Modulation of activity of known cytotoxic ruthenium(III) compound (KP418) with hampered transmembrane transport in electrochemotherapy in vitro and in vivo.

To increase electrochemotherapy (ECT) applicability, the effectiveness of new drugs is being tested in combination with electroporation. Among them two ruthenium(III) compounds, (imH)[trans-RuCl4(im)(DMSO-S)] (NAMI-A) and Na[trans-RuCl4(ind)2] (KP1339), proved to possess increased antitumor effectiveness when combined with electroporation. The objective of our experimental work was to determine influence of electroporation on the cytotoxic and antitumor effect of a ruthenium(III) compound with hampered transmembrane transport, (imH)[trans-RuCl4(im)2] (KP418) in vitro and in vivo and to determine changes in metastatic potential of cells after ECT with KP418 in vitro. In addition, platinum compound cisplatin (CDDP) and ruthenium(III) compound NAMI-A were included in the experiments as reference compounds. Our results show that electroporation leads to increased cellular accumulation and cytotoxicity of KP418 in murine melanoma cell lines with low and high metastatic potential, B16-F1 and B16-F10, but not in murine fibrosarcoma cell line SA-1 in vitro which is probably due to variable effectiveness of ECT in different cell lines and tumors. Electroporation does not potentiate the cytotoxicity of KP418 as prominently as the cytotoxicity of CDDP. We also showed that the metastatic potential of cells which survived ECT with KP418 or NAMI-A does not change in vitro: resistance to detachment, invasiveness, and re-adhesion of cells after ECT is not affected. Experiments in murine tumor models B16-F1 and SA-1 showed that ECT with KP418 does not have any antitumor effect while ECT with CDDP induces significant dose-dependent tumor growth delay in the two tumor models used in vivo.

Speciation of cisplatin in environmental water samples by hydrophilic interaction liquid chromatography coupled to inductively coupled plasma mass spectrometry.

Cisplatin is still widely used for treatment of numerous types of tumours. Different speciation methods have been applied to study behaviour of the intact drug and its individual biotransformation species in various clinical samples. These methods are mainly based on electrophoresis, size exclusion (SEC) or ion chromatography (IC) techniques coupled to inductively coupled plasma mass spectrometry (ICP-MS). Hydrophilic interaction liquid chromatography (HILIC), which is a common technique for separation of polar substances, was rarely applied for separation of cisplatin and its hydrolysed metabolites. There is also a lack of information available on the occurrence of cisplatin and its hydrolysed complexes in the environmental waters. In the present study the concentrations of Pt were determined in hospital wastewaters by ICP-MS. A procedure for separation of cisplatin and its aqueous hydrolysed complexes by the use of HILIC column was optimized. Quantification of separated Pt species was performed by isotope dilution (ID)-ICP-MS procedure. Low limits of detection (LODs) and quantification (LOQs) were obtained for cisplatin and its hydrolysed complexes ranging from 0.0273 to 0.1726 ng Pt/mL and from 0.0909 to 0.5753 ng Pt/mL, respectively. Good repeatability of the procedure with relative standard deviation (RSD) lower than ±2.3% was obtained. The column recoveries, which ranged from 95 to 101%, indicated that the procedure developed enabled quantitative speciation analysis of aqueous cisplatin complexes. The ZIC-HILIC-ID-ICP-MS procedure was successfully applied in speciation of cisplatin in spiked hospital wastewater samples.

New method for the speciation of ruthenium-based chemotherapeutics in human serum by conjoint liquid chromatography on affinity and anion-exchange monolithic disks.

An important step in pharmacological characterisation of a candidate drug is the study of the drugs interactions with serum proteins. In the present work, conjoint liquid chromatography (CLC) was used for separation of ruthenium (Ru)-based drug candidates in human serum. CIM Protein G and CIM DEAE disks were assembled together in a single housing forming a CLC monolithic column. By applying isocratic elution with Tris-HCl-NaHCO3 buffer (pH 7.4) in the first min, followed by gradient elution with 1 mol L(-1) NH4Cl (pH 7.4) in the next 9 min, immunoglobulins (IgG) were retained by the Protein G disk enabling subsequent separation of unbound Ru species from Ru species bound to human serum transferrin (Tf) and albumin (HSA) on the CIM DEAE disk. Finally, elution with acetic acid (AcOH) in the next 3 min allowed separation of Ru species associated with IgG. Protein elution was followed on-line with UV detection at 278nm, while the separated Ru species were quantified by post-column isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS). The instrumental set-up enabled fast two-dimensional separation by affinity and ion-exchange modes to be carried out in a single chromatographic run. Two Ru-based chemotherapeutics: a newly synthesised compound chlorido(η6-p-cymene)(nalidixicato-κ2O,O)Ru(II) (1) and (H2im)[trans-Ru(III)Cl4(Him)2] (2; KP418), which is currently undergoing preclinical studies, were investigated. The CLC procedure applied is sensitive with low limit of detection (LOD) (0.027 µg Ru mL(-1) for (1)) and good method repeatability (RSD±3.5%). The experimental data revealed that it enables investigation of the kinetics of interaction of positively charged and neutral complexes of metallodrugs with serum proteins as well as the distribution of metallodrug species in human serum. However, negatively charged metallic complexes co-eluted with Tf and HSA and thus hindered their speciation analysis. An example of successful application of the kinetic studies on the CLC column is (1), a neutral Ru complex that hydrolyses to a positively charged species. For comparison, speciation data obtained for serum samples spiked with cisplatin are also shown.

