Defining the next generation of severe malaria treatment: a target product profile.

BACKGROUND: Severe malaria is a life-threatening infection, particularly affecting children under the age of 5 years in Africa. Current treatment with parenteral artemisinin derivatives is highly efficacious. However, artemisinin partial resistance is widespread in Southeast Asia, resulting in delayed parasite clearance after therapy, and has emerged independently in South America, Oceania, and Africa. Hence, new treatments for severe malaria are needed, and it is prudent to define their characteristics now. This manuscript focuses on the target product profile (TPP) for new treatments for severe malaria. It also highlights preparedness when considering ways of protecting the utility of artemisinin-based therapies. TARGET PRODUCT PROFILE: Severe malaria treatments must be highly potent, with rapid onset of antiparasitic activity to clear the infection as quickly as possible to prevent complications. They should also have a low potential for drug resistance selection, given the high parasite burden in patients with severe malaria. Combination therapies are needed to deter resistance selection and dissemination. Partner drugs which are approved for uncomplicated malaria treatment would provide the most rapid development pathway for combinations, though new candidate molecules should be considered. Artemisinin combination approaches to severe malaria would extend the lifespan of current therapy, but ideally, completely novel, non-artemisinin-based combination therapies for severe malaria should be developed. These should be advanced to at least phase 2 clinical trials, enabling rapid progression to patient use should current treatment fail clinically. New drug combinations for severe malaria should be available as injectable formulations for rapid and effective treatment, or as rectal formulations for pre-referral intervention in resource-limited settings. CONCLUSION: Defining the TPP is a key step to align responses across the community to proactively address the potential for clinical failure of artesunate in severe malaria. In the shorter term, artemisinin-based combination therapies should be developed using approved or novel drugs. In the longer term, novel combination treatments should be pursued. Thus, this TPP aims to direct efforts to preserve the efficacy of existing treatments while improving care and outcomes for individuals affected by this life-threatening disease.

Lipid Compositions in Infant Formulas Affect the Solubilization of Antimalarial Drugs Artefenomel (OZ439) and Ferroquine during Digestion.

Recent studies have shown that the solubilization of two antimalarial drug candidates, artefenomel (OZ439) and ferroquine (FQ), designed to provide a single-dose combination therapy for uncomplicated malaria can be enhanced using milk as a lipid-based formulation. However, milk as an excipient faces significant quality and regulatory hurdles. We therefore have investigated infant formula as a potential alternative formulation approach. The significance of the lipid species present in a formula with different lipid compositions upon the solubilization of OZ439 and FQ during digestion has been investigated. Synchrotron small-angle X-ray scattering was used to measure the diffraction from a dispersed drug during digestion and thereby determine the extent of drug solubilization. High-performance liquid chromatography was used to quantify the amount of drug partitioned into the digested lipid phases. Our results show that both the lipid species and the amount of lipids administered were key determinants for the solubilization of OZ439, while the solubilization of FQ was independent of the lipid composition. Infant formulas could therefore be designed and used as milk substitutes to tailor the desired level of drug solubilization while circumventing the variability of components in naturally derived milk. The enhanced solubilization of OZ439 was achieved during the digestion of medium-chain triacylglycerols (MCT), indicating the potential applicability of MCT-fortified infant formula powder as a lipid-based formulation for the oral delivery of OZ439 and FQ.

Spray drying OZ439 nanoparticles to form stable, water-dispersible powders for oral malaria therapy.

