Versatile analytical methodology for evaluation of drug-like properties of potentially multi-targeting anticancer metallodrugs.

Using inductively coupled plasma mass spectrometry (in combination with ultrafiltration) and microemulsion electrokinetic chromatography, the drug properties of two new, potentially multi-targeting Ru(III) and Pt(IV) compounds, containing biologically active ligands, were evaluated. The ruthenium complex with bexarotene was shown to bind to albumin faster than to transferrin and exhibits much the same (to albumin) binding profile in human serum. The Pt(IV)-lonidamine complex interacts with albumin relatively slowly but possesses high stability and lipophilicity (log P 1.

How versatile is the use of ultrafiltration to study biointeractions of therapeutic metallodrugs?

For a representative number of approved or investigational anticancer metallodrugs varying in lipophilicity, unspecific adsorption onto ultracentrifugal filter units was studied. It was found that for fairly hydrophilic compounds, such as cisplatin and oxaliplatin, the binding to filters does not substantially affect their amount measured (by ICP-MS) after ultrafiltration (>95%). In the case of metal complexes with moderate lipophilicity (log P > -0.

Combination of ICP-MS, capillary electrophoresis, and their hyphenation for probing Ru(III) metallodrug-DNA interactions.

Determination of the DNA-binding reactivity and affinity is an important part of a successful program for the selection of metallodrug candidates. For such assaying, a range of complementary analytical techniques was proposed and tested here using one of few anticancer metal-based drugs that are currently in clinical trials, indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III), and a DNA oligonucleotide. A high reactivity of the Ru drug was confirmed in affinity capillary electrophoresis (CE) mode, where adduct formation takes place in situ (i.

Can neutral analytes be concentrated by transient isotachophoresis in micellar electrokinetic chromatography and how much?

Transient isotachophoresis (tITP) is a versatile sample preconcentration technique that uses ITP to focus electrically charged analytes at the initial stage of CE analysis. However, according to the ruling principle of tITP, uncharged analytes are beyond its capacity while being separated and detected by micellar electrokinetic chromatography (MEKC). On the other hand, when these are charged micelles that undergo the tITP focusing, one can anticipate the concentration effect, resulting from the formation of transient micellar stack at moving sample/background electrolyte (BGE) boundary, which increasingly accumulates the analytes.

Metallomics for drug development: an integrated CE-ICP-MS and ICP-MS approach reveals the speciation changes for an investigational ruthenium(III) drug bound to holo-transferrin in simulated cancer cytosol.

A method based on combining inductively coupled plasma mass spectrometry (ICP-MS) with capillary electrophoresis (CE) or an ultrafiltration step was developed to study the speciation of the serum-protein adducts of a ruthenium anticancer drug under in vitro intracellular conditions. The formation of a reactive Ru species in the cell, following the metal release from the protein, is thought to play an important role in the drug's mode of action. Glutathione and ascorbic acid at their cancer cytosol concentrations were shown to be capable of altering the metal speciation in the drug adduct with holo-transferrin but not that with albumin.

Metallomics for drug development: serum protein binding and analysis of an anticancer tris(8-quinolinolato)gallium(III) drug using inductively coupled plasma mass spectrometry.

The application of an inductively coupled plasma mass spectrometry (ICP-MS) assay for quantifying in vitro binding of a gallium-based anticancer drug, tris(8-quinolinolato)gallium(III), to serum albumin and transferrin and in human serum is described. The distribution of the drug between the protein-rich and protein-free fractions was assessed via ICP-MS measurement of total gallium in ultrafiltrates. Comparative kinetic studies revealed that the drug exhibits a different reactivity toward individual proteins.

Advances of CE-ICP-MS in speciation analysis related to metalloproteomics of anticancer drugs.

The mode of action of metal-based anticancer drugs, including their accumulation in blood, transport, delivery to cancer cell, and cell processing (together with release of an active form and possibly targeting) is largely dependent on protein binding. Among analytical methods capable of providing a better understanding of metallodrug-protein interactions, capillary electrophoresis (CE) with inductively coupled plasma mass spectrometry (ICP-MS) detection is arguably a premier technique. Since its advent to the area of metallodrug proteomics in 2004, the benefits of CE-ICP-MS became evident, stimulating further research and methodological developments.

Metall(prote)omic studies by capillary electrophoresis using separation capillary as an in-line reactor.

This perspective article highlights the potential of capillary electrophoresis (CE) in in-line monitoring of biomolecular reactions related to in vivo transformations of metal species. In such scrutinizing, the capillary is regarded as a nanolitre-volume reactor in which electrical field-driven reactants are mixed to produce a response that enables in situ following-up and characterization of non-covalent molecular interactions. The concept of a CE reactor has been extended here to the investigation of processes that are responsible for the formation and decomposition of metal-bioligand species under simulated physiological conditions.

A quantitative structure-activity approach for lipophilicity estimation of antitumor complexes of different metals using microemulsion electrokinetic chromatography.

Microemulsion electrokinetic chromatography (MEEKC) offers a valuable tool for the rapid and highly productive determination of lipophilicity for metal-based anticancer agents. In this investigation, the MEEKC technique was applied for estimation of n-octanol-water partition coefficient (logP(oct)) of a series of antiproliferative complexes of gallium(III) and iron(III) with (4)N-substituted α-N-heterocyclic thiosemicarbazones. Analysis of relationships between the experimental logP(oct) and the retention factors of compounds showed their satisfactory consistency in the case of single metal sets, as well as for both metals.

