Synthesis, Characterisation and In Vitro Permeation, Dissolution and Cytotoxic Evaluation of Ruthenium(II)-Liganded Sulpiride and Amino Alcohol.

Sulpiride (SPR) is a selective antagonist of central dopamine receptors but has limited clinical use due to its poor pharmacokinetics. The aim of this study was to investigate how metal ligation to SPR may improve its solubility, intestinal permeability and prolong its half-life. The synthesis and characterisation of ternary metal complexes [Ru(p -cymene)(L)(SPR)]PF6 (L1 = (R)-(+)-2-amino-3-phenyl-1-propanol, L2 = ethanolamine, L3 = (S)-(+)-2-amino-1-propanol, L4 = 3-amino-1-propanol, L5 = (S)-(+)-2-pyrrolidinemethanol) are described in this work. The stability constant of the [Ru(p -cymene)(SPR)] complex was determined using Job's method. The obtained value revealed higher stability of the metal complex in the physiological pH than in an acidic environment such as the stomach. The ternary metal complexes were characterised by elemental analysis, Fourier transform infrared spectroscopy (FT-IR), 1H and 13C nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), thermal analyses, Ultraviolet-Visible (UV-Vis). Solubility studies showed higher aqueous solubility for complexed SPR than the free drug. Dissolution profiles of SPR from the metal complexes exhibited slower dissolution rate of the drug. Permeation studies through the pig's intestine revealed enhanced membrane permeation of the complexed drug. In vitro methyl thiazolyl tetrazolium (MTT) assay showed no noticeable toxic effects of the ternary metal complexes on Caco-2 cell line.

Ultradeformable liposome loaded with zinc phthalocyanine and [Ru(NH.NHq)(tpy)NO]3+ for photodynamic therapy by topical application.

Ultradeformable liposomes (UDLs) as a drug delivery system (DDS), prepared from the unsaturated phospholipid, dioleylphosphocholine (DOPC), and containing the non-ionic surfactant Tween 20 as edge activator, have been explored as topical vehicles for zinc phthalocyanine (ZnPc) and the nitrosyl ruthenium complex [Ru(NH.NHq)(tpy)NO]3+ (RuNO) as a photosensitizers for co-generation of 1O2 and NO as reactive species, respectively. However, in order to ensure that ZnPc was present in the UDLs in its monomeric form - essential for maximal ZnPc photophysical properties - it was necessary to replace 40wt% of the DOPC with the saturated phospholipid, dimyristoylphosphocholine (DMPC). The resultant ZnPc and complex [Ru(NH.NHq)(tpy)NO]3+ containing UDLs were stable for at least a month when stored at 4°C, six times more elastic/deformable than conventional liposome (c-Ls), i.e. liposome prepared using the same weight ratio of lipids but in the absence of Tween 20, and to significantly enhance the in vitro permeation of ZnPc across fresh pig ear skin. The UDLs DDS incorporating ZnPc and [Ru(NH.NHq)(tpy)NO]3+ were toxic (by the MTT assay) towards B16-F10 melanoma cells when irradiated with visible light at 670nm, the maximum absorption of ZnPc, and at a dose of 3.18J/cm2, but not when applied in the absence of light as expected. Based on these results it is proposed that the novel topical UDLs formulation developed is a suitable delivery vehicle for photodynamic therapy.

A ruthenium(II) complex inhibits tumor growth in vivo with fewer side-effects compared with cisplatin.

The antitumor activity of a ruthenium(II) polypyridyl complex, Δ-[Ru(bpy)2(HPIP)](ClO4)2 (Δ-Ru1, where bpy=2,2'-bipyridine, HPIP=2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline), was evaluated. The in vivo experiments showed that Δ-Ru1 inhibited the growth of a human cervical carcinoma cell line (HeLa) xenotransplanted into nude mice with efficiency similar to that of cisplatin. Histopathology examination of the tumors from treated xenograft models was consistent with apoptosis in tumor cells. Importantly, in striking contrast with cisplatin, Δ-Ru1 did not cause any detectable side effects on the kidney, liver, peripheral neuronal system, or the hematological system at the pharmacologically effective dose. The preclinical studies reported here provide support for the clinical use of Δ-Ru1 as an exciting new drug candidate with lower toxicity than cisplatin, endowed with proapoptotic properties.

Contrasting cellular uptake pathways for chlorido and iodido iminopyridine ruthenium arene anticancer complexes.

