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. Indeed, no measurable effect of extracellular concentration levels of glutathione incorporated into the BGE on the UV signals of albumin and transferrin adducts was observed over 30 min of examination. Incubation of the KP1019-albumin adduct with the major blood reducing agent, ascorbic acid, revealed no changes in the continuously monitored peak areas (average corrected responses were 9.56 +/- 0.86 and 9.87 +/- 0.60 mAU for the adduct and its mixtures with ascorbic acid in the physiological range of 1 x 10(-5) -8 x 10(-5) M, respectively). On the other hand, both the transferrin adduct and transferrin itself accelerated the oxidation of ascorbic acid; however, the oxidation rate constants measured by CE were virtually the same: (19.1 +/- 4.4) x 10(-3) and (18.2 +/- 5.0) x 10(-3) min(-1), respectively. In order to confirm more unambiguously the stability of KP1019-protein adducts in the presence of ascorbic acid (UV absorbance detection does not distinguish the adduct and protein signals), CE with inductively coupled plasma (ICP) MS detection was applied to follow metal-selectively the signal of bound ruthenium, which remained unaffected by this reducing agent. This work appears the first to present the application of CE to the stability studies of the protein-bound metallodrugs.
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. The appearance of the additional peaks in ICP-MS electropherograms (by recording both Ru- and Fe-specific signals) was found to be dependent on time which allowed for kinetic assessment of the evolution of novel metal species. On the contrary, after the addition of citric acid the ruthenium ion (within the appropriately complexed scaffold) remained sequestered in the adduct. This was inferred as a proof of the speciation changes taking place by a virtue of a redox mechanism rather than due to ligand-exchange transformations. The protein-bound metallodrug was further characterized by direct ICP-MS assaying so as to confirm a partial release of ruthenium induced by glutathione.
Abstract:The synthesis and in vitro cytotoxicity of a series of Pt IV complexes with lonidamine as a ligand coordinated in axial position are described. Lonidamine was found to affect strongly the in vitro cytotoxic activity of these new complexes, lowering
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. Micellar and microemulsion electrokinetic chromatographic techniques are tested as tools for the rapid estimation of the n-octanol-water partition coefficient (log P) that provides a rationale estimate of a drug's ability to cross biological membranes. A range of electrolyte buffer systems with varying (both in the nature and concentration) organic modifiers are examined to evaluate their effect on the relationship between experimental or calculated log P and the retention factors of compounds (log k'). Both methods were found to be better applicable for neutral than for cationic Ga complexes, the microemulsion mode demonstrating superior lipophilicity estimations as well as statistically meaningful log P versus log k' correlations when all the complexes were included in one regression set.
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.
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. The origin of the blank signal at masses of gallium isotopes, 71 and 69, was investigated using high-resolution ICP-MS and attributed, respectively, to the formation of (36)Ar(35)Cl(+) and (40)Ar(31)P(+) ions and, tentatively, to a triplet of doubly charged ions of Ba, La, and Ce. The accuracy and precision performance was tested by evaluating a set of parameters for analytical method validation. The developed assay has been applied for the determination of gallium in urine samples spiked with GaQ(3). The achieved recoveries (95-102%) and quantification limit of 0.2 μg L(-1) emphasize the practical applicability of the presented analytical approach to monitor renal elimination of GaQ(3) at all dose levels in clinical trials that are currently in progress.
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