(W1130-01-04) Biodegradable Nanogels for Controlled Release of Artemisinin-Derived Drug to Treat Leukemia

Purpose: Acute myeloid leukemia (AML), the most common acute leukemia, affects both adults and children, with 20,050 new cases and 11,540 deaths in the United States in 2022. The 5-year survival rate is only 30%, even with new regimens that include targeted inhibitors of mutated AML driver molecules. Cases which harbor Mixed Lineage Leukemia Rearranged (MLLr) chromosomal aberrations have an especially poor prognosis. Due to AML’s constantly evolving cell/molecular heterogeneity, non-cross-resistant agents must be integrated, ideally based on MOA, into highly synergistic combination regimens to improve current unacceptable cure rates. Since >10% of patients die from treatment-related toxicity, optimal novel drug regimens must be more effective while causing less toxicity. With low/absent toxicity in worldwide use as the cornerstone of malaria treatment, ARTs are active against leukemias resistant to antineoplastic drugs. We developed potent and long-lasting ART analogs that synergize strongly with kinase inhibitors and BCL2 inhibitors. Indeed, combination regimens consisting of ART838 + venetoclax + a FLT3 inhibitor were tolerable and induced long, deep remissions with potential cures in xenograft models, most impressively in MOLM14 and MV4;11 MLLr AML models. We found that several 2-carbon linked dimeric artemisinin analogs (2C-ARTs: US9487538B2) effectively killed 9 human leukemia cell lines at half maximal inhibitory concentrations (IC₅₀) < 50 nM. However, many of these ARTs including ART631 have poor water solubility and shorter than desired half-life in vivo. The objective of this project is to develop an ART631-loaded biodegradable nanogel system (NanoART631, PCT/US2023/019974) with enhanced water solubility and sustained release of ART631, as a potential antileukemic.

Methods: Nanogels composed of poly(N-isopropylacrylamide) and dextran-poly(lactate-2-hydroxyethyl-methacrylate), developed previously in the Lowe lab, were used to load ART631, a 2C-ART. The structures of these ART631-containing nanogels (“NanoART631”) were confirmed using FTIR and NMR techniques; their encapsulation efficiencies were quantified by NMR; and their size and zeta potential were measured by dynamic light scattering (DLS), atomic force microscopy (AFM), and zeta potential techniques. In vitro cytotoxicity of nanogels, with and without ART631, against the MOLM14, MV4;11, and THP1 human MLLr AML cell lines, normal adult retinal pigment epithelium (ARPE-19) cells and dental pulp stem cells (DPSCs) was evaluated via MTT and WST1 in vitro cytotoxicity assays over 5 days. In vitro cellular uptake by MOLM14 and MV4;11 cells was determined by flow cytometry at different time points. In vitro ART631 release was determined by NMR. In vivo toxicology of the ART631-loaded nanogels was evaluated by intravenous (IV) injection of the nanogel-drug system into highly immunodeficient NSG mice.

Results: ART631 was successfully polymerized in the nanogel, up to > 15 wt% with encapsulation efficiencies >97%. The nanogels increased ART631’s water solubility ˃ 400-fold, from < 10 µg/mL for ART631 alone (without nanogel) to > 4 mg/mL for the NanoART631. The z-average diameter of NanoART631 was 100-200 nm in water at 37°C, with a polydispersity index < 0.2 and a 9 - 16 mV zeta potential. Single particle size was estimated to be 22- 50 nm. In vitro cytotoxicity assays determined that NanoART631 had IC₅₀ < 40 nM after two-day incubation with MOLM14, MV4;11, and THP1 cell lines and inhibited the proliferation of > 95% of the MOLM14 AML cells at < 30 nM after 5-day incubation. In contrast, NanoART631 was not toxic to ARPE-19 cells or DPSCs at a nanoparticle concentration of 1 mg/mL, which contained ~79 µM ART631. MOLM14 and MV4;11 cell took up >98% of NanoART631 after 24 hours of incubation. At 35 days, 68% - 84% of the initially loaded ART631 was released in vitro from NanoART631 formulations. The single-dose IV maximal tolerated dose (MTD) of NanoART631 in NSG mice (n=3) was 500 mg/kg of the nanoparticle (containing ~25 mg/kg ART631). Empty nanoparticles (without ART631) had no clinical toxicity to mice at concentrations ≥ 800 mg/kg IV doses.

Conclusion: We have developed a nanogel system that can significantly increase the effective water solubility of ART631 without destroying ART631’s activity against human AML cells. Nanogels with or without ART631 were not toxic to two nonmalignant cell lines and were well tolerated by NSG mice at a dose of 500 mg/kg IV. In contrast, NanoART631 was potent against 3 AML cell lines. Therefore, NanoART631 has potential as a novel active antileukemic agent that increased ART631’s water solubility and sustained its release.

References: 1. Posner, G.H. and Mott, B.T. (2014). Two-carbon linked artemisinin-derived trioxane dimers. U.S. Patent No. 9,487,538.
2. Kagan, A. B.; Moses, B. S.; Mott, B. T.; Rai, G.; Anders, N. M.; Rudek, M. A.; Civin, C. I. (2021), A Novel 2-Carbon-Linked Dimeric Artemisinin With Potent Antileukemic Activity and Favorable Pharmacology. Front Oncol
3. Lowe TL and Civin CI. Platform Nanoparticle Technology for Sustained Delivery of Hydrophobic Drugs. PCT Application PCT/US2023/019974. Filed on 4/26/23

Acknowledgements: Bryan T. Mott for the synthesis of ART631. Supported in part by Maryland’s Cancer Moonshot Initiative in Pediatric Cancer Research and the Frederick G. Smith, MS, DDS and Venice K. Paterakis, DDS Endowment Fund.

In vitro cytotoxicity of NanoART631 against MOLM14, MV4-11, and THP-1 leukemia cell lines using WST-1 proliferation assay