RESUMEN
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.
Asunto(s)
Antineoplásicos/metabolismo , Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Compuestos Organometálicos/metabolismo , Administración Oral , Antineoplásicos/administración & dosificación , Antineoplásicos/sangre , Antineoplásicos/química , Galio/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Compuestos Organometálicos/administración & dosificación , Compuestos Organometálicos/sangre , Compuestos Organometálicos/química , Factores de Tiempo , Transferrina/metabolismoRESUMEN
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). For such kinetic assaying, capillary electrophoresis (CE) interfaced online with inductively coupled plasma mass spectrometry (ICP-MS) to specifically monitor changes in the metal speciation following the formation of ruthenium-protein adducts was applied. The rate constants of interaction with albumin and transferrin were determined at pharmacologically fitting drug-to-protein ratios as on average 0.0319+/-0.0021 min(-1) and 0.0931+/-0.0019 min(-1) (KP1019) and 0.0316+/-0.0018 min(-1) and 0.0935+/-0.0053 min(-1) (KP1339), respectively. The results of this brief study showed that changing from organic to inorganic counter-ion at the stage of formulation could commonly be recommended for improving ruthenium-based drug solubility and bioavailability.
Asunto(s)
Antineoplásicos/química , Proteínas Sanguíneas/química , Compuestos de Rutenio/química , Algoritmos , Electrocromatografía Capilar , Humanos , Indazoles/química , Indicadores y Reactivos , Cinética , Espectrometría de Masas , Compuestos Organometálicos , Albúmina Sérica/química , Soluciones , Transferrina/químicaRESUMEN
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.
Asunto(s)
Proteínas Sanguíneas/química , Electroforesis Capilar/métodos , Indazoles/química , Compuestos de Rutenio/química , Ácido Ascórbico/química , Tampones (Química) , Estabilidad de Medicamentos , Electrólitos/química , Glutatión/química , Indazoles/sangre , Espectrometría de Masas , Compuestos Organometálicos , Oxidación-Reducción , Compuestos de Rutenio/sangre , Albúmina Sérica/química , Espectrofotometría Ultravioleta , Transferrina/químicaRESUMEN
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.