A low molecular weight multifunctional theranostic molecule for the treatment of prostate cancer

Abstract

Rationale: Although surgery and radiation therapy in patients with low risk prostate cancer appear appropriate and effective, those with high-risk localized disease almost always become hormone refractory and then rapidly progress. A new treatment strategy is urgently needed for patients with high-risk localized prostate cancer, particularly an approach that combines two drugs with different mechanisms. Combinations of photodynamic therapy (PDT) and chemotherapy have shown synergistic effects in clinical trials, but are limited by off-target toxicity. Prostate specific membrane antigen (PSMA) is a well-established biomarker for prostate cancer. Here we describe the use of a PSMA ligand to selectively and simultaneously deliver a potent microtubule inhibiting agent, monomethyl auristatin E (MMAE), and a PDT agent, IR700, to prostate cancers. Methods: Using a bifunctional PSMA ligand PSMA-1-Cys-C6-Lys, we created a novel theranostic molecule PSMA-1-MMAE-IR700. The molecule was tested in vitro and in vivo for selectivity and antitumor activity studies. Results: PSMA-1-MMAE-IR700 showed selective and specific uptake in PSMA-positive PC3pip cells, but not in PSMA-negative PC3flu cells both in vitro and in vivo. In in vitro cytotoxicity studies, when exposed to 690 nm light, PSMA-1-MMAE-IR700 demonstrated a synergistic effect leading to greater cytotoxicity for PC3pip cells when compared to PSMA-1-IR700 with light irradiation or PSMA-1-MMAE-IR700 without light irradiation. In vivo antitumor activity studies further showed that PSMA-1-MMAE-IR700 with light irradiation significantly inhibited PC3pip tumor growth and prolonged survival time as compared to mice receiving an equimolar amount of PSMA-1-IR700 with light irradiation or PSMA-1-IR700-MMAE without light irradiation. Conclusion: We have synthesized a new multifunctional theranostic molecule that combines imaging, chemotherapy, and PDT for therapy against PSMA-expressing cancer tissues. This work may provide a new treatment option for advanced prostate cancer.

Keywords: IR700; MMAE; PSMA; multifunctional molecule; prostate cancer.

Figure 1

Schematic illustration of mechnaism of the multifunctional theranostic molecule PSMA-1-MMAE-IR700. After administration, PSMA-1-MMAE-IR700 will selectivly bind to PSMA receptors on prostate cancer cells and enter cancer cells through receptor mediated endocytosis. The fluorescence signal emitted by IR700 can be used for diagnosis and image-guided surgery of prostate cancer by detecting residual cancer (A). Internalized PSMA-1-MMAE-IR700 will be delivered to lysosomes, the conjugate will be digested by cathepsins to generate free MMAE and PSMA-1-IR700. Delivered as a prodrug , protease released MMAE will exert its chemotherapeutic effect (B), while PSMA-1-IR700 will generated reactive oxygen species and deploy photodynamic therapy when illuminated by 690 nm light (C).

Figure 2

Characterization of PSMA-1-MMAE-IR700. (A) Structure of PSMA-1-MMAE-IR700. (B) Absorbance spectrum of PSMA-1-MMAE-IR700. (C) In vitro competition binding results of PSMA-1-MMAE-IR700. Values are mean ± SD of triplicates. (D) In vitro cathepsin cleavage of PSMA-1-MMAE-IR700. Values are mean ± SD of triplicates.

Figure 3

In vitro uptake studies of PSMA-1-MMAE-IR700 in PSMA-positive PC3pip and PSMA-negative PC3flu cells. Cells were incubated with 50 nM of PSMA-1-MMAE-IR700 for various times. Blocking experiments were performed by co-incubation of cells with 50 nM of PSMA-1-MMAE-IR700 and 100 × of PSMA-1 ligand. Nuclei were stained by DAPI and false colored blue. PSMA-1-MMAE-IR700 signal was false colored red. Images were taken at 40 ×. Scale bar = 50 μm. Representative images are shown from three independent studies.

