Biodistribution of the novel anticancer drug sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] KP-1339/IT139 in nude BALB/c mice and implications on its mode of action.

The ruthenium complex sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP-1339/IT139) has entered clinical trials as the more soluble alternative to the indazolium compound KP1019. In order to get insight into its distribution and accumulation throughout a living organism, KP-1339/IT139 was administered intravenously in non-tumor bearing nude BALB/c mice and the Ru content in blood cells and plasma, bone, brain, colon, kidneys, liver, lung, muscle, spleen, stomach and thymus was determined at several time points. The Ru concentration in blood cells and plasma was found to increase slightly within the first hours of analysis, with the Ru concentration being 3-times higher in plasma compared to blood cells. The plasma samples were subjected to analysis by capillary zone electrophoresis (CZE) and size exclusion/anion exchange chromatography (SEC-IC) both coupled to inductively coupled plasma-mass spectrometry (ICP-MS) and a large majority of the total Ru content was found attached to mouse serum albumin (MSA), confirming similar behavior to KP1019 in an in vivo setting. Within 1h, the peak ratio of approximately 1.2-1.5 Ru per albumin molecule was reached which declined to about 1 Ru per albumin molecule within 24h. Beside the MSA adduct a higher molecular weight species was observed probably stemming from MSA conjugates. In addition, the tissue samples were mineralized by microwave digestion and analyzed for their Ru content. The highest Ru levels were found in colon, lung, liver, kidney and notably in the thymus. The peak Ru concentrations in these tissues were reached 1-6h after administration and declined slowly over time.

Characterization of the binding sites of the anticancer ruthenium(III) complexes KP1019 and KP1339 on human serum albumin via competition studies.

Indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1019) and its Na(+) analogue (KP1339) are two of the most prominent non-platinum antitumor metal complexes currently undergoing clinical trials. After intravenous administration, they are known to bind to human serum albumin (HSA) in a noncovalent manner. To elucidate their HSA binding sites, displacement reactions with the established site markers warfarin and dansylglycine as well as bilirubin were monitored by spectrofluorimetry, ultrafiltration-UV-vis spectrophotometry, and/or capillary zone electrophoresis. Conditional stability constants for the binding of KP1019 and KP1339 to sites I and II of HSA were determined, indicating that both Ru(III) compounds bind to both sites with moderately strong affinity (log K(1)' = 5.3-5.8). No preference for either binding site was found, and similar results were obtained for both metal complexes, demonstrating low influence of the counter ion on the binding event.

Capillary electrophoretic methods in the development of metal-based therapeutics and diagnostics: new methodology and applications.

In recent years, capillary electrophoresis (CE) has matured to a standard method in medicinal inorganic chemistry. More and more steps of the drug discovery process are followed by CE. However, not only the number of applications has steadily increased but also the variety of used methodology has significantly broadened and, as compared to a few years ago, a wider scope of separation modes and hyphenated systems has been used. Herein, a summary of the newly utilized CE methods and their applications in metallodrug research in the timeframe 2006-2011 is presented, following related reviews from 2003 and 2007 (Electrophoresis, 2003, 24, 2023-2037; Electrophoresis 2007, 28, 3436-3446). Areas covered include impurity profiling, quality control of pharmaceutical formulations, lipophilicity estimation, interactions between metallodrugs and proteins or nucleotides, and characterization and also quantification of metabolites in biological matrices and real-world samples.

Anticancer activity of methyl-substituted oxaliplatin analogs.

Oxaliplatin is successfully used in systemic cancer therapy. However, resistance development and severe adverse effects are limiting factors for curative cancer treatment with oxaliplatin. The purpose of this study was to comparatively investigate in vitro and in vivo anticancer properties as well as the adverse effects of two methyl-substituted enantiomerically pure oxaliplatin analogs [[(1R,2R,4R)-4-methyl-1,2-cyclohexanediamine] oxalatoplatinum(II) (KP1537), and [(1R,2R,4S)-4-methyl-1,2-cyclohexanediamine]oxalatoplatinum(II) (KP1691)] and to evaluate the impact of stereoisomerism. Although the novel oxaliplatin analogs demonstrated in multiple aspects activities comparable with those of the parental compound, several key differences were discovered. The analogs were characterized by reduced vulnerability to resistance mechanisms such as p53 mutations, reduced dependence on immunogenic cell death induction, and distinctly attenuated adverse effects including weight loss and cold hyperalgesia. Stereoisomerism of the substituted methyl group had a complex and in some aspects even contradictory impact on drug accumulation and anticancer activity both in vitro and in vivo. To summarize, methyl-substituted oxaliplatin analogs harbor improved therapeutic characteristics including significantly reduced adverse effects. Hence, they might be promising metal-based anticancer drug candidates for further (pre)clinical evaluation.

