The challenges of performing ring test studies to evaluate the efficacy of complex drug combinations in vitro

Within the REMEDi4ALL project reproducibility received special attention as a key barrier to successful drug repurposing (DR) in different fields from their original application.

Three different partners of the REMEDi4ALL community (Germany, Sweden and Italy) performed, in the frame of Demonstrator2, a ring test study to analyze the critical steps necessary to achieve robust and reproducible preclinical outcomes.

The DR hypothesis of Demonstrator-2 is based on the original results obtained in the Italian center demonstrating the synergistic effect of the histone deacetylase inhibitor (HDACi), valproic acid (VPA), combined with the lowering-cholesterol drug, simvastatin (SIM), in potentiating the antitumoral activity of the first-line gemcitabine/nab-paclitaxel chemotherapy in pancreatic ductal adenocarcinoma (PDAC).

To ensure reproducibility, an experimental workflow was developed following three main steps: material sharing (compounds, cells, legal documentation); alignment of experimental conditions (visiting scientists, protocols); synergy screening, data sharing and analysis.

A first drug screening was performed across three PDAC cell lines (BxPC3, MiaPaca-2, PanC-1) testing the combinations of HDACis (vorinostat, entinostat, panobinostat, VPA) plus statins (lovastatin, atorvastatin, SIM). A second drug screening was performed to assess the ability of VPA and SIM to enhance the cytotoxic effects of chemotherapy in the same cell lines. Across the tested compound concentrations, we obtained the MSA score by Synergy Finder tool, corresponding to the highest synergistic area.

Despite the initial development of a common workflow, technical and experimental challenges occurred, highlighting the need to realign the experimental conditions. Through the full alignment of the three institutes, we achieved consistent reproducibility of Demonstrator-2 data.

This success enabled us to develop standard operative procedures (SOPs) as a valuable resource for the REMEDi4ALL.

Author and article information

Conference

Publisher: ScienceOpen

Publication date (Electronic, pub): 12 April 2025

Affiliations

[1 ] Istituto Nazionale Tumori IRCSS G.Pascale ( https://ror.org/0506y2b23)

[2 ] Fraunhofer Institute for Translational Medicine and Pharmacology (ITMP) ( https://ror.org/01s1h3j07)

[3 ] Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland ( https://ror.org/030sbze61)

[4 ] Chemical Biology Consortium Sweden, Division of Translational Medicine and Chemical Biology, Department of Medical Chemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden ( https://ror.org/056d84691)

[5 ] Experimental Pharmacology Unit-Laboratory of Naples and Mercogliano (AV), Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione G. Pascale” – IRCCS, Naples; Italy; ( https://ror.org/0506y2b23)

[6 ] Experimental Pharmacology Unit-Laboratory of Naples and Mercogliano (AV), Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione G. Pascale” – IRCCS, Naples; Italy ( https://ror.org/0506y2b23)

[7 ] Chemical Biology Consortium Sweden, Division of Translational Medicine and Chemical Biology, Department of Medical Chemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden ( https://ror.org/056d84691)

[8 ] Experimental Pharmacology Unit-Laboratory of Naples and Mercogliano (AV), Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione G. Pascale” – IRCCS, Naples; Italy ( https://ror.org/0506y2b23)

[9 ] Experimental Animal Unit; Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione G. Pascale” – IRCCS, Naples; Italy ( https://ror.org/0506y2b23)

[10 ] Fraunhofer Institute for Translational Medicine and Pharmacology (ITMP), Schnackenburgallee 114, D-22525 Hamburg, Germany ( https://ror.org/01s1h3j07)

[11 ] Scientific Directorate, Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione G. Pascale” – IRCCS, Naples; Italy ( https://ror.org/0506y2b23)

Author information

Veronica Barile https://orcid.org/0009-0008-7319-2724

Article

DOI: 10.14293/iDR.25.006VB

SO-VID: c125d16b-1dc3-4c14-a8d6-6584a841d9ad

License:

Published under Creative Commons Attribution 4.0 International ( CC BY 4.0). Users are allowed to share (copy and redistribute the material in any medium or format) and adapt (remix, transform, and build upon the material for any purpose, even commercially), as long as the authors and the publisher are explicitly identified and properly acknowledged as the original source.

Conference name: International Drug Repurposing Conference 2025

Conference acronym: iDR25

Conference number: 2

Conference location: Amsterdam, The Netherlands

Conference date: 7-8 May 2025

History

Product

ScienceOpen

Keywords: ring test studies, complex drug combinations