(T1430-12-77) Development of a Dual-Drug Conjugate for Site-Specific Delivery to Prostate Cancer

Purpose: Prostate cancer is the most frequently diagnosed cancer and the second leading cause of cancer deaths in American men. Although. localized prostate cancer is curable via surgery or local radiation, once metastatic, no curative therapy is available. Challenges to the success of treating advanced prostate cancer include the high toxicity, poor selectivity of anticancer agents, the adverse effects of current treatment strategies, the continuous generation of cancer cells by cancer stem cells and tumor heterogeneity. To overcome these challenges, an effective drug delivery system that targets cytotoxic agents specifically to sites of metastatic prostate cancer to improve therapeutic efficacy while decreasing systemic toxicity is essential.

Methods: In this study, we developed and characterized a dual-drug SN-38-cabazitaxel conjugate. This was done by synthesizing SN-38 and cabazitaxel drug conjugates intermediates using a prostate specific antigen (PSA) recognition peptide sequence, His-Ser-Ser-Lys-Leu-Glu (HSSKLQ) and leucine as a spacer. HSSKLQ terminated with DBCO at the N-terminus was synthesized and conjugated to SN-38 (DBCO-PEG₃-HSSKLQ//L-SN-38). Similarly, HSSKLQ terminated with azido polyethylene glycol functional group was synthesized and conjugated to cabazitaxel (Azide-PEG₄-HSSKLQ//L-cabazitaxel). The two conjugates intermediates were then linked together via SPAAC to form the SN-38-cabazitaxel conjugate.

Results: The conjugates and intermediates were characterized by proton nuclear magnetic resonance, mass spectrometry and analytical high performance liquid chromatography. The synthesis of the SN-38-Cabazitaxel conjugate was confirmed by ESI-MS 3593g/mol. Cleavage studies in the presence of exogenous PSA showed that at 2hours, over 75% of the conjugate had been cleaved with over 85% cleaved at 24hours to release the drug constructs L-SN38 and L-cabazitaxel. The cytotoxicity of the conjugate was tested in vitro in LNCaP cells (PSA expressing) and in PC-3 cells (non-PSA expressing). The IC₅₀ values obtained were 5.61nM in LNCaP cells and 31.2nM in PC-3 cells. The combination of SN-38 and Cabazitaxel also shows strong synergism with a combination index < 0.4.

Conclusion: Evaluation of the cytotoxic potential of the SN-38-Cabazitaxel conjugate show excellent cytotoxicity and exhibit similar cytotoxicity compared to the free drugs. The data confirms that the integrity of the drugs was not compromised by the conjugation process and that the free drugs were released when in culture with cells. These findings reveal the versatility and potential of utilizing peptide-drug conjugates to improve drug delivery. The novel approach of PSA targeted combination of SN-38 and cabazitaxel delivery via conjugate design and development showcases the potential of designing peptide-drug conjugates for targeted combination therapy.

Figure showing percent conjugate remaining to be cleaved in the presence of exogeneous PSA to release the free drugs.

Figure showing cell viability data showing comparison of the effect of SN38+cab, Leu-SN38+Leu-cab and the cabazitaxel-SN-38 conjugate on LNCaP cell line after 48-hour exposure to treatment (left) and after 72-hour exposure to treatment (right). Controls represent 0.05% DMSO in medium and growth medium only. Data represent mean ± S.D. (n=4).

Figure showing cell viability data showing comparison of the effect of SN-38+cab, Leu-SN38+Leu-cab and the cabazitaxel-SN-38 conjugate on PC-3 cell line after 48-hour exposure to treatment (left) and after 72-hour exposure to treatment (right). Controls represent 0.05% DMSO in medium and growth medium only. Data represent mean ± S.D. (n=4).