(M1030-06-36) Creating an Ultra-Long-Acting Parenteral Atovaquone Prodrug Nanosuspension for Malaria Chemoprophylaxis

Purpose: Despite recent advancements in therapeutics, malaria remains a life-threatening disease to the local communities as well as U.S. service members deployed to endemic regions and those travelling to such areas. The World Health Organization (WHO) reported 263 million malaria cases and 597,000 deaths globally in 2023, underscoring the continued threat posed by the disease [1]. While existing antimalarial drugs effectively prevent malaria infections, their efficacy is limited by the need for daily oral administration and the risk of developing drug resistance [2]. Vaccine development is inherently challenging and limited by the parasite's unique mechanisms of immune evasion [3]. Therefore, there is a significant unmet medical need for long-acting (LA) malaria chemoprophylaxis therapies to address existing challenges, particularly in high-transmission settings. Notably, a recent survey in such regions indicates a strong preference for LA injectable formulations over existing oral therapies and other methods of antimalarial administration [4]. Atovaquone (ATQ) is a promising candidate for LA therapy due to its high barrier to resistance, broad activity against both liver and erythrocytic stage Plasmodium parasites, and minimal drug-drug interactions [5]. We hypothesize that scalable, reversible prodrug modifications of ATQ could potentially alter the drug’s physicochemical properties and improve upon its pharmacokinetics, safety, and efficacy profiles. This strategy aims to develop an extended-duration injectable formulation for malaria chemoprevention and to simplify dosing regimens.

Methods: The prodrugs (M2ATQ, M3ATQ, and M4ATQ) were synthesized through esterification of ATQ. The synthesized prodrugs were nanoformulated using high-pressure homogenization to produce aqueous solid drug nanosuspensions stabilized by non-ionic surfactants (NM2ATQ, NM3ATQ, NM4ATQ). The parent drug formulation (NATQ) was used as a control. The nanoformulations were characterized for physicochemical properties by nuclear magnetic resonance (NMR), fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). Stability of nanoformulations was monitored over seven months using dynamic light scattering (DLS). These formulations were then tested for cytotoxicity in monocyte-derived macrophages (MDMs). Cellular uptake and retention were evaluated by measuring intracellular drug levels using HPLC over 24 hours and 21 days, respectively. Intracellular localization of the nanocrystals after 24 hours of treatment was further visualized using transmission electron microscopy (TEM). Pharmacokinetic (PK) profiles of the prodrug nanoformulations were evaluated in Sprague Dawley (SD) rats following a single intramuscular (IM) injection of 90 mg ATQ-equivalent/kg. Based on the PK results, the lead candidate (NM2ATQ) was further tested at a reduced dose of 20 mg/kg and compared to NATQ.

Results: Chemical modification of ATQ enhanced its hydrophobicity and lipophilicity. The produced nanoformulations remained stable for at least seven months, as determined by size, homogeneity, and surface charge (Fig. 1). In vitro cell vitality assessments showed that the ATQ prodrug nanoformulations were non-toxic to MDMs across a wide concentration range (1.95 μM to 500 μM) and demonstrated significantly improved intracellular drug delivery and retention compared to the parent compound (NATQ) at 50 μM (Fig. 2). Notably, a single IM injection of the lead ATQ prodrug formulation (NM2ATQ) sustained ATQ concentrations above therapeutic concentration (200 ng/ml) [5] for a year at a 90 mg/kg dose and four months at a 20 mg/kg dose (Fig. 3). The formulation exhibited a crystalline morphology, was well tolerated at the injection site, and did not show any adverse effects.

Conclusion: ATQ prodrugs exhibited distinct physicochemical properties from the parent drug. The prodrug nanosuspensions facilitated enhanced drug delivery and retention in macrophages in vitro compared to NATQ. A single intramuscular injection of the lead NM2ATQ nanosuspension at 90 mg and 20 mg ATQ eq./kg exhibited monthslong sustained plasma ATQ levels above the effective concentration in rats. NM2ATQ nanosuspension improved the PK profile of ATQ, demonstrating the potential for extended dosing intervals of every four months to once a year, tailored to the target population for malaria chemoprophylaxis.

References: 1. Venkatesan, P., WHO world malaria report 2024. The Lancet Microbe, 2025.
2. Hanboonkunupakarn, B. and N.J. White, The threat of antimalarial drug resistance. Tropical diseases, travel medicine and vaccines, 2016. 2: p. 1-5.
3. El-Moamly, A.A. and M.A. El-Sweify, Malaria vaccines: the 60-year journey of hope and final success—lessons learned and future prospects. Tropical Medicine and Health, 2023. 51(1): p. 29.
4. Scarsi, K.K., et al., Preferences of Patients and Providers in High-Burden Malaria Settings for Long-Acting Malaria Chemoprevention. The American journal of tropical medicine and hygiene, 2023. 109(4): p. 752.
5. Bakshi, R.P., et al., Long-acting injectable atovaquone nanomedicines for malaria prophylaxis. Nature communications, 2018. 9(1): p. 315.

Acknowledgements: This research is supported by the University of Nebraska Foundation, which includes donations Dr. Carol Swarts, Ronald and Christina Blumkin, the Community Pride of Nebraska Professorship.


Figure 1. Nanoformulation Stability. The stability of ATQ and prodrug nanoformulations was evaluated at 4ºC over 214 days following manufacture, as determined by (A) size (nm), (B) Polydispersity index (PDI), and (C) Zeta potential (mV).


Figure 2. Cellular Uptake and Retention. (A) Uptake and (B) Retention of prodrug formulations compared with NATQ at 50 μM in MDMs. Results are expressed as mean ± SEM where N = 3 biological replicates. (C) Transmission Electron Microscopy (TEM) images of MDMs at 24 hrs treated with 50 μM of NATQ, NM2ATQ, NM3ATQ or NM4ATQ. The uptake was seen for NM2ATQ, NM3ATQ and NM4ATQ (Red arrowheads), while NATQ showed minimal uptake.


Figure 3. ATQ prodrug nanoformulations PK testing. SD rats were administered a single intramuscular injection of 90 mg ATQ equivalent/kg of either NATQ, NM2ATQ, NM3ATQ or NM4ATQ (A). The lead (NM2ATQ) and parent (NATQ) formulations were further screened at single injections of 20 mg ATQ equivalent/kg (B). The dotted line indicates the effective ATQ plasma concentration of 200 ng/ml. Drug levels were determined by LC-MS/MS. Data are presented as mean ± SEM for N=5.