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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.

We report here that the established anticancer ruthenium(iii) complex NAMI-A induces potent and selective cytotoxic effects in a few leukaemia cell lines. These results sound very surprising after 20 years of intense studies on NAMI-A, commonly considered as a "non-cytotoxic" antimetastatic agent. In addition, evidence is given for selective inhibition of KCa 3.1 channels. The implications of these findings are discussed. © 2014 the Partner Organisations.

The reaction of Na[transRuCl4Me2SO(Im)] (NAMI; where Im is imidazole), a novel anti-neoplastic ruthenium(III) complex, with BSA, was studied in detail by various physico-chemical techniques. It is shown that NAMI, following chloride hydrolysis, binds bovine serum albumin tightly; spectrophotometric and atomic absorption data point out that up to five ruthenium ions are bound per albumin molecule when BSA is incubated for 24 h with an eightfold excess of NAMI. CD and electronic absorption results show that the various ruthenium centers bound to albumin exhibit well distinct spectroscopic features. The first ruthenium equivalent produces a characteristic positive CD band at 415 nm whereas the following NAMI equivalents produce less specific and less marked spectral effects. At high NAMI/BSA molar ratios a broad negative CD band develops at 590 nm. Evidence is provided that the bound ruthenium centers remain in the oxidation state +3. By analogy with the case of transferrins it is proposed that the BSA-bound ruthenium ions are ligated to surface histidines of the protein; results from chemical modification experiments with diethylpyrocarbonate seem to favor this view. Spectral patterns similar to those shown by NAMI are observed when BSA is reacted with two strictly related ruthenium(III) complexes Na[transRuCl4(Me2SO)2] and H(Im)[transRuCl4(Im)2] (ICR), implying a similar mechanism of interaction in all cases. It is suggested that the described NAMI-BSA adducts may form in vivo and may be relevant for the biological properties of this complex; alternatively NAMI/BSA adducts may be tested as specific carriers of the ruthenium complex to cancer cells. Implications of these findings for the mechanism of action of NAMI and of related ruthenium(III) complexes are discussed.

A crystallographic study of the adduct formed between hen egg white lysozyme (HEWL) and NAMI-A, an established ruthenium(III) anticancer agent in clinical trials, is presented here. The X-ray structure reveals that NAMI-A coordinates the protein, as a naked ruthenium ion, at two distinct sites (namely Asp101 or Asp119) after releasing all its original ligands (DMSO, imidazole and Cl(-)). Structural data of the HEWL/NAMI-A adduct are compared with those previously obtained for the HEWL adduct of AziRu, a NAMI-A analogue bearing a pyridine in place of imidazole. The present results further support the view that NAMI-A exerts its biological effects acting as a classical "prodrug" first undergoing activation and then causing extensive metalation of relevant protein targets. It is also proposed that the original Ru-ligands, although absent in the final adduct, play a major role in directing the ruthenium center to its ultimate anchoring site on the protein surface.

Novel macromolecular ruthenium chemotherapeutics were designed, synthesised and investigated in comparison to small drugs. Ruthenium possesses unique properties that have led to the development of exceptionally promising anticancer and antimetastatic therapeutics. The ruthenium(III) and ruthenium(II) drugs, NAMI-A [trans-RuCl4(DMSO)Im]-ImH+ and RAPTA-C [RuCl2(p-cymene)(PTA)], respectively, are two such candidates. The amplified benefit that can be gained by incorporating drugs into macromolecules has not previously been investigated for ruthenium agents. Two novel approaches to the synthesis of two types of rationally designed polymers, both containing ruthenium, were developed based on the ruthenium anticancer drugs NAMI-A and RAPTA-C. Both approaches utilised a distinct ligand of each drug. The first approach relied on the polymerisation of 4-vinyl imidazole via RAFT polymerisation. This created a macromolecular ligand for ruthenium(III) to which a ruthenium precursor could be subsequently conjugated. The second approach used the inherent activity of the amide group on the PTA ligand of RAPTA-C to allow for conjugation to poly(2-iodoethyl methacrylate) via the substitution of the reactive halide. Two pathways were investigated to conjugate RAPTA-C to the polymer and both routes were tested using n-butyl iodide as a model compound. 1D and 2D NMR experiments were used to assess the conjugation and elucidate the superior pathway: a two-step reaction involving the conjugation of PTA via its reactive amine and subsequent complexation to form RAPTA-C. Nano-sized carriers were designed to increase the cell uptake of these macromolecular drugs micelles and nanotubes. Amphiphilic block copolymers incorporating NAMI-A or RAPTA-C were developed and self-assembled into micelles. RAPTA-C was also attached to cyclopeptide-polymer conjugates and self-assembled into nanotubes. A 1.5-times increase in cytotoxicity was found for NAMI-A micelles, and an outstanding 10-fold increase was found for both RAPTA-C micelles and nanotubes, when compared to the free drugs. An initial exploration into the antimetastatic activity revealed that the NAMI-A and RAPTA-C polymeric micelles significantly improved the inhibitory effects on both the migration and invasion of human breast cancer cells, indicating that it is highly probable that these nanoparticles will inhibit metastases in vivo. Thus, this work provides a substantial basis for the progression of these macromolecular chemotherapeutics into advanced biological studies.