BACKGROUND: OZ439 is a new chemical entity which is active against drug-resistant malaria and shows potential as a single-dose cure. However, development of an oral formulation with desired exposure has proved problematic, as OZ439 is poorly soluble (BCS Class II drug). In order to be feasible for low and middle income countries (LMICs), any process to create or formulate such a therapeutic must be inexpensive at scale, and the resulting formulation must survive without refrigeration even in hot, humid climates. We here demonstrate the scalability and stability of a nanoparticle (NP) formulation of OZ439. Previously, we applied a combination of hydrophobic ion pairing and Flash NanoPrecipitation (FNP) to formulate OZ439 NPs 150 nm in diameter using the inexpensive stabilizer hydroxypropyl methylcellulose acetate succinate (HPMCAS). Lyophilization was used to process the NPs into a dry form, and the powder's in vitro solubilization was over tenfold higher than unprocessed OZ439. METHODS: In this study, we optimize our previous formulation using a large-scale multi-inlet vortex mixer (MIVM). Spray drying is a more scalable and less expensive operation than lyophilization and is, therefore, optimized to produce dry powders. The spray dried powders are then subjected to a series of accelerated aging stability trials at high temperature and humidity conditions. RESULTS: The spray dried OZ439 powder's dissolution kinetics are superior to those of lyophilized NPs. The powder's OZ439 solubilization profile remains constant after 1 month in uncapped vials in an oven at 50 °C and 75% RH, and for 6 months in capped vials at 40 °C and 75% RH. In fasted-state intestinal fluid, spray dried NPs achieved 80-85% OZ439 dissolution, to a concentration of 430 µg/mL, within 3 h. In fed-state intestinal fluid, 95-100% OZ439 dissolution is achieved within 1 h, to a concentration of 535 µg/mL. X-ray powder diffraction and differential scanning calorimetry profiles similarly remain constant over these periods. CONCLUSIONS: The combined nanofabrication and drying process described herein, which utilizes two continuous unit operations that can be operated at scale, is an important step toward an industrially-relevant method of formulating the antimalarial OZ439 into a single-dose oral form with good stability against humidity and temperature.

Impact of Ferroquine on the Solubilization of Artefenomel (OZ439) during in Vitro Lipolysis in Milk and Implications for Oral Combination Therapy for Malaria.

Milk is an attractive lipid-based formulation for the delivery of poorly water-soluble drugs to pediatric populations. We recently observed that solubilization of artefenomel (OZ439) during in vitro intestinal lipolysis was driven by digestion of triglycerides in full-cream bovine milk, reflecting the ability of milk to act as an enabling formulation in the clinic. However, when OZ439 was co-administered with a second antimalarial drug, ferroquine (FQ) the exposure of OZ439 was reduced. The current study therefore aimed to understand the impact of the presence of FQ on the solubilization of OZ439 in milk during in vitro intestinal digestion. Synchrotron small-angle X-ray scattering was used for in situ monitoring of drug solubilization (inferred via decreases in the intensity of drug diffraction peaks) and polymorphic transformations that occurred during the course of digestion. Quantification of the amount of each drug solubilized over time and analysis of their distributions across the separated phases of digested milk were determined using high-performance liquid chromatography. The results show that FQ reduced the solubilization of OZ439 during milk digestion, which may be due to competitive binding of FQ to the digested milk products. Interactions between the protonated FQ-H+ and ionized liberated free fatty acids resulted in the formation of amorphous salts, which removes the low-energy crystalline state as a barrier to dissolution of FQ, while inhibiting the solubilization of OZ439. We conclude that although milk could enhance the solubilization of poorly water-soluble OZ439 during in vitro digestion principally due to the formation of fatty acids, the solubilization efficiency was reduced by the presence of FQ by competition for the available fatty acids. Assessment of the solubilization of both drugs during digestion of fixed-dose combination lipid formulations (such as milk) is important and may rationalize changes in bioavailability when compared to that of the individual drugs in the same formulation.

Amorphous nanoparticles by self-assembly: processing for controlled release of hydrophobic molecules.

