A PSMA-targeted theranostic agent for photodynamic therapy

Abstract

Prostate-specific membrane antigen (PSMA) is over-expressed in the epithelium of prostate cancer and in the neovasculature of many non-prostate solid tumors. PSMA has been increasingly used as a target for cancer imaging and therapy. Here we describe a low-molecular-weight theranostic photosensitizer, YC-9, for PSMA-targeted optical imaging and photodynamic therapy (PDT). YC-9 was synthesized by conjugating IRDye700DX N-hydroxysuccinimide (NHS) ester with a PSMA targeting Lys-Glu urea through a lysine-suberate linker in suitable yield. Optical imaging in vivo demonstrated PSMA-specific tumor uptake of YC-9 with rapid clearance from non-target tissues. PSMA-specific cell kill was demonstrated with YC-9in vitro through PDT in PSMA⁺ PC3-PIP and PSMA^- PC3-flu cells. In vivo PDT in mice bearing PSMA⁺ PC3-PIP tumors at 4h post-injection of YC-9 (A total of four PDT sessions were performed, 48h apart) resulted in significant tumor growth delay, while tumors in control groups continued to grow. PDT with YC-9 significantly increased the median survival of the PSMA⁺ PC3-PIP tumor mice (56.5days) compared to control groups [23.5-30.0days, including untreated, light alone, YC-9 alone (without light) and non-targeted IRDye700DX PDT treatment groups], without noticeable toxicity at the doses used. This study proves in principle that YC-9 is a promising therapeutic agent for targeted PDT of PSMA-expressing tissues, such as prostate tumors, and may also be useful against non-prostate tumors by virtue of neovascular PSMA expression.

Keywords: Molecular imaging; Optical imaging; PDT; Prostate cancer; Prostate-specific membrane antigen.

Figure 1. Synthesis of YC-9

Figure 2

YC-9 efficiently and specifically kills PSMA-expressing cells in vitro. PSMA⁺ PC3-PIP and PSMA^- PC3-flu cells were incubated with no dye, IRDye700DX (100 nM), and YC-9 (100 nM) for 1 h at 37°C. After washing twice with media, cells were either irradiated with 2J/cm^z of 690 nm NIR light or were not irradiated. Cell kill was measured by XTT assay 24 hr post irradiation. Error bars represents standard deviation (n = 4). Student's t-test demonstrated a significant difference in cell kill only for the group treated with Light+YC-9, NS: not significant.

Figure 3

Top row: Images after administration of 10 nmol of YC-9 at 6 and 24 h post-injection, as well as images of the excised organs at 24 h post-injection. Bottom row: Image after administration of 10 nmol of YC-9 and 1 mmol of DCIBzL, a high-affinity ligand forPSMA (blocker) at the same time points as above. Note lack of uptake in the mouse with DCIBzL, indicating binding specificity.

Figure 4. In vivo PDT study with YC-9

Mice with ∼50 mm³ PSMA⁺ PC3-PIP tumors were randomized into five treatment groups (n=6) : 1) YC-9 + light, 2) Light alone, 3) non-targeted IRDye700DX + light, 4) YC-9 without light and 5) untreated control without light. Four doses of YC-9 or non-targeted IRDye700DX (10 nmol each, in PBS) were injected intravenously, 48 h apart. Four hours after injection, the tumor surface of each mouse was superficially irradiated with an LED light source (wavelength: 690 ± 20 nm, total fluence: 100 J/cm²), while YC-9 without light and the untreated control group received no light treatment. (A) Tumor growth curves in different treatment groups. Statistical significance was calculated using one-way ANOVA. (B) Kaplan-Meier plot showing delayed tumor growth in the YC-9+ light treatment group. Tumor growth progression was plotted as number of days taken for a tumor to reach a ten-fold increase of the initial tumor volume. (C) Median time to reach a ten-fold increase of the initial tumor size in different treatment groups, derived from the Kaplan-Meier plot. (D) Mouse body weights are shown as a percentage of initial pretreatment weights. Four red arrows on the X-axis indicate the start of PDT treatment (**, P < 0.01; ***, P < 0.001).

Figure 5. Histological assessment of tumor sections after PDT treatment

(A) PSMA⁺ PC3-PIP untreated control tumor and one or four doses of YC-9 PDT-treated tumor sections were stained with anti-PSMA, (B) anti-Ki-67, (C) anti-cleaved caspase-3 and (D) H&E. All images were acquired at 40× magnification. Scale bars represent 50 μm for all panels. The graphs at the right most column describe quantification of the corresponding immunohistochemical staining. PDT-treated PSMA⁺ PC3-PIP tumor showed reduced PSMA expression, increased cell death (H&E and anti-caspase-3) and lower cellular proliferation (Ki-67) compared to control tumor (**, P < 0.01; ***, P < 0.001).