glutathione (gamma-glutamylcysteinylglycine, GSH), a peptide believed to play an important role in driving the cellular effects of platinum drugs. The reaction led to different products, the phen complexes forming a stable binuclear mu-thiol-bridged species still containing the phenanthroline and the Me(2)phen complexes releasing the neocuproine ligand and forming an insoluble material. In vitro tests confirmed that the greater cell toxicity of complex 1 is due to the displacement of the neocuproine ligand by GSH. The results highlight the great dependence of the glutathione reactivity upon relatively small changes in the platinum coordination sphere.

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

Frausin F, Scarcia V, Cocchietto M, Furlani A, Serli B, Alessio E, Sava G.
Department of Biomedical Sciences, University of Trieste, Italy.
J. Pharmacol. Exp. Ther.; 313(1):227-33, Apr 2005. Epub 2004 Dec 3.

ABSTRACT

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

Ruthenium antimetastatic agents.

Alessio E, Mestroni G, Bergamo A, Sava G.
Department of Chemical Sciences, University of Trieste, Via L. Giorgieri 1, 34127 Trieste, Italy.
Curr. Top. Med. Chem.; 4(15):1525-35, 2004.

ABSTRACT

NAMI-A, i. e. (imH)[trans-RuCl(4)(dmso-S)(im)] (im = imidazole, dmso = dimethylsulfoxide), is a Ru(III) complex that, after extensive preclinical investigations that evidenced its remarkable and specific activity against metastases, has recently and successfully completed a Phase I trial (first ruthenium complex ever to reach clinical testing). This review article, after a brief summary of the main chemical and pharmacological aspects of NAMI-A, focuses on the development of new classes of ruthenium complexes originated from the NAMI-A frame. In particular, the chemical and biological features of the following classes of compounds will be treated: i) NAMI-A-type complexes, derived from NAMI-A by changing the nature of the N-ligand, ii) dinuclear NAMI-A-type compounds containing heterocyclic bridging N-N ligands, iii) new Ru-dmso nitrosyls broadly derived from NAMI-A-type complexes. Several of these new compounds were found to have antimetastatic activity comparable to, or even better than, NAMI-A; however, the nature of the target(s) responsible for the antimetastatic activity remains unclear. Common to any type of NAMI-A-type compound, both monomeric and dimeric, cell cytotoxicity (which is generally very low) is not sufficient to explain their potent and peculiar antitumor activity. All active NAMI-A-type compounds share the capacity to modify important parameters of metastasis such as tumor invasion, matrix metallo proteinases activity and cell cycle progression.

TGFß1 regulation and collagen-release-independent connective tissue re-modelling by the ruthenium complex NAMI-A in solid tumours.

Casarsa C, Mischis MT, Sava G.
Callerio Foundation Onlus, via Fleming 22/31, 34127 Trieste, Italy.
J Inorg Biochem.;98(10):1648-54,

ABSTRACT

Purpose. The objective of this study is to evaluate the fibrotic process induced in vivo by NAMI-A in mice with solid tumours. In addition, the in vitro effects of NAMI-A on collagen fibres and the expression of TGF [Formula: see text] in TS/A adenocarcinoma cells, NIH/3T3 fibroblasts and co-culture of fibroblasts and tumour cells have also been studied. Methods. Collagen fibres release was assayed in supernatant of culture cells treated with 0.1 and 0.01 mM NAMI-A. TGF [Formula: see text] was detected by RT-PCR and immunoblot on cellular lysates. Results. NAMI-A, given to mice bearing MCa mammary carcinoma at advanced stages of growth, increased the thickness of connective tissue and induced recruitment of leukocytes, particularly in the peritumour capsule. In vitro NAMI-A stimulated collagen production by NIH/3T3 fibroblasts and decreased collagen release by TS/A tumour cells after prolonged exposure, either after single cell treatment or in co-cultures. In co-cultures, NAMI-A, in a dose-dependent manner, down-regulated the expression of TGF [Formula: see text] mRNA and protein in tumour cells and up-regulated it in fibroblasts. The isoform of this cytokine is involved in fibrosis, invasion and metastatic processes. Conclusions. These data emphasize the ability of NAMI-A to evoke beneficial effects from healthy cells against tumour growth and metastases. The contribution of fibroblasts to the fibrosis arising in tumour masses is due to TGF [Formula: see text], and its down-regulation in tumour cells might explain the documented reduction of gelatinase release.