Atovaquone-HSA nano-drugs enhance the efficacy of PD-1 blockade immunotherapy by alleviating hypoxic tumor microenvironment.

BACKGROUND: Hypoxia is inherent character of most solid malignancies, leading to the failure of chemotherapy, radiotherapy and immunotherapy. Atovaquone, an anti-malaria drug, can alleviate tumor hypoxia by inhibiting mitochondrial complex III activity. The present study exploits atovaquone/albumin nanoparticles to improve bioavailability and tumor targeting of atovaquone, enhancing the efficacy of anti-PD-1 therapy by normalizing tumor hypoxia. METHODS: We prepared atovaquone-loaded human serum albumin (HSA) nanoparticles stabilized by intramolecular disulfide bonds, termed HSA-ATO NPs. The average size and zeta potential of HSA-ATO NPs were measured by particle size analyzer. The morphology of HSA-ATO NPs was characterized by transmission electron microscope (TEM). The bioavailability and safety of HSA-ATO NPs were assessed by animal experiments. Flow cytometry and ELISA assays were used to evaluate tumor immune microenvironment. RESULTS: Our data first verified that atovaquone effectively alleviated tumor hypoxia by inhibiting mitochondrial activity both in vitro and in vivo, and successfully encapsulated atovaquone in vesicle with albumin, forming HSA-ATO NPs of approximately 164 nm in diameter. We then demonstrated that the HSA-ATO NPs possessed excellent bioavailability, tumor targeting and a highly favorable biosafety profile. When combined with anti-PD-1 antibody, we observed that HSA-ATO NPs strongly enhanced the response of mice bearing tumor xenografts to immunotherapy. Mechanistically, HSA-ATO NPs promoted intratumoral CD8+ T cell recruitment by alleviating tumor hypoxia microenvironment, thereby enhancing the efficacy of anti-PD-1 immunotherapy. CONCLUSIONS: Our data provide strong evidences showing that HSA-ATO NPs can serve as safe and effective nano-drugs to enhance cancer immunotherapy by alleviating hypoxic tumor microenvironment.

A mitochondrial-metabolism-regulatable carrier-free nanodrug to amplify the sensitivity of photothermal therapy.

The oxidative phosphorylation inhibitor atovaquone (ATO) and the photosensitizer new indocyanine green (IR820) were self-assembled into carrier-free nanodrugs (IR820/ATO NPs) to achieve superior photothermal therapy (PTT), offering an attractive mitochondrial metabolism-regulatable approach for breast cancer treatment, where adenosine triphosphate (ATP) was downregulated along with downregulating the expression of heat shock proteins (HSPs) to amplify the sensitivity of PTT.

Synthesis and comparison of in vitro dual anti-infective activities of novel naphthoquinone hybrids and atovaquone.

A principal factor that contributes towards the failure to eradicate leishmaniasis and tuberculosis infections is the reduced efficacy of existing chemotherapies, owing to a continuous increase in multidrug-resistant strains of the causative pathogens. This accentuates the dire need to develop new and effective drugs against both plights. A series of naphthoquinone-triazole hybrids was synthesized and evaluated in vitro against Leishmania (L.) and Mycobacterium tuberculosis (Mtb) strains. Their cytotoxicities were also evaluated, using the human embryonic kidney cell line (HEK-293). The hybrids were found to be non-toxic towards human cells and had demonstrated micromolar cellular antileishmanial and antimycobacterial potencies. Hybrid 13, i.e. 2-{[1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl]methoxy}naphthalene-1,4-dione was the most active of all. It was found with MIC90 0.5 µM potency against Mtb in a protein free medium, and with half-maxima inhibitory concentrations (IC50) of 0.81 µM and 1.48 µM against the infective promastigote parasites of L. donavani and L. major, respectively, with good selectivity towards these pathogens (SI 22 - 65). Comparatively, the clinical naphthoquinone, atovaquone, although less cytotoxic, was found to be two-fold less antimycobacterial potent, and six- to twelve-fold less active against leishmania. Hybrid 13 may therefore stand as a potential anti-infective hit for further development in the search for new antitubercular and antileishmanial drugs. Elucidation of its exact mechanism of action and molecular targets will constitute future endeavour.

