Abstract
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Objectives Targeted therapy has become an effective strategy of precision medicine for better treatments of cancer. Based on the encouraging results from the therapies of lymphoma and breast cancer by antibody-drug conjugates (ADCs), here we describe a theranostic small-molecule drug conjugate (T-SMDC) design for prostate cancer therapy by targeting prostate specific membrane antigen (PSMA) for drug delivery and incorporating a positron emission tomography (PET) imaging component enabled by 68Ga (β+, t1/2 = 68 min) for concurrent noninvasive PET evaluation of the therapy.
Methods The design of T-SMDC, NO1A-DM1, consists of a 68Ga chelating moiety (NO1A) and a lysine-glutamate urea PSMA specific ligand conjugated to a polyethylene based di-amino linker, in which a cytotoxic DM1 drug is embedded via a disulfide bond, which is expected to render the conjugate with controlled DM1 release. The radiolabeling of NO1A-DM1 with 68Ga was optimized. The binding affinity of NO1A-DM1 was determined by a competitive binding assay in LNCaP cells. The cytotoxicity of NO1A-DM1 and the free DM1 drug was measured by the crystal violet method in three different prostate cancer cell lines (LNCaP, C4-2, and PC3) with distinct PSMA expression levels. The in vivo evaluation of the 68Ga-labeled T-SMDC was conducted by PET/CT imaging in mouse models bearing PSMA+ (PC3-PIP) and PSMA- (PC3-Flu) xenografts of prostate cancer.
Results In order to validate the T-SMDC design, the properties of each major component of NO1A-DM1 were evaluated independently. The conjugation chemistry only slightly altered the binding affinity of the lysine-glutamate urea ligand to PSMA (NO1A-DM1: IC50: 187 nM; free ligand: 96 nm). The 68Ga-labeled NO1A-DM1 displayed an appreciable level of internalization in a time-dependent manner, which allows the PSMA-targeted drug delivery and release within tumor cells. As expected, the anti-tumor activity of NO1A-DM1 was found increasing with the expression level of PSMA (p < 0.01). Compared to the free DM1 drug, T-SMDC was less toxic to all of the three prostate cancer cell lines. These toxicity data along with the specific PSMA binding, PSMA-mediated internalization, and controlled release mechanism of T-SMDC, demonstrate that the theranostic conjugate would have an alleviated toxicity profile in non-PSMA expressing tissues. Further, quantitative analysis of in vivo PET/CT images revealed that 68Ga-NO1A-DM1 had significantly higher uptake in PC3-PIP tumors (4.30 ± 0.20 %ID/g) than in PC3-Flu tumors (1.12 ± 0.42 %ID/g) at 1 h post-injection (p < 0.01). No significant accumulation of 68Ga-NO1A-DM1 was observed in most non-target organs except kidneys and bladder (urine) through which the conjugate was excreted. Conclusions: In summary, we have successfully synthesized a small molecular conjugate, NO1A-DM1. Preliminary data from the proof-of-concept experiments with the conjugate convincingly support our rational design of the T-SMDC system for theranostics of prostate cancer. Acknowledgement This study was partially supported by a grant from the National Institutes of Health (R01 CA159144), a Synergistic Idea Award from the Department of Defense (W81XWH-12-1-0336), the Simmons Cancer Center Grant (NIH 5P30 CA 142543), the CPRIT Grant RP110771, and the Dr. Jack Krohmer Professorship Funds. The PC3-PIP and PC3-Flu cell lines were kindly provided by Dr. Martin G. Pomper from John Hopkins University.