A Theranostic Small-Molecule Prodrug Conjugate for Neuroendocrine Prostate Cancer

Abstract

After androgen deprivation therapy, a significant number of prostate cancer cases progress with a therapy-resistant neuroendocrine phenotype (NEPC). This represents a challenge for diagnosis and treatment. Based on our previously reported design of theranostic small-molecule prodrug conjugates (T-SMPDCs), herein we report a T-SMPDC tailored for targeted positron emission tomography (PET) imaging and chemotherapy of NEPC. The T-SMPDC is built upon a triazine core (TZ) to present three functionalities: (1) a chelating moiety (DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) for PET imaging when labeled with ⁶⁸Ga (t_1/2 = 68 min) or other relevant radiometals; (2) an octreotide (Octr) that targets the somatostatin receptor 2 (SSTR2), which is overexpressed in the innervated tumor microenvironment (TME); and (3) fingolimod, FTY720-an antagonist of sphingosine kinase 1 that is an intracellular enzyme upregulated in NEPC. Polyethylene glycol (PEG) chains were incorporated via conventional conjugation methods or a click chemistry reaction forming a 1,4-disubstituted 1,2,3-triazole (Trz) linkage for the optimization of in vivo kinetics as necessary. The T-SMPDC, DOTA-PEG₃-TZ(PEG₄-Octr)-PEG₂-Trz-PEG₃-Val-Cit-pABOC-FTY720 (PEG_n: PEG with n repeating ethyleneoxy units (n = 2, 3, or 4); Val: valine; Cit: citrulline; pABOC: p-amino-benzyloxycarbonyl), showed selective SSTR2 binding and mediated internalization of the molecule in SSTR2 ^high cells. Release of FTY720 was observed when the T-SMPDC was exposed to cathepsin B, and the released FTY720 exerted cytotoxicity in cells. In vivo PET imaging showed significantly higher accumulation (2.1 ± 0.3 %ID/g; p = 0.02) of [⁶⁸Ga]Ga-DOTA-PEG₃-TZ(PEG₄-Octr)-PEG₂-Trz-PEG₃-Val-Cit-pABOC-FTY720 in SSTR2^high prostate cancer xenografts than in the SSTR2^low xenografts (1.5 ± 0.4 %ID/g) at 13 min post-injection (p.i.) with a rapid excretion through the kidneys. Taken together, these proof-of-concept results validate the design concept of the T-SMPDC, which may hold a great potential for targeted diagnosis and therapy of NEPC.

Keywords: controlled drug release; drug delivery; neuroendocrine prostate cancer; positron emission tomography (PET); prodrug conjugate; targeted therapy; theranostics; tumor innervation.

Figure 1

Design of a neuroendocrine prostate cancer (NEPC)-targeted chemotheranostic prodrug conjugate, DOTA-PEG₃-TZ(PEG₄-Octr)-PEG₂-Trz-PEG₃-Val-Cit-pABOC-FTY720. DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; PEG_n: polyethylene glycol chain with n repeating ethyleneoxy units (n = 2, 3, or 4); TZ: 1,3,5-triazine; Octr: octreotide; Trz: 1,4-disubstituted 1,2,3-triazole; Val: valine; Cit: citrulline; pABOC: p-amino-benzyloxycarbonyl; FTY720: fingolimod; T-SMPDC: theranostic small-molecule prodrug conjugate.

Scheme 1

Synthetic route to DOTA-PEG₃-TZ(PEG₄-Octr)-PEG₂-Trz-PEG₃-Val-Cit-pABOC-FTY720. (a) DOTA chelator and PEG₃ diamine linker condensation reaction. (b) Triazine core SN2 reaction with a propargyl-PEG₂-amine group. (c) Activation of the succinimidyl-ester- PEG₄ linker. (d) The FTY720 SN2 reaction with the cathepsin-B-cleavable linker followed by its addition to the triazine core through the propargyl-PEG₂ linker using click chemistry, the DOTA-PEG₃ (compound 2) SN2 reaction with compound 11, the octreotide-PEG₄ molecule substitution reaction with the triazine core, and hydrolysis of the tertbutyl groups from the DOTA chelator to yield the designed T-SMPDC, DOTA-PEG₃-TZ(PEG₄-Octr)-PEG₂-Trz-PEG₃-Val-Cit-pABOC-FTY720, compound 13. DIPEA: N,N-diisopropylethylamine; DMF: N,N-dimethylformamide; HBTU: (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; TFA: trifluoroacetic acid, THF: tetrahydrofuran.

Figure 2

In vitro cell assays of [⁶⁴Cu]Cu-DOTA-PEG₃-TZ(PEG₄-Octr)-PEG₂-Trz-PEG₃-Val-Cit-pABOC-FTY720: (a) Normalized SSTR2-selective uptake of [⁶⁴Cu]Cu-DOTA-PEG₃-TZ(PEG₄-Octr)-PEG₂-Trz-PEG₃-Val-Cit-pABOC-FTY720 (uptake in SSTR2^low cells was set at 1). (b) Internalization of [⁶⁴Cu]Cu-DOTA-PEG₃-TZ(PEG₄-Octr)-PEG₂-Trz-PEG₃-Val-Cit-pABOC-FTY720 in SSTR2^high AR42J cells. Data presented as average counts ± s.d. (n = 3).

Figure 3

Cytotoxicity studies using PC3 cells incubated with different concentrations of FTY720 and Val-Cit-pABOC-FTY720 after (a) 48 h and (b) 72 h of treatment. Data are presented as average counts ± s.d. (n = 3) (unpaired t-tests performed using GraphPad Prism 9.4.1).

Figure 4

(a) Representative PET/CT images of SCID/NOD mice bearing SSTR2^high PC3_VC and SSTR2^low PC3_sgPTP1B xenografts during a 60-min dynamic PET scan with [⁶⁸Ga]Ga-DOTA-PEG₃-TZ(PEG₄-Octr)-PEG₂-Trz-PEG₃-Val-Cit-pABOC-FTY720 (left) and [⁶⁸Ga]Ga-PSMA-11 (right). Yellow arrows indicate the tumors. (b) Quantitative PET imaging analysis in tissues of interest. (c) Western blot showing high expression of SSTR2 in PC3_VC but low expression in PC3_sgPTP1B (β-actin as a loading control). (d) IHC staining of the excised tumor sections to assess the presence of SSTR2 and PSMA in the tumors. Data are presented as the average %ID/g ± s.d. (n = 4) (unpaired t-tests performed by GraphPad Prism 9.4.1).