Enzyme Models-From Catalysis to Prodrugs.

Enzymes are highly specific biological catalysts that accelerate the rate of chemical reactions within the cell. Our knowledge of how enzymes work remains incomplete. Computational methodologies such as molecular mechanics (MM) and quantum mechanical (QM) methods play an important role in elucidating the detailed mechanisms of enzymatic reactions where experimental research measurements are not possible. Theories invoked by a variety of scientists indicate that enzymes work as structural scaffolds that serve to bring together and orient the reactants so that the reaction can proceed with minimum energy. Enzyme models can be utilized for mimicking enzyme catalysis and the development of novel prodrugs. Prodrugs are used to enhance the pharmacokinetics of drugs; classical prodrug approaches focus on alternating the physicochemical properties, while chemical modern approaches are based on the knowledge gained from the chemistry of enzyme models and correlations between experimental and calculated rate values of intramolecular processes (enzyme models). A large number of prodrugs have been designed and developed to improve the effectiveness and pharmacokinetics of commonly used drugs, such as anti-Parkinson (dopamine), antiviral (acyclovir), antimalarial (atovaquone), anticancer (azanucleosides), antifibrinolytic (tranexamic acid), antihyperlipidemia (statins), vasoconstrictors (phenylephrine), antihypertension (atenolol), antibacterial agents (amoxicillin, cephalexin, and cefuroxime axetil), paracetamol, and guaifenesin. This article describes the works done on enzyme models and the computational methods used to understand enzyme catalysis and to help in the development of efficient prodrugs.

A porphysome-based photodynamic O2 economizer for hypoxic tumor treatment by inhibiting mitochondrial respiration.

A porphysome-based photodynamic O2 economizer (P-PAT) is prepared for hypoxic tumor therapy. The self-assembled porphyrin bilayers of P-PAT possess high loading capacity to atovaquone (ATO) (nearly 70%), which could restrain mitochondrial respiration to relieve hypoxia and enhance photodynamic therapy.

Decoding the Molecular Effects of Atovaquone Linked Resistant Mutations on Plasmodium falciparum Cytb-ISP Complex in the Phospholipid Bilayer Membrane.

Atovaquone (ATQ) is a drug used to prevent and treat malaria that functions by targeting the Plasmodium falciparum cytochrome b (PfCytb) protein. PfCytb catalyzes the transmembrane electron transfer (ET) pathway which maintains the mitochondrial membrane potential. The ubiquinol substrate binding site of the protein has heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors that act as redox centers to aid in ET. Recent studies investigating ATQ resistance mechanisms have shown that point mutations of PfCytb confer resistance. Thus, understanding the resistance mechanisms at the molecular level via computational approaches incorporating phospholipid bilayer would help in the design of new efficacious drugs that are also capable of bypassing parasite resistance. With this knowledge gap, this article seeks to explore the effect of three drug resistant mutations Y268C, Y268N and Y268S on the PfCytb structure and function in the presence and absence of ATQ. To draw reliable conclusions, 350 ns all-atom membrane (POPC:POPE phospholipid bilayer) molecular dynamics (MD) simulations with derived metal parameters for the holo and ATQ-bound -proteins were performed. Thereafter, simulation outputs were analyzed using dynamic residue network (DRN) analysis. Across the triplicate MD runs, hydrophobic interactions, reported to be crucial in protein function were assessed. In both, the presence and absence of ATQ and a loss of key active site residue interactions were observed as a result of mutations. These active site residues included: Met 133, Trp136, Val140, Thr142, Ile258, Val259, Pro260 and Phe264. These changes to residue interactions are likely to destabilize the overall intra-protein residue communication network where the proteins' function could be implicated. Protein dynamics of the ATQ-bound mutant complexes showed that they assumed a different pose to the wild-type, resulting in diminished residue interactions in the mutant proteins. In summary, this study presents insights on the possible effect of the mutations on ATQ drug activity causing resistance and describes accurate MD simulations in the presence of the lipid bilayer prior to conducting inhibitory drug discovery for the PfCytb-iron sulphur protein (Cytb-ISP) complex.

