Lisa Xie

Development and validation of flow cytometric measurement for parasitaemia using autofluorescence and YOYO-1 in rodent malaria.

An automated flow cytometric (FCM) detection method has been developed and validated with a simple diagnostic procedure in parasitized erythrocytes of Plasmodium berghei-infected rats using the nucleic acid-binding fluorescent dye YOYO-1. High levels of reticulocytes were detected during the course of the infection, ranging from 1.2-51.2%, but any RNA potentially confounding the assay could be removed by digestion with RNAse. The cell counts of uninfected, infected, and nucleated cells occurred with high precision. The cells were divided into different populations according to their physical or chemical properties but various factors within the assay such as cell fixation, RNA digestion, and compensation required optimization. In this study, FCM greatly simplified and accelerated parasite detection, with a mean precision of 4.4%, specificity of 98.9% and accuracy of 101.3%. The detection and quantitation limits in the assay were 0.024% and 0.074% parasitaemia, respectively. Overall, the parasite counts by FCM measurement correlated highly (r2=0.954-0.988) with the parasitaemia measured by light microscopical analysis when animals treated with suppressive, clearance, and curative doses of novel antimalarial drugs were examined. The lower levels of parasitaemia (30%) detected by microscopy compared to FCM may be related to a number of schizonts externally attached to the erythrocyte membranes that normally would not be included in microscopy counting. Lower sampling error and reliable identification of rodent erythrocyte parasites based on the principles of FCM have replaced the traditional blood smear in our laboratory.

In vitro Biotransformation, in vivo Efficacy and Pharmacokinetics of Antimalarial Chalcones

4′-n-Butoxy-2,4-dimethoxy-chalcone (MBC) has been described as protecting mice from an otherwise lethal infection with Plasmodium yoelii when dosed orally at 50 mg/kg/dose, daily for 5 days. In contrast, we found that oral dosing of MBC at 640 mg/kg/dose, daily for 5 days, failed to extend the survivability of P. berghei-infected mice. The timing of compound administration and metabolic activation likely contribute to the outcome of efficacy testing in vivo. Microsomal digest of MBC yielded 4′-n-butoxy-4-hydroxy-2-methoxy-chalcone and 4′-(1-hydroxy-n-butoxy)-2,4-dimethoxy-chalcone. We propose that the latter will hydrolyze in vivo to 4′-hydroxy-2,4-dimethoxy-chalcone, which has greater efficacy than MBC in our P. berghei-infected mouse model and was detected in plasma following oral dosing of mice with MBC. Pharmacokinetic parameters suggest that poor absorption, distribution, metabolism and excretion properties contribute to the limited in vivoefficacy observed for MBC and its analogs.

Efficacy comparison of intravenous artelinate and artesunate in Plasmodium berghei-infected Sprague-Dawley rats.

This paper reports the comparative antimalarial efficacy of intravenous artelinate and artesunate in rats. Prior to efficacy experiments, a Plasmodium berghei-Sprague-Dawley rat model of malaria was developed, in which the clearance effects of intravenous drugs could be readily compared. In efficacy experiments, groups of P. berghei-infected rats were given 3 daily intravenous treatments of artelinate or artesunate at molar equivalent dose rates (total of 0-191.2 micromoles/kg). Artelinate was superior to artesunate in terms of clearance (100% clearance dose of 95.6 micromoles/kg (40 mg/kg) versus 191.2 micromoles/ kg for AS (73.4 mg/kg)) and parasite clearance time (1.7 +/- 0.5 days for AL versus 2.7 +/- 0.5 days for AS at a dose rate of 191.2 micromoles/kg, P < 0.01). No frank clinical toxicity was observed, though both artesunate and artelinate induced dose-related vascular necrosis at the site of injection. The necrosis was less severe and reversible when the drugs were administered via femoral, rather than tail/foot veins. The data suggest that the P. berghei-7-week-old Sprague-Dawley rat model of malaria is reproducible and useful for assessing the efficacy of antimalarials and that artelinate is at least as potent, and safe, as artesunate, the leading clinical treatment for severe malaria.