Single drug-based cancer therapies are frequently associated with the development of drug resistance. To overcome this problem, combination therapy with two or more anticancer drugs is a promising strategy, but clinical studies are logistically challenging and costly. Intermediary in vitro studies, however, can provide critical insight to decide whether one should proceed to in vivo studies. To this end, cisplatin and the Ru-based anticancer drug NAMI-A were added to human plasma and the size distribution of Pt-containing and Ru-containing entities was determined over a 2 h period. The results revealed a dramatically different rate of plasma protein binding for each drug and/or their hydrolysis products. Both drugs bound to the same apparent plasma proteins, but crucially they did not adversely affect each other’s metabolism. Therefore, combination therapy of patients with these metallodrugs should be further assessed in clinical studies in order to systematically develop an effective combination therapy protocol to prevent the resurgence of cancer.

The binding to human lactoferrin of three Ru(III) complexes with anti-tumor activity has been investigated by X-ray crystallography in order to gain insights into how such complexes might be carried during transferrin-mediated delivery to cells. The complexes, HIm[RuIm2Cl4], HInd[RuInd2Cl4] and (HInd)2 [RuIndCl5], where Im = imidazole and Ind = indazole, were diffused into crystals of apo-lactoferrin (apoLf). X-ray diffraction data were collected to 2.6 A, 2.2 A and 2.4 A respectively. The binding sites for the Ru complexes were determined from difference Fouriers, in comparison with native apoLf; the two indazole-apoLf complexes were also refined crystallographically to final R factors of 0.202 (for 8.0 to 2.3 A data) and 0.192 (for 8.0 to 2.4 A data) respectively. Two types of binding site were identified, a high-affinity site at His 253 in the open N-lobe iron-binding cleft of apoLf (and by analogy a similar one at His 597 in the C-lobe), and lower-affinity sites at surface-exposed His residues, primarily His 590 and His 654. The exogenous heterocyclic ligands remain bound to Ru, at least at the His 253 site, and modelling suggests that the nature and number of these ligands may determine whether the closed structure that is required for receptor binding could be formed or not. The results also highlight the importance of His residues for binding such complexes and the value of heavy atom binding studies from crystallographic analyses for identifying non-specific binding sites on proteins.

The anti-metastatic ruthenium complex NaCtransRuCI4(DMSO)lm] was given i.p. at 22 and 44 mg/kg/day, on days 8-13 after tumour implantation, to mice carrying S.C. implants of MCa mammary carcinoma. The aim ofthe study was to compare the effects on lung metastasis formation with those on primary tumour cells. This investigation was based on flow cytometry analysis after propidium iodide and acridine orange staining, histology of tumour parenchyma and RT-PCR analysis for the type-IV collagenases MMP-9 and MMP-2 and their respective inhibitors TIMP-I and TIMP-2 mRNAs. NactransRuCl,(DMSO)lm] is not cytotoxic for tumour cells but has the capacity of interacting with nucleic acids, giving a general reduction of nucleic acid content as shown by a marked reduction of acridine orange staining and a tendency to a reduction of DNA polyploidy with marked reduction of 8n and 4n cell populations. Na[trans-RuCI~(DMSO)lm] also influences a proteolytic system which has the potential of degrading the basement membrane and has been related to metastatic aggressiveness: it markedly reduces, in a dose-dependent manner, MMP-2/TIMP-2 balance, but not that of MMP-9/TIMP-I. The different enzyrne/inhibitor mRNA levels between untreated and treated tumours seem to be unaffected by tumourinfiltrating lymphocytes and are paralleled by the maintenance of connective tissue around blood vessels in the tumour mass. Correspondingly, lung metastasis formation is markedly reduced, to less than 10% of that seen in conbols. o 1996 Wiley-Liss, Inc. Basic research on synthetic drugs, effective against tumour metastases, has recently highlighted the effects of a ruthenium complex, namely sodium trans-rutheniumtetrachloridedimethylsulphoxideimidazole (hereafter indicated by Na[transRuCI4(DMSO)Im]) (Sava er al., 1992a, b, 1993, 1994). The effects of this new generation ruthenium(II1) complex on solid metastasizing tumour are particularly evident on the formation of spontaneous metastases. The selectivity of Na[transRuCI4(DMSO)Im] on lung metastases is also marked on advanced metastases and accounts for a significant prolongation of the host's survival time; combined with surgical removal of primary tumour, Na[trans-RuC14(DMSO)Im] prevents the formation of metastases and inhibits the growth of those already formed (Sava et al., 1994). The histological analysis of tumour growth and of healthy host tissues such as lung and kidney epithelia, muscle and liver cells, splenocytes and bone-marrow cells, by light microscopy and by SEM, shows a lack of significant cytotoxicity (Gagliardi et al., 1994). It thus appears that the selective anti-metastatic effects do not result directly from histological modification of the primary tumour structure.