Novel molecule combinations and corresponding hybrids targeting artemisinin-resistant Plasmodium falciparum parasites.

Malaria is still considered as the major parasitic disease and the development of artemisinin resistance does not improve this alarming situation. Based on the recent identification of relevant malaria targets in the artemisinin resistance context, novel drug combinations were evaluated against artemisinin-sensitive and artemisinin-resistant Plasmodium falciparum parasites. Corresponding hybrid molecules were also synthesized and evaluated for comparison with combinations and individual pharmacophores (e.g. atovaquone, mefloquine or triclosan). Combinations and hybrids showed remarkable antimalarial activity (IC50 = 0.6 to 1.1 nM for the best compounds), strong selectivity, and didn't present any cross-resistance with artemisinin. Moreover, the combination triclosan + atovaquone showed high activity against artemisinin-resistant parasites at the quiescent stage but the corresponding hybrid lost this pharmacological property. This result is essential since only few molecules active against quiescent artemisinin-resistant parasites are reported. Our promising results highlight the potential of these combinations and paves the way for pharmacomodulation work on the best hybrids.

Potent inhibition of tumour cell proliferation and immunoregulatory function by mitochondria-targeted atovaquone.

The FDA-approved prophylactic antimalarial drug atovaquone (ATO) recently was repurposed as an antitumor drug. Studies show that ATO exerts a profound antiproliferative effect in several cancer cells, including breast, ovarian, and glioma. Analogous to the mechanism of action proposed in parasites, ATO inhibits mitochondrial complex III and cell respiration. To enhance the chemotherapeutic efficacy and oxidative phosphorylation inhibition, we developed a mitochondria-targeted triphenylphosphonium-conjugated ATO with varying alkyl side chains (Mito4-ATO, Mito10-ATO, Mito12-ATO, and Mito16-ATO). Results show, for the first time, that triphenylphosphonium-conjugated ATO potently enhanced the antiproliferative effect of ATO in cancer cells and, depending upon the alkyl chain length, the molecular target of inhibition changes from mitochondrial complex III to complex I. Mito4-ATO and Mito10-ATO inhibit both pyruvate/malate-dependent complex I and duroquinol-dependent complex III-induced oxygen consumption whereas Mito12-ATO and Mito16-ATO inhibit only complex I-induced oxygen consumption. Mitochondrial target shifting may have immunoregulatory implications.

Sustainable Dissolution Performance of a Carrier Tailored Electrospun.

PURPOSE: This study aims to conduct an impact investigation in the hydrophobic-hydrophilic balance as an important factor for dissolution improvement of a hydrophilic carrier-based solid dispersion system. METHODS: Polymeric carriers with different hydrophobic to hydrophilic ratios were used to prepare several electrospun solid dispersion formulations. Physicochemical properties and surface morphology of the samples were assessed using Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR), polarized light microscopy, Differential Scanning Calorimetry (DSC), X-ray Powder Diffraction (XRPD) and Scanning Electron Microscopy (SEM). Dissolution study was conducted in a non-sink condition to assess the drug release. RESULTS: Incorporation of a higher amount of hydrophilic component showed an improvement in formulating a fully amorphous system based on XRPD, yet the dissolution rate increment showed no significant difference from the lower. Hence, the degree of crystallinity is proven not to be the crucial factor contributing to dissolution rate improvement. The presence of a concomitant hydrophobic component, however, showed ability in resisting precipitation and sustaining supersaturation. CONCLUSION: Hydrophobicity in a binary carrier system plays an important role in achieving and maintaining the supersaturated state particularly for an amorphous solid dispersion. Graphical Abstract.

