LC-MS/MS for Pharmacokinetic Evaluation of Potential Anti-Cancer Compounds

One common research goal is the development of novel compounds that inhibit cancerous cells without affecting the surrounding healthy tissues. Thiosemicarbazones (DpT series) represent a class of anti-cancer compounds with the potential to meet this standard; however, the well-documented DpT di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) presents a degree of cardiotoxicity when administered at non-ideal levels.¹ For this reason, researchers recently turned to a related compound, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), which has potential as both an anti-tumoral and anti-retroviral agent.^2,3

Recently, Stariat et al. (2013) applied liquid chromatography–tandem mass spectrometry (LC-MS/MS) to ascertain the concentration-time profile and pharmacokinetic parameters of Bp4eT and two of its metabolites, semicarbazone and amidrazone, in rat plasma.⁴ The researchers used a C18 column with mobile phase consisting of 2 mM ammonium formate and ACN (40:60 v/v) at a 0.4 ml/min flow rate to separate and quantify plasma analytes within a 12-minute timeframe. They used the ligand di-2-pyridylketone 4-phenyl-3-thiosemicarbazone as an internal standard and aqueous EDTA (20 mM) injections before each run to prevent complex formation with transition ions.

In a previous study, Stariat and co-workers noted complications when using electrospray ionization mass spectrometry (ESI-MS) settings.⁵ In particular, the metabolic isomers formed [M + Na]+ adducts that led to poor quantification of Bp4eT. This time, the researchers dropped the capillary temperature to 150°C to reduce adduct formation and used the sum of the peak areas for both isomers to quantify the parent compound. They also optimized and reformatted the previous solid phase extraction (SPE) method to a more convenient 96-well plate system.

Stariat et al. used six calibration standards to verify the linearity of their calibration curves for the following ranges of concentration: 0.18–2.80 μm for Bp4eT, 0.02–0.37 μm for semicarbazone, and 0.10–1.60 μm for amidrazone. Accuracy percentiles within the acceptable range and reasonable matrix factors indicate that the method is highly selective. They also demonstrated long-term stability of the parent compound, metabolites, and internal standard, as well as acceptable dilution integrity. In this way, the authors determined that the method is competent for the analysis of Bp4eT.

For the pharmacokinetic evaluation, the researchers used the LC-MS/MS method from their pilot study. Bp4eT and amidrazone demonstrated adequate responses, while both isomers of semicarbazone fell below the lower limits of quantitation. Bp4eT presented a high apparent distribution volume (2.41 L/kg), likely due to the lipophilic nature of the compound that allows it to permeate membranes and distribute to the tissues. The researchers noted that the parent compound demonstrated a longer half-life of elimination (t1/2=84.9 min) when compared to previous studies involving rabbits. This more closely mimics the half-life observed in humans.^6,7

Amidrazone was present at 30 minutes after delivery, increased until 180 minutes, and then plateaued and surpassed Bp4eT at 300 minutes. Stariat et al. identified exposure to amidrazone as 20% in comparison to the parent compound and indicated that this key biotransformation pathway warrants further study.

 

References 

1. Whitnall, M., et al. (2006) “A class of iron chelators with a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics,” Proceedings of the National Academy of Sciences of the USA, 103 (pp. 14901–06).

2. Kalinowski, D., et al. (2007) “Design, synthesis, and characterization of novel iron chelators: Structure-activity relationships of the 2-benzoylpiridine thiosemicarbazone series and their 3-nitrobenzoyl analogues as potent anti-tumor agents,” Journal of Medicinal Chemistry, 50 (pp. 3716–29).

3. Debebe, Z., et al. (2012) “Development of a sensitive HPLC method to measure in vitro permeability of E+ and Z- isometric forms of thiosemicarbazones in Caco-2 monolayers,” Journal of Chromatography B, 906 (pp. 25–32).

4. Stariat, J., et al. (2013) “Simultaneous determination of the novel thiosemicarbazone anti-cancer agent, Bp4eT, and its main phase I metabolites in plasma: Application to a pilot pharmacokinetic study in rats,” Biomedical Chromatography, doi: 10.1002/bmc.3080.

5. Stariat, J., et al. (2012) “LC-MS/MS identification of the principal in vitro and in vivo phase I metabolites of the novel thiosemicarbazone anti-cancer drug, Bp4eT,” Analytical and Bioanalytical Chemistry, 403 (309–21).

6. Kovarikova, P., et al. (2006) “HPLC determination of a novel aroylhydrazone iron chelator (o-108) in rabbit plasma and its application to a pilot pharmacokinetic study,” Journal of Chromatography, 838 (pp. 107–12).

7. Feun, L., et al. (2002) “Phase I and pharmacokinetic study of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) using a single intravenous dose schedule,” Cancer Chemotherapy and Pharmacology, 50 (pp. 223–9).

Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.