Date of Award

Summer 8-2024

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Pharmaceutical Sciences

First Advisor

Dr. Keykavous Parang

Second Advisor

Dr. Rakesh Tiwari

Third Advisor

Dr. Aftab Ahmed

Abstract

Despite the introduction of molecularly targeted drugs, chemotherapy remains at the frontline of cancer therapy. However, multi-drug resistance and the non-specific distribution of anticancer drugs to healthy cells pose challenges that limit the clinical application of these drugs.

Epirubicin (EPI) is a topoisomerase II Inhibitor and an antineoplastic agent used to treat acute myeloid leukemias, multiple sclerosis (relapsing or secondary progressive), and prostate cancer. However, this agent causes myocardial toxicity and potentially fatal heart failure (HF). It is also important to note that this side effect increases with cumulative dosing. Cardiotoxicity may occur during therapy or may be delayed (months or years after completion of treatment). Furthermore, enhanced resistance to EPI has been shown to reduce intracellular drug accumulation and activity.

We have previously shown that conjugation of doxorubicin (DOX), another topoisomerase inhibitor that is also known for its cardiotoxicity and development of resistance, with cyclic peptides containing tryptophan (W) and arginine (R) residues showed comparable or higher activity against cancer cells and reduced the toxicity to heart cells when compared to free DOX. [(WR)4WK]bA-DOX conjugate exhibited enhanced uptake, retention, and sustained intracellular hydrolysis of DOX. The objective of this work was to conjugate EPI to cyclic peptide [(WR)4WK]bA by a linker to enhance drug delivery and efficacy against cancer cells and drug-resistant cells and diminish cardiotoxicity. We hypothesized that an optimized peptide-EPI conjugate would have potential applications for selective delivery to different cancer cells as a molecular transporter of EPI.

The cyclic peptide was synthesized through Fmoc solid-phase peptide synthesis, followed by cyclization in the solution phase. Since EPI has one free amine group, it was protected with Fmoc as a protecting group before conjugation with [(WR)4WK]bA through a glutarate linker.

The cytotoxicity of the conjugate, physical mixture, and EPI alone were compared at different concentrations in ovarian adenocarcinoma (SK-OV-3), breast adenocarcinoma (MCF-7) triple-negative breast (MDA-MB-231), DOX/EPI-resistant uterine sarcoma (MES-SA/MX2), and normal rat cardiomyoblast (H9C2) cells. The conjugate (5 µM) exhibited significantly higher antiproliferative activity against breast cancer cells and DOX/EPI-resistant cells and less antiproliferative activity against SK-OV-3 cells, while it showed minimal cytotoxicity against heart cells after 24 and 72 h incubation when compared to EPI (5 µM).

Flow cytometry and confocal microscopy were used to evaluate the cellular uptake of the conjugate in MDA-MB-231, MES-SA/MX2, and H9C2 cells compared to EPI. Confocal microscopy images revealed that at a concentration of 5 µM, the conjugate exhibited enhanced nuclear uptake compared to EPI in MDA-MB-231 and MES-SA/MX cells. Notably, there was no significant uptake in H9C2 cells after 24 h incubation. The cellular uptake was not significantly changed in the presence and absence of endocytosis inhibitors in MDA-MB-231 and MES-SA/MX cells, suggesting an endocytosis-independent cell entry.

Overall, the data suggest that the [W(RW)4K]βA-EPI conjugate offers a potential therapeutic advantage over free EPI and physical mixtures by improving efficacy, reducing cardiotoxicity, and exhibiting selective targeting to cancer cells. These findings highlight the promise of peptide-drug conjugates as a novel approach for enhancing cancer therapy effectiveness and reducing side effects.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

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