Combining Stage Specificity and Metabolomic Profiling to Advance Antimalarial Drug Discovery.

We report detailed susceptibility profiling of asexual blood stages of the malaria parasite Plasmodium falciparum to clinical and experimental antimalarials, combined with metabolomic fingerprinting. Results revealed a variety of stage-specific and metabolic profiles that differentiated the modes of action of clinical antimalarials including chloroquine, piperaquine, lumefantrine, and mefloquine, and identified late trophozoite-specific peak activity and stage-specific biphasic dose-responses for the mitochondrial inhibitors DSM265 and atovaquone. We also identified experimental antimalarials hitting previously unexplored druggable pathways as reflected by their unique stage specificity and/or metabolic profiles. These included several ring-active compounds, ones affecting hemoglobin catabolism through distinct pathways, and mitochondrial inhibitors with lower propensities for resistance than either DSM265 or atovaquone. This approach, also applicable to other microbes that undergo multiple differentiation steps, provides an effective tool to prioritize compounds for further development within the context of combination therapies.

Development of Atovaquone Nanosuspension: Quality by Design Approach.

OBJECTIVE: The present study reports the use of MicrofluidizerTM technology to form a stable nanosuspension of atovaquone (ATQ) using quality by design (QbD) approach. METHODS: The patient-centric quality target product profile and critical quality attributes (CQAs) were identified. A Box-Behnken design was employed for the optimization of dependent variables, while CQAs like particle size and PDI were evaluated as response variables. Effective optimization of ATQ nanosuspension preparation using Microfluidizer processor as a novel green technology was achieved using QbD approach. RESULT: The prepared nanosuspension had a mean particle size of 865 nm ± 5%, PDI of 0.261 ± 3%, and zeta potential of -1.79 ± 5 mV. The characterization of the prepared nanosuspension by SEM, DSC, and XRD revealed its nano-crystalline nature whereas FTIR spectroscopic analysis confirmed the absence of any physicochemical interaction because of process parameters between the drug and excipients. CONCLUSION: In vitro dissolution studies of the nanosuspension using USP-IV exhibited a 100% cumulative drug release over 90 minutes, which is significantly better than that of ATQ pure API. In vivo pharmacokinetic studies revealed bioequivalence of ATQ nanosuspensions by Microfluidizer homogenization process to the marketed formulation1.

Cyclization-blocked proguanil as a strategy to improve the antimalarial activity of atovaquone.

Atovaquone-proguanil (Malarone®) is used for malaria prophylaxis and treatment. While the cytochrome bc1-inhibitor atovaquone has potent activity, proguanil's action is attributed to its cyclization-metabolite, cycloguanil. Evidence suggests that proguanil has limited intrinsic activity, associated with mitochondrial-function. Here we demonstrate that proguanil, and cyclization-blocked analogue tBuPG, have potent, but slow-acting, in vitro anti-plasmodial activity. Activity is folate-metabolism and isoprenoid biosynthesis-independent. In yeast dihydroorotate dehydrogenase-expressing parasites, proguanil and tBuPG slow-action remains, while bc1-inhibitor activity switches from comparatively fast to slow-acting. Like proguanil, tBuPG has activity against P. berghei liver-stage parasites. Both analogues act synergistically with bc1-inhibitors against blood-stages in vitro, however cycloguanil antagonizes activity. Together, these data suggest that proguanil is a potent slow-acting anti-plasmodial agent, that bc1 is essential to parasite survival independent of dihydroorotate dehydrogenase-activity, that Malarone® is a triple-drug combination that includes antagonistic partners and that a cyclization-blocked proguanil may be a superior combination partner for bc1-inhibitors in vivo.