Figure 4

In vitro cytotoxicity of PSMA-1-MMAE-IR700. (A) Dark cytotoxicity of PSMA-1-MMAE-IR700. Cells were incubated with drugs for 24 h in the dark, and then cell viability was determined. NA means IC₅₀ value is not available. Values are mean ± SD of six replicates. (B) Cytotoxicity of PSMA-1-MMAE-IR700 with 690 nm light treatment. Cells were incubated with 5 nM of drugs for 24 h in the dark. Drugs were then washed off and cell were exposed to 1 J/cm² or 3 J/cm² light. Cell viability was measured 24 h later. Values are mean ± SD of six replicates. (*: P < 0.0001, PSMA-1-MMAE-IR700 with 1 J/cm² light versus PSMA-1-MMAE-IR700 with no light, or PSMA-1-IR700 with 1 J/cm² light. $: P < 0.0001, PSMA-1-MMAE-IR700 with 1 J/cm² light to PC3pip cells versus that treatment to PC3flu cells. #: P < 0.0001, PSMA-1-MMAE-IR700 with 3 J/cm² light versus PSMA-1-MMAE-IR700 with no light, or PSMA-1-IR700 with 3 J/cm² light. &: P < 0.0001, PSMA-1-MMAE-IR700 with 3 J/cm² light to PC3pip cells versus that treatment to PC3flu cells).

Figure 5

In vivo fluorescence images of PSMA-1-MMAE-IR700. (A) In vivo Maestro imaging of a typical mouse bearing heterotopic PC3pip and PC3flu tumors treated with 100 nmol/kg of PSMA-1-MMAE-IR700 delivered through i.v. injection. Representative images are shown of n = 5. Selective uptake was observed in PC3pip tumors. (B) Quantification of fluorescent signal intensity in PC3pip and PC3flu tumors. Values are mean ± SD of 5 animals. (C) Ex vivo imaging of mouse organs at 48 h post injection. Fluorescence from PC3pip tumors was significantly higher than in other organs. Representative images are shown of n = 5. (D) Quantification of fluorescent signal intensity in tissues. Values are mean ± SD of 5 animals.

Figure 6

Detection of primary orthotopic prostate tumor and lymph node metastases by PSMA-1-MMAE-IR700. Representative images are shown from 3 animals. (A) In vivo and ex vivo fluorescence image of PSMA-1-MMAE-IR700 in mice bearing orthotopic PC3pipGFP tumor. Mice received 100 nmol of PSMA-1-MMAE-IR700 through tail vein injection. Images were taken at 1 h post injection. White arrow indicates lymph node, and red arrow indicates residual primary tumor. (B) Histological analysis of dissected primary tumor and lymph nodes. Presence of tumor cells was confirmed by H&E staining, GFP signal (false colored green), and IR700 signal from PSMA-1-MMAE-IR700 (false colored red). Nuclear stain, DAPI, is false colored blue. Scale bar = 100 μm in the lower panel. Black or white dashed lines indicate the borderline between normal tissue and cancer tissues. “T” is for tumor tissues; “N” is for normal prostate; and “L” is for lymphocytes.

Figure 7

In vivo antitumor activity of PSMA-1-MMAE-IR700 in mice bearing heterotopic PC3pip tumors. For survival experiments, mice received drugs through tail vein injection. PDT was performed at 1 hour post injection. Treatment was scheduled every 4 days with a total of five doses as indicated by the red arrows. Each group had 5 mice. For tumor growth curves and body weight curves, values are mean ± SD of 5 animals. The plots stopped when animals died during the experiments since values are represent as mean ± SD of 5 animals. (A) Tumor growth curves of mice. (B) Kaplan-Meier survival curves of treated mice. (*, P < 0.05, PSMA-1-MMAE-IR700+PDT versus the other 6 groups. Δ, P < 0.05, PSMA-1-MMAE-IR700 without PDT versus PBS, PSMA-1-IR700 without PDT and PSMA-1-IR700 + MMAE without PDT. #, P < 0.05, PSMA-1-IR700 with PDT versus PBS, PSMA-1-IR700 without PDT and PSMA-1-IR700 + MMAE without PDT; Ø, P < 0.05, PSMA-1-IR700 + MMAE with PDT versus PBS, PSMA-1-IR700 without PDT and PSMA-1-IR700 + MMAE without PDT). P values between different groups are summarized in Table S1. (C) Body weight changes of mice treated with PSMA-1-VcMMAE. (D) Induction of apoptosis by the treatment. Tumors were dissected at 4 days post treatment and examined by H&E staining and caspase-3 assay. DAPI is false colored blue and caspase-3 is false colored red. Scale bar = 100 μm. Pictures are representative images of five mice.