Encapsulation of OZ439 into Nanoparticles for Supersaturated Drug Release in Oral Malaria Therapy

Malaria poses a major burden on human health and is becoming increasingly difficult to treat due to the development of antimalarial drug resistance. The resistance issue is further exacerbated by a lack of patient adherence to multi-day dosing regimens. This situation motivates the development of new antimalarial treatments that are less susceptible to the development of resistance. We have applied Flash NanoPrecipitation (FNP), a polymer-directed self-assembly process, to form stable, water-dispersible nanoparticles (NPs) of 50–400 nm in size containing OZ439, a poorly orally bioavailable but promising candidate for single-dose malaria treatment developed by Medicines for Malaria Venture (MMV). During the FNP process, a hydrophobic OZ439 oleate ion paired complex was formed and was encapsulated into NPs. Lyophilization conditions for the NP suspension were optimized to produce a dry powder. The in vitro release rates of OZ439 encapsulated in this powder were determined in biorelevant media and compared with the release rates of the unencapsulated drug. The OZ439 NPs exhibit a sustained release profile and several-fold higher release concentrations compared to that of the unencapsulated drug. In addition, XRD suggests the drug was stabilized into an amorphous form within the NPs, which may explain the improvement in dissolution kinetics. Formulating OZ439 into NPs in this way may be an important step toward developing a single-dose oral malaria therapeutic, and offers the possibility of reducing the amount of drug required per patient, lowering delivery costs, and improving dosing compliance.


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The most stable of these stabilizer-free formulations was at a ratio of 1:4 OZ439:OA.
These nanoparticles did not show significant changes in size and maintained a narrow size distribution with only a small amount of precipitate evident at 28 hours ( Figure S2-A S2-B).
Although these NPs were stable enough to allow for further processing, zeta potential measurements showed that they possessed a highly-charged surface ( Figure S1-C). Particles with charged surfaces are prone to flocculation in environments with varying pH and high salt Figure S1: Characteristic DLS properties over time of OZ439 nanoparticles with varying molar ratios of OA and no stabilizer; size distribution at t=0 hours (A), average size as a function of molar ratio of OA to OZ439 (B), zeta potential distribution (C) and average zeta potential as a function of molar ratio of OA to OZ439 (D). (A) demonstrates that ratios of OZ439 to OA of 1:2, 1:4 and 1:8 initially formed nanoparticles, while 1:1 produced nanoparticles with some aggregates. OZ439 nanoparticles without OA immediately formed aggregates and are therefore not included (A). The size of the nanoparticles decreased with increasing OA; this could be the result of increased nucleation (B). All nanoparticle formulations had a negative surface charge (C). Increasing the molar ratio of OA led to more negative surface charges (D).