Managing emerging mutagenicity risks: Late stage mutagenic impurity control within the atovaquone second generation synthesis.

The mutagenic-impurity control strategy for a second generation manufacturing route to the non-mutagenic antipneumocystic agent atovaquone (2-((1R,4R)-4-(4-chlorophenyl)cyclohexyl)-3-hydroxynaphthalene-1,4-dione) 1 is described. Preliminary assessment highlighted multiple materials of concern which were largely discharged either through returning a negative bacterial mutagenicity assay or through confidence that the impurity would be purged during the downstream processing from when it was first introduced. Additional genotoxicity testing highlighted two materials of concern where initial assessment suggested that testing for these impurities at trace levels within the drug substance would be required. Following a thorough review of process purging detail, spiking and purging experimentation, and an understanding of the process parameters to which they were exposed an ICH M7 Option 4 approach could be justified for their control. The development of two 1H NMR spectroscopy methods for measurement of these impurities is also described as well as a proposed summary table for describing the underlying rationale for ICH M7 control rationales to regulators. This manuscript demonstrates that process purging of potential mutagenic impurities can be realised even when they are introduced in the later stages of a process and highlights the importance of scientific understanding rather than relying on a stage-counting approach.

Hepatobiliary Disposition of Atovaquone: A Case of Mechanistically Unusual Biliary Clearance.

Atovaquone, an antiprotozoal and antipneumocystic agent, is predominantly cleared by biliary excretion of unchanged parent drug. Atovaquone is ≥10,000-fold concentrated in human bile relative to unbound plasma. Even after correcting for apparent nonspecific binding and incomplete solubility in bile, atovaquone is still concentrated ≥100-fold in bile, consistent with active biliary excretion. Mechanisms of atovaquone hepatobiliary disposition were studied using a multiexperimental in vitro and in vivo approach. Atovaquone uptake was not elevated in HEK293 cells singly overexpressing OATP1B1, OATP1B3, OATP2B1, OCT1, NTCP, or OAT2. Hepatocyte uptake of atovaquone was not impaired by OATP and OCT inhibitor cocktail (rifamycin and imipramine). Atovaquone liver-to-blood ratio at distributional equilibrium was not reduced in Oatp1a/1b and Oct1/2 knockout mice. Atovaquone exhibited efflux ratios of approximately unity in P-gp and BCRP overexpressing MDCK cell monolayers and did not display enhanced uptake in MRP2 vesicles. Biliary and canalicular clearance were not decreased in P-gp, Bcrp, Mrp2, and Bsep knockout rats. In the present study, we rule out the involvement of major known basolateral uptake and bile canalicular efflux transporters in the hepatic uptake and biliary excretion of atovaquone. This is the first known example of a drug cleared by biliary excretion in humans, with extensive biliary concentration, which is not transported by the mechanisms investigated herein.

Nanoemulsion of atovaquone as a promising approach for treatment of acute and chronic toxoplasmosis.

Treatment of toxoplasmosis is necessary in congenital form and immunocompromised patients. Atovaquone is a powerful suppressor of protozoan parasites with a broad-spectrum activity, but an extremely low water solubility and bioavailability. In this study, nanoemulsion of this drug was prepared with grape seed oil using spontaneous emulsification method to increase bioavailability and efficacy of atovaquone for treatment of toxoplasmosis. In vitro activity of atovaquone nanoemulsion against T. gondii, RH and Tehran strains, was assessed in HeLa cell culture. For in vivo assessment, BALB/c mice were infected with RH and Tehran strains and then treated with nanoemulsion of atovaquone, compared to that treated with free atovaquone. Concentration of atovaquone nanoemulsion showed in vitro anti-parasitic effects in both strains of T. gondii. Furthermore, oral administration of atovaquone nanoemulsion increased oral bioavailability, tissue distribution and mice survival time and reduced parasitemia and number and size of the brain cysts. Decrease of cyst numbers was verified by down regulation of BAG1 using real-time polymerase chain reaction (real-time PCR) assay. Effective therapeutic activity of atovaquone at a reduced dose is the major achievement of this study.

