Nanocarrier-Based Delivery of SN22 as a Tocopheryl Oxamate Prodrug Achieves Rapid Tumor Regression and Extends Survival in High-Risk Neuroblastoma Models

Abstract

Despite the use of intensive multimodality therapy, the majority of high-risk neuroblastoma (NB) patients do not survive. Without significant improvements in delivery strategies, anticancer agents used as a first-line treatment for high-risk tumors often fail to provide clinically meaningful results in the settings of disseminated, recurrent, or refractory disease. By enhancing pharmacological selectivity, favorably shifting biodistribution, strengthening tumor cell killing potency, and overcoming drug resistance, nanocarrier-mediated delivery of topoisomerase I inhibitors of the camptothecin family has the potential to dramatically improve treatment efficacy and minimize side effects. In this study, a structurally enhanced camptothecin analog, SN22, reversibly coupled with a redox-silent tocol derivative (tocopheryl oxamate) to allow its optimally stable encapsulation and controlled release from PEGylated sub-100 nm nanoparticles (NP), exhibited strong NB cell growth inhibitory activity, translating into rapid regression and durably suppressed regrowth of orthotopic, MYCN-amplified NB tumors. The robust antitumor effects and markedly extended survival achieved in preclinical models recapitulating different phases of high-risk disease (at diagnosis vs. at relapse with an acquired loss of p53 function after intensive multiagent chemotherapy) demonstrate remarkable potential of SN22 delivered in the form of a hydrolytically cleavable superhydrophobic prodrug encapsulated in biodegradable nanocarriers as an experimental strategy for treating refractory solid tumors in high-risk cancer patients.

Keywords: SN22; bioluminescent imaging; drug resistance; high-risk disease; mitocan; nanoparticle; neuroblastoma; orthotopic xenograft model; prodrug; topoisomerase I inhibitor.

Figure 1

Schematically shown molecular design of SN22-tocol prodrugs with varying hydrolytic labilities and in vitro characterization of their nanoparticle formulations: size distributions (A) and release kinetics determined using an external sink method (B). Data in (B) are presented as mean ± SD.

Figure 2

Spectral properties of NP/prodrug formulations and Förster Resonance Energy Transfer studies of NP disassembly. Changes in emission spectra (A,B) and N_FRET (C) as a function of the NP integrity status were simulated by combining NP co-labeled with donor and acceptor fluorophores with a 1:1 mixture of singly labeled NP at ratios representing respective disintegration levels. Disassembly of NP/prodrug formulations was monitored fluorimetrically in fetal bovine serum at 37 °C (D) based on energy transfer efficiency measurements and the N_FRET/NP integrity status correlation shown in (C). Data in (C,D) are presented as mean ± SD.

Figure 3

In vitro growth inhibition studies in cultured MYCN-amplified neuroblastoma (IMR-32) cells. The effect of NP loaded with SN22 prodrugs (5 ng/well, 24-h exposure) is shown in comparison to ‘no treatment’ or free SN22 and blank NP controls applied to cells at equivalent doses (A). The effect of prodrug-loaded NP on IMR-32 cell growth measured at 6 days post-treatment is shown as a function of the drug dose and exposure duration in comparison to blank NP (B). Data are presented as mean ± SD.

Figure 4

Therapeutic efficacy of the NP-encapsulated SN22-tocopheryl oxamate prodrug in an orthotopic xenograft model of the MYCN-amplified, newly diagnosed NB. Mice bearing xenografts established using luciferase-expressing IMR-32 cells were administered with one of five weekly NP doses equivalent to 10 mg SN22 per kg. Tumor growth was continuously monitored by bioluminescent imaging over the course of treatment and after treatment cessation. Data are presented as mean ± SD.

Figure 5

The growth inhibitory effect of SN22 on IMR-32 (A) and BE(2)C (B) MYCN-amplified NB cells derived pre-therapy and at relapse shown at 6 days post-treatment as a function of drug concentration and exposure duration. Cell growth was monitored by bioluminescence using untreated cells as a reference. The weaker response of the BE(2)C cell line in comparison to the chemo-naïve IMR-32 cells reflects a loss of chemosensitivity due to a mutation in the tumor suppressor protein p53 acquired following a course of intensive chemoradiotherapy. Data are presented as mean ± SD.

Figure 6

Tumor regression and survival extension in an orthotopic xenograft model of recurrent MYCN-amplified NB with acquired chemoresistance in response to the SN22-tocopheryl oxamate prodrug formulated in sub-100 nm PLA-PEG-based NP. Mice orthotopically inoculated with using luciferase-expressing BE(2)C cells were administered with four weekly doses of the NP/prodrug formulation equivalent to 10 mg SN22 per kg, and irinotecan administered twice a week at the equivalent dose (15 mg/kg) was included as a control (5 animals per group). Tumor growth quantitatively determined by bioluminescent imaging (A) is shown until the elimination of the first animal in the cohort. Event-free survival was monitored as another therapeutically relevant endpoint (B). Data in (A) Ftabare presented as mean ± SD.