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Cisplatin and its second and third generation analogues are widely used in the treatment of cancer. To study their reactions with proteins, we present a method based on SDS-PAGE separation and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) for platinum detection in the reaction between human serum albumin (HSA) and cisplatin. We developed matrix-matched standards of HSA/cisplatin mixtures and used them to quantify the amount of adducts formed at different HSA:cisplatin ratios. We noted that cisplatin incubation with HSA resulted in the formation of higher order HSA n-mers, depending on the amount of cisplatin added. This caused a depletion of the HSA dimer bands, while the majority of HSA was present as the monomer. Inducing the formation of such higher molecular weight species may have an impact on the mode of action of metallodrugs., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Capillary electrophoretic analyses benefit significantly from hyphenation to mass spectrometric techniques. While the coupling to ESI-MS is routinely performed, for example by using a coaxial sheath-flow interface, hyphenating it to inductively coupled plasma mass spectrometry is more technically challenging. We use a commercially available coaxial sheath-flow interface (CSFI) and a simple PTFE-based end-cap for easy, inexpensive CE-ICP-MS hyphenation with improved sensitivity and analytical performance compared to commercially available interfaces. We have optimized key nebulizer parameters such as capillary position, sheath liquid flow rate, and carrier gas flow rate, and compared the CSFI with a commercially available interface. In a set of proof-of-principle experiments employing the anticancer agent cisplatin it was demonstrated that the signal to noise response and sensitivity were considerably improved leading to detection limits for 195 Pt of 0.08 μM., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We present here the first comprehensive study on the lipophilicity of ruthenium anticancer agents encompassing compounds with broad structural diversity, ranging from octahedral Ru III (azole) through to Ru II (arene) complexes. MEEKC was used to determine the capacity factors of the Ru complexes, and after a complex peak was unambiguously assigned using MEEKC-ICP-MS, the results were validated through comparison with the log P determined by octanol/water partitioning experiments. Correlation of the two data sets demonstrated a close relationship despite the limited structural overlap of the compounds studied. The capacity factors found by MEEKC allowed for the clustering of complexes based on their structure and this could be used to rationalize the observed cytotoxicity in the human colon carcinoma HCT116 cell line. It was demonstrated that rather than modification of the mono- or bidentate coordinated ligands much tighter control over a complexes lipophilic properties could be achieved through modification of the Ru(arene) ligand, with minimal detriment to cytotoxicity. This demonstrates the flexibility and potential of the Ru piano-stool scaffold. MEEKC proved to be a highly efficient means of screening the anticancer potential of preclinical ruthenium complex candidates for their lipophilic properties and correlate them with their biological activity and structural properties., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Electrophoretic methods have been widely applied in research on the roles of metal complexes in biological systems. In particular, CE, often hyphenated to a sensitive MS detector, has provided valuable information on the modes of action of metal-based pharmaceuticals, and more recently new methods have been added to the electrophoretic toolbox. The range of applications continues to expand as a result of enhanced CE-to-MS interfacing, with sensitivity often at picomolar level, and evolved separation modes allowing for innovative sample analysis. This article is a followup to previous reviews about CE methods in metallodrug research (Electrophoresis, 2003, 24, 2023-2037; Electrophoresis, 2007, 28, 3436-3446; Electrophoresis, 2012, 33, 622-634), also providing a comprehensive overview of metal species studied by electrophoretic methods hyphenated to MS. It highlights the latest CE developments, takes a sneak peek into gel electrophoresis, traces biomolecule labeling, and focuses on the importance of early-stage drug development., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mass spectrometry (MS) has emerged as an important tool for studying anticancer metallodrugs in complex biological samples and for characterising their interactions with biomolecules and potential targets on a molecular level. The exact modes-of-action of these coordination compounds and especially of next generation drug candidates have not been fully elucidated. Due to the fact that DNA is considered a crucial target for platinum chemotherapeutics, metallodrug-DNA binding studies dominated the field for a long time. However, more recently, alternative targets were considered, including enzymes and proteins that may play a role in the overall pharmacological and toxicological profile of metallodrugs. This review focuses on MS-based techniques for studying anticancer metallodrugs in vivo, in vitro and in situ to delineate their modes-of-action.

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., (This journal is © The Royal Society of Chemistry 2011)

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.

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.

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.

The application of capillary electrophoresis (CE) combined with highly sensitive inductively-coupled-plasma mass spectrometric (ICP-MS) detection allows the interactions of metal complexes with biomolecules to be characterized. This technique has been used to provide new insights into the mode of action of the ruthenium-based anticancer drug candidate indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019). While the compound binds rapidly and efficiently to serum proteins, especially albumin, its reactivity towards the model DNA compound 2'-deoxyguanosine 5'-monophosphate (5'-dGMP) is moderate.

