Search

It is reported that NAMI-A and other Ru-anticancer complexes preferably bind with the N7 site of guanine and can also form DNA inter-strand cross-links. Therefore, in order to understand the DNA cross-link formation capability of NAMI-A, we have investigated here the structure and energetics of the reactions of the GN7-NAMI-A (a monofunctional adduct of NAMI-A with the N7 site of guanine) with the N3, N7, and O6 sites of guanine; the N1, N3, and N7 sites of adenine; the O2 and N3 sites of cytosine; and the O2 and O4 sites of thymine, using the M06-2X functional of density functional theory. It is found that the GN7-NAMI-A can form stable cross-linked products at all the sites studied here except at the N3 site of cytosine and O2 site of thymine. The calculated reaction free energies and reaction enthalpies indicate that the N3 site of adenine (AN3) and N7 site of guanine (GN7) are most exothermic among all the studied reactions. This study shows that NAMI-A would favorably form the cross-linked products involving the N7 site of guanine at one side and the N7 site of guanine or the N3 site of adenine at the other side. [ABSTRACT FROM AUTHOR]

The cytotoxic activities of KP1019 and other Ru(III) drugs are believed to be associated with their binding with DNA. Here, we report the density functional theory (DFT) study of reactions of KP1019 drug at the O6 and O8 sites of 8-oxoguanine (8-oxoG) and the O8 site of 8-oxoadenine (8-oxoA). 8-OxoG is predominantly formed in oxidative stress and can cause mutation and cancer. It is found that the barrier free energies (ΔGb) of these reactions obey the following trend: O8 (8-oxoG) < O8 (8-oxoA) < O6 (8-oxoG), at different levels of theory in gas phase and aqueous media. The ΔGb of reaction at the O8 (8-oxoG) is found to be 10.96 (13.81) kcal/mol at the M06-2X/(LanL2DZ+6-311+G**) level of theory in gas phase (aqueous media). The rate constant of reaction at the O8 (8-oxoG) site in aqueous media is 4.6 × 102 s−1. The reaction free energies (ΔGf) and reaction enthalpies (ΔHf) of all the reactions are appreciably negative in both gas phase and aqueous media which indicate that the reaction of mono-aquated KP1019 at the O6 and O8 sites of 8-oxoG as well as at the O8 site of 8-oxoA would occur spontaneously. Further, our calculations demonstrate that KP1019 would react with 8-oxoG more favourably as compared with guanine. Thus, it predicts that the main mechanism of the action of KP1019 drug might be due to its binding with the O8 site of 8-oxoG in biological media. [ABSTRACT FROM AUTHOR]

Although the mechanisms of action of Ru(III) anticancer drugs including NAMI-A and KP1019 are not properly understood, it is reported that they are readily aquated in vivo before reacting to the biological targets and their cytotoxic activities are mainly attributed to their binding with DNA. In the present contribution in order to understand how changing the axial ligands affects the mechanisms of action of Ru(III)-based drugs which are structurally similar to NAMI-A, we study here the aquation reactions (substitution of one chloride anion (Cl−) ligand by a water molecule) of six structurally similar Ru(III) complexes (1–6) including NAMI-A (complex 1) and KP1019 (complex 2) as well as the reactions of their mono-aquated products with the N3, N7 and O6 sites of guanine at the M06-2X/(LanL2DZ+6-311+G**)//M06-2X/(LanL2DZ+6-31G**) level of density functional theory in gas phase and aqueous media. It is found that the aquation reaction of every Ru(III) complex occurs through a single transition state where a chloride ligand of the complex is replaced by a H2O molecule. The activation free energies (ΔGb) and rate constants of aquation reactions show that the complex 6 would get aquated in biological media more readily than other complexes. The rate constant of aquation of complex 6 is 7.98 × 10−3 s−1. It is evident from our calculations that axial ligands play important role in the aquation of Ru(III) complexes structurally similar to NAMI-A, but they do not obey any specific trends. Both the calculated reaction enthalpies (ΔHf) and reaction free energies (ΔGf) show that reactions of Ru(III) complexes (1–6) at the N3, N7 and O6 sites of guanine are exothermic and would occur spontaneously. It is found that KP1019 would react more favourably than NAMI-A at the N3, N7 and O6 of guanine, in agreement with experimental observations. Further, among all the complexes studied here, reactions of complex 6 at the different sites of guanine are most exothermic in aqueous media. [ABSTRACT FROM AUTHOR]