The ruthenium-based compound imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate (NAMI-A) is free of cytotoxicity up to 1. mM concentration after 1. h in vitro exposure of the LLC-PK1 renal tubule cells. In vivo, one cycle of i.p. administrations of 35. mg/kg/day NAMI-A (1. cycle. =. 6 consecutive days), is free of a measurable toxicity on mouse kidneys. After two cycles with a one-week drug-free washout between cycles, mitochondrial membrane potential of the renal cells drops by 37% (p

We investigated the molecular events of the ruthenium complex NAMI-A (0.1 mM for 1 h) on cell cycle G2-M arrest in KB carcinoma cells. Flow cytometry analysis showed a progressive accumulation of cells in S phase at 16 h, and in G2-M phase at 20 h after the end of treatment. NAMI-A pre-mitotic stop to cell proliferation was due to the maintenance of the phosphorylated, inactive, form of Cdk1, caused by the activation of the ATM/ATR checkpoint, as confirmed by the up-regulation and phosphorylation of Chk1. All these events are related to intracellular ruthenium accumulation, as confirmed by the lack of similar effects in cell lines unable to take the ruthenium compound up. Considering the dependence of NAMI-A cell cycle arrest on the dose and on the length of cell challenge, and considering the prolonged NAMI-A t1/2 in vivo in the lungs, we proved an even greater perturbation of the cell cycle regulating pathways in lung metastases of NAMI-A treated mice. The ex-vivo data confirm the interaction of the ruthenium compound NAMI-A with the ATM/ATR pathway, leading to the modulation of cell cycle regulating proteins, that can break the metastases cell cycle progression off.

We investigated the molecular events of the ruthenium complex NAMI-A (0.1 mM for 1 h) on cell cycle G2-M arrest in KB carcinoma cells. Flow cytometry analysis showed a progressive accumulation of cells in S phase at 16 h, and in G2-M phase at 20 h after the end of treatment. NAMI-A pre-mitotic stop to cell proliferation was due to the maintenance of the phosphorylated, inactive, form of Cdk1, caused by the activation of the ATM/ATR checkpoint, as confirmed by the up-regulation and phosphorylation of Chk1. All these events are related to intracellular ruthenium accumulation, as confirmed by the lack of similar effects in cell lines unable to take the ruthenium compound up. Considering the dependence of NAMI-A cell cycle arrest on the dose and on the length of cell challenge, and considering the prolonged NAMI-A t1/2 in vivo in the lungs, we proved an even greater perturbation of the cell cycle regulating pathways in lung metastases of NAMI-A treated mice. The ex-vivo data confirm the interaction of the ruthenium compound NAMI-A with the ATM/ATR pathway, leading to the modulation of cell cycle regulating proteins, that can break the metastases cell cycle progression off.

In this paper we report a review of the results obtained in the last few years by our group in the development of ruthenium(III) complexes characterized by the presence of sulfoxide ligands and endowed with antitumor properties. In particular, we will focus on ruthenates of general formula Na[trans-RuCl4(R1R2SO)(L)], where R1R2SO = dimethylsulfoxide (DMSO) or tetramethylenesulfoxide (TMSO) and L = nitrogen donor ligand. The chemical behavior of these complexes has been studied by means of spectroscopic techniques both in slightly acidic distilled water and in phosphate buffered solution at physiological pH. The influence of biological reductants on the chemical behavior is also described. The antitumor properties have been investigated on a number of experimental tumors. Out of the effects observed, notheworthy appears the capability of the tested ruthenates to control the metastatic dissemination of solid metastasizing tumors. The analysis of the antimetastatic action, made in particular on the MCa mammary carcinoma of CBA mouse, has demonstrated a therapeutic value for these complexes which are able to significantly prolong the survival time of the treated animals. The antimetastatic effect is not attributable to a specific cytotoxicity for metastatic tumor cells although in vitro experiments on pBR322 double stranded DNA has shown that the test ruthenates bind to the macromolecule, causing breaks corresponding to almost all bases, except than thymine, and are able to cause interstrand bonds, depending on the nature of the complex being tested, some of which results active as cisplatin itself.