Cellular accumulation and DNA interaction studies of cytotoxic trans-platinum anticancer compounds.

Forty years after the discovery of the anticancer effects of cisplatin, scientists are still pursuing the development of platinum complexes with improved properties regarding side effects and resistance, which are two main problems in cisplatin treatment. Among these compounds, trans-configured platinum complexes with oxime ligands emerged as a new class with features distinct from those of established anticancer agents, including different DNA binding behavior, increased cellular accumulation, and a different pattern of protein interaction. We report herein on the reactivity with biomolecules of three novel pairs of cis- and trans-configured acetone oxime platinum(II) complexes and one pair of 3-pentanone oxime platinum(II) complexes. Cellular accumulation experiments and in vitro DNA platination studies were performed and platinum contents were determined by inductively coupled plasma mass spectrometry. The trans-configured complexes were accumulated in SW480 cells in up to 100 times higher amounts than cisplatin and up to 50 times higher amounts than their cis-configured counterparts; r (b) values (number of platinum atoms per nucleotide) were more than tenfold increased in cells treated with trans complexes compared with cells treated with cisplatin. The interaction of the complexes with DNA was studied in cell-free experiments with plasmid DNA (pUC19), in capillary zone electrophoresis with the DNA model 2-deoxyguanosine 5'-monophosphate, and in in vitro experiments showing the degree of DNA damage in the comet assay. Whereas incubation with cis compounds did not induce degradation of DNA, the trans complexes led to pronounced strand cleavage.

LC- and CZE-ICP-MS approaches for the in vivo analysis of the anticancer drug candidate sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1339) in mouse plasma.

Ruthenium-indazole complexes are promising anticancer agents undergoing clinical trials. KP1339 is administered intravenously (i.v.), where serum proteins are the first available biological binding partners. In order to gain a better insight into the mode of action, mice were treated with different doses of KP1339 i.v. and sacrificed at different time points. The blood plasma was isolated from blood samples and analyzed by capillary zone electrophoresis (CZE) and size exclusion/anion exchange chromatography (SEC-IC) both combined on-line to inductively coupled plasma-mass spectrometry (ICP-MS). The performance of the analytical methodology was compared and the interaction of KP1339 with mouse plasma proteins characterized in vivo. Interestingly, the samples of the mice treated with 50 mg kg(-1) and terminated after 24 h showed a ca. 4-fold lowered albumin content and increased ruthenation of albumin aggregates as compared to the untreated control group and the 40 mg kg(-1) group. The majority of Ru was bound to albumin and the stoichiometry of the KP1339 protein binding was determined through the molar Ru/S ratio. In general, good agreement of the data obtained with both techniques was achieved and the SEC-IC method was found to be more sensitive as compared to the CZE-ICP-MS approach, whereas the latter benefits from the shorter analysis time and lower sample consumption.

Physicochemical Studies and Anticancer Potency of Ruthenium η-p-Cymene Complexes Containing Antibacterial Quinolones.

With the aim of exploring the anticancer properties of organometallic compounds with bioactive ligands, Ru(arene) compounds of the antibacterial quinolones nalidixic acid (2) and cinoxacin (3) were synthesized, and their physicochemical properties were compared to those of chlorido(η(6)-p-cymene)(ofloxacinato-κ(2)O,O)ruthenium(II) (1). All compounds undergo a rapid ligand exchange reaction from chlorido to aqua species. 2 and 3 are significantly more stable than 1 and undergo minor conversion to an unreactive [(cym)Ru(µ-OH)(3)Ru(cym)](+) species (cym = η(6)-p-cymene). In the presence of human serum albumin 1-3 form adducts with this transport protein within 20 min of incubation. With guanosine 5'-monophosphate (5'-GMP; as a simple model for reactions with DNA) very rapid reactions yielding adducts via its N7 atom were observed, illustrating that DNA is a possible target for this compound class. A moderate capacity of inhibiting tumor cell proliferation in vitro was observed for 1 in CH1 ovarian cancer cells, whereas 2 and 3 turned out to be inactive.

From hydrolytically labile to hydrolytically stable Ru(II)-arene anticancer complexes with carbohydrate-derived co-ligands.

The synthesis, characterization, reactivity and in vitro anticancer activity of a series of Ru(II)-arene complexes with carbohydrate-derived phosphite and biscarboxylato co-ligands are reported. The compounds were characterized by NMR spectroscopy and electrospray ionization (ESI) mass spectrometry, and the molecular structures of oxalato(η(6)-p-cymene)(3,5,6-bicyclophosphite-1,2-O-isopropylidene-α-D-glucofuranoside)ruthenium(II) and oxalato(η(6)-p-cymene)(3,5,6-bicyclophosphite-1,2-O-cyclohexylidene-α-D-glucofuranoside)ruthenium(II) were determined by X-ray diffraction analysis. In contrast to their dichlorido counterparts, the biscarboxylato complexes did not exhibit significant reactivity towards biomolecules, such as cysteine, methionine, ubiquitin or the DNA model 5'-GMP, and resist hydrolysis; no hydrolytic species were detected by (1)H and (31)P{(1)H} NMR spectroscopy over several days. These structural alterations led to a decrease in the tumor-inhibiting potency of the compounds in human cancer cell lines.