Cancer research is among the key challenges in current medicine and biology. Many decades of investigations have brought measurable benefits in both areas with regard to expanding the knowledge of the molecular mechanism of cancer and developing treatment strategies. Despite that cancers are still among diseases with the highest mortality rate, and cancer treatment is often unsuccessful and connected with severe side effects. The development of therapeutic strategies in both targeting the primary tumor origin and preventing metastasis is largely based on testing newly synthesized chemical agents, including a group of metal-containing complexes. It seems that ruthenium-containing complexes are of high potential in cancer therapy, and our work presents the current data about the application of ruthenium-based complexes - NAMI-A and KP1019 in cancer therapy. [ABSTRACT FROM AUTHOR]

Nitrosylation reaction mechanisms of the hydrolysates of NAMI‐A and hydrolysis reactions of ruthenium nitrosyl complexes were investigated in the triplet state and the singlet state. Activation free energies were calculated by combining the QM/MM(ABEEM) method with free energy perturbation theory, and the explicit solvent environment was simulated by an ABEEMσπ polarizable force field. Our results demonstrate that nitrosylation reactions of the hydrolysates of NAMI‐A occur in both the triplet and the singlet states. The Ru‐N‐O angle of the triplet ruthenium nitrosyl complexes is in the range of 132.0°–138.2°. However, all the ruthenium nitrosyl complexes at the singlet state show an almost linear Ru‐N‐O angle. The nitrosylation reaction happens prior to the hydrolysis reaction for the first‐step hydrolysates. The activation free energies of the nitrosylation reactions show that the H2O‐NO exchange reaction of [RuCl4(Im)(H2O)] in the singlet spin sate is the most likely one. Comparing with the activation free energies of the hydrolysis reactions of the ruthenium nitrosyl complexes, the results indicate that the rate of the DMSO–H2O exchange reaction of [RuCl3(NO)(Im)(DMSO)] is faster than that of [RuCl3(H2O)(Im)(DMSO)] in both the triplet spin state and the singlet spin state. © 2018 Wiley Periodicals, Inc. Ab initio method and ABEEM/MM‐FEP method have been combined to investigate reaction mechanism of the interactions of NO with NAMI‐A hydrolysates in both the triplet and singlet spin states. The combined QM/MM(ABEEM polarizable force field) method with MD‐FEP was employed to calculate the activation free energies. The rates of nitrosylation reactions are faster than those of further hydrolysis reactions for the first step hydrolysates of NAMI‐A. The activation free energies of the nitrosylation reactions show that the H2O‐NO exchange reaction of [RuCl4(Im)(H2O)] in the singlet spin sate is the most likely one. [ABSTRACT FROM AUTHOR]

The ruthenium-based anticancer agent NAMI-A (ImH[ trans -RuCl 4 (dmso)(Im)], where Im = imidazole) has been shown to interact with RNA in vivo and in vitro. We hypothesized that the similarly structured drug KP1019 (IndH[ trans -RuCl 4 (Ind) 2 ], where Ind = indazole) binds to RNA as well. Fluorescence spectroscopy was employed to assay the interactions between either NAMI-A or KP1019 and tRNA Phe through an intrinsic fluorophore wybutosine (Y) base and by extrinsic displacement of the intercalating agent ethidium bromide. In both the intrinsic Y-base and extrinsic ethidium bromide studies, KP1019 exhibited tighter binding to phenylalanine-specific tRNA (tRNA Phe ) than NAMI-A. In the ethidium bromide study, reducing both drugs from Ru III to Ru II resulted in a significant decrease in binding. Our findings suggest that the relatively large heteroaromatic indazole ligands of KP1019 intercalate in the π-stacks of tRNA Phe within structurally complex binding pockets. In addition, NAMI-A appears to be sensitive to destabilizing electrostatic interactions with the negative phosphate backbone of tRNA Phe . Interactions with additional tRNA molecules and other types of RNA require further evaluation to determine the role of RNA in the mechanisms of action for KP1019 and to better understand how Ru drugs fundamentally interact with biomolecules that are more structurally sophisticated than short DNA oligonucleotides. To the best of our knowledge, this is the first study to report KP1019 binding interactions with RNA. [ABSTRACT FROM AUTHOR]