Two generations of lipophilic pyrenyl functionalized poly(benzyl ether) dendrimers (P1 and P2) have been synthesized. The thermal properties of the two functionalized dendrimers have been investigated, and the pyrenyl group of the dendritic molecules encapsulated in the arene–ruthenium metalla-cage, [Ru6(p-cymene)6(tpt)2(donq)3]6+ ([1]6+) (tpt=2,4,6-tri(pyridin-4-yl)-1,3,5-triazine; donq=5,8-dioxydo-1,4-naphthoquinonato). The host–guest properties of [P1⊂1]6+ and [P2⊂1]6+ were studied in solution by NMR and UV/Vis spectroscopic methods, thus allowing the determination of the affinity constants. Moreover, the cytotoxicity of these water-soluble host–guest systems was evaluated on human ovarian cancer cells.

[ImH][trans-Ru(III)Cl(4)(DMSO)(Im)] (where DMSO is dimethyl sulfoxide and Im is imidazole) (NAMI-A) is an antimetastatic prodrug currently in phase II clinical trials. The mechanisms of action of this and related Ru-based anticancer agents are not well understood, but several cellular targets have been suggested. Although Ru has been observed to bind to DNA following in vitro NAMI-A exposure, little is known about Ru-DNA interactions in vivo and even less is known about how this or related metallodrugs might influence cellular RNA. In this study, Ru accumulation in cellular RNA was measured following treatment of Saccharomyces cerevisiae with NAMI-A. Drug-dependent growth and cell viability indicate relatively high tolerance, with approximately 40% cell death occurring at 6 h for 450 μM NAMI-A. Significant dose-dependent accumulation of Ru in cellular RNA was observed by inductively coupled plasma mass spectrometry measurements on RNA extracted from yeast treated with NAMI-A. In vitro, binding of Ru species to drug-treated model DNA and RNA oligonucleotides at pH 6.0 and 7.4 was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the presence and absence of the reductant ascorbate. The extent of Ru-nucleotide interactions increases slightly with lower pH and significantly in the presence of ascorbate, with differences in observed species distribution. Taken together, these studies demonstrate the accumulation of aquated and reduced derivatives of NAMI-A on RNA in vitro and in cellulo, and enhanced binding with nucleic acid targets in a tumorlike acidic, reducing environment. To our knowledge, this is also the first study to characterize NAMI-A treatment of S. cerevisiae, a genetically tractable model organism.

A series of organometallic compounds of general formula [(arene)M(PTA)(n)X(m)]Y (arene = eta(6)-C(10)H(14), eta-C(5)Me(5)); M = Ru(ii), Os(ii), Rh(iii) and Ir(iii); X = Cl, mPTA; Y = OTf, PF(6)) have been screened for their cytotoxicity and ability to inhibit cathepsin B in vitro, in comparison to the antimetastatic compound NAMI-A. The Ru and Os analogues and NAMI-A showed similar enzyme inhibition properties (with IC(50) values in the low muM range), whereas the Rh(iii) and Ir(iii) compounds were inactive. In order to build up a rational for the observed differences, DFT calculations of the metal complexes adducts with N-acetyl-l-cysteine-N'-methylamide, a mimic for the Cys residue in the cathepsin B active site, were performed to provide insights into binding thermodynamics in solution. Initial structure-activity relationships have been defined with the calculated binding energies of the M-S bonds correlating well with the observed inhibition properties of the compounds.

A novel ruthenium(II) compound, trans-cis-cis-[Ru(II)Cl-2(DMSO)(2)(2-amino-5-methyl-thiazole)(2)], (I), PMRu52 hereafter, that may be obtained from the previously described (cis and trans)[Ru(II)Cl-2(DMSO)(4)] complexes, was designed, synthesized and characterised. The single crystal X-ray structure shows a roughly regular octahedral environment for the ruthenium(II) center with the two chloride ligands in trans and the other two pairs of identical ligands in cis. The behaviour of PMRu52 in phosphate buffer, at pH = 7.4, was characterised spectroscopically as well as its interactions with a few representative biomolecules. Tight ruthenium binding to serum albumin was established by joint use of spectroscopic and separation methods. Afterward, the reactions of PMRu52 with the model proteins ubiquitin and cytochrome c were monitored through electrospray ionisation mass spectrometry (ESI-MS) methods: the formation of metallodrug-protein adducts was documented in detail and the fragmentation patterns of PMRu52 were defined. Finally, the ability of PMRu52 to affect the activity of cathepsin B, a well known cysteine protease, was evaluated in vitro and a pronounced enzyme inhibition highlighted, with an IC50 value of 5.5 mu M. This latter finding is of particular interest as cathepsin B constitutes an attractive "druggable" target for cancer, rheumatoid arthritis and other important diseases. (C) 2009 Elsevier Inc. All rights reserved.

