(M1530-01-03) Targeted Cell-Penetrating Peptide-Drug Conjugates for the Treatment of Pancreatic Cancer

Purpose: Pancreatic cancer (PC) is one of the deadliest and most difficult cancers to treat. PC is projected to become the second leading cause of cancer-related death by the year 2030.  Recently, efforts have been geared towards developing targeted therapeutics such as peptide-drug conjugates (PDCs) that aim to facilitate specificity, improve drug delivery to difficult-to-reach solid tumors, and increase overall patient survival. Their small size of PDCs enables enhanced penetration of both primary and metastatic tumor sites making it a suitable approach for difficult-to-penetrate desmoplastic PC. We have synthesized and characterized an iRGD-functionalized PDC using emetine as the model drug. Our PDC consisted of a protease-cleavable linker (GFLG), a cell-penetrating peptide (iRGD), and a cytotoxic agent (emetine). GFLG is a cathepsin-B cleavable substrate overexpressed in PC and is extensively recognized for facilitating targeted drug delivery within the tumor microenvironment. GFLG will ensure the conjugate remains inactive until it reaches the tumor microenvironment where enzymatic cleavage occurs in the presence of cathepsin B. iRGD has been reported to improve vascular and tissue penetration more effectively in a tumor-specific and neuropilin-1-dependent fashion. Previous reports have proven emetine to be a highly potent cytotoxic agent. Thus, our conjugate is hypothesized to ensure targeted delivery of emetine and reduce exposure to healthy tissues.

Methods: The targeted conjugate iRGD-GFLG-Eme was synthesized via a revised Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) method for the end-to-end joining. A modified cell-penetrating peptide; azide-terminated cyclic iRGD was synthesized by solid-phase peptide synthesis (SPPS) and conjugated to the emetine-bearing linker. Analytical HPLC and high-resolution mass spectroscopy confirmed the synthesis of the compounds. The in vitro release profile of emetine from the conjugate in the presence of exogenous cathepsin B at pH 6.0, 37 °C was performed and analyzed by analytical HPLC. The stability of the conjugate in phosphate buffer saline at pH 7.4 was evaluated.

Results: The iRGD-GFLG-Eme conjugate synthesis was confirmed by mass spectrometry (m/2:1405.48; calculated exact mass: 1404.65). In the presence of cathepsin B, the in vitro release profile of the conjugates showed that approximately 50% and 80% of the free drug was released at 8 hours and 24 hours, respectively. For the control group in the absence of exogenous cathepsin B, no cleavage was observed in the absence of cathepsin B. These findings highlighted the requirement of cathepsin B for the specific release of the drug. For the stability studies, no degradation of the conjugate was observed for 48 h at 37 °C (pH 7.4) as determined by ESI-MS and analytical HPLC. Additionally, there was no change in the pattern and retention time (5.53 min) of the conjugate peak across various time intervals when analyzed by HPLC, suggesting that the conjugate would be stable in the blood

Conclusion: A targeted peptide conjugate was synthesized and characterized to treat pancreatic cancer. Our preliminary findings showed that the conjugate may offer significant therapeutic advantages in PC in vitro and in vivo models. iRGD peptide will ensure tumor penetration of the conjugate while specific cathepsin B cleavage of the conjugate will allow selective drug release within the tumor microenvironment.


Cathepsin-B mediated release of emetine from the conjugate iRGD-GFLG-Eme at pH 6.0, 37 °C. Conjugate cleavage in the presence of cathepsin-B (blue line), the appearance of emetine (orange line), the control conjugate without exogenous cathepsin-B (grey line) and the appearance of emetine in the absence of cathepsin-B (yellow line)


Stability studies data showing the area plot versus time of iRGD-GFLG-Eme (blue line) in PBS (pH 7.4) and appearance of free emetine (orange line).