The pathways involved in cellular uptake and accumulation of iminopyridine complexes of general formula [Ru(η(6)-p-cymene)(N,N-dimethyl-N'-[(E)-pyridine-2-ylmethylidene]benzene-1,4-diamine)X]PF(6) bearing two different halido ligands X = Cl or I, have been explored. The ratio of passive/active cellular accumulation of Ru in A2780 human ovarian cancer cells is compared and contrasted with cisplatin. Also, saturation of cellular uptake, time-dependence of cellular influx/efflux equilibria, together with endocytotic pathways such as caveolae and facilitated diffusion are investigated and discussed. Temperature dependence studies of Ru accumulation in the A2780 cells show that in contrast to cisplatin (CDDP) and chlorido complex , which are taken up largely through active transport, the iodido complex enters cells via passive transport. The cellular efflux of Ru is slow (ca. 25% retained after 72 h) and is partially inhibited by verapamil, implicating the P-gp protein in the efflux mechanism. Ouabain inhibition experiments suggest that the cellular uptake of these ruthenium complexes relies at least in part on facilitated diffusion, and in particular is dependent on the membrane potential. In addition the finding that depletion of cellular ATP with antimycin A had little effect on cellular Ru accumulation from iodido complex is consistent with passive diffusion. In contrast, ATP depletion caused a major increase in cellular accumulation of ruthenium from chlorido complex .

Ruthenium anticancer compounds: myths and realities of the emerging metal-based drugs.

Ruthenium anticancer drugs have attracted an increasing interest in the last 20 years and two of them have entered clinical trials. Compared to platinum drugs, the complexes based on ruthenium are often identified as less toxic and capable of overcoming the resistance induced by platinum drugs in cancer cells. These activities were attributed to the transportation to tumour cells by transferrin and to the selective activation to more reactive species by the reducing environment of solid tumours as compared to healthy tissues. Ruthenium anticancer drugs have been almost always designed to mimic platinum drugs, particularly for targeting DNA. Indeed, none of the above properties has never been clearly demonstrated even for the ruthenium drugs that entered clinical trials. The suggestion for the future is to change the perspective when designing new chemical entities, abandoning the philosophy that guided the actual panel of ruthenium drugs and to look further into the fine mechanism by which the most relevant ruthenium complexes available kill the target tumour cells, then focusing on targets selective of tumour cells and responsible for cell growth and malignancy.

Tailoring NO donors metallopharmaceuticals: ruthenium nitrosyl ammines and aliphatic tetraazamacrocycles.

The discovery of the involvement of nitric oxide (NO) in several physiological and pathophysiological processes launched a spectacular increase in studies in areas such as chemistry, biochemistry, and pharmacology. As a consequence, the development of NO donors or scavengers for regulation of its concentration and bioavailability in vivo is required. In this sense, ruthenium nitrosyl ammines and aliphatic tetraazamacrocyles have attracted a lot of attention due to their unique chemical properties. These complexes are water soluble and stable in solution, not to mention that they can deliver NO when photochemically or chemically activated by the reduction of the coordinated nitrosonium (NO+). The tuning of the energies of the charge transfer bands, the redox potential, and the specific rate constants of NO liberation, in both solution and matrices, is desirable for the achievement of selective NO delivery to biological targets, hence making the ruthenium ammines and aliphatic tetraazamacrocyles a quite versatile platform for biological application purposes. These ruthenium nitrosyls have shown to be active in firing neurons in mouse hippocampus, performing redox reactions in mitochondria, acting in blood pressure control, exhibiting cytotoxic activities against trypanosomatids (T.cruzi and L.major) and tumor cells. This tailoring approach is explored here, being heavily supported by the accumulated knowledge on the chemistry and photochemistry of ruthenium complexes, which allows NO donors/scavengers systems to be custom made designed.

Pharmacokinetics of a novel anticancer ruthenium complex (KP1019, FFC14A) in a phase I dose-escalation study.

A phase I and pharmacokinetic study was carried out with the new ruthenium complex indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019, FFC14A). Seven patients with various types of solid tumours refractory to standard therapy were treated with escalating doses of KP1019 (25-600 mg) twice weekly for 3 weeks. No dose-limiting toxicity occurred. Ruthenium plasma concentration-time profiles after the first dose and under multiple-dose conditions were analysed using a compartmental approach. The pharmacokinetic disposition was characterised by a small volume of distribution, low clearance and long half-life. Only a small fraction of ruthenium was excreted renally. The area under the curve values increased proportionally with dose indicating linear pharmacokinetics.

Reactivity of an antimetastatic organometallic ruthenium compound with metallothionein-2: relevance to the mechanism of action.

The reaction of metallothionein-2 (MT-2) with the organometallic antitumour compound [Ru(η(6)-p-cymene)Cl(2)(pta)], RAPTA-C, was investigated using ESI MS and ICP AES. The studies were performed in comparison to cisplatin and significant differences in the binding of the two complexes were observed. RAPTA-C forms monoadducts with MT-2, at variance with cisplatin, that has been observed to form up to four adducts. These data, combined with ICP AES analysis, show that binding of both RAPTA-C and cisplatin to MT-2 requires the displacement of an equivalent amount of zinc, suggesting that Cys residues are the target binding sites for the two metallodrugs. The competitive binding of RAPTA-C and cisplatin towards a mixture of ubiquitin (Ub) and MT-2 was also studied, showing that MT-2 can abstract RAPTA-C from Ub more efficiently than it can abstract cisplatin. The mechanistic implications of these results are discussed.