More than 40% of newly developed drug molecules are highly hydrophobic and, thus, suffer from low bioavailability. Kinetically trapping the drug as a nanoparticle in an amorphous state enhances solubility. However, enhanced solubility can be compromised by subsequent recrystallization from the amorphous state during drying processes. We combine Flash NanoPrecipitation (FNP) to generate nanoparticles with spray-drying to produce stable solid powders. We demonstrate that the continuous nanofabrication platform for nanoparticle synthesis and recovery does not compromise the dissolution kinetics of the drug. Lumefantrine, an anti-malaria drug, is highly hydrophobic with low bioavailability. Increasing the bioavailability of lumefantrine has the potential to reduce the dose and number of required administrations per treatment, thus reducing cost and increasing patient compliance. The low melting temperature of lumefantrine (Tm = 130 °C) makes the drying of amorphous nanoparticles at elevated temperatures potentially problematic. Via FNP, we produced 200-400 nm nanoparticles using hydroxypropyl methylcellulose acetate succinate (HPMCAS), lecithin phospholipid, and zein protein stabilizers. Zein nanoparticles were spray-dried at 100 °C and 120 °C to study the effect of the drying temperature. For zein powders, at two hours the dissolution kinetics under fasted conditions reached 85% release for the 100 °C sample, but only 60% release for the 120 °C sample. Powder X-ray diffraction, differential scanning calorimetry, and solid state nuclear magnetic resonance indicate that the lumefantrine in the nanoparticle core is amorphous for samples spray-dried at 100 °C. Dissolution under fed state conditions showed similar release kinetics for both temperatures, with 90-95% release at two hours. Zein and HPMCAS nanoparticles spray-dried at 100 °C showed release profiles in fasted and fed state media that are identical to those of lyophilized samples, i.e. those dried at cryogenic conditions where no transformation to the crystalline state can occur. Thus, spray drying 30 °C below the melting transition of lumefantrine is sufficient to maintain the amorphous state. These inexpensive formulations have potential to be developed into future therapies for malaria, and the results also highlight the potential of combining FNP and spray-drying as a versatile platform to assemble and rapidly recover amorphous nanoparticles in a solid dosage form.

Partnering to fight malaria in India: Past, present and future.

The global fight against malaria requires continual development of new tools. Collaborations in India have played a key role in MMV's partnerships to discover, develop and deliver new medicines. Over the last decade, India has become a focal point of global medicinal chemistry, and combined with investments in basic science, this has led to the discovery of new potential drugs. India also brings significant experience to drug development, in clinical trials, but also in formulation and manufacturing. Finally, innovative new approaches in case management have streamlined impact at the level of communities and the patients.

Interactions of Artefenomel (OZ439) with Milk during Digestion: Insights into Digestion-Driven Solubilization and Polymorphic Transformations.

Milk has been used as a vehicle for the delivery of antimalarial drugs during clinical trials to test for a food effect and artefenomel (OZ439) showed enhanced oral bioavailability with milk. However, the nature of the interaction between milk and OZ439 in the gastrointestinal tract remains poorly understood. To understand the role of milk digestion on the solubilization of OZ439 and polymorphism, we conducted real-time monitoring of crystalline drug in suspension during in vitro intestinal lipolysis of milk containing OZ439 using synchrotron X-ray scattering. OZ439 formed an unstable solid-state intermediate free base form (OZ439-FB form 1) at intestinal pH and was partially solubilized by milk fat globules prior to lipolysis. Dissolution of the free base form 1 and recrystallization of OZ439 in a more stable polymorphic form (OZ439-FB form 2) occurred during in vitro lipolysis in milk. Simply stirring the milk/drug suspension in the absence of lipase or addition of lipase to OZ439 in a lipid-free buffer did not induce this polymorphic transformation. The formation of OZ439-FB form 2 was therefore accelerated by the solubilization of OZ439-FB form 1 during the digestion of milk. Our findings confirmed that although crystalline precipitates of OZ439-FB form 2 could still be detected after in vitro digestion, milk-based lipid formulations provided a significant reduction in crystalline OZ439 compared to lipid-free formulations, which we attribute to the formation of colloidal structures by the digested milk lipids. Milk may therefore be particularly suited as a form of lipid-based formulation (LBF) for coadministration with OZ439, from which both an enhancement in OZ439 oral bioavailability and the delivery of essential nutrients should result.

Injectable anti-malarials revisited: discovery and development of new agents to protect against malaria.

