University of Hamburg (Germany) researchers created a method based on hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) to simultaneously quantify antitumor prodrugs and their polar metabolites. Their findings were published in the Journal of Chromatography B (1).
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Nucleotides and nucleosides play vital roles in various cellular processes, such as the synthesis of RNA and DNA. If these structures are structurally modified compared to naturally occurring nucleosides, they are referred to as nucleoside analogues. These analogues are metabolized in a manner similar to those of natural nucleosides, allowing the inhibition of metabolic pathways (and making them be known as anti-metabolites). Due to their similar structures, nucleoside analogues can be recognized by cellular or viral enzymes and can be incorporated into DNA or RNA. That said, structural differences can result in the interruption or termination of replication.
The TriPPPro approach was developed in 2015, being a nucleoside triphosphate (NTP) delivery system where NTP is masked by two lipophilic moieties which are enzymatically cleaved off after successful cellular uptake (1,2). These masks can enable membrane permeability of compounds, and on an intracellular level, the masks are enzymatically cleaved, leading to the release of active nucleoside triphosphates (NTAs).
In this study, the scientists created a HILIC–MS/MS method for quantifying TriPPPro-prodrugs, derived from the anticancer drug fluorouracil (5-FU) and all resulting metabolites—FdU, FdU-monophosphate (MP), FdU-diphosphate (DP), and FdU-triphosphate (TP)—in cancer cell lysate. FdU-TP and FdU-MP were considered the responsible metabolites for antitumor activity. FdU-TP can be incorporated into the DNA, leading to pathological DNA structures and eventual cell death. FdU-MP, on the other hand, inhibits thymidylate synthase (TS), which is the enzyme that catalyzes the transformation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (TMP). TS inhibition causes an accumulation of deoxyuridine triphosphate (dUTP) and the subsequent depletion of thymidine triphosphate (TTP), causing an imbalance that ultimately leads to cell death. Further, the method should include sample preparation of the cell lysate after celluar uptake; this would facilitate greater understandings of the prodrug’s uptake efficiency and intracellular metabolism.
Since the lipophilic prodrugs and the hydrophilic metabolites involve different chemical properties, sample preparation and liquid chromatography method development proved to be challenging factors. A liquid-liquid extraction protocol was employed, and when HILIC was used, the simultaneous retention of all analytes was guaranteed.
The method was validated for the 2.0–1000 ng/mL concentration ranges, specifically in cancer cell lysate and the associated supernatant. Further, it was successfully applied to quantify prodrugs and metabolites in HT29 cancer cell lysate and supernatant samples after cellular uptake studies with two different TriPPPro-prodrugs.
According to the researchers, this is the first method that can be used to determine the concentration levels of lipophilic prodrugs and their hydrophilic metabolites. Unlike other approaches, this method can simultaneously quantify prodrugs and their various metabolites within a single run. Overall, the researchers found that their method allowed for higher sensitivity, broader linear ranges, and the inclusion of more analytes compared to earlier approaches. Future research efforts may involve testing two TriPPPro-compounds in other cancer cell lines, or even by testing antiviral compounds in addition to antitumor compounds in cellular uptake studies with virus cells to gain more data concerning cellular uptakes and metabolic conversions.
(1) Vogts, M.; Witt, J.; Riedner, M.; Meier, C. Development of a HILIC-MS/MS Method for Simultaneous Quantification of Lipophilic Antitumor TriPPPro-Prodrugs and Their Polar Metabolites in HT29 Cell Extracts. J. Chromatogr. B 2025, 1263, 124673. DOI: 10.1016/j.jchromb.2025.124673
(2) Jia, X.; Schols, D.; Meier, C. Lipophilic Triphosphate Prodrugs of Various Nucleoside Analogues. J. Med. Chem. 2020, 63 (13), 6991–7007. DOI: 10.1021/acs.jmedchem.0c00358
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