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concentrations, such as the GI tract; furthermore, high surface charge increases protein binding, which leads to faster clearance in vivo 12 . Thus such charge-stabilized NPs are less desirable than sterically-stabilized particles, which do not display such high stability dependence on pH and salt concentration. Figure S2: The average size and PDI over time of nanoparticles with varying molar ratios of OZ439 to OA in water (A&B), PBS (C&D) and FaSSGF (E&F). A ratio of OZ439 to OA of 1:4 was the most stable in water over 28 hours. A molar ratio of OZ439 to OA of 1:0 formed large aggregates immediately, while 1:2 was stable for 3 hours and 1:1 and 1:8 were stable for 6 hours. The high concentration of ions in PBS shielded the negative surface charge of the nanoparticles with 1:1 OZ439 to OA, leading to faster aggregation in PBS than water (C). The lower concentration of ions in gastric fluid did not lead to charge shielding and aggregation (E&F). The stability in gastric fluid could be the result of the lower solubility of OZ439 in FaSSGF of 9.8g/mL compared to 4.25mg/mL in water.   The molar ratio of OZ439:OA of 1:1 with HPMC 126 produced the most stable nanoparticles. The size distribution over time of 1:2 OZ439 to OA nanoparticles showed that although the size increased over 24 hours from an average size of 152  3nm to 348  2nm no aggregates above 1000nm appeared (B).
S6 Figure S5: Effect on nanoparticle stability of changing the ratio of OZ FB to VitE-S, with HPMC 126. The initial size distribution of each formulation tested, with ratios of OZ FB to VitE-S of 1:0.5, 1:1, 1:2 and 1:4 demonstrate that increasing concentrations of VitE-S led to larger nanoparticles (A). The size distribution over time for nanoparticles with 1:2 OZ FB to VitE-S and HPMC 126 shows the stability of the nanoparticles, at 24 hours there was a small fraction of nanoparticles greater than 1000nm (B). The average size over time, demonstrates the increase in size with increasing concentrations of VitE-S, as well as the stability of nanoparticles with OZ FB to VitE-S ratios of 1:1, 1:2 and 1:4 (C). Nanoparticles with a ratio of 1:0.5 swelled in size over 3 hours and reached a PDI of 0.47  0.18, while the PDI of the other formulations all remained below 0.2 over 24 hours (D). Together these figures demonstrate that OZ FB to VitE-S ratios of 1:1, 1:2 and 1:4 produced stable nanoparticles, while a ratio of 1:0.5 did not produce stable nanoparticles. Figure S6: OZ FB nanoparticles with HPMC 716 and varying molar ratios of OZ FB to VitE-S. The initial size distribution of each formulation tested, with ratios of OZ FB to VitE-S of 1:1, 1:2 and 1:4 demonstrates that increasing concentrations of VitE-S led to larger nanoparticles (A). The size distribution over time for nanoparticles with 1:2 OZ FB to VitE-S and HPMC 126 shows the stability of the nanoparticles; at 3 hours, there was a small fraction of nanoparticles greater than 1000nm (B). The average size over time, demonstrates the increase in size with increasing concentrations of VitE-S, as well as the stability of nanoparticles with OZ FB to VitE-S ratios of 1:2 and 1:4 (C). Nanoparticles with OZ FB to VitE-S ratio of 1:1 were stable for the first 6 hours, but there was a moderate increase in size at 24 hours and increase in PDI (C&D).
S8 Figure S7: The effect of species and amount of cryoprotectant on average size (A) and PDI (B) of re-dispersed lyophilized powder of 1:2 OZ439 to OA nanoparticles with the stabilizer HPMC 126. HPMC E3 was the most promising cryoprotectant, the correlogram (C) and size distribution (D) of the re-dispersed powders with varying mass ratios of nanoparticle to HPMC E3 are shown. For nanoparticles:cryoprotectant mass ratios above 1:1 the average size of the re-dispersed nanoparticles was relatively constant, at 195 2nm, 209 28nm and 227 5nm for sucrose, HPMC E3 and mannitol respectively (A). The PDI decreased with increasing amounts of cryoprotectant, indicating that higher levels of cryoprotectant protected nanoparticles against aggregation or instability in the lyophilization process (B). The correlogram demonstrates that for all levels of HPMC E3 cryoprotectant added the re-dispersed suspension primarily contained nanoparticles of a tight size distribution (C). Increasing the amount of HPMC E3 cryoprotectant generally reduced the level of aggregates present on re-dispersion, there was only a small tail of aggregates present in the size distribution at 1:1, 1:3 and 1:10 mass of nanoparticles to HPMC E3 (D). Figure S8: Release rates of lyophilized nanoparticles and unencapsulated OZ439. The powders were added to a total concentration of 140g/mL and incubated in either water (A), FaSSGF (B), FaSSIF (C) or FeSSIF (D). The concentration of free OZ439 was measured in the solution over 24 hours to indicate the release of OZ439 in different bio-media. In water and FaSSGF the maximum concentration achieved by both nanoparticles and unencapsulated powder was 45g/mL (A&B). The maximum concentration of OZ439 released from nanoparticles was 142 and 131g/mL in FaSSIF and FeSSIF respectively (C&D). In FaSSIF, the concentration of OZ439 in solution was on average 9 times higher from nanoparticles than unencapsulated powder and all OZ439 was released from the nanoparticles (C). The concentration after 24 hours approached 140g/mL for both the nanoparticles and unencapsulated powder in FeSSIF (D). Figure S9: Differential scanning calorimetry profiles for sodium oleate (green), OZ439 mesylate (yellow), a physical mixture of sodium oleate and OZ439 mesylate with masses corresponding to the 1:2 molar ratio used in the text (blue), isolated OZ439 oleate hydrophobic ion paired complex (red), and OZ439 free base (purple). The physical mixture contains elements of the individual component curves that are not present in the ion paired complex; for example, the slight peaks at 60°C and 140°C. The ion paired complex's profile contains a peak note present in any of the other profiles (80°C). The differences among the profiles suggest the formation of a hydrophobic ion paired complex between OZ439 cation and oleate anion.
DSC measurements were performed using a TA Instruments Q200 (New Castle, DE). 5-10 mg of sample was weighed into an aluminum pan and equilibrated at 20 °C under nitrogen (50 mL/min). Subsequently, the samples were heated from 20 to 250 °C at a heating rate of 5 °C/min. The scan was analyzed by TA Instruments Universal Analysis 2000 software.