Recent developments in ruthenium anticancer drugs.

Interest in Ru anticancer drugs has been growing rapidly since NAMI-A ((ImH(+))[Ru(III)Cl(4)(Im)(S-dmso)], where Im = imidazole and S-dmso = S-bound dimethylsulfoxide) or KP1019 ((IndH(+))[Ru(III)Cl(4)(Ind)(2)], where Ind = indazole) have successfully completed phase I clinical trials and an array of other Ru complexes have shown promise for future development. Herein, the recent literature is reviewed critically to ascertain likely mechanisms of action of Ru-based anticancer drugs, with the emphasis on their reactions with biological media. The most likely interactions of Ru complexes are with: (i) albumin and transferrin in blood plasma, the former serving as a Ru depot, and the latter possibly providing active transport of Ru into cells; (ii) collagens of the extracellular matrix and actins on the cell surface, which are likely to be involved in the specific anti-metastatic action of Ru complexes; (iii) regulatory enzymes within the cell membrane and/or in the cytoplasm; and (iv) DNA in the cell nucleus. Some types of Ru complexes can also promote the intracellular formation of free radical species, either through irradiation (photodynamic therapy), or through reactions with cellular reductants. The metabolic pathways involve competition among reduction, aquation, and hydrolysis in the extracellular medium; binding to transport proteins, the extracellular matrix, and cell-surface biomolecules; and diffusion into cells; with the extent to which individual drugs participate in various steps along these pathways being crucial factors in determining whether they are mainly anti-metastatic or cytotoxic. This diversity of modes of action of Ru anticancer drugs is also likely to enhance their anticancer activities and to reduce the potential for them to develop tumour resistance. New approaches to metabolic studies, such as X-ray absorption spectroscopy and X-ray fluorescence microscopy, are required to provide further mechanistic insights, which could lead to the rational design of improved Ru anticancer drugs.

KP1019, a new redox-active anticancer agent--preclinical development and results of a clinical phase I study in tumor patients.

The promising drug candidate indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) is the second Ru-based anticancer agent to enter clinical trials. In this review, which is an update of a paper from 2006 (Hartinger et al., J. Inorg. Biochem. 2006, 100, 891-904), the experimental evidence for the proposed mode of action of this coordination compound is discussed, including transport into the cell via the transferrin cycle and activation by reduction. The results of the early clinical development of KP1019 are summarized in which five out of six evaluated patients experienced disease stabilization with no severe side effects.

Functionalization of PAMAM dendrimers with [Ru(III)(edta)(H2O)](-).

The anchoring of K[Ru(III)(edta)(Cl)] on poly(amidoamine) dendrimers (PAMAM of three generations G(x)/Ru (x=0, 2 and 3)) through a peptide type bond yielded the aquo species, [Ru(III)(edta)(H2O)] on dendrimer surface, and upon NO exposure, yielded their nitrosyl analogues, G(x)/RuNO. Characterization of these compounds by elemental analysis, and a UV-vis, IR and 13C NMR spectroscopies indicated the immobilization of 4, 12 and 29 molecules of [Ru(III)(edta)(H2O)] or of the nitrosyl complex [Ru(II)(edta)NO] on the dendrimer surface for G(X)=0, 2 and 3, respectively. For each complex the electrochemical spectrum presented only one redox process with redox potential values of -0.20 and -0.32 V(vs SCE) attributed to the Ru(III)/Ru(II) and NO+/NO(0) couples in G(x)/Ru and G(x)/RuNO, respectively. The one-electron reduction of G(x)/RuNO+ generates G(x)/RuNO(0), which undergoes aquation with a k(-NO) of 2.1+/-0.7 x 10(-3)s(-1) (pH 1.0, mu=0.2 mol/L, CF3COOH/NaCF3COO, 25 degrees C). The G(x)/RuNO species induced a relaxing effect in aortic rings denuded of endothelium and exhibited in vitro assay trypanocidal activity.

Influence of the chemical form on the plasma clearance of ruthenium in humans.

The radioisotopes of ruthenium (103Ru and 106Ru) are abundant fission products and represent a radiological risk for the population in case of nuclear accidents. Few biokinetic studies have been performed on humans up to now and consequently the current model recommended by ICRP for ruthenium is derived mainly by extrapolation from animal data. The stable isotope 101Ru and proton activation analysis have been used to study the biokinetics of Ru in blood plasma samples taken during 8 studies in three healthy volunteers. The results obtained demonstrated that complexed Ru (in the form of citrate Ru(IV) complexes) is cleared from blood plasma very rapidly (characteristic half time of 17+/-2 min), while inorganic Ru remains longer in the systemic circulation, and is transferred to other organs and/or excreted with a biological half time of 23+/-2h. Good reproducibility of the clearance curves indicated no evidence of inter- or intra-individual variability when the same Ru solution was injected in repeated experiments to different subjects.

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.

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.