(M1330-07-42) Development of a Theranostic Agent for Potential Diagnosis and Treatment of High-Risk Neuroblastoma in Children

Purpose: Etoposide (VP-16), a topoisomerase II inhibitor, is a commonly used drug for the treatment of high-risk neuroblastoma. Due to low aqueous solubility, systemic delivery of etoposide is limited and thus requiring slow infusion and harmful solubilizers. Meta-iodobenzylguanidine (MIBG) is an analog of norepinephrine that specifically targets neuroendocrine cells including some neuroendocrine tumors. MIBG is taken up by the norepinephrine transporter and accumulates in the cytoplasm and mitochondria. Most neuroblastoma cells express the norepinephrine transporter. Unlike etoposide, MIBG is soluble in water. Iodine 123 (¹²³I) meta-iodobenzylguanidine plays an important role in the diagnostic workup of patients with neuroblastoma and is the first-line functional imaging agent for disease assessment. The goal of this work is to improve upon the aqueous solubility of etoposide by complexation with MIBG for additive antitumor effects and to ensure the resulting formulation does not antagonize the in vitro anti-cancer activities of etoposide. This work shows for the first time a proof of concept of the complexation of etoposide and MIBG for treatment, and potential diagnosis of neuroblastoma.

Methods: Firstly, a predetermined amount of MIBG was dissolved in 5 to 10 mL of aqueous solvent. Then, 5 to 10 mg of etoposide was added to the solution. The solution was then mixed at room temperature at 1000 rpm for 12 to 24 h. Finally, the solution was vacuum dried with a lab-scale speed vacuum dryer and stored at 4℃ for further analysis. The physicochemical properties of the complex formulation were analyzed by X-ray powder diffraction (P-XRD), and fourier transform infrared spectroscopy (FTIR). Size and charge analysis was performed using dynamic light scattering (DLS). VP-16 and VP-16/MIBG cytotoxicity were evaluated via cell proliferation assay (MTS), to assess the cellular uptake of drug and the antitumor activity. Triton-X 100 and serum free media was used as positive and negative control, respectively. To measure the apparent solubility, two milligrams of etoposide and VP-16/MIBG complex mixture were dissolved in 2 mL aqueous solution. The final concentration of both etoposide and VP-16/MIBG was measured using UV spectrometry. Decay rate was estimated using curve fitting modelling at varying concentrations of VP-16 alone and VP-16/MIBG. Statistical analysis was completed using multi-factorial ANOVA and an independent t-test for cell viability and solubility data, respectively. All statistical analysis was conducted using SPSS.

Results: The mean particle size and zeta potential measurements are 458.5 ± 0.417 nm and 0.951 ± 6.11 mV respectively. In the spectra of crystalline etoposide, a broad distinct peak at 3440 cm^-1 denotes the phenol OH stretch; while bands at 1610, 1500, and 1459 cm^-1 were assigned to the aromatic C=C stretching vibration. The peak at 1760 cm^-1 was thought to be indicative of the ester bond present in native etoposide. Similarly, the characteristic peak at 3400 cm^-1 corresponds to the N-H amine stretch, and 1350-1250 cm^-1 represents C-N aromatic stretches in MIBG spectra. P-XRD analysis was performed to elucidate the structural characterization and measure the crystalline nature of VP-16-MIBG complex formulation. the interaction between MIBG and etoposide can be seen to evidently alter the drug solid state from a crystalline structure to an amorphous salt. The solubility of native etoposide in aqueous solvent is 81.28 ± 1.14 µg/mL, whereas the formulation of VP-16-MIBG complex increased the solubility to 946.2 ± 3.30 µg/mL in 1:10 ratio of etoposide to MIBG. The solubility enhancement fold has been calculated to be ~12 fold of solubility increase. The MTS assay data revealed that treatment of etoposide alone at 50 µM had approximately 50% cell viability on BE2C neuroblast cell line. Similarly, when used alone as a treatment, MIBG at 50 µM had about the same cancer killing effect on BE2C cell line. However, when the cells were exposed to 50 µM of VP-16-MIBG complex, the antitumor activity observed was more than that of the native drug, with a cell viability of less than 20%. A statistically significant difference was found between the 3 drug treatments, at the 10 µM and 50 µM concentrations.

Conclusion: In this study, we investigated the ability of MIBG to improve the solubility of etoposide and enhance its anti-tumor activity. It was observed that MIBG improves the solubility of etoposide by approximately 12 folds compared to etoposide alone. P-XRD data indicated MIBG modified the solid state of the drug from crystalline to amorphous form. At a relatively moderate VP-16 + MIBG concentration (50 µM), the antitumor activity was higher than the VP-16 alone, probably due to a synergistic effect between etoposide and MIBG, and the complex of etoposide with MIBG increased the aqueous solubility of etoposide. Future studies include investigating the toxicity of combined VP-16 and MIBG, PK/PD analysis, in-vivo efficacy study and bioavailability of this new VP-16 and MIBG complex. Additional further studies will include labeling the optimized formulation with ¹²³I, for simultaneous diagnosis  and treatment of neuroblastoma.

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Acknowledgments: This study was supported by the University of Missouri-Kansas City Technology Jump and University of Missouri System Fast Track grants. Omowumi Akinjole is a recipient of The University of Missouri Kansas City Women’s Council 2021 and 2022 Graduate Assistant Fund (GAF).

Solubility fold of VP-16+MIBG complex compared to drug alone



Figure 2: Cell viability data of treatment with MIBG alone at varying concentrations (A); VP-16 alone, VP-16 + MIBG complex at varying concentrations (B) and Exponential decay models of cell viability fitted to (a) VP-16 data, (b) MIBG data, (c) combined VP-16 and MIBG data with the same concentrations, (d) combined VP-16 and MIBG data with different concentrations (C).



FTIR and P-XRD summary of VP-16, MIBG, and VP-16+MIBG formulation