Imidazoliumtrans-tetrachloridodimethylsulfoxideimidazoleruthenate(III), NAMI-A, is a ruthenium drug exhibiting unique properties under clinical studies such as ability to inhibit the process of metastases without exerting tumor toxicity. Its place of action is concentrated at the extracellular level, therefore, the transformation and fate of this drug upon injection is of great importance. This study focuses on evaluation of the reducing potency of blood stream on interaction with two serum proteins: albumin and transferrin. It was investigated by applying various simplified serum models preserving physiological concentration of proteins and the amount of Ru complex as found in patients. It was shown that ruthenation of albumin is slightly increased while transferrin decreased upon addition of reductant in blood stream (ascorbate, glutathione, and cysteine) at physiological concentration. Interestingly, in serum models comprising low-molecular-mass components the amount of the reductant in the solution had a pronounced effect on the Ru content, in particular in transferrin. Supplementation of serum models with glutathione at concentration enough for complete reduction of NAMI-A selectively enhanced the binding of Ru complex to transferrin while ruthenation of albumin was almost unchanged. Spectrofluorimetric studies revealed that reduction has a marginal effect on binding affinity, therefore, both Ru(III) and (II) derivatives equally can compete for binding to transferrin. [ABSTRACT FROM AUTHOR]

Imidazolium trans-tetrachloridodimethylsulfoxideimidazolruthenate(III), NAMI-A, a novel antimetastatic ruthenium complex was investigated towards affinity to transferrin (Tf), whether Tf-Ru adducts might be formed after its intravenous injection. Studies were focused on the holotransferrin due to its preferential binding to transferrin receptor. Here, we showed that holotransferrin is able to bind NAMI-A as readily as apotransferrin. The simulation of biological conditions of human serum performed by application of simplified serum models allowed to analyse ruthenium distribution between transferrin and albumin. The presence of physiological concentration of albumin (ca. 18-fold excess over Tf) resulted in a twofold decrease of ruthenium binding to Tf. Interestingly, the introducing of low-molecular-mass components of serum dramatically increased the ruthenation of Tf. Intermolecular competition binding studies between transferrin and albumin showed that both proteins bound similar amount of ruthenium species. Investigation of NAMI-A binding to Tf in human serum showed that this protein was not the major binding partner for Ru complex. However, in spite of many competing proteins still the ruthenation of Tf was observed. The lack of free Ru species (protein unbounded) after incubation with human serum allowed to make an assumption of high affinity of NAMI-A towards serum proteins. [ABSTRACT FROM AUTHOR]

Here we present a split-enzyme sensor approach for the sequence-specific detection of metal-based drug adducts of DNA. Split β-lactamase reporters were constructed using domain A of the High Mobility Group Box 1 protein (HMGB1a) in conjunction with zinc finger DNA-binding domains. As a proof of concept, the sensors were characterized with the well-known drug cisplatin, which forms 1,2-intrastrand crosslinks with DNA that are recognized by HMGB1a. After promising results with cisplatin, five ruthenium-based drugs were studied, four of which produced significant signal over background. These results highlight the utility of our approach for rapid screening of novel metal-based chemotherapeutic drug candidates and provide evidence that HMGB1a likely binds to DNA adducts formed by NAMI-A (imidazolium trans -tetrachlorodimethylsulfoxideimidazoleruthenate(III)), KP1019 (indazolium trans -tetrachlorodiindazoleruthenate(III)), KP418 (imidazolium trans -tetrachlorodiimidazoleruthenate(III)), and RAPTA-C (dichloro(η6- p -cymene)(1,3,5-triaza-7-phosphaadamantane)ruthenium(II)). These results thus imply a potential biologically relevant mode of action for the ruthenium-based drugs investigated herein. Split-enzyme sensors with the capacity to detect metal-based adducts of DNA have been developed. The sensors use a split β-lactamase reporter coupled to High Mobility Group Box 1 protein domain A (HMGB1a) partnered with a zinc finger DNA binding domain. Several dsDNA adducts of cisplatin and ruthenium-based drugs were investigated. [Display omitted] • Domain A of the High Mobility Group Box 1 protein (HMGB1a) binds to cisplatin-modified DNA. • Split protein sensors using HMGB1a can detect modification of DNA by cisplatin. • Sensors for cisplatin-modified DNA are modular and can detect various dsDNA targets. • Sensors that detect cisplatin-modified DNA also respond to DNA reacted with Ru drugs. • HMGB1a likely binds to DNA modified by some ruthenium-based drugs. [ABSTRACT FROM AUTHOR]