A novel method for speciation of Pt in human serum incubated with cisplatin, oxaliplatin and carboplatin by conjoint liquid chromatography on monolithic disks with UV and ICP-MS detection.

Conjoint liquid chromatography (CLC) on monolithic convective interaction media (CIM) disks coupled on-line to UV and inductively coupled plasma mass spectrometry (ICP-MS) detectors was used for the first time in speciation analysis of Pt in human serum spiked with Pt-based chemotherapeutics. CIM Protein G and CIM DEAE disks were assembled together in a single housing forming a CLC monolithic column. Such a set-up allows rapid two-dimensional separation by affinity and ion-exchange (IE) modes to be carried out in a single chromatographic run. By applying isocratic elution with Tris-HCl-NaHCO3 buffer (pH 7.4) in the first minute, followed by gradient elution with 1 mol L(-1) NH4Cl (pH 7.4) in the next 9 min, immunoglobulins (IgG) were retained by the Protein G disk enabling subsequent separation of unbound Pt from Pt bound to transferrin (Tf) and albumin (HSA) on the CIM DEAE disk. Further elution with acetic acid (AcOH) in the next 3 min allowed separation of Pt associated with IgG. Separated Pt species were quantified by post-column isotope dilution-ICP-MS. Pt recovery on the CLC column was close to 100%. In comparison to commonly applied procedures that involve separation of protein peaks by size-exclusion chromatography (SEC) followed by IE separation of metal-based chemotherapeutic fractions bound to serum proteins, the CLC method developed is much faster and simpler. Its sensitivity (LOQs adequate for quantification of all separated Pt species, lower than 2.4 ng Pt mL(-1)), good selectivity and method repeatability (RSD±3%) enabled investigation of the kinetics of interaction of Pt-based chemotherapeutics with serum proteins and the distribution of Pt species in spiked human serum. Pt species present in spiked serum were bound preferentially to HSA. The proportion of Pt associated with IgG and Tf was lower than 13%. Cisplatin and especially oxaliplatin react rapidly with serum proteins, while carboplatin much less. The method developed may be reliably applied in preclinical and clinical studies of the kinetics of the interaction and distribution of different metallodrugs with proteins in blood serum.

Preparation of Cr(VI) and Cr(III) isotopic spike solutions from 50Cr and 53Cr enriched oxides without the use of oxidizing and/or reducing agents.

The use of enriched stable isotopes as tracers in speciation procedures by ion-exchange chromatography coupled to ICP-MS enables to follow the oxidation-reduction processes of Cr. The most commonly available Cr stable isotopes are (50)Cr and (53)Cr enriched oxides or metallic Cr. For application of Cr enriched stable isotopes, adequate preparation of isotopic spike solutions is necessary. To ensure that Cr species present in the sample investigated are not compromised, no excess of the reducing neither oxidizing agents should remain in the isotopic spike solutions. Cr(VI) isotopic solutions are mostly prepared by dissolving of Cr oxide in HClO(4), followed by the addition of ammonia and H(2)O(2) to quantitatively oxidize Cr, while the excess of H(2)O(2) is removed by boiling or UV irradiation. If traces of H(2)O(2) still remains, such isotopic spike solution may cause artefacts in Cr speciation in the sample investigated. In the present work, new procedure based on alkaline melting of (50)Cr enriched oxide for preparation of pure (50)Cr(VI) spike solution was developed. Cr(III) was quantitatively oxidized to Cr(VI) with air oxygen without use of other oxidizing agents. Moreover, the microwave assisted digestion procedure of (53)Cr enriched oxide was applied for preparation of (53)Cr(III) spike solution without use of reducing agents. The purity of (50)Cr(VI) and (53)Cr(III) isotopic spike solutions was verified by the speciation analysis applying hyphenation of anion-exchange FPLC to ICP-MS. Speciation analysis demonstrated suitability of the proposed procedures for preparation of Cr isotopic spike solutions. In addition, the artefacts in Cr speciation, which may be initiated by traces of oxidizing and/or reducing agents present in Cr spike solutions, were demonstrated. The outcomes of our investigation highlighted the importance of the adequate preparation of spike solutions of Cr isotopes that may be used as reliable tracers in the investigations of the oxidation-reduction processes of Cr in wide range of environmentally relevant pH values.