Liver-targeted polymeric prodrugs delivered subcutaneously improve tafenoquine therapeutic window for malaria radical cure.

Approximately 3.3 billion people live with the threat of Plasmodium vivax malaria. Infection can result in liver-localized hypnozoites, which when reactivated cause relapsing malaria. This work demonstrates that an enzyme-cleavable polymeric prodrug of tafenoquine addresses key requirements for a mass administration, eradication campaign: excellent subcutaneous bioavailability, complete parasite control after a single dose, improved therapeutic window compared to the parent oral drug, and low cost of goods sold (COGS) at less than $1.50 per dose. Liver targeting and subcutaneous dosing resulted in improved liver:plasma exposure profiles, with increased efficacy and reduced glucose 6-phosphate dehydrogenase-dependent hemotoxicity in validated preclinical models. A COGS and manufacturability analysis demonstrated global scalability, affordability, and the ability to redesign this fully synthetic polymeric prodrug specifically to increase global equity and access. Together, this polymer prodrug platform is a candidate for evaluation in human patients and shows potential for P. vivax eradication campaigns.

Plasmodium falciparum Malaria Is Associated With Increased Kaposi Sarcoma-Associated Herpesvirus (KSHV) Seropositivity and Higher KSHV Antibody Breadth and Magnitude: Results of a Case-Control Study From Rural Uganda.

BACKGROUND: Previously, we showed that children with asymptomatic Plasmodium falciparum (Pf) malaria infection had higher Kaposi sarcoma-associated herpesvirus (KSHV) viral load, increased risk of KSHV seropositivity, and higher KSHV antibody levels. We hypothesize that clinical malaria has an even larger association with KSHV seropositivity. In the current study, we investigated the association between clinical malaria and KSHV seropositivity and antibody levels. METHODS: Between December 2020 and March 2022, sick children (aged 5-10 years) presenting at a clinic in Uganda were enrolled in a case-control study. Pf was detected using malaria rapid diagnostic tests (RDTs) and subsequently with quantitative real-time polymerase chain reaction (qPCR). Children with malaria were categorized into 2 groups: RDT+/PfPCR+ and RDT-/PfPCR+. RESULTS: The seropositivity of KSHV was 60% (47/78) among Pf-uninfected children, 79% (61/77) among children who were RDT-/PfPCR+ (odds ratio [OR], 2.41 [95% confidence interval {CI}, 1.15-5.02]), and 95% (141/149) in children who were RDT+/PfPCR+ (OR, 10.52 [95% CI, 4.17-26.58]; Ptrend < .001). Furthermore, RDT+/PfPCR+ children followed by RDT-/PfPCR+ children had higher KSHV IgG and IgM antibody levels and reacted to more KSHV antigens compared to uninfected children. CONCLUSIONS: Clinical malaria is associated with both increased KSHV seropositivity and antibody magnitude, suggesting that Pf is affecting KSHV immunity.

Mechanisms of 8-aminoquinoline induced haemolytic toxicity in a G6PDd humanized mouse model.

Primaquine (PQ) and Tafenoquine (TQ) are clinically important 8-aminoquinolines (8-AQ) used for radical cure treatment of P. vivax infection, known to target hepatic hypnozoites. 8-AQs can trigger haemolytic anaemia in individuals with glucose-6-phosphate dehydrogenase deficiency (G6PDd), yet the mechanisms of haemolytic toxicity remain unknown. To address this issue, we used a humanized mouse model known to predict haemolytic toxicity responses in G6PDd human red blood cells (huRBCs). To evaluate the markers of eryptosis, huRBCs were isolated from mice 24-48 h post-treatment and analysed for effects on phosphatidylserine (PS), intracellular reactive oxygen species (ROS) and autofluorescence. Urinalysis was performed to evaluate the occurrence of intravascular and extravascular haemolysis. Spleen and liver tissue harvested at 24 h and 5-7 days post-treatment were stained for the presence of CD169+ macrophages, F4/80+ macrophages, Ter119+ mouse RBCs, glycophorin A+ huRBCs and murine reticulocytes (muRetics). G6PDd-huRBCs from PQ/TQ treated mice showed increased markers for eryptosis as early as 24 h post-treatment. This coincided with an early rise in levels of muRetics. Urinalysis revealed concurrent intravascular and extravascular haemolysis in response to PQ/TQ. Splenic CD169+ macrophages, present in all groups at day 1 post-dosing were eliminated by days 5-7 in PQ/TQ treated mice only, while liver F4/80 macrophages and iron deposits increased. Collectively, our data suggest 8-AQ treated G6PDd-huRBCs have early physiological responses to treatment, including increased markers for eryptosis indicative of oxidative stress, resulting in extramedullary haematopoiesis and loss of splenic CD169+ macrophages, prompting the liver to act as the primary site of clearance.

Liver-targeted polymeric prodrugs of 8-aminoquinolines for malaria radical cure.

Primaquine and tafenoquine are the two 8-aminoquinoline (8-AQ) antimalarial drugs approved for malarial radical cure - the elimination of liver stage hypnozoites after infection with Plasmodium vivax. A single oral dose of tafenoquine leads to high efficacy against intra-hepatocyte hypnozoites after efficient first pass liver uptake and metabolism. Unfortunately, both drugs cause hemolytic anemia in G6PD-deficient humans. This toxicity prevents their mass administration without G6PD testing given the approximately 400 million G6PD deficient people across malarial endemic regions of the world. We hypothesized that liver-targeted delivery of 8-AQ prodrugs could maximize liver exposure and minimize erythrocyte exposure to increase their therapeutic window. Primaquine and tafenoquine were first synthesized as prodrug vinyl monomers with self-immolative hydrolytic linkers or cathepsin-cleavable valine-citrulline peptide linkers. RAFT polymerization was exploited to copolymerize these prodrug monomers with hepatocyte-targeting GalNAc monomers. Pharmacokinetic studies of released drugs after intravenous administration showed that the liver-to-plasma AUC ratios could be significantly improved, compared to parent drug administered orally. Single doses of the liver-targeted, enzyme-cleavable tafenoquine polymer were found to be as efficacious as an equivalent dose of the oral parent drug in the P. berghei causal prophylaxis model. They also elicited significantly milder hemotoxicity in the humanized NOD/SCID mouse model engrafted with red blood cells from G6PD deficient donors. The clinical application is envisioned as a single subcutaneous administration, and the lead tafenoquine polymer also showed excellent bioavailability and liver-to-blood ratios exceeding the IV administered polymer. The liver-targeted tafenoquine polymers warrant further development as a single-dose therapeutic via the subcutaneous route with the potential for broader patient administration without a requirement for G6PD diagnosis.