Ruthenium-based compounds are an attractive alternative to clinically used platinum drugs due to several features including a wide range of accessible oxidation states, varied synthetic chemistry and typically lower general toxicities. One series of ruthenium(II)-arene-pta, RAPTA (pta = 1,3,5-triaza-7-phosphatricyclo[3.3.1.1] decane) compounds has been found to show particularly high selectivity towards cancer cells in both in vitro and in vivo studies.

The synthesis, molecular structure and chemical behavior of two chloride-dimethyl sulfoxide-ruthenium (III) derivatives, namely [(DMSO) 2 H][trans-Ru(DMSO) 2 Cl 4 ] (1) and mer-Ru(DMSO) 3 Cl 3 (2), are reported. They represent the first examples of fully characterized halogen-dimethyl sulfoxide-ruthenium (III) complexes

(ImH)[trans-RuCl(4)(DMSO-S)(Im)], (Im = imidazole, DMSO-S = S-bonded dimethylsulfoxide), NAMI-A, is the first anticancer ruthenium compound that successfully completed Phase I clinical trials. NAMI-A shows a remarkable activity against lung metastases of solid tumors, but is not effective in the reduction of primary cancer. The structurally similar (ImH)[trans-RuCl(4)(Im)(2)], ICR (or KP418), and its indazole analog (KP1019) are promising candidate drugs in the treatment of colorectal cancers, but have no antimetastatic activity. Despite the pharmacological relevance of these compounds, no rationale has been furnished to explain their markedly different activity. While the nature of the chemical species responsible for their antimetastatic/anticancer activity has not been determined, it has been suggested that the difference between reduction potentials of NAMI-A and ICR may be the key to the different biological responses they induce. In this work, Density Functional Theory calculations were performed to investigate the hydrolysis of NAMI-A and ICR in both Ru(III) and Ru(II) oxidation states, up to the third aquation. In line with experimental findings, our calculations provide a picture of the hydrolysis of NAMI-A and ICR mainly as a stepwise loss of chloride ligands. While dissociation of Im is unlikely under neutral conditions, that of DMSO becomes competitive with the loss of chloride ions as the hydrolysis proceeds. Redox properties of NAMI-A and ICR and of their most relevant hydrolytic intermediates were also studied in order to monitor the effects of biological reductants on the mechanism of action. Our findings may contribute to the identification of the active compounds that interact with biological targets, and to explain the different biological activity of NAMI-A and ICR.

We carried out density functional theory (DFT) calculations to investigate the thermodynamics and the kinetics of the double aquation reaction of the anticancer drug NAMI-A. Three explicit water molecules were included in the calculations to improve the PB solvation energies. Our calculations show that the chloride substitution reactions on the considered Ru(III) octahedral complex follow a dissociative interchange mechanism, I(d), passing through a loose heptacoordinate transition state. We calculated an activation enthalpy and free energy for the first aquation step of 101.5 and 103.7 kJ mol(-1), respectively, values that are in good agreement with the available experimental results. The activation enthalpy and free energy for the second aquation step were found significantly higher, 118.7 and 125.0 kJ mol(-1), again in agreement with the experimental evidence indicating a slower rate for the second aquation.

The interactions with protein targets of the ruthenium(III) complex imidazolium trans-[tetrachloro(dimethyl sulfoxide)(imidazole)ruthenate(III)], NAMI-A, an effective anticancer and antimetastatic agent now in clinical trials, deserve great attention as they are believed to be at the basis of the mechanism of action of this innovative molecule. Here, we report on the reactions of NAMI-A with two well-known model proteins, namely, hen egg white lysozyme and horse heart cytochrome c; these reactions were investigated by a variety of physicochemical methods, including optical spectroscopy, (1)H NMR and electrospray ionization mass spectrometry. The combined use of the analytical techniques mentioned resulted in a rather exhaustive description of the NAMI-A-protein interactions; in particular, the formation of fairly stable metal-protein adducts was clearly documented and the nature of the resulting protein-bound metallic fragments ascertained in most cases. Notably, greatly different patterns of interaction were found to be operative for NAMI-A toward these two proteins. The biological implications of the present findings are discussed.

