Therapeutic efficacy of generic artemether-lumefantrine in the treatment of uncomplicated malaria in Ghana: assessing anti-malarial efficacy amidst pharmacogenetic variations.

BACKGROUND: Despite efforts made to reduce morbidity and mortality associated with malaria, especially in sub-Saharan Africa, malaria continues to be a public health concern that requires innovative efforts to reach the WHO-set zero malaria agenda. Among the innovations is the use of artemisinin-based combination therapy (ACT) that is effective against Plasmodium falciparum. Generic artemether-lumefantrine (AL) is used to treat uncomplicated malaria after appropriate diagnosis. AL is metabolized by the cytochrome P450 family of enzymes, such as CYP2B6, CYP3A4 and CYP3A5, which can be under pharmacogenetic influence. Pharmacogenetics affecting AL metabolism, significantly influence the overall anti-malarial activity leading to variable therapeutic efficacy. This study focused on generic AL drugs used in malarial treatment as prescribed at health facilities and evaluated pharmacogenomic influences on their efficacy. METHODS: Patients who have been diagnosed with malaria and confirmed through RDT and microscopy were recruited in this study. Blood samples were taken on days 1, 2, 3 and 7 for parasite count and blood levels of lumefantrine, artemisinin, desbutyl-lumefantrine (DBL), and dihydroartemisinin (DHA), the active metabolites of lumefantrine and artemether, respectively, were analysed using established methods. Pharmacogene variation analysis was undertaken using iPLEX microarray and PCR-RFLP. RESULTS: A total of 52 patients completed the study. Median parasite density from day 1 to 7 ranged from 0-2666/µL of blood, with days 3 and 7 recording 0 parasite density. Highest median plasma concentration for lumefantrine and desbutyl lumefantrine, which are the long-acting components of artemisinin-based combinations, was 4123.75 ng/mL and 35.87 ng/mL, respectively. Day 7 plasma lumefantrine concentration across all generic ACT brands was ≥ 200 ng/mL which potentially accounted for the parasitaemia profile observed. Monomorphism was observed for CYP3A4 variants, while there were observed variations in CYP2B6 and CYP3A5 alleles. Among the CYP3A5 genotypes, significant differences in genotypes and plasma concentration for DBL were seen on day 3 between 1/*1 versus *1/*6 (p = 0.002), *1/*3 versus *1/*6 (p = 0.006) and *1/*7 versus *1/*6 (p = 0.008). Day 7 plasma DBL concentrations showed a significant difference between *1/*6 and *1/*3 (p = 0.026) expressors. CONCLUSIONS: The study findings show that CYP2B6 and CYP3A5 pharmacogenetic variations may lead to higher plasma exposure of AL metabolites.

LC-HRMS methods for the quantification of two artemisinin drugs and their metabolites in rat blood and plasma.

As first-line antimalarials used in the artemisinin combination therapy, artemisinin drugs exert their action inside red blood cells. However, the blood pharmacokinetic characteristics of artemisinin drugs have not been fully revealed owing to their built-in chemical instability initiated by Fe2+ released from hemoglobin, with limited information on their metabolites. In this study, liquid chromatography tandem high-resolution mass spectrometric (LC-HRMS) methods were developed for the quantification of two representative artemisinin drugs (artemisinin, ART; dihydroartemisinin, DHA) and their respective metabolite (deoxyartemisinin, D-ART; dihydroartemisinin glucuronide, DHA-Glu) in rat blood/plasma. The blood samples were pretreated with the stabilizer (0.4 m potassium dichromate and 3% EDTA-2Na). The methods displayed excellent specificity, linearity, accuracy and precision for ART (17.7-709.2 nm) and its metabolite D-ART (18.8-751.9 nm), and the linear range was 40.0-4,000.0 nm for both DHA and DHA-Glu. The methods were successfully applied to the pharmacokinetic studies of ART and DHA in rats. The blood-to-plasma ratio was 0.8-1.5 for ART, 1.0-1.5 for D-ART, 1.2-2.2 for DHA and 0.9-1.3 for DHA-Glu, which was time dependent. The results indicated that artemisinin drugs and their metabolites showed a high but different blood-to-plasma ratio, which should be considered when optimizating their dosing regimens or evaluating their clinical outcomes.