Atovaquone oral bioavailability enhancement using electrospraying technology.

Atovaquone in combination with proguanil hydrochloride, marketed as Malarone® tablets by GlaxoSmithKline (GSK), is prescribed for the treatment of malaria. High dose and poor bioavailability are the main hurdles associated with atovaquone oral therapy. The present study reports development of atovaquone nanoparticles, using in house designed and fabricated electrospraying equipment, and the assessment of bioavailability and therapeutic efficacy of the nanoparticles after oral administration. Solid nanoparticles of atovaquone were successfully produced by electrospraying and were characterized for particle size and flow properties. Differential Scanning Calorimetry, X-ray Diffraction, Fourier Transform Infrared Spectroscopy studies were also carried out. Atovaquone nanoparticles along with proguanil hydrochloride and a suitable wetting agent were filled in size 2 hard gelatin capsules. The formulation was compared with Malarone® tablets (GSK) and Mepron® suspension (GSK) in terms of in vitro release profile and in vivo pharmacokinetic studies. It showed 2.9-fold and 1.8-fold improved bioavailability in rats compared to Malarone® tablets and Mepron® suspension respectively. Therapeutic efficacy of the formulation was determined using modified Peter's 4-day suppressive tests and clinical simulation studies using Plasmodium berghei ANKA infected Swiss mice and compared to Malarone®. The developed formulation showed a 128-fold dose reduction in the modified Peter's 4-day suppressive tests and 32-fold dose reduction in clinical simulation studies. Given that only one capsule a day of developed formulation is required to be administered orally compared to 4 Malarone® tablets once a day and that too at a significantly reduced dose, this nanoparticle formulation will definitely reduce the side-effects of the treatment and is also likely to increase patient compliance.

Gene expression-based discovery of atovaquone as a STAT3 inhibitor and anticancer agent.

The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is frequently activated inappropriately in a wide range of hematological and solid cancers, but clinically available therapies targeting STAT3 are lacking. Using a computational strategy to identify compounds opposing the gene expression signature of STAT3, we discovered atovaquone (Mepron), an antimicrobial approved by the US Food and Drug Administration, to be a potent STAT3 inhibitor. We show that, at drug concentrations routinely achieved clinically in human plasma, atovaquone inhibits STAT3 phosphorylation, the expression of STAT3 target genes, and the viability of STAT3-dependent hematological cancer cells. These effects were also observed with atovaquone treatment of primary blasts isolated from patients with acute myelogenous leukemia or acute lymphocytic leukemia. Atovaquone is not a kinase inhibitor but instead rapidly and specifically downregulates cell-surface expression of glycoprotein 130, which is required for STAT3 activation in multiple contexts. The administration of oral atovaquone to mice inhibited tumor growth and prolonged survival in a murine model of multiple myeloma. Finally, in patients with acute myelogenous leukemia treated with hematopoietic stem cell transplantation, extended use of atovaquone for Pneumocystis prophylaxis was associated with improved relapse-free survival. These findings establish atovaquone as a novel, clinically accessible STAT3 inhibitor with evidence of anticancer efficacy in both animal models and humans.

Bioavailability enhancement of atovaquone using hot melt extrusion technology.