Aim of the study The tumor metastases targeting ruthenium complex NAMI-A synergistically improves the activity of gemcitabine in combination therapies. High-throughput screening was used to identify other potential drug combinations from a library of FDA approved drugs. Doxorubicin was identified as a hit compound and was therefore evaluated in combination with NAMI-A in vitro and in a preclinical in vivo model. Results High-throughput screening identified eight structurally diverse compounds that synergize with NAMI-A including doxorubicin. The combination index on MCF-7 cells showed synergism as the concentration of NAMI-A increases independent of the doxorubicin concentration. In MCa mammary carcinoma of CBA mice, NAMI-A (35 mg/kg/day i.p. on days 7-12) followed by doxorubicin (10 mg/kg i.p. on day 16), significantly increased the effects of the individual drugs on metastases with 70 % animals resulting free of macroscopically detectable tumor nodules in the lungs at sacrifice. NAMI-A, unlike doxorubicin, cured 60 % of the treated mice but the combination therapy was toxic to the animals. Conclusions The combined therapy of NAMI-A with doxorubicin synergizes on lung metastasis in a preclinical mouse model. The combination therapy at the maximum tolerated doses of the two drugs is toxic. Hence, this combination is not suitable for clinical studies using maximum tolerated doses. [ABSTRACT FROM AUTHOR]

Background This phase I/II study determined the maximal tolerable dose, dose limiting toxicities, antitumor activity, the pharmacokinetics and pharmacodynamics of ruthenium compound NAMI-A in combination with gemcitabine in Non-Small Cell Lung Cancer patients after first line treatment. Methods Initial dose escalation of NAMI-A was performed in a 28 day cycle: NAMI-A as a 3 h infusion through a port-a-cath at a starting dose of 300 mg/m at day 1, 8 and 15, in combination with gemcitabine 1,000 mg/m at days 2, 9 and 16. Subsequently, dose escalation of NAMI-A in a 21 day schedule was explored. At the maximal tolerable dose level of this schedule an expansion group was enrolled of which 15 patients were evaluable for response. Results Due to frequent neutropenic dose interruptions in the third week, the 28 day schedule was amended into a 21 day schedule. The maximal tolerable dose was 300 and 450 mg/m of NAMI-A (21 day schedule). Main adverse events consisted of neutropenia, anemia, elevated liver enzymes, transient creatinine elevation, nausea, vomiting, constipation, diarrhea, fatigue, and renal toxicity. Conclusion NAMI-A administered in combination with gemcitabine is only moderately tolerated and less active in NSCLC patients after first line treatment than gemcitabine alone. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: The ruthenium-based compound imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate (NAMI-A) is free of cytotoxicity up to 1mM concentration after 1h in vitro exposure of the LLC-PK1 renal tubule cells. In vivo, one cycle of i.p. administrations of 35mg/kg/day NAMI-A (1cycle=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 <0.05), serum creatinine increases by 30% (p <0.05) and a significant decrease of body weight of 12% (p <0.05) occurs. These parameters return to normal within 7days after the end of treatment. A cycle-dependent alteration of glomeruli and a diffused swelling of renal tubules are also evident leading to a significant alteration of these structures after the third cycle. These effects are completely prevented if a 2-week drug free washout is used between two consecutive cycles. These data support the toxic accumulation of NAMI-A or of its products of transformation in the kidneys and stress the need of at least 14days washout between two treatment cycles when the drug is given daily for 6 consecutive days. [Copyright &y& Elsevier]

