(W1030-05-25) Targeted Peptide Dual Drug Conjugate for the Treatment of Glioblastoma

Purpose: Glioblastoma is the most aggressive and fatal brain tumor. GBM patients survive for 12 to 18 months, and the disease has high recurrence rates. Current treatment approaches include chemotherapy and radiotherapy, but their effectiveness is limited by high toxicity, poor selectivity, and limited drug penetration and accumulation in brain tumors because of the barrier properties of the blood-brain barrier (BBB). To overcome these challenges, researchers have explored strategies involving spatial placement and temporal delivery of novel drug delivery systems. One such approach is the development of targeted peptide drug conjugates for selective drug delivery to the brain using targeting peptides possessing specific affinity for overexpressed cell surface receptors on the blood brain barrier and glioma cells. These targeting peptides are linked to chemotherapeutic agents via the targeted prodrug approach. Rucaparib was chosen as one of the drugs due to its efficacy in combination with DNA damaging agents like SN-38, however, rucaparib use in glioma treatment is currently not possible due to its inability to cross the BBB hindering its therapeutic efficacy. Thus, the peptide drug conjugate approach will aid the transport of rucaparib to the brain for improved accumulation in brain tumor cells.

Methods: A dual drug conjugate - T7-SN-38-Rucaparib - was synthesized and characterized to achieve tumor delivery of rucaparib and SN-38, with a view to improve therapeutic efficacy and reduction in adverse effects in the treatment of glioblastoma. Our approach involves leveraging the specific binding affinity of the HAIYPRH (T7) peptide for the transferrin receptor (TfR) on the blood-brain barrier and also overexpressed on glioma cells to improve accumulation of targeted chemotherapy to the brain tumor site. Intermediates and the T7-SN-38-Rucaparib conjugate were synthesized and characterized by proton nuclear magnetic resonance, mass spectrometry and analytical high performance liquid chromatography. Conjugate stability at physiological pH and cleavage studies in the presence of exogenous cathepsin B were evaluated. Evaluation of the cytotoxic potential of the dual drug conjugate was completed in U87MG cells and non-cancerous HEK 293 cells.

Results: The dual drug conjugate displayed high stability in buffer at physiological pH. Cleavage of the conjugate to release free drugs was observed in the presence of exogenous cathepsin B. T7-SN-38-Rucaparib showed cytotoxic effects in U87MG cells with an IC₅₀ value of 22.27 nM. Additionally, blocking studies was done to determine whether the observed cytotoxicity is facilitated by receptor mediated endocytosis after binding to the transferrin receptor. Blocking the TfR receptors on U87MG cells resulted in a notable inhibition of cytotoxicity of the conjugate which was reversed when exogeneous cathepsin B was added to the cells. Furthermore, the toxicity of the T7-SN-38-Rucaparib conjugate in HEK 293 cells reveal that the T7-SN-38-Rucaparib conjugate was toxic to non-cancerous cells at higher concentrations with IC₅₀ values of 115.78nM compared to cancerous U87MG cells. Thus, the conjugate is 5x more toxic to cancerous than non-cancerous cells. These allows the selective killing of cancer cells at lower IC₅₀ values.

Conclusion: Receptor mediated transcytosis after binding to the transferrin receptor will facilitate traversal across the blood-brain barrier, enabling preferential accumulation of drugs within the neoplastic microenvironment, followed by selective targeting of cancerous cells, thereby improving therapeutic efficacy, and minimizing toxicity. Our findings reveal the versatility and potential of utilizing transferrin receptor targeted conjugates to improve brain drug delivery in the treatment of glioblastoma.