Ruthenium complex NAMI-A [ImH][trans-RuCl(4)(DMSO-S)Im] (Im = imidazole) is a potential chemotherapeutic drug in cancer treatment. Electroporation can be used to facilitate delivery of NAMI-A into cells. Suspension of B16F1 tumour cells from mouse melanoma in NAMI-A solution was exposed to a train of electric pulses. The effect of NAMI-A was determined by examining cell viability in clonogenic test. Our results show that electroporation increases the otherwise scarce in vitro effects of NAMI-A, i.e. reduces cell viability. At the conditions chosen for experiments 90% of cells survived in the presence of 1 microM NAMI-A, whereas in a combined treatment with 1 microM NAMI-A and electroporation only about 10% of cells survived.

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.

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 (IC50 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.

Biotransformation of ruthenium(III) anticancer complexes as hypothesized in the activation-by-reduction theory is the central topic of the present paper. The redox behavior of tetrachlorobis(azole)ruthenate(III)-type complexes was studied by NMR spectroscopy and square wave voltammetry. The influence of reducing agents on the binding behavior toward the DNA-modeling nucleotide GMP was determined by capillary electrophoresis, accompanied by identification of arising peaks by online coupling to electrospray ionization mass spectrometry. The determination of redox potentials revealed that the biologically relevant reductants ascorbic acid and glutathione are capable of reducing the studied Ru(III) complexes under physiological conditions. Characteristic differences in reduction kinetics dependent on the pH value can be explained by higher reduction strength of ascorbic acid and glutathione at higher pH compared to the pH-independent redox response of ruthenium(III) complexes. Binding behavior of (H2ind)[trans-RuCl4(Hind)2] (Hind = 1H-indazole) toward GMP was found to be increased upon addition of two equivalents of glutathione but not of ascorbic acid. In contrast, only a minor influence on the GMP-binding under reductive conditions was found for (H2im)[trans-RuCl4(Him)2] (KP418, Him = 1H-imidazole).

Transition-metal-based compounds constitute a discrete class of chemotherapeutics, widely used in the clinic as antitumor and antiviral agents. Examples of established antitumor metallodrugs, routinely used in the clinic, are cisplatin [cis-diamminedichloroplatinum(II)] and its analogues carboplatin and oxaliplatin. However, drug resistance and side effects have limited their clinical utility. These limitations have prompted a search for more effective and less toxic metal-based antitumor agents. Some of the efforts have been directed in the design of non-platinum, transition-metal-based antitumor agents and ruthenium complexes have attracted much interest as alternative drugs to cisplatin in cancer chemotherapy. Ruthenium complexes demonstrate similar ligand exchange kinetics to those of platinum(II) antitumor drugs already used in the clinic while displaying only low toxicity. This is in part due to the ability of ruthenium complexes to mimic the binding of iron to molecules of biological significance, exploiting the mechanisms that the body has evolved for transport of iron. In addition, the redox potential between the different accessible oxidation states occupied by ruthenium complexes enables the body to catalyze oxidation and reduction reactions, depending on physiological environment. The biochemical changes that accompany cancer alter physiological environment, enabling ruthenium complexes to be selectively activated in cancer tissues. Due to differing ligand geometry between their complexes, ruthenium compounds bind to DNA affecting its conformation differently than cisplatin and its analogues. In addition, non-nuclear targets, such as the mitochondrion and the cell surface, have also been implicated in the antineoplastic activity of some ruthenium complexes. Thus, ruthenium compounds offer the potential over antitumor platinum(II) complexes currently used in the clinic of reduced toxicity, a novel mechanism of action, the prospect of non-cross-resistance and a different spectrum of activity. In other words, some chemical properties make ruthenium compounds well suited for medicinal applications and as an alternative to platinum antitumor drugs in the treatment of cancer cells resistant to cisplatin. Although the pharmacological target for antitumor ruthenium compounds has not been unequivocally identified, there is a large body of evidence indicating that the cytotoxicity of many ruthenium complexes correlates with their ability to bind DNA although few exceptions have been reported. This review summarizes results demonstrating that several ruthenium compounds that exhibit antitumor effects different from cisplatin or its analogues bind DNA and modify it differently than cisplatin or its analogues.

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 alpha 2 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.

Inibizione delle metastasi di un melanoma murino da complessi di rutenio e down-regulation dell'invasione cellulare.

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.

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.

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.

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.