Inter-Species Pharmacokinetic Modeling and Scaling for Drug Repurposing of Pyronaridine and Artesunate.

Even though several new targets (mostly viral infection) for drug repurposing of pyronaridine and artesunate have recently emerged in vitro and in vivo, inter-species pharmacokinetic (PK) data that can extend nonclinical efficacy to humans has not been reported over 30 years of usage. Since extrapolation of animal PK data to those of humans is essential to predict clinical outcomes for drug repurposing, this study aimed to investigate inter-species PK differences in three animal species (hamster, rat, and dog) and to support clinical translation of a fixed-dose combination of pyronaridine and artesunate. PK parameters (e.g., steady-state volume of distribution (Vss), clearance (CL), area under the concentration-time curve (AUC), mean residence time (MRT), etc.) of pyronaridine, artesunate, and dihydroartemisinin (an active metabolite of artesunate) were determined by non-compartmental analysis. In addition, one- or two-compartment PK modeling was performed to support inter-species scaling. The PK models appropriately described the blood concentrations of pyronaridine, artesunate, and dihydroartemisinin in all animal species, and the estimated PK parameters in three species were integrated for inter-species allometric scaling to predict human PKs. The simple allometric equation (Y = a × Wb) well explained the relationship between PK parameters and the actual body weight of animal species. The results from the study could be used as a basis for drug repurposing and support determining the effective dosage regimen for new indications based on in vitro/in vivo efficacy data and predicted human PKs in initial clinical trials.

Parasite Viability as a Measure of In Vivo Drug Activity in Preclinical and Early Clinical Antimalarial Drug Assessment.

The rate at which parasitemia declines in a host after treatment with an antimalarial drug is a major metric for assessment of antimalarial drug activity in preclinical models and in early clinical trials. However, this metric does not distinguish between viable and nonviable parasites. Thus, enumeration of parasites may result in underestimation of drug activity for some compounds, potentially confounding its use as a metric for assessing antimalarial activity in vivo. Here, we report a study of the effect of artesunate on Plasmodium falciparum viability in humans and in mice. We first measured the drug effect in mice by estimating the decrease in parasite viability after treatment using two independent approaches to estimate viability. We demonstrate that, as previously reported in humans, parasite viability declines much faster after artesunate treatment than does the decline in parasitemia (termed parasite clearance). We also observed that artesunate kills parasites faster at higher concentrations, which is not discernible from the traditional parasite clearance curve and that each subsequent dose of artesunate maintains its killing effect. Furthermore, based on measures of parasite viability, we could accurately predict the in vivo recrudescence of infection. Finally, using pharmacometrics modeling, we show that the apparent differences in the antimalarial activity of artesunate in mice and humans are partly explained by differences in host removal of dead parasites in the two hosts. However, these differences, along with different pharmacokinetic profiles, do not fully account for the differences in activity. (This study has been registered with the Australian New Zealand Clinical Trials Registry under identifier ACTRN12617001394336.).

Piperaquine Pharmacokinetic and Pharmacodynamic Profiles in Healthy Volunteers of Papua New Guinea after Administration of Three-Monthly Doses of Dihydroartemisinin-Piperaquine.

Mass drug administration (MDA) with monthly dihydroartemisinin-piperaquine (DHA-PQP) appears useful in malaria control and elimination strategies. Determining the relationship between consecutive piperaquine phosphate (PQP) exposure and its impact on QT interval prolongation is a key safety consideration for MDA campaigns. Healthy volunteers from Papua New Guinea received a 3-day course of DHA-PQP (2.1/17.1 mg/kg) monthly for 3 consecutive months in a single arm longitudinal study. Plasma PQP concentrations were measured after the third dose of each course (at 52-54 h) and at 0 h of course 3. Twelve-lead electrocardiographic readings were conducted at 0 h, 48 h, 52 h, and day 7 of each course. QT interval corrected by Fridericia's formula (QTcF) was measured at each time point. A pharmacokinetic-pharmacodynamic model using nonlinear mixed effects models was developed to correlate PQP concentrations with QTcF. Ten thousand female and 10,000 male individuals were simulated at each treatment course. Eighty-two participants were included; mean age was 28.3 years (standard deviation [SD] ±12.3 years), and 36 (44%) were female. Pharmacokinetic-pharmacodynamic models were determined with 290 PQP concentrations and 868 QTcF observations. The average baseline QTcF was 392 ms with a between-subject variability SD ±14.4 ms and between-occasion variability SD ±3.64 ms. From the population modeled, only 0.08% of males and 0.45% of females would be at risk of an absolute QTcF of >500 ms. DHA-PQP is safe at standard doses in consecutive months, and the likelihood of severe cardiac events occurring during an MDA campaign is very low. This study has been registered at ClinicalTrials.gov under identifier NCT02605720.

