(T1330-01-04) Pre-Formulation Characterization and Stability Evaluation of a Novel Peptide-Based Drug for Triple-Negative Breast Cancer Treatment

Purpose: B4-007 is a novel Ephrin agonist peptide comprised of 15 amino acids. Discovered in Dr. Chun Li’s laboratory, this peptide has a high pico-molar binding affinity for EphB4, a membrane-bound tyrosine receptor overexpressed in many solid tumors including triple-negative breast cancer (TNBC). TNBC is a highly aggressive, heterogenous subtype, and when metastasized, the 5-year survival rate drops from over 90% to only 10-20%. EphB4 simultaneously activates EphB4-initiated Abl/Crk1 tumor suppressive signaling in tumor cells and hinders EFNB2-regulated angiogenic signaling. B4-007 is capable of activating this bi-directional signaling, making it a promising first-in-class therapy agent against TNBC. This study investigates known challenges faced by peptide-based drugs by evaluating the metabolic stability of B4-007 in a range of matrices including plasma and cell culture media in order to optimize chemical structure modifications and predict in vivo disposition of the peptide.

Methods: B4-007 was tested in the following matrices: mouse plasma, human plasma, human cell line (A2780) with fetal bovine serum (FBS), non-cell media without FBS, and cell-conditioned media without FBS. Prepared triplicates of each were incubated in a 37⁰C water bath with a beginning B4-007 concentration of 10 μg/mL. Serial samples were collected at multiple time points up to 24 hours. Protein precipitation with acetonitrile was used to extract B4-007 from the collected samples. Quantification of B4-007 concentrations was conducted on a SCIEX 4000 LC-MS/MS System with chromatographic separation performed by a Kinetic HILIC column (50 x 2.1 mm, 2.6 µm, 100 Å). Using gradient elution and a stable isotope of L-Proline as the internal standard (IS), the mobile phases consisted of 0.25% formic acid in water and 0.25% formic acid in acetonitrile. In positive ionization mode, the Multiple Reaction Monitoring transitions were at m/z 854à1215 for B4-007 and m/z 122à75 for IS. Metabolite identification was conducted on SCIEX X500B accurate LC-MS/MS instrument.

Results: A variation in degradation patterns was observed between mouse and human plasma, indicating potential differences in species-specific enzymatic activity. In the 24-hour stability study, B4-007 degraded more rapidly in mouse plasma with an initial half-life of 1.3 hours. The concentration declined quickly, leaving 20% of B4-007 after 6 hours and 4% after 22 hours. In contrast, B4-007 was more stable in human plasma, revealing slower degradation with an initial half-life of 29.7 hours. After 6 hours, about 80% of B4-007 remained, and by 22 hours, 65% remained. Additionally, for the three cell culture media, the presence of cellular byproducts and FBS correlated with increased degradation of B4-007, suggesting the potential influence of enzymatic or protein factors. In 6-hour stability studies, B4-007 demonstrated the greatest stability in non-cell media without FBS (~95% remaining), moderate stability in cell-conditioned media without FBS (~68% remaining), and the least stability in A2780 media with FBS (~60% remaining). Among the three cell culture media, B4-007 exhibited greater stability in non-cell media without FBS than that of human plasma. Then using SCIEX X500B accurate LC-MS/MS instrument, two major metabolites (M1 and M2) were predicted and identified for B4-007 in mouse plasma. From the in vitro stability studies, M1 and M2 were detected in mouse plasma, while only M1 was detected in human plasma.

Conclusion: These studies provide insights into the stability of B4-007 across various matrices and establish initial degradation profiles. Together, these findings emphasize the need to evaluate multiple biological environments when assessing peptide drug stability, highlighting the influence of systemic and extracellular environments on degradation. These preliminary results will support the next steps of this project, which include investigating key peptidases contributing to degradation and exploring the use of targeted inhibitors. Moreover, this knowledge can be used to guide the development of metabolically stable analogs, synthesized by our collaborators, for the selection of candidates for further screening and optimizing this peptide’s potential as a therapeutic agent for TNBC.

Acknowledgements: This study was funded in part by Cancer Prevention & Research Institute of Texas (CPRIT) Core Facilities Support Awards (RP240430, Liang), the NIH/NIMHD grant U54MD007605 (Xie, Liang), Emerson Collective (Li), and DoD Ovarian Cancer Research Program (HT9425-23-1-0500, Li).