In silico and in vitro investigations of the drug–drug interaction mechanisms between fludarabine and busulfan

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Abstract
Background: Understanding drug interactions in hematopoietic stem cell transplantation (HSCT) is crucial given the narrow therapeutic index of busulfan (BU), which is a key conditioning agent. In this study, we investigate the potential impacts of fludarabine (Flu) as a frequently co-administered agent in HSCT on BU pharmacokinetics (PK). Specifically, we examine whether Flu can alter BU metabolism by affecting the predominantly expressed cytosolic glutathione-S-transferases (GSTs), particularly GSTA1, GSTM1, and GSTP1, which are essential for BU detoxification. Methods: We conducted molecular docking and atomistic molecular dynamics simulations using the crystal structures of GSTA1, GSTM1, and GSTP1 to study the estimated binding affinity of Flu as well as its metabolites to these target enzymes. We then performed in vitro assays on human recombinant GST enzymes and HepaRG hepatocyte cells by focusing on enzymatic inhibition, GST expression analysis, and glutathione level measurements. Enzymatic assays were conducted using 1-chloro-2,4-dinitrobenzene as a substrate alongside Western blotting and cell-viability-corrected glutathione (GSH) assays to determine the influences of Flu on the enzymatic activities and expression of GSTs. Results: In silico analysis predicted the binding affinities of Flu to the GST isoforms, with estimated Ki values of 0.2 µM, 5.2 µM, and 23.9 µM for GSTA1, GSTM1, and GSTP1, respectively; although these are micromolar (µM) inhibition values that indicate relatively weak or moderate inhibition, they are still insightful for understanding the potential interactions. However, the in vitro assays revealed no significant inhibition of the GST enzymes by Flu, even at concentrations up to ten times the clinical peak (C max ). Further, Flu did not notably alter GSTA1 expression or affect cellular glutathione levels in the HepaRG cells. These experimental findings suggest that Flu may have minimal influence on the GST-mediated detoxifying pathway in the context of cancer treatment. Conclusion: The present study underscores the importance of empirically validating in silico observations in pharmacological research by emphasizing the minimal effect of Flu on GST activity and its implications in clinical oncology. Moreover, the findings suggest that Flu is not likely to alter BU pharmacokinetics via the GSH-conjugation pathway.
Funding Information
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (153389)
  • Oak Spring Garden Foundation (OCAY-17-642)

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