Artemisinin Cocrystals for Bioavailability Enhancement. Part 1: Formulation Design and Role of the Polymeric Excipient.

Artemisinin (ART) is a most promising antimalarial agent, which is both effective and well tolerated in patients, though it has therapeutic limitations due to its low solubility, bioavailability, and short half-life. The objective of this work was to explore the possibility of formulating ART cocrystals, i.e., artemisinin-orcinol (ART-ORC) and artemisinin-resorcinol (ART2-RES), as oral dosage forms to deliver ART molecules for bioavailability enhancement. This is the first part of the study, aiming to develop a simple and effective formulation, which can then be tested on an appropriate animal model (i.e., mouse selected for in vivo study) to evaluate their preclinical pharmacokinetics for further development. In the current work, the physicochemical properties (i.e., solubility and dissolution rate) of ART cocrystals were measured to collect information necessary for the formulation development strategy. It was found that the ART solubility can be increased significantly by its cocrystals, i.e., 26-fold by ART-ORC and 21-fold by ART2-RES, respectively. Screening a set of polymers widely used in pharmaceutical products, including poly(vinylpyrrolidone), hydroxypropyl methylcellulose, and hydroxypropyl methylcellulose acetate succinate, based on the powder dissolution performance parameter analysis, revealed that poly(vinylpyrrolidone)/vinyl acetate (PVP-VA) was the most effective crystallization inhibitor. The optimal concentration of PVP-VA at 0.05 mg/mL for the formulation was then determined by a dissolution/permeability method, which represented a simplified permeation model to simultaneously evaluate the effects of a crystallization inhibitor on the dissolution and permeation performance of ART cocrystals. Furthermore, experiments, including surface dissolution of single ART cocrystals monitored by Raman spectroscopy, scanning electron microscopy and diffusion properties of ART in solution measured by 1H and diffusion-ordered spectroscopy nuclear magnetic resonance spectroscopy, provided insights into how the excipient affects the ART cocrystal dissolution performance and bioavailability.

Artemisinin Cocrystals for Bioavailability Enhancement. Part 2: In Vivo Bioavailability and Physiologically Based Pharmacokinetic Modeling.

We report the evaluation and prediction of the pharmacokinetic (PK) performance of artemisinin (ART) cocrystal formulations, that is, 1:1 artemisinin/orcinol (ART-ORC) and 2:1 artemisinin/resorcinol (ART2-RES), using in vivo murine animal and physiologically based pharmacokinetic (PBPK) models. The efficacy of the ART cocrystal formulations along with the parent drug ART was tested in mice infected with Plasmodium berghei. When given at the same dose, the ART cocrystal formulation showed a significant reduction in parasitaemia at day 4 after infection compared to ART alone. PK parameters including Cmax (maximum plasma concentration), Tmax (time to Cmax), and AUC (area under the curve) were obtained by determining drug concentrations in the plasma using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), showing enhanced ART levels after dosage with the cocrystal formulations. The dose-response tests revealed that a significantly lower dose of the ART cocrystals in the formulation was required to achieve a similar therapeutic effect as ART alone. A PBPK model was developed using a PBPK mouse simulator to accurately predict the in vivo behavior of the cocrystal formulations by combining in vitro dissolution profiles with the properties of the parent drug ART. The study illustrated that information from classical in vitro and in vivo experimental investigations of the parent drug of ART formulations can be coupled with PBPK modeling to predict the PK parameters of an ART cocrystal formulation in an efficient manner. Therefore, the proposed modeling strategy could be used to establish in vitro and in vivo correlations for different cocrystals intended to improve dissolution properties and to support clinical candidate selection, contributing to the assessment of cocrystal developability and formulation development.

Artemisinin co-delivery system based on manganese oxide for precise diagnosis and treatment of breast cancer.

