Long-acting injectable atovaquone nanomedicines for malaria prophylaxis

Abstract

Chemoprophylaxis is currently the best available prevention from malaria, but its efficacy is compromised by non-adherence to medication. Here we develop a long-acting injectable formulation of atovaquone solid drug nanoparticles that confers long-lived prophylaxis against Plasmodium berghei ANKA malaria in C57BL/6 mice. Protection is obtained at plasma concentrations above 200 ng ml^-1 and is causal, attributable to drug activity against liver stage parasites. Parasites that appear after subtherapeutic doses remain atovaquone-sensitive. Pharmacokinetic-pharmacodynamic analysis indicates protection can translate to humans at clinically achievable and safe drug concentrations, potentially offering protection for at least 1 month after a single administration. These findings support the use of long-acting injectable formulations as a new approach for malaria prophylaxis in travellers and for malaria control in the field.

Fig. 1

Utility of long-acting antimalarial prophylaxis. Pharmacokinetic exposure to atovaquone monotherapy via oral administration blocks liver and erythrocytic stages of the parasite life cycle within the host (causal and suppressive activity, respectively). However, there is a vulnerability of oral dosing to non-adherence. The current work reports the preclinical development of an intramuscular long-acting nanomedicine, which provides sustained protection to parasite exposure in a preclinical model, expected to provide at least 1-month protection in humans

Fig. 2

Creation and screening of atovaquone nanoformulations. a Hydrophobic hydroxynaphthoquinone atovaquone was formulated to generate candidate ^ATQSDN nanoparticles. b ^ATQSDN library was screened for Z-average nanoparticle diameter after dispersion into water (0.5 mg mL⁻¹, 25 °C); 'hits' (solid bars) were selected for further evaluation. c Eleven ^ATQSDN 'hits' were evaluated using an in vitro rapid equilibrium dialysis intramuscular depot model assay (SIF) using ^ATQSDN formulation prepared with ³H-labelled atovaquone

Fig. 3

Efficacy testing of ^ATQSDN₇. a Experimental scheme. Mice dosed on day 0 with placebo, oral atovaquone or intramuscular nanoparticle atovaquone formulation were challenged once with intravenous P. berghei sporozoites at a given interval after dosing (28 days is depicted). For 42 days after challenge, blood samples were obtained and monitored for parasitemia (dots). b Prophylactic efficacy of intramuscular ^ATQSDN₇. Cohorts of mice treated on day 0 with indicated doses of ^ATQSDN₇ were challenged at a given interval after dosing (red arrows). Black lines, dose-to-challenge interval; grey lines, 42 days follow-up monitoring period. Prophylaxis was successful (circles) if, in two independent experiments (each with a cohort of 3–5 mice) all animals remained parasite-free for 42 days after challenge. Prophylaxis failed (x) if patent parasitemia was detected in any mouse in the cohort. For failed regimens the actual intervals between challenge and failure are not depicted. Not shown, all concurrent placebo recipients developed parasitemia, and all concurrent oral atovaquone controls failed challenge on or before 7 days after dosing. In all comparisons, successful prophylaxis with intramuscular ^ATQSDN₇ was superior to no-drug control at p ≤ 0.003 (Fisher’s exact test)

Fig. 4

Causal vs suppressive prophylactic antimalarial activity. On day 0 mice were injected with 36 mg kg^{−1 ATQ}SDN₇, then four paired cohorts (three mice per cohort) were challenged at the indicated times with 5000 P. berghei sporozoites (upward black arrows) or 150,000 infected erythrocytes (upward red arrows). In keeping with the 48 h duration of P. berghei ANKA liver stages in C57BL/6 mice, the challenges for each pair were staggered by 48 h. For all four pairs, the sporozoite-challenged mice (green bar with red arrowhead, to indicate liver then possible erythrocytic phases) remained parasite-free at 42 days after challenge (hatched). All blood stage-challenged mice (red arrows) and placebo controls (not depicted) developed parasitemia (solid)

Fig. 5

Pharmacokinetics and pharmacokinetic–pharmacodynamic relationship. a Plasma was collected at indicated intervals for assay of atovaquone concentrations in mice dosed intramuscularly with 200 mg kg^{−1 ATQ}SDN₇. Log-transformed concentrations yield a plasma half-life of 105 h (using 4–42 days values, inclusive; R², 0.985); data obtained in six independent experiments. Insert, At 14 days after intramuscular injection, plasma concentrations (y-axis, ng mL⁻¹) were linear with dose (x-axis, mg kg⁻¹). b Plasma atovaquone concentrations > 200 ng mL⁻¹ at the time of challenge correlate well with efficacy. Each dot represents a cohort of 3–5 mice, 7–42 days after a single intramuscular dose of 36, 50, 100 or 200 mg kg^{−1 ATQ}SDN₇. Data from six independent experiments

Fig. 6

Experimental scheme and results of resistance testing. Mice treated with 36 mg kg^{−1 ATQ}SDN₇ (n = 6) or no drug (n = 2) were challenged with P. berghei ANKA sporozoites. Erythrocytic parasites appeared in both cohorts, and were harvested and subinoculated into naive mice. At the same time a third cohort of naive mice was infected with atovaquone-resistant parasites, to serve as positive controls (n = 2). All animals were then treated orally once daily for 3 sequential days with 10 mg kg⁻¹ atovaquone, and monitored for parasitemia. Mice infected with parasites from the ^ATQSDN₇ prophylaxis failures, or from the no prophylaxis controls, remained parasite-free for 30 days after oral treatment (green bars). Mice infected with atovaquone-resistant P. berghei became parasitemic by 2 days after oral treatment and died (pink bars)