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Abstract

According to the World Health Organization (WHO), cancer represents the second leading cause of mortality, with almost 10 million deaths in 2020. A broad spectra of cancer treatments are being developed, among them are metal-based drugs. Consistent with the available data, platinum (Pt)-based chemotherapeutics are still widely used to treat various cancers. The main drawback of Pt-based chemotherapeutics is their high toxicity, which leads to severe side effects. So, this research is oriented towards the development of new anticancer drugs. Two of the promising drug candidates are Ru (II) and Ru (III) complexes.
An important step in a candidate drug’s characterization is the pharmacological evaluation of its interactions with serum proteins. It provides data for the elucidation of the mechanism of antitumor therapy, the behaviour of the intact drug, its biotransformation in clinical samples at physiologically relevant concentrations and, as the final goal, its dosage adjustment. In investigations of the performance of new candidate drugs, it is also important to elucidate the kinetics of drug bindings to human serum proteins. A speciation analysis can largely acquire this information. Two ruthenium (Ru) complexes, i.e., [(η6-p-cymene)Ru(1-hydroxypyridine-2(1H)-thionato)Cl] (1) and [(η6-p-cymene)Ru(1-hydroxypyridine-2(1H)-thionato)pta]PF6 (2) (where pta = 1,3,5-triaza-7-phosphaadamantane), were studied. Conjoint liquid chromatography (CLC) monolithic column, assembling convective interaction media (CIM) protein G and diethylaminoethyl (DEAE) disks, was used for the separation of unbound Ru species from those bound to human serum transferrin (Tf), albumin (HSA) and immunoglobulins G (IgG). Eluted proteins were monitored by UV spectrometry at 278 nm, while Ru species were quantified by post-column isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS). The binding kinetics of chlorido (1) and pta complex (2) to serum proteins was followed from 5 min to 48 h after incubation with human serum. Speciation analysis data revealed that the interaction with serum proteins was faster and more extensive in the chlorido (1) complex.
Ceruloplasmin (Cp) is human plasma's major copper-carrying (Cu) protein. Due to copper’s important physiological functions and its role in various diseases, it is necessary to quantify the concentration bound to Cp and the exchangeable form of Cu. In the present work, CLC on short-bed CIM monolithic disks was used to separate the Cu bound to low molecular mass (LMM) species and the Cu bound to Cp and HSA in human serum. Two immunoaffinity CIMmic albumin depletion (α-HSA) disks and one CIMmic weak anion-exchange DEAE disk were assembled in a single housing, forming a CLC monolithic column. The separated Cu species were quantified by post-column ID-ICP-MS, while the elution profile of the proteins was followed by UV detection at 278 nm. This novel developed method was successfully applied in the determination of Cu-Cp, Cu-HSA and a fraction that most probably corresponds to the Cu-LMM species in the human serum of healthy individuals, transplanted renal patients and cancer patients. The data from the present research provide an important new analytical tool that can be used to assess Cu metabolic disorders in many diseases.