Neurotoxicity and Toxicokinetics of Artelinic Acid Following Repeated Oral Administration in Rats

Neurotoxicity secondary to oil-soluble artemisinins has been reported in various animal species. The onset of neurotoxicity and toxicokinetics of oral artelinic acid (AL), a water-soluble artemisinin, were investigated. After dose range study, rats were dosed at either 160 mg/kg daily for 9 consecutive days or at 288 mg/kg once every other day for five doses, so that the total dose (1440 mg/kg) and duration (9 days) were identical. Neuronal damage of varying severity was identified beginning as early as 1 day after completing dosing and continued for up to 10 days post dosing. Neuronal injury was most severe 7 days after the last treatment in each of the two dosing regimens. The rats dosed with 160 mg/kg of AL daily showed moderate neurotoxicity and lost 22% of their body weight during treatment. Compared with the first dose, the toxicokinetic profile of this regimen changed significantly, with the elimination half-life increasing 3.82-fold and the volume of distribution increasing 5.23-fold on the last day of dosing. In the animals treated with AL at 288 mg/kg every other day for 5 doses, minimal neuronal degeneration (severity score 1.17) was identified and the body weight was only 8% loss. Furthermore, there were no obvious differences in the pharniacokinetic parameters between first and last dosing days with this regimen. Additionally, a progressively drug retention in stomach and drug accretion in blood were only found in rats treated with 160 mg/kg daily for 9 days. These results imply that delayed gastric emptying resulted in AL accumulation in blood and prolonged a neurotoxic exposure time (186 h) in 160 mg/kg rats when compared to that (75 h) in 288 mg/kg animals. Therefore, the drug exposure time is a key factor in the neurotoxicity induced by AL.

Severe embryolethality of artesunate related to pharmacokinetics following intravenous and intramuscular doses in pregnant rats.

Artesunate (AS), a rapid, effective, and safe antimalarial drug, has been used for the treatment of malaria for decades. However, severe embryolethality was found for injectable AS in pregnant animals. In the present study, pregnant rats were selected and dosed with AS (GMP product) intravenously (IV) and intramuscularly (IM) at varied doses daily for 13 days from gestation day (GD) 6 to 18. In addition, a toxic dose of 1.2 mg/kg/day was subsequently tested in the GD 6-10, GD 11-15, and GD 16-20 periods of rat pregnancy. A pharmacokinetic study was also conducted to evaluate the bioavailability of AS following the IM administrations. Results showed that no significant adverse effects were found in maternal rats. All of the fetuses were either damaged or reabsorbed by placentas in treated pregnant rats, but doses did not show an adverse effect at 0.4 and 0.5 mg/kg after IV and IM administrations, respectively. The survival rate of fetuses is dose-dependent and the 50% fetus re-absorption doses (FRD(50)) were 0.61 and 0.60 mg/kg following the IV and IM, respectively. The most drug-sensitive period, showing severe embryotoxicity, was between GD 11 and 15 for injectable AS. When calculated with total concentrations of AS and dihydroartemisinin, an active metabolite of AS, the bioavailability of 97.8% after intramuscular injection was fulfilled to a bioequivalence of that in intravenous treatment. The fact that injectable AS exhibited severe embryolethality after both IV and IM injections seems related to their comparable pharmacokinetic profiles that indicate high peak concentrations in pregnant animals.

The distribution pattern of intravenous [14C] artesunate in rat tissues by quantitative whole-body autoradiography and tissue dissection techniques

Quantitative whole-body autoradiography (QWBA) and liquid scintillation counting (LSC) have been conducted to determine the metabolic profiles and tissue distribution of [(14)C] labeled artesunate (AS) injection in rats. The QWBA technique showed more accurate results in the quantification of radioactivity in 40 organs and tissues, compared to 19 organs with the LSC technique. The benefit of QWBA was especially apparent on measurements of bile, bone marrow, and gland organs; however, the LSC method produced more relevant findings than QWBA. Particularly, the LSC method allowed access to the following distribution patterns that were unavailable via QWBA performance: such as pharmacokinetic evaluation of radiolabeled AS in blood and plasma, tissue/plasma partition coefficients, conversion pathway of AS to dihydroartemisinin (DHA, an active and major metabolite of AS), unchanged AS and DHA in plasma, mass balance assessment, urinary and faecal eliminations, drug pathway with conjugation, [(14)C] AS binding with RBC and plasma protein, and metabolites identification. Even though the each method has its own advantages, common profiles were obtained from the two processes as shown in the results of the biliary metabolism, long-lasting metabolites, tissue distribution profiles, and multiple concentration peaks, which indicate a [(14)C] AS enterohepatic circulation.