Strategies for the covalent conjugation of a bifunctional chelating agent to albumin: synthesis and characterization of potential MRI contrast agents.

With the purpose to develop macromolecular magnetic resonance imaging contrast agents, we herein report three different synthetic approaches to the covalent attachment of bifunctional chelating agents to human serum albumin followed by coordination to contrast enhancing gadolinium(III). Applied methods cover active ester-mediated conjugation, linkage through glutaryl spacer, as well as the connection by the employment of glutaraldehyde. The content of gadolinium(III) was evaluated by inductively-coupled-plasma mass-spectrometry (ICP-MS) measurements and indicated reproducible amounts of conjugated contrast enhancing material. Small angle X-ray scattering (SAXS) experiments provided the size and altered shape of the gadolinium loaded proteins in comparison to unmodified albumin. Finally, the magnetic resonance properties of the protein conjugates were evaluated. The results indicated suitability of the gadolinium(III) loaded protein conjugates for use as macromolecular contrast agents in magnetic resonance imaging (MRI).

Tuning of lipophilicity and cytotoxic potency by structural variation of anticancer platinum(IV) complexes.

A series of bis(carboxylato)dichlorido(ethane-1,2-diamine)platinum(IV) compounds with IC(50) values ranging between 142 µM and 18 nM was investigated with respect to their lipophilicity (by the shake flask method as well as microemulsion electrokinetic chromatography), reduction potential, as well as their cellular accumulation in cancer cells in vitro. In general, the antiproliferative properties of the complexes correlated with their lipophilicity as well as their accumulation, whereas differences in antiproliferative potency could not be explained by reduction potentials since they do not vary significantly within the investigated series of compounds. Only minor effects for complexes featuring polar end groups were detected.

The first example of MEEKC-ICP-MS coupling and its application for the analysis of anticancer platinum complexes.

MEEKC is a powerful electrodriven separation technique with many applications in different disciplines, including medicinal chemistry; however, up to now the coupling to highly sensitive and selective MS detectors was limited due to the ion suppressive effect of the commonly used surfactant SDS. Herein, the first example of the coupling of MEEKC to ICP-MS is presented and an MEEKC method for the separation of Pt(II) and Pt(IV) anticancer drugs and drug candidates was developed. Different compositions of microemulsions were evaluated and the data were compared with those collected with standard ultraviolet/visible (UV/vis) spectroscopy detection. The MEEKC-ICP-MS system was found to be more sensitive than MEEKC-UV/vis and the analysis of UV/vis silent compounds is now achievable. The migration behavior of the Pt(II) and Pt(IV) compounds under investigation is correlated to their different chemical structures.

Biodistribution of anti-diabetic Zn(II) complexes in human serum and in vitro protein-binding studies by means of CZE-ICP-MS.

Application of modern analytical technology for studying the fate of metallodrugs after administration to the blood is of utmost importance for drug development. Zn(II) compounds are under development as insulin-enhancing drugs with potential use in the treatment of diabetes. In comparison to the well-established vanadium compounds, especially the lower risk of adverse effects due to the essentiality of the element in biological processes is advantageous. Herein, CZE-ICP-MS studies on the interaction of Zn(II)-maltolato, -2-picolinato and -2,6-dipicolinato complexes with human serum proteins are discussed and modeling calculations were confirmed by experimental results. Studies with human serum reveal preference for HSA over other less abundant proteins and serum components.

The serum protein binding of pharmacologically active gallium(III) compounds assessed by hyphenated CE-MS techniques.

Transition metal-based drugs exhibit high affinity to the soft donors of human serum proteins, especially of the high-abundance protein HSA and of transferrin (Tf), whereas Ga(III) salts are known to bind to Tf and other iron-containing metalloproteins, thereby interfering with the iron metabolism. Herein, the utilization of CE-MS methods for studying the binding behavior of a therapeutic gallium nitrate formulation and the anticancer drug candidate Tris(8-oxyquinolinato)gallium(III) to Tf and HSA under simulated physiological conditions is described. Both the Ga(III) salt and the complex were found to bind to Tf exclusively in the presence of carbonate, however, at different kinetics and to a different extent. Fe(III) induces the release of the Ga ions due to the higher affinity constant and also prevents the Ga(III) species from accessing the iron-binding pockets of Tf. In contrast, only low affinity to HSA was observed and even when present at ca. 20-fold excess, the majority of the Ga was attached to Tf.