Tumor microenvironment (TME) responsive intelligent system can realize the specific release and uniform distribution of chemotherapy drugs in tumor tissues, to achieve high-efficiency and low-toxic treatment of tumors. In this paper, drug delivery system TKD@RBCm-Mn2O3-ART with the above characteristics was constructed. We synthesized hollow mesoporous manganese trioxide (Mn2O3) nanoparticles and firstly found that they owned time-dependent size transformation feature in simulated TME. The particle size decreased from 318 nm to 50 nm and 6 nm at 1 h and 4 h in simulated TME, respectively. Then artemisinin (ART) was loaded into Mn2O3to realize the co-delivery of Mn2+and ART. The modification of homologous red cell membrane (RBCm) and TKD peptide was aimed at long circulation and tumor targeting in the body.In vitroresults demonstrated that in the presence of GSH, the cumulative drug release percentage could achieve 97.5%. Meanwhile, Mn2O3exhibited a good imaging capability in tumor, with the relaxation rate of 6.3113 mM-1s-1. After entering into MCF-7 cells, TKD@RBCm-Mn2O3/ART synchronously released Mn2+and ART to generate large amount of ROS and induce DNA damage.In vivoresults proved TKD@RBCm-Mn2O3/ART could arrive the deep area of solid tumors and achieve accurate diagnosis and treatment of breast cancer.

Murine pharmacokinetics and antimalarial pharmacodynamics of dihydroartemisinin trimer self-assembled nanoparticles.

Currently, conjugation of artemisinin-derived dimers, trimers, and tetramers is a viable strategy for developing new effective antimalarial candidates. Furthermore, nanotechnology is an effective means to achieve intravenous administration of hydrophobic drugs. In this paper, an ester-linked dihydroartemisinin trimer (DHA3) was synthesized and further prepared as self-assembled nanoparticles (DHA3NPs) by a one-step nanoprecipitation method. The pharmacokinetics and antimalarial pharmacodynamics of DHA3NPs were studied in rats and mice infected with Plasmodium yoelii BY265 (PyBY265). DHA3NPs had a regular spherical shape with a uniform size distribution of 140.27 ± 3.59 nm, entrapment efficiency (EE) of 99.63 ± 0.17%, and drug loading efficiency (DL) of 79.62 ± 0.11%. The in vitro release characterization revealed that DHA3NPs were easily hydrolysed into DHA in an esterase environment. The pharmacokinetics study demonstrated that the area under the concentration-time curve (AUC0-t) of DHA in DHA3NPs group was 2070.52 ± 578.76 h×ng×mL-1, which was higher than that of DHA and artesunate (AS) control groups (AUC0-t values of 724.18 ± 94.32 and 448.40 ± 94.45 h×ng×mL-1, respectively) (P < 0.05). The antimalarial pharmacodynamics in vivo suggested that DHA3NPS (ED90 7.82 ± 1.16 µmol×(kg×day)-1) had a superior antimalarial effect compared with that of control groups (ED90 values of 14.68 ± 0.98 (DHA) and 14.34 ± 1.96 (AS) µmol×(kg×day)-1) (P < 0.05). In addition, DHA3NPS reduced the recurrence ratio and improved the cure ratio and survival time. In summary, DHA3NPs exhibited promising pharmacokinetic characteristics and antimalarial pharmacodynamics in vivo.

Pharmacokinetics and Ex Vivo Antimalarial Activity of Artesunate-Amodiaquine plus Methylene Blue in Healthy Volunteers.