Over the last 15 years, the majority of malaria drug discovery and development efforts have focused on new molecules and regimens to treat patients with uncomplicated or severe disease. In addition, a number of new molecular scaffolds have been discovered which block the replication of the parasite in the liver, offering the possibility of new tools for oral prophylaxis or chemoprotection, potentially with once-weekly dosing. However, an intervention which requires less frequent administration than this would be a key tool for the control and elimination of malaria. Recent progress in HIV drug discovery has shown that small molecules can be formulated for injections as native molecules or pro-drugs which provide protection for at least 2 months. Advances in antibody engineering offer an alternative approach whereby a single injection could potentially provide protection for several months. Building on earlier profiles for uncomplicated and severe malaria, a target product profile is proposed here for an injectable medicine providing long-term protection from this disease. As with all of such profiles, factors such as efficacy, cost, safety and tolerability are key, but with the changing disease landscape in Africa, new clinical and regulatory approaches are required to develop prophylactic/chemoprotective medicines. An overall framework for these approaches is suggested here.

A randomized, open-label two-period crossover pilot study to evaluate the relative bioavailability in the fed state of atovaquone-proguanil (Atoguanil™) versus atovaquone-proguanil hydrochloride (Malarone®) in healthy adult participants.

Atoguanil™ is a novel complex of atovaquone (ATV) and proguanil (PG) with enhanced ATV bioavailability compared to Malarone®. This pilot study assessed whether the relative bioavailability (Frel) of ATV, PG, and the primary PG metabolite cycloguanil (CG) following a single oral dose in the fed state of Atoguanil was similar to Malarone despite a 50% lower ATV dose. This open-label, single-dose, randomized 2-period, 2-treatment, balanced crossover study was conducted between 17th November 2021 and 18th March 2022. Eligible participants (aged 18-55 years) were randomized (1:1) in period 1 to Atoguanil (ATV/PG 500/348 mg) or Malarone (ATV/PG hydrochloride 1000/400 mg) administered following a high-fat, high caloric meal. After a 24-day washout period, participants crossed treatment arms. For the doses tested, Frel was assumed similar if 90%CIs were between 80 and 125% for the geometric mean ratio of the least square mean differences for each exposure parameter. In 15 evaluable participants, Frel was similar for ATV Cmax (93.6% [90%CI 83.6, 104.9]) but not AUC0-inf (77.8% [67.4, 89.8]), for PG AUC0-inf (95.6% [92.1, 99.2]) but not Cmax (82.4% [75.8, 89.5]), and for both CG Cmax (100.8% [95.0, 107.0]) and AUC0-inf (102.9% [98.4, 107.7]). Nine adverse events occurred; all were of mild severity and not considered treatment related. At the doses tested, ATV Frel was lower following Atoguanil versus Malarone based on AUC0-inf, though when adjusted for dose Frel increased by 156%. Both drugs were well tolerated with no safety concerns. ClinicalTrials.gov: NCT04866602 (April 26th, 2021).

A Prodrug Strategy to Reposition Atovaquone as a Long-Acting Injectable for Malaria Chemoprotection.

Recent malaria drug discovery approaches have been extensively focused on the development of oral, smallmolecule inhibitors for disease treatment whereas parenteral routes of administration have been avoided due to limitations in deploying a shelf-stable injectable even though it could be dosed less frequently. However, an updated target candidate profile from Medicines for Malaria Venture (MMV) and stakeholders have advocated for long-acting injectable chemopreventive agents as an important interventive tool to improve malaria prevention. Here, we present strategies for the development of a long-acting, intramuscular, injectable atovaquone prophylactic therapy. We have generated three prodrug approaches that are contrasted by their differential physiochemical properties and pharmacokinetic profiles: mCBK068, a docosahexaenoic acid ester of atovaquone formulated in sesame oil, mCKX352, a heptanoic acid ester of atovaquone formulated as a solution in sesame oil, and mCBE161, an acetic acid ester of atovaquone formulated as an aqueous suspension. As a result, from a single 20 mg/kg intramuscular injection, mCKX352 and mCBE161 maintain blood plasma exposure of atovaquone above the minimal efficacious concentration for >70 days and >30 days, respectively, in cynomolgus monkeys. The differences in plasma exposure are reflective of the prodrug strategy, which imparts altered chemical properties that ultimately influence aqueous solubility and depot release kinetics. On the strength of the pharmacokinetic and safety profiles, mCBE161 is being advanced as a first-in-class clinical candidate for first-in-human trials.

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.