(T1130-05-30) Development of a Targeted Peptide-Drug Conjugate for the Treatment of Glioblastoma

Purpose: Glioblastoma is the deadliest and most common primary brain tumor with an incidence of approximately 10,000 Americans per year. Despite decades of research, survival for patients with glioblastoma is a modest 12 to 18 months while recurrence rates remain at about 90%. Chemotherapy and radiotherapy are the main treatment options for such brain tumors. However, the high toxicity, poor selectivity, and limited accumulation of therapeutic concentrations of anticancer agents in brain tumors remain challenges in the treatment of glioblastoma. To overcome these challenges, an effective drug delivery system for glioblastoma therapy should consist of a targeted drug delivery approach that is able to selectively deliver the drug to the tumor. Peptides with specific affinity for overexpressed cell-surface receptors are conjugated to chemotherapeutic agents to make peptide-drug conjugates (PDCs). Peptide T7 (sequence His-Ala-Ile-Tyr-Pro-Arg-His) is a heptapeptide that targets overexpressed transferrin receptors on the BBB and the glioblastoma cells. As such the conjugate of a chemotherapeutic agent such as SN-38 to T7 via a cathepsin B cleavable substrate will bind to the transferrin receptors and selectively cleave in the tumor where cathepsin B is overexpressed.

Methods: The T7 targeting peptide was synthesized using an Fmoc solid-phase method. The T7 peptide was terminated with azide-terminated polyethylene glycol. SN-38 as the toxic payload was conjugated to Valine-Alanine dipeptide as a cathepsin B cleavable substrate via an ether bond. The VA linker also serves as the mechanism of drug release and was terminated with a dibenzocyclooctyne group. The targeting peptide and the drug conjugate were coupled using Strain-Promoted Azide Alkyne Click Chemistry (SPAAC) reaction. Molecular docking studies using Glide SP-Peptide Docking was completed to evaluate the binding affinity of the drug conjugate compared to T7 peptide. In addition, the stability of the drug conjugate in pH 6.0 and 7.0 to simulate the pH of the tumor microenvironment and plasma was evaluated followed by in vitro release studies using exogenous cathepsin B. Cell culture experiments were conducted to evaluate the cytotoxicity and targeting efficiency of the T7-SN-38 in U-87 glioblastoma cell lines.

Results: The T7 peptide drug conjugate was successfully characterized using ¹H NMR, Fourier transform infrared spectroscopy (FT-IR), mass spectrometry and reversed-phase high-performance liquid chromatography (RP-HPLC). The successful conjugation of SN-38 to the T7 moiety via a cathepsin B linker was confirmed by mass spectrometry (2454.40g/mol). The molecular docking studies illustrate no significant difference between the binding of T7 and that of the T7 -SN-38 to the transferrin receptor. Cathepsin B-mediated release of drug from the conjugate showed over 90% of the conjugate cleaved in 24 hours. Mass analysis of the samples showed that SN-38 (m/z =393.47g/mol) was efficiently released from the T7-SN38- conjugate (m/2=1226.61g/mol) in the presence of cathepsin B. The release of free SN-38 by cathepsin B cleavage showed a rapid decrease in the area of the drug conjugate peak (RT=5.2minutes) with an appearance of peaks consistent with the retention time of the parent drug (RT=4.8minutes). The stability of the T7 targeted peptide drug conjugate was analyzed in sodium phosphate (PBS) buffer (pH. 7.4) to simulate the pH of the blood at 37°C for 24hours. There was no change in the pattern and retention time (RT=5.142minutes) of the peaks at different time points when analyzed by HPLC, indicating that the conjugate was stable and there was no decomposition or cleavage of the conjugate. To determine the efficacy of T7-SN-38 as a therapeutic, cell viability assay was conducted on U-87 cells and compared against free SN-38. Untreated cells served as controls. The IC₅₀ values of SN-38 and T7-SN-38 in U -87 cells were 26.41nM and 70.07nM respectively. The targeting efficacy of the drug conjugate was determined via blocking studies. U87-MG cells were pre-incubated with T7 followed by treatment with free drug and the T7-SN-38 drug conjugate. Our data shows that the efficacy of T7-SN-38 ( < 50% cell viability) was markedly higher in the cells that did not receive free T7 peptide, suggesting that the transferrin receptors are required for the uptake of the T7-SN-38 drug conjugate.

Conclusion: A T7-SN-38 peptide drug conjugate was successfully synthesed and characterized. In vitro studies suggest that the conjugate can bind to the transferrin receptors overexpressed on glioma cells and accumulate in the tumor environment where it is cleaved by cathepsin B to release the free drug. The peptide drug conjugate may provide a promising targeting chemotherapy strategy for glioblastoma.

Acknowledgements: Howard University