Determination of gallium originated from a gallium-based anticancer drug in human urine using ICP-MS.

Urine analysis gives an insight into the excretion of the administered drug which is related to its reactivity and toxicity. In this work, the capability of inductively coupled plasma mass spectrometry (ICP-MS) to measure ultratrace metal levels was utilized for rapid assaying of gallium originating from the novel gallium anticancer drug, tris(8-quinolinolato)gallium(III) (GaQ(3)), in human urine. Sample dilution with 1% (v/v) HNO(3) as the only required pre-treatment was shown to prevent contamination of the sample introduction system and to reduce polyatomic interferences from sample components.

Focusing of anionic micelles using sample-induced transient isotachophoresis: computer simulation and experimental verification in MEKC.

The specific phenomenon of high-salt micelle focusing used as a preconcentration method for uncharged solutes in MEKC has been interpreted in terms of complementary transient isotachophoresis (tITP) process. High matrix chloride can focus anionic SDS micelles not only due to a field-enhanced effect but also by creating the tITP state in which chloride acts as a leading ion. The latter micelle-enrichment event was simulated by SIMUL software in order to understand tITP conditions under which micelle stacking can be expected to occur.

A versatile approach for assaying in vitro metallodrug metabolism using CE hyphenated with ICP-MS.

An informative analytical method, based on commercially available capillary electrophoresis (CE), inductively coupled plasma mass spectrometry (ICP-MS) and interface units, was developed for ascertaining possible metabolic transformations of metal-based drugs. Using a novel anticancer gallium compound, it was demonstrated that the drug remains intact in the simulated intestine juice. On the contrary, it undergoes a marked change in speciation (mostly, due to binding to transferrin) in human serum.

Capillary electrophoretic assay for the stability of tris(8-quinolinolato)gallium(III) in tablet formulations.

A capillary electrophoresis (CE) method for testing the stability of a novel oral anticancer metallodrug, tris(8-quinolinolato)gallium(III) (KP46), is proposed. As both the intact drug and its eventual impurity or/and decomposition product, 8-quinolinol, are not charged (at most of the pH range), the micellar-mediated CE mode based on using micellar concentrations of sodium dodecyl sulfate was employed. The running electrolyte conditions were optimized in order to resolve the peak of KP46 from the signal of 8-quinolinol, as well as from these of tablet matrix components.

Toward high-throughput monitoring of metallodrug-protein interaction using capillary electrophoresis in chemically modified capillaries.

The performance of capillary electrophoresis (CE) operating with a sulfonated capillary for the separation of protein adducts of anticancer ruthenium(III)-based drugs was evaluated. The coated capillary was shown to yield improved resolution of albumin- and transferrin-bound species of ruthenium compared with that attained with the bare fused-silica capillary. The coating also showed an increased reproducibility of migration times and peak areas and allowed reasonably high efficiency separation of analytes (up to 1300 theoretical plates per meter), which display high affinity toward a fused-silica surface.

Application of capillary electrophoresis-inductively coupled plasma mass spectrometry to comparative studying of the reactivity of antitumor ruthenium(III) complexes differing in the nature of counter-ion toward human serum proteins.

Varying the counter-ion is a highly supportive practice in tackling the problem of poor water-solubility of metal complexes of pharmaceutical importance. As a matter of fact, the relevant structural modification may alter the metabolic pathways and possibly the mode of action of a drug. To prove that this does not take place for one of the lead anticancer metal-based developmental compounds, indazolium trans-[RuCl(4)(1H-indazole)(2)] (KP1019), its reactivity toward human serum proteins was assessed under simulated physiological conditions and compared to that of a much more soluble analogue, sodium trans-[RuCl(4)(1H-indazole)(2)] (KP1339).

Probing the stability of serum protein-ruthenium(III) drug adducts in the presence of extracellular reductants using CE.

A CE kinetic assay was developed to study the stability of the adducts of a novel ruthenium(III)-based anticancer agent with serum proteins under simulated reductive physiological conditions. Formation of the reactive Ru(II) species and their release from the serum proteins are thought to play an important role in the mode-of-action of indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) which has successfully finished a clinical phase I study. The CE method was adapted, in zone electrophoresis and affinity CE modes, to make obvious that such transformation would take place in the hypoxic tumor tissue rather than in the bloodstream.

Application of micellar and microemulsion electrokinetic chromatography for characterization of gallium(III) complexes of pharmaceutical significance.

CE with conventional UV detection has recently been shown as a highly effective means to assaying cytotoxic gallium(III)-based compounds with regard to desirable drug-like properties such as the stability and binding to serum proteins. In this extension of that work, different CE techniques are used to further characterize a given set of gallium coordination compounds with established antiproliferating efficacy. Using free-zone CE mode, the electrophoretic profiles of complexes are recorded in order to assess their actual charge state under physiological buffer conditions.

Preclinical characterization of anticancer gallium(III) complexes: solubility, stability, lipophilicity and binding to serum proteins.

The discovery and development of gallium(III) complexes capable of inhibiting tumor growth is an emerging area of anticancer drug research. A range of novel gallium coordination compounds with established cytotoxic efficacy have been characterized in terms of desirable chemical and biochemical properties and compared with tris(8-quinolinolato)gallium(III) (KP46), a lead anticancer gallium-based candidate that successfully finished phase I clinical trials (under the name FFC11), showing activity against renal cell cancer. In view of probable oral administration, drug-like parameters, such as solubility in water, saline and 0.