The ruthenium complex salt indazolium trans-[tetrachlorobisindazole-ruthenate(III)] (KP1019) and the analogous sodium salt KP1339 are effective tumor-inhibiting drugs in experimental therapy of autochthonous colorectal carcinomas in rats. This paper examines the cell biological mechanisms underlying their antineoplastic effects.Colorectal tumor cell lines were used to analyze uptake of the ruthenium(III) complexes into the cells and the mechanism as well as the efficacy of their cytotoxic effects.KP1019 and KP1339 are efficiently taken up into the cells: 100 microM ruthenium(III) complex in the growth medium led to the uptake of 120-160 ng ruthenium per 10(6) cells within 30 min. Uptake of KP418 was tenfold lower correlating with its lower cytotoxic efficiency. KP1019 and KP1339 induced apoptosis in SW480 and HT29 cells predominantly by the intrinsic mitochondrial pathway as indicated by loss of mitochondrial membrane potential. Correspondingly sensitivity of the cells paralleled expression of bcl(2) while it was only slightly affected by mutations in Ki-ras.Our data demonstrate that trans-[tetrachlorobisindazole-ruthenate(III)] complex salts are promising candidate drugs in the second-line treatment of colorectal cancers resistant to other cytostatic drugs and has been introduced into phase I clinical trials.

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.

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.

NAMI-A (imidazolium trans-imidazole dimethylsulfoxide tetrachlororuthenate (III)) is a novel antimetastatic ruthenium complex currently undergoing Phase I clinical trials. It is pharmaceutically formulated as a lyophilized product to be reconstituted and diluted before infusion. The aim of this study was to determine the reconstitution and dilution fluid of choice, and to investigate the stability and compatibility of NAMI-A in solution under different storage conditions and with several container materials. Furthermore, the hemolytic potential and buffer capacity of NAMI-A infusion solution were investigated in vitro. Normal saline or Water for Injections (WfI) can be used for reconstitution of NAMI-A, and the reconstituted solution should be diluted with normal saline to infusion concentration immediately after dissolution if stored at room temperature (1 20 - 258C), but can be stored for up to 48 hours at refrigerated conditions (1 2 - 88C). Infusion solutions can be stored for a maximum of four hours at room temperature or 24 hours at refrigerated conditions before administration to the patient. The infusion solutions can be prepared in either glass or PVC containers, although for practical reasons PVC containers are recommended. The infusion solutions are compatible with a PVC infusion system consisting of a PVC container, PVC infusion line, and needle. Furthermore, NAMI-A infusion solutions have a low buffer capacity and do not cause hemolysis in vitro. Therefore, no pain or hemolysis upon infusion of NAMI-A in the clinical setting is expected.

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.

25 years after the first approval of cisplatin in the clinic against a number of cancer diseases, cisplatin and related compounds continue to be among the most efficient anticancer drugs used so far. Efforts are focused to develop novel platinum- and non-platinum-based antitumor drugs to improve clinical effectiveness, to reduce general toxicity and to broaden the spectrum of activity. In the field of non-platinum compounds exhibiting anticancer properties, ruthenium complexes are very promising, showing activity on tumors which developed resistance to cisplatin or in which cisplatin is inactive. Furthermore, general toxicity was found to be very low. The first ruthenium compound NAMI-A entered phase I clinical trials in 1999 as an antimetastatic drug, whereas the ruthenium complex KP1019 will enter phase I clinical trials in 2003 as an anticancer drug which is among others very active against colon carcinomas and their metastases. Remarkable progress is also seen in developing tumor inhibiting gallium compounds. One of them, KP46, will also enter phase I clinical trials in 2003. This article reviews briefly the achievements in the field of anticancer metal complexes focusing the discussion onto the impact of the group of Bioinorganic Chemistry at the Department of Inorganic Chemistry at the University of Vienna. The development of pH sensitive platinum prodrugs, platinum-based drug targeting strategies with low-molecular-weight carriers, kinetically inert platinum(IV) complexes, as well as tumor inhibiting non-platinum anticancer drugs based on ruthenium and gallium is covered in the following sections.

