Novel SPME fibers based on a plastic support for determination of plasma protein binding of thiosemicarbazone metal chelators: a case example of DpC, an anti-cancer drug that entered clinical trials
Solid-phase microextraction (SPME) is an alternative method to dialysis and ultrafiltration for the determination of plasma protein binding (PPB) of drugs. It is particularly advantageous for complicated analytes where standard methods are not applicable. Di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) is a lead compound of novel thiosemicarbazone anti-cancer drugs, which entered clinical trials in 2016. However, this agent exhibited non-specific binding on filtration membranes and had intrinsic chelation activity, which precluded standard PPB methods. In this study, using a simple and fast procedure, we prepared novel SPME fibers for extraction of DpC based on a metal-free, silicon string support, covered with C18 sorbent. Reproducibility of the preparation process was demonstrated by the percent relative standard deviation (RSD) of ≤ 9.2% of the amount of DpC extracted from PBS by several independently prepared fibers. The SPME procedure was optimized by evaluating extraction and desorption time profiles. Suitability of the optimized protocol was verified by examining reproducibility, linearity, and recovery of DpC extracted from PBS or plasma. All samples extracted by SPME were analyzed using an optimized and validated UHPLC-MS/MS method. The developed procedure was applied to the in vitro determination of PPB of DpC at two clinically relevant concentrations (500 and 1000 ng/mL). These studies showed that DpC is highly bound to plasma proteins (PPB ≥ 88%) and this did not differ significantly between both concentrations tested. This investigation provides novel data in the applicability of SPME for the determination of PPB of chelators, as well as useful information for the clinical development of DpC.
KeywordsDi-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone Anti-cancer agents Sample preparation Solid-phase microextraction
This study was supported by the project EFSA-CDN (No.CZ.02.1.01/0.0/0.0/16_019/0000841) cofunded by ERDF to P.S.K and D.R.R., the Charles University (the Project SVV 260 401) to P.S.K and P.R., and a National Health and Medical Research Council (NHMRC) Project Grant (No. 1060482) and Senior Principal Research Fellowship (APP1062607) to D.R.R.
Compliance with ethical standards
Plasma from Wistar rats was obtained from in vivo experiments that were approved and supervised by the Animal Welfare Committee of the Faculty of Pharmacy, Charles University (Czech Republic). All experiments were performed in accordance with Directive 2010/63/EU on the Protection of Animals Used for Scientific Purposes.
Conflict of interest
The authors declare that they have no conflict of interest.
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