Elucidating the mode of action and thereby opening the way to the design of chemotherapeutic agents is one of the major goals of metal-based anticancer research. Hydrolysis and DNA binding play an important role for pharmaceutical formulation and for exerting anticancer activity. Herein, for the first time the application of capillary zone electrophoresis-inductively-coupled plasma mass spectrometry (CZE-ICP-MS) for studying the hydrolytic stability and the binding of the ruthenium anticancer drug candidates KP418, KP1019, and RAPTA-C to dGMP is described. RAPTA-C was found to hydrolyze fastest and showed the highest reactivity toward the DNA model compound, whereas KP418 was the most stable compound in both these respects.

The development of metal-based chemotherapeutics lacks methods which are capable of providing early indication on the potential of new metal complexes as future anticancer drugs. Since most of these compounds are administered intravenously, serum proteins are the first available biological binding partners in the bloodstream. For platinum-based anticancer drugs the interaction with serum proteins is regarded as an important contribution to the side effects accompanying chemotherapy. In contrast, newly developed ruthenium compounds are thought to be transported into the tumor in a protein-bound form. In here, the application of CE hyphenated to inductively coupled plasma (ICP)-MS, applying Polybrene-coated capillaries, is demonstrated for studying the interaction of indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) with HSA and transferrin, which are important transport proteins. Furthermore, the applicability of the method to human serum and plasma and, more importantly, to real-world patient samples was proven. KP1019 was found to bind to a high degree to HSA both in serum, plasma and the patient samples. Only minor fractions of ruthenium were found attached to other proteins.

Biochemical speciation analysis has become a hot area of CE research due largely to growing emergence of inductively coupled plasma (ICP)-MS as a proper detection technique. A benefit of CE-ICP-MS coupling in species-selective analysis of anticancer metal-based drugs is the possibility of distinguishing the signals of the intact drug and its metabolites and hence of quantifying them independently. This advantage (over CE with UV-vis detection) was exploited here in order to gain better knowledge about the rate and degree of the transformation of indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019), a promising tumor-inhibiting agent that successfully finished phase I clinical studies, upon its binding toward individual serum transport proteins. At increasing the KP1019/protein molar ratio, the reaction rate expressed by an evolving peak of the protein adduct became faster, with the equilibrium state being reached after about 40 and 60 min of incubation at 37 degrees C for transferrin and albumin, respectively. The binding reaction was shown to obey the first-order character that enabled for reliable calculation of the corresponding rate constants as (28.7 +/- 1.5) x 10(-4) and (10.6 +/- 0.7) x 10(-4)/s, respectively. When incubated with a ten-fold excess of KP1019, albumin and transferrin bound, respectively, up to 8 and 10 equiv. of ruthenium (Ru). Relative affinity of KP1019 toward different proteins under simulated physiological conditions was also characterized in terms of the overall binding constants (5600 and 10 600/M, respectively). To emphasize the difference in the protein-binding behavior, a competitive interaction of KP1019 was followed by CE-ICP-MS at the actual molar ratio of proteins in blood, i.e. a ten-fold excess of albumin over transferrin. The fact that KP1019 binds to albumin stronger than to transferrin was manifested by finding almost all ruthenium (98-99%) in the albumin fraction.

Characterizing how platinum metallocomplexes bind to human serum albumin (HSA) is essential in evaluating anticancer drug candidates. Using cisplatin as a reference complex, the application of capillary electrophoresis (CE) to reliably assess drug/HSA interactions was validated. Since this complex is small compared to the size of the protein, the binding response could only be recognized when applying CE coupled to a (platinum) metal-specific mode of detection, namely inductively coupled plasma-mass spectrometry (ICP-MS). This coupling allowed for confirmation of a specific affinity of cisplatin and novel Pt complexes to HSA, measurement of the kinetics of binding reactions, and determination of the number of drug molecules attached to the protein. As the cisplatin/HSA molar ratio increased, the reaction rate became faster with a maximum on the kinetic curve appearing at about 50 h of incubation at 20 times excess of cisplatin. The reaction was characterized as a pseudo-first order reaction with the rate constant k = 0.003 min(-1) at 37 degrees C. When incubated with a 20-fold excess of cisplatin, HSA bound up to 10 mol of Pt per mol of the protein. This is indicative for a strong metal-protein coordination occurring at several HSA sites other than the only protein cysteine residue. Structural analogs of cisplatin, bearing aminoalcohol ligands, showed comparable protein binding reactivity and stoichiometry but a common equilibrium was not reached even after one week of incubation. Also apparent was a two-step mechanism of the binding reaction. Results demonstrated the suitability of CE-ICP-MS as a rapid assay for high-throughput studying of drug/HSA interactions.