Abstract: NAMI-A i.e. (ImH)[trans-RuCl4(DMSO)(Im)] (where Im is imidazole) is a ruthenium(III) complex with promising antimetastatic activity, which has been classified for II phase clinical trial. In this study, its binding properties toward apo-transferrin (apo-Tf) with regard to its hydrolytic and redox behavior are systematically investigated by the use of fluorescence spectroscopy. The reaction of NAMI-A and its reduced form with apo-Tf is proceeded by formation of aqua derivatives and the presence of at least one labile aqua ligand is sufficient to form adducts. It is found that presence of bicarbonate is not necessary for interaction of studied ruthenium complexes with apo-Tf. The calculated association constants for both NAMI-A and its reduced form are very similar with the values of 1.28×104 M−1 and 1.36×104 M−1 at 37°C, respectively however, the reduced derivatives reach the equilibrium ca. 8–10 times slower. The percentage of ruthenium content in protein fractions separated from protein-unbounded ruthenium by using FPLC (fast protein liquid chromatography) method is rather high and depends on redox state of the complex, for most samples is found higher for reduced species. [Copyright &y& Elsevier]

[ImH][ trans-RuCl(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. [ABSTRACT FROM AUTHOR]

Since the discovery of cis-platinum, many transition metal complexes have been synthesized and assayed for antineoplastic activity. In recent years, ruthenium-based molecules have emerged as promising antitumor and antimetastatic agents with potential uses in platinum-resistant tumors or as alternatives to platinum. Ruthenium compounds theoretically possess unique biochemical features allowing them to accumulate preferentially in neoplastic tissues and to convert to their active state only after entering tumor cells. Intriguingly, some ruthenium agents show significant activity against cancer metastases but have minimal effects on primary tumors. Two ruthenium-based drugs, NAMI-A and KP1019, have reached human clinical testing. This review will highlight the chemical properties, mechanism of action, preclinical data, and early phase clinical results of these two lead ruthenium compounds. Other promising ruthenium agents will also be reviewed with emphasis on the novel ruthenium compound ONCO4417, and DW1/2 that has demonstrated Pim-1 kinase inhibition in preclinical systems. Further development of these and other ruthenium agents may rely on novel approaches including rational combination strategies as well as identification of potential pharmacodynamic biomarkers of drug activity aiding early phase clinical studies. [ABSTRACT FROM AUTHOR]

A systematic study of the reduction of (ImH)[ trans-RuCl4(dmso)(Im)] (NAMI-A; dmso is dimethyl sulfoxide, Im is imidazole), a promising antimetastasing agent, by l-ascorbic acid under physiological conditions is reported. Under blood plasma conditions (pH 7.4, 0.1–0.15 M NaCl , 37 °C) the rapid reduction of trans-[RuIIICl4(dmso)(Im)]− results in the formation of trans-[RuIICl4(dmso)(Im)]2− within seconds, and is followed by successive dissociation of the chloride ligands, whereas neither dmso nor imidazole ligands are released during the reaction. Under our experimental conditions, the formation of the ascorbate dianion is the rate-determining step, and once it has formed it reacts rapidly with NAMI-A. Moreover, the NAMI-A complex is very unstable at physiological pH (7.4); therefore, the hydrolysis of NAMI-A cannot be excluded as a competing reaction. During hydrolysis, aquated derivatives via stepwise dissociation of chloride and dmso ligands are formed, and most of these species have a higher redox potential and are expected to be even more easily reduced by ascorbic acid. Thus, it is very likely that the reduced form of NAMI-A or the reduction products of its hydrolytic derivatives react with albumin. The reaction of reduced NAMI-A with human serum albumin leads to the formation of stable adducts, with a binding efficiency very similar to that of the parent complex, viz., 3.2 ± 0.3 and 4.0 ± 0.4 mol of Ru(II) and Ru(III) per mole of albumin, respectively, however with a significantly higher reactivity. [ABSTRACT FROM AUTHOR]

Abstract: Ruthenium complex NAMI-A [ImH][trans-RuCl4(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 μM NAMI-A, whereas in a combined treatment with 1 μM NAMI-A and electroporation only about 10% of cells survived. [Copyright &y& Elsevier]

