(W1030-02-09) Design and Evaluation of a Cyclic Peptide-Epirubicin Conjugate to Enhance Anticancer Activity and Reduce Cardiac Toxicity

Purpose: The purpose of this study is to explore a novel approach in cancer treatment by designing and evaluating a cyclic peptide-epirubicin conjugate. This conjugate aims to enhance the anticancer efficacy of epirubicin, a widely used anthracycline drug, while significantly reducing its associated cardiac toxicity. By conjugating epirubicin with a specific peptide, the study seeks to address the limitations of epirubicin, such as cardiotoxic side effects and resistance, offering a potentially more effective and safer therapeutic option for treating certain types of solid tumors. Epirubicin, an anthracycline anticancer drug, disrupts DNA and RNA synthesis by inserting itself into DNA strands. This insertion leads to DNA cleavage through topoisomerase II, resulting in cell death. However, its use is associated with serious and dose limiting adverse reactions, including cardiotoxicity. Furthermore, there is a growing concern about resistance to epirubicin via different mechanisms, such as reducing its uptake by the targeted neoplastic cells. Previous studies in our laboratory have indicated that conjugating doxorubicin (Dox), a prototype anthracycline chemotherapeutic agent, to cyclic peptides containing tryptophan (W) and arginine (R), such as [W5R4K], enhanced its anti-cancer activity, while reducing cardiac toxicity.

Methods: We conjugated epirubicin with a specific peptide [W(RW)₄KA] that we have designed, to form a peptide-epirubicin conjugate. This conjugate was evaluated for its anti-cancer activity using antiproliferative assays across various cancer cell lines. The cellular uptake and uptake mechanism were evaluated using confocal microscopy and flow cytometry in the absence and presence of endocytosis inhibitors.

Results: At a concentration of 5 µM, the conjugate reduced the viability of ovarian adenocarcinoma (SK-OV-3) by 22% and breast cancer cell lines (MDA-MB-231 and MCF-7) by 20% and 43%, respectively (Figure 1). In the case of doxorubicin/epirubicin-resistant cell line (MES-SA), cell viability was reduced by 40%, which was significantly higher than that with the parent drug and the corresponding physical mixture at 72 h (Figure 2). At 10 μM concentration, the inhibition rates after 72 h for SK-OV-3, MDA-MB-231, MCF-7, and MES-SA were 80%, 77%, 76%, and 70%, respectively. Additionally, the conjugate at the concentration of 5 µM after 72 h incubation demonstrated significantly lower toxicity towards healthy cardiomyocytes (H9C2), maintaining cell viability of approximately 100%, compared to 58% cell viability observed for free epirubicin (without peptide conjugation)(Figure 3). W(RW)4KA-EPI conjugate at the concentration of 10 uM showed a time-dependent cytotoxicity effect compared to the free form of EPI except for the MDA-MB-23.

Conclusion: These promising findings suggest that peptide-epirubicin conjugate has the potential to be used as an effective therapeutic agent with minimal cardiotoxicity for certain solid tumors.

References: Nasrolahi Shirazi A, Tiwari R, Chhikara BS, Mandal D, Parang K. Design and biological evaluation of cell-penetrating peptide–doxorubicin conjugates as prodrugs. Molecular pharmaceutics. 2013;10(2):488-99.
Darwish S, Sadeghiani N, Fong S, Mozaffari S, Hamidi P, Withana T, Yang S, Tiwari RK, Parang K. Synthesis and antiproliferative activities of doxorubicin thiol conjugates and doxorubicin-SS-cyclic peptide. Eur J Med Chem. 2019 Jan 1;161:594-606. doi: 10.1016/j.ejmech.2018.10.042.
Mozaffari S, Salehi D, Mahdipoor P, Beuttler R, Tiwari R, Aliabadi HM, et al. Design and application of hybrid cyclic-linear peptide-doxorubicin conjugates as a strategy to overcome doxorubicin resistance and toxicity. European Journal of Medicinal Chemistry. 2021;226:113836.
Zoghebi K, Aliabadi HM, Tiwari RK, Parang K. Overcome Multi-Drug Resistance against Doxorubicin. 2022.

Acknowledgements: The authors are grateful to Dr. Ahmed Aftab and Chapman University School of Pharmacy Core Lab for their support.

Figure 1: Comparative Analysis of MCF-7 Cell Viability Post-Treatment with Epirubicin and Peptide-Conjugated Epirubicin. The chart displays the percentage of viable MCF-7 cells following exposure to Epirubicin (EPI) alone and in combination or conjugation of W(RW)4KA cyclic peptide (in various concentration) over a period of 24 hours (red) and 72 hours (gray). Non-treated (NT) and DMSO-treated cells are included as controls. Error bars represent the standard deviation of triplicate samples.

Figure 2: Comparative Analysis of MES-SA Cell Viability Post-Treatment with Epirubicin and Peptide-Conjugated Epirubicin. The chart displays the percentage of viable MES-SA cells following exposure to Epirubicin (EPI) alone and in combination or conjugation of W(RW)4KA cyclic peptide (in various concentration) over a period of 24 hours (red) and 72 hours (gray). Non-treated (NT) and DMSO-treated cells are included as controls. Error bars represent the standard deviation of triplicate samples.

Figure 3: Comparative Analysis of H9c2 Cell Viability Post-Treatment with Epirubicin and Peptide-Conjugated Epirubicin. The chart displays the percentage of viable H9c2 cells following exposure to Epirubicin (EPI) alone and in combination or conjugation of W(RW)4KA cyclic peptide (in various concentration) over a period of 24 hours (red) and 72 hours (gray). Non-treated (NT) and DMSO-treated cells are included as controls. Error bars represent the standard deviation of triplicate samples.