The cytotoxicity, intracellular accumulation and DNA adduct formation of the ruthenium complex imidazolium trans-imidazoledimethylsulfoxide tetrachlororuthenate (ImH[ trans-RuCl(4)(DMSO)Im], Nami-A) were compared in vitro with those of cisplatin in four human tumor cell lines: Igrov-1, 2008, MCF-7, and T47D.Cytotoxicity was assessed in vitro using a growth inhibition assay. Accumulation was determined by flameless atomic absorption spectroscopy (AAS). GG and AG intrastrand adducts were measured using the (32)P-postlabeling assay.Nami-A was on average 1053 times less cytotoxic than cisplatin. The cytotoxicity of cisplatin was linearly related to both intracellular platinum accumulation and DNA binding, while the cytotoxicity of Nami-A was significantly related only to DNA binding and not to intracellular ruthenium accumulation. The levels of accumulation of Nami-A measured as ruthenium and of cisplatin measured as platinum were correlated linearly with the incubation concentration over a concentration range of 0 to 600 micro M of both drugs. Ruthenium intracellular accumulation and DNA binding were on average 4.8 and 42 times less, respectively, than those of cisplatin. In addition, the numbers of GG and AG intrastrand adducts induced by Nami-A were 418 and 51 times fewer, respectively. Nami-A and cisplatin had the same binding capacity to calf thymus DNA. Nami-A was 25-40% less bound to cellular proteins than cisplatin.There was no saturation of the uptake and DNA binding capacity of either Nami-A or cisplatin. Furthermore, the low binding of Nami-A to cellular DNA cannot simply be explained by a lower capacity to bind to DNA, because the absolute level of binding in vitro to calf thymus DNA was the same for Nami-A and cisplatin. Finally, the lower cytotoxicity of Nami-A on a molar basis than that of cisplatin can at least partly be explained by its reduced reactivity to DNA in intact cells.

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.

The pharmaceutical development of the two novel metal-based anticancer agents NAMI-A and AP 5280 is described in this thesis, starting with the development of analytical methods for the quality control of drug substance and final product, via the formulation process leading to a stable, intravenous administration form, and ending with the stability and compatibility studies of the infusion solutions. This research could also serve as the basis for the pharmaceutical development of other novel, metal-based anticancer agents. Because many metals exchange ligands easily, the compounds will usually not be stable in solution, necessitating preparation of a lyophilized product. NAMI-A and AP 5280 are examples of this. The experiments described in this thesis enabled the clinical evaluation of NAMI-A and AP 5280. At this time, 23 patients have been treated with NAMI-A and 30 with AP 5280 in the clinical Phase I study. Subsequent Phase II studies will provide insight into the clinical efficacy of these agents

The pharmaceutical development of the two novel metal-based anticancer agents NAMI-A and AP 5280 is described in this thesis, starting with the development of analytical methods for the quality control of drug substance and final product, via the formulation process leading to a stable, intravenous administration form, and ending with the stability and compatibility studies of the infusion solutions. This research could also serve as the basis for the pharmaceutical development of other novel, metal-based anticancer agents. Because many metals exchange ligands easily, the compounds will usually not be stable in solution, necessitating preparation of a lyophilized product. NAMI-A and AP 5280 are examples of this. The experiments described in this thesis enabled the clinical evaluation of NAMI-A and AP 5280. At this time, 23 patients have been treated with NAMI-A and 30 with AP 5280 in the clinical Phase I study. Subsequent Phase II studies will provide insight into the clinical efficacy of these agents

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 pH6 and zero-order kinetics at pHor =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.

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.

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.

NAMI-A is a ruthenium-based compound with selective anti-metastasis activity in experimental 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 well as its in vivo effects in the chick embryo chorioallantoic membrane model. Endothelial cell proliferation, chemotaxis, and secretion of the matrix-degrading enzyme metalloproteinase-2 were inhibited by NAMI-A in a dose-dependent manner, and without morphologic signs of cell apoptosis or necrosis. Lastly, 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. British Journal of Cancer (2002) 86, 993–998. DOI: 10.1038/sj/bjc/6600176 www.bjcancer.com © 2002 Cancer Research UK

NAMI-A is a novel ruthenium-containing experimental anticancer agent. We have developed and validated a rapid and sensitive analytical method to determine NAMI-A in human plasma, plasma ultrafiltrate and urine using atomic absorption spectrometry with Zeeman correction. The sample pretreatment procedure is straightforward, involving only dilution with an appropriate hydrochloric acid buffer-solution. Because the response signal of the spectrometer depended on the composition of the sample matrix, in particular on the amount of human plasma in the sample, all unknown samples were diluted to match the matrix composition in which the standard line was prepared (plasma-buffer 1:10 v/v). This procedure enabled the measurement of samples of different biological matrices in a single run. The validated range of determination was 1.1-220 μM NAMI-A for plasma and urine, and 0.22-44 μM for plasma ultrafiltrate. The lower limit of detection was 0.85 μM in plasma and urine and 0.17 μM in plasma ultrafiltrate. The lower limit of quantitation was 1.1 and 0.22 μM, respectively. The performance of the method, in terms of precision and accuracy, was according to the generally accepted criteria for validation of analytical methodologies. The applicability of the method was demonstrated in a patient who was treated in a pharmacokinetic phase I trial with intravenous NAMI-A. © Springer-Verlag 2001.