The effects of NAMI-A, [H2im][trans-RuCl4(dmso-S)(Him)], a new metal-based agent for treating tumor metastases, have been investigated in vitro on splenocytes, ConA- or LPS-activated T and B lymphoblasts, and thymocytes. Splenocytes and thymocytes exposed for 1 h to 0.01-0.1-mM NAMI-A do not change their mitochondrial functionality, cell cycle distribution, protein synthesis, and CD44 expression in comparison to untreated control samples. Instead, mitochondrial functionality increased 24 h after treatment in a fraction of splenocytes. The same treatment reduced mitochondrial functionality and S phase of the cell cycle in T and B blasts (already after 1 h treatment) and reduced CD44 expression on B blasts, 24 h after treatment. On cocultures of splenocytes and metastatic cells (metGM) (1:1), NAMI-A induces a selective depolarization of mitochondrial membrane potential of metGM cells, while it stimulates splenocytes (mainly lymphocytes), as shown by the increase of the S phase, nitric oxide production, and adhesion onto metastatic cells. This, in turn, reduces the number of metastatic cells and results in the increased ratio between splenocytes and metGM in favor of diploid cells (doubling from one to two). Rosetting of leukocytes onto metastatic cells correlates with induction of CD54 expression on tumor cells after NAMI-A in vivo treatment, which in turn, might contribute to metastasis recognition by cytotoxic lymphocytes. The overall antimetastatic activity displayed by NAMI-A might therefore be the result of complex interactions with tumor cells, on which it displays selective antitumor activity, and with host immune cells through which it promotes activation of host immune defenses involved in tumor suppression. [ABSTRACT FROM AUTHOR]

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 TGFβ1 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. TGFβ1 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 TGFβ1 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 TGFβ1, and its down-regulation in tumour cells might explain the documented reduction of gelatinase release. [Copyright &y& Elsevier]

Purpose. The cytotoxicity, intracellular accumulation and DNA adduct formation of the ruthenium complex imidazolium trans-imidazoledimethylsulfoxide tetrachlororuthenate (ImH[trans-RuCl4(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. Methods. 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 32P-postlabeling assay. Results. 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 μM 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. Conclusions. 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. [ABSTRACT FROM AUTHOR]

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 (+20-25°C), but can be stored for up to 48 hours at refrigerated conditions (+2-8°C). 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. [ABSTRACT FROM AUTHOR]

The coordination of the antimetastatic agent NAMI-A, [H2im][trans-RuCl4(dmso-S)(Him)], (Him=imidazole; dmso=dimethyl sulfoxide), to the DNA model base 9-methyladenine (9-MeAde) was investigated in water. NMR spectroscopy was first applied for the study of the molecular stability and hydrolysis of NAMI-A in aqueous solution over a range of pH (3.0–7.4) and chloride ion concentrations (0–1 M) at 37.0 °C. In physiological conditions (phosphate buffer, pH 7.4) NAMI-A disappears from the solution in 15 min due to chloride and dmso hydrolysis, leading to uncharacterised poly-oxo Ru species. Conversely, at lower pH (3.0–6.0) and in water (pH ∼ 5.5), only a partial dmso hydrolysis occurs, slowly forming the [trans-RuCl4(H2O)(Him)]− complex. This latter species coordinates to 9-MeAde (via the N7 of 9-MeAde), forming the [trans-RuCl4(9-MeAde)(Him)]− complex. NAMI-A and [trans-RuCl4(H2O)(Him)]− give comparable intracellular ruthenium concentrations and accumulate in KB cells (human mouth carcinoma) and accumulate these at the G2/M phase, while poly-oxo Ru species do not, and their cell uptake is reduced to 50%. On the contrary, G2/M arrest and protein content in the murine metastatic cell line metGM, are not influenced by NAMI-A hydrolysis. Hydrolysed NAMI-A species apparently are easier taken up by the metGM cells, showing intracellular ruthenium concentrations one order of magnitude greater than those of intact NAMI-A. Therefore, it is proposed that the selective antimetastatic activity of NAMI-A during in vivo experiments can be attributed to its hydrolysed species. [Copyright &y& Elsevier]

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

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. [Copyright &y& Elsevier]

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

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