Emerging parasite resistance and poor oral bioavailability of anti-malarials are the two cardinal issues which hinder the clinical success of malaria chemotherapy. Atovaquone-Proguanil is a WHO approved fixed dose combination used to tackle the problem of emerging resistance. However, Atovaquone is a highly lipophilic drug having poor aqueous solubility (less than 0.2 µg/ml) thus reducing its oral bioavailability. The aim of the present investigation was to explore hot melt extrusion (HME) as a solvent-free technique to enhance solubility and oral bioavailability of Atovaquone and to develop an oral dosage form for Atovaquone-Proguanil combination. Solid dispersion of Atovaquone was successfully developed using HME. The solid dispersion was characterized for DSC, FTIR, XRD, SEM, and flow properties. It was filled in size 2 hard gelatin capsules. The formulation showed better release as compared to Malarone® tablets, and 3.2-fold and 4.6-fold higher bioavailability as compared to Malarone® tablets and Atovaquone respectively. The enhanced bioavailability also resulted in 100% anti-malarial activity in murine infection model at 1/8(th) therapeutic dose. Thus the developed methodology shows promising potential to solve the problems associated with Atovaquone therapy, namely its high cost and poor oral bioavailability, resulting in increased therapeutic efficacy of Atovaquone.

Formulation and characterization of atovaquone nanosuspension for improved oral delivery in the treatment of malaria.

AIM: The objective of the present study was to develop an atovaquone (ATQ) nanosuspension and evaluate its ability to improve the pharmacokinetic and therapeutic efficacy on oral administration. MATERIALS & METHODS: The ATQ nanosuspension was prepared by a combination of microprecipitation and high-pressure homogenization. It was freeze dried and characterized for various physiochemical properties. In vivo pharmacokinetics was performed in rats whereas antimalarial efficacy was assessed in mice using a 4-day suppressive test. RESULTS: The ATQ nanosuspension stabilized with Solutol(®) HS 15 (BASF India Ltd, Mumbai, India) and Capryol™ 90 (Gattefosse, Mumbai, India) exhibited a z-average diameter of 371.50 nm and a polydispersity index of 0.19. X-ray diffraction and differential scanning calorimetry analysis indicated no substantial changes in the crystalline state of ATQ nanocrystals. The aqueous solubility and in vitro dissolution rate were significantly increased by reducing the particle size. An in vivo pharmacokinetics study of the nanosuspension compared with a drug suspension and Malarone(®) (GlaxoSmithKline, Brentford, UK) exhibited an approximately 4.6-3.2-fold improvement in area under plasma concentration. A significant increase in Cmax and decrease in time to reach peak plasma concentration after administration was also observed. ATQ in nanosized form, even at one-quarter lower doses, exhibited greater reduction in parasitemia and prolonged survival compared with its reference formulations. CONCLUSION: Results of this pilot study highlight the potential of nanosuspension as an efficient and commercially viable strategy for improving delivery of ATQ for malaria treatment.

Reconstructing the Qo site of Plasmodium falciparum bc 1 complex in the yeast enzyme.

The bc 1 complex of the mitochondrial respiratory chain is essential for Plasmodium falciparum proliferation, the causative agent of human malaria. Therefore, this enzyme is an attractive target for antimalarials. However, biochemical investigations of the parasite enzyme needed for the study of new drugs are challenging. In order to facilitate the study of new compounds targeting the enzyme, we are modifying the inhibitor binding sites of the yeast Saccharomyces cerevisiae to generate a complex that mimics the P. falciparum enzyme. In this study we focused on its Qo pocket, the site of atovaquone binding which is a leading antimalarial drug used in treatment and causal prophylaxis. We constructed and studied a series of mutants with modified Qo sites where yeast residues have been replaced by P. falciparum equivalents, or, for comparison, by human equivalents. Mitochondria were prepared from the yeast Plasmodium-like and human-like Qo mutants. We measured the bc 1 complex sensitivity to atovaquone, azoxystrobin, a Qo site targeting fungicide active against P. falciparum and RCQ06, a quinolone-derivative inhibitor of P. falciparum bc 1 complex.The data obtained highlighted variations in the Qo site that could explain the differences in inhibitor sensitivity between yeast, plasmodial and human enzymes. We showed that the yeast Plasmodium-like Qo mutants could be useful and easy-to-use tools for the study of that class of antimalarials.