Nanoparticle formulations of decoquinate increase antimalarial efficacy against liver stage Plasmodium infections in mice

UNLABELLED Decoquinate has potent activity against both Plasmodium hepatic development and red cell replication when tested in vitro. Decoquinate, however, is practically insoluble in water. To achieve its maximal in vivo efficacy, we generated nanoparticle formulations of decoquinate with a mean particle size less than 400 nm. Three separate preparations at doses of decoquinate 0.5-5 mg/kg were examined in mice infected with Plasmodium berghei. Oral administration of nanoparticle decoquinate at a dose of 1.25 mg/kg effectively inhibited the liver-stage parasite growth and provided complete causal prophylactic protection. This efficacy is 15 fold greater than that observed for microparticle decoquinate, which requires minimal dose of 20 mg/kg for the same inhibitory effect. Further in vitro studies utilizing dose-response assays revealed that decoquinate nanoformulation was substantially more potent than decoquinate microsuspension in killing both liver and blood stage malarial parasites, proving its potential for therapeutic development. FROM THE CLINICAL EDITOR In this study, a nanoparticle formulation of decoquinate is shown to have superior bioavailability and efficacy in a mouse model of malaria, paving the way to the development of novel, potentially less toxic and more effective therapeutics to combat a disease that still has an enormous impact on a global scale despite the available partially effective therapies.

Formulation and Particle Size Reduction Improve Bioavailability of Poorly Water-Soluble Compounds with Antimalarial Activity

Decoquinate (DQ) is highly effective at killing malaria parasites in vitro; however, it is extremely insoluble in water. In this study, solid dispersion method was used for DQ formulation which created a suitable physical form of DQ in aqueous phase for particle manipulation. Among many polymers and surfactants tested, polyvinylpyrrolidone 10, a polymer, and L-α-phosphatidylcholine or polysorbate, two surfactants, were chosen as DQ formulation components. The formulation particles were reduced to a mean size between 200 to 400 nm, which was stable in aqueous medium for at least three weeks. Pharmacokinetic (PK) studies showed that compared to DQ microparticle suspension, a nanoparticle formulation orally dosed to mice showed a 14.47-fold increase in area under the curve (AUC) of DQ plasma concentration and a 4.53-fold increase in AUC of DQ liver distribution. WR 299666, a poorly water-soluble compound with antimalarial activity, was also tested and successfully made into nanoparticle formulation without undergoing solid dispersion procedure. We concluded that nanoparticles generated by using appropriate formulation components and sufficient particle size reduction significantly increased the bioavailability of DQ and could potentially turn this antimalarial agent to a therapeutic drug.

Comparative Susceptibility of Different Mouse Strains to Liver-Stage Infection With Plasmodium berghei Sporozoites Assessed Using In Vivo Imaging

BACKGROUND The liver stages of Plasmodium parasites are important targets for the discovery and development of prophylactic drugs. METHODS A real-time in vivo imaging system was used to determine the level of luminescence measured from firefly luciferase expression by sporozoites developing in hepatocytes in different strains of mice. RESULTS The luminescence values (photon counts/sec) measured from the anatomical liver location in the untreated mice infected with 10,000 Plasmodium berghei sporozoites were 8.15 × 105 for C57BL/6 Albino, 2.12 × 105 for C3H/HeNCrL, 0.91 × 105 for C57BL/6 WT, 0.28 × 105 for BALB/c, and 0.16 × 105 for ICR/CD-1 mice. This data suggests that the C57BL/6 Albino strain is most susceptible to luminescent photon, mainly because the less light scattering and absorption from deeper tissues and the skin in the strain of mouse. The photon count observed in black C57BL/6 wild type mice was shown to be 88.83% lower compared to C57BL/6 Albino mice. Although the highest growth rate of sporozoites in hepatocytes was found for C57BL/6 wild type mice in this study, the black skin of this mouse significantly reduced parasite-associated bioluminescence. CONCLUSIONS The minimal light scattering and absorption and also enhanced susceptibility to liver infection of C57BL/6 Albino mice makes this strain preferable sensitivity for discovery and development of prophylactic antimalarial drugs.

Long-Term Prophylaxis and Pharmacokinetic Evaluation of Intramuscular Nano- and Microparticle Decoquinate in Mice Infected with P. berghei Sporozoites

Decoquinate nanoparticle and microparticle suspended in an oily vehicle to retard drug release are evaluated for long-term malaria prophylaxis. Pharmacokinetic studies in normal animals and antimalarial efficacy in liver stage malaria mice were conducted at various single intramuscular-decoquinate doses for 2, 4, 6, or 8 weeks prior to infection with P. berghei sporozoites. The liver stage efficacy evaluation was monitored by using an in vivo imaging system. Full causal prophylaxis was shown in mice with a single intramuscular dose at 120 mg/kg of nanoparticle decoquinate (0.43 μm) for 2-3 weeks and with microparticle decoquinate (8.31 μm) injected 8 weeks earlier than inoculation. The time above MIC of 1,375 hr observed with the microparticle formulation provided a 2.2-fold longer drug exposure than with the nanoparticle formulation (624 hr). The prophylactic effect of the microparticle formulation observed in mice was shown to be 3-4 times longer than the nanoparticle decoquinate formulation.