High rates of artemisinin-based combination therapy (ACT) failures in the treatment of Plasmodium falciparum malaria in Southeast Asia have led to triple-drug strategies to extend the useful life of ACTs. In this study, we determined whether methylene blue [MB; 3,7-bis(dimethylamino)phenothiazin-5-ium chloride hydrate] alters the pharmacokinetics of artesunate-amodiaquine (ASAQ) and enhances the ex vivo antimalarial activity of ASAQ. In an open-label, randomized crossover design, a single oral dose of ASAQ (200 mg AS/540 mg AQ) alone or with MB (325 mg) was administered to 15 healthy Vietnamese volunteers. Serial blood samples were collected up to 28 days after dosing. Pharmacokinetic properties of the drugs were determined by noncompartmental analysis. After drug administration, plasma samples from seven participants were assessed for ex vivo antimalarial activity against the artemisinin-sensitive MRA1239 and the artemisinin-resistant MRA1240 P. falciparum lines, in vitro MB significantly increased the mean area under the curve of the active metabolite of AS, dihydroartemisinin (1,246 ± 473 versus 917 ± 405 ng·h/ml, P = 0.009) but did not alter the pharmacokinetics of AQ, AS, or desethylamodiaquine. Comparing the antimalarial activities of the plasma samples from the participants collected up to 48 h after ASAQ plus MB (ASAQ+MB) and ASAQ dosing against the MRA1239 and MRA1240 lines, MB significantly enhanced the blood schizontocidal activity of ASAQ by 2.0-fold and 1.9-fold, respectively. The ring-stage survival assay also confirmed that MB enhanced the ex vivo antimalarial activity of ASAQ against MRA1240 by 2.9-fold to 3.8-fold, suggesting that the triple-drug combination has the potential to treat artemisinin-resistant malaria and for malaria elimination. (This study has been registered in the Australian New Zealand Clinical Trials Registry [https://anzctr.org.au/] under registration number ACTRN12612001298808.).

In vivo antimalarial activity and pharmacokinetics of artelinic acid-choline derivative liposomes in rodents.

It is urgent to develop new antimalarial drugs with good therapeutic effects to address the emergence of drug resistance. Here, the artelinic acid-choline derivative (AD) was synthesized by dehydration reaction and esterification reaction, aimed to avoid the emergence of drug resistance by synergistic effect of artemisinins and choline derivative, which could compete with choline for rate-limiting enzymes in the phosphatidylcholine (PC) biosynthetic pathway. AD was formulated into liposomes (ADLs) by the thin-film hydration method. Efficacy of ADLs was evaluated by Peters 4-day suppression test. The suppression percentage against Plasmodium yoelii BY265 (PyBY265) in ADLs group was higher than those of positive control groups (dihydroartemisinin liposomes, P < 0.05) and other control groups (P ⩽ 0.05) at the doses of 4.4, 8.8, 17.6 µmol (kg·d)-1, respectively. The negative conversion fraction, recrudescence fraction and survival fraction of ADLs group were superior to other control groups. Pharmacokinetics in rats after intravenous injection suggested that ADLs exhibited higher exposure levels (indexed by area under concentration-time curve) than that of AD solution, artelinic acid liposomes or artelinic acid solution (P < 0.01). Taken together, ADLs exhibited promising antimalarial efficacy and pharmacokinetic characteristics.

Quantitative determination of artemisinin in rat hemolyzed plasma by an HPLC-HRMS method.

Iron present in hemolyzed plasma could cause the degradation of artemisinin by reductively cleaving the peroxide bridge of artemisinin during sample preparation, which is a significant technical challenge for artemisinin determination. In this paper, this issue was resolved by using sodium nitrite as methemoglobin-forming agent to oxidize hemoglobin to methemoglobin in the presence of acetic acid and prevent the degradation of artemisinin in hemolyzed plasma during the sample preparation procedure. Then, a high-performance liquid chromatography tandem high-resolution mass spectrometry method was developed and validated for the determination of artemisinin in normal and hemolyzed plasma. The linear range was validated over the concentration range of 5-500 ng ml-1 . The matrix effect and stability were also evaluated. This robust and sensitive assay was successfully applied to a pharmacokinetic study in rats after an oral administration of Artemisia annua L. extract.

Sequential Open-Label Study of the Safety, Tolerability, and Pharmacokinetic Interactions between Dihydroartemisinin-Piperaquine and Mefloquine in Healthy Thai Adults.

Artemisinin-based combination therapies (ACTs) have contributed substantially to the global decline in Plasmodium falciparum morbidity and mortality, but resistance to artemisinins and their partner drugs is increasing in Southeast Asia, threatening malaria control. New antimalarial compounds will not be generally available soon. Combining three existing antimalarials in the form of triple ACTs, including dihydroartemisinin (DHA)-piperaquine + mefloquine, is a potential treatment option for multidrug-resistant Plasmodium falciparum malaria. In a sequential open-label study, healthy Thai volunteers were treated with DHA-piperaquine (120 to 960 mg), mefloquine (500 mg), and DHA-piperaquine + mefloquine (120 to 960 mg + 500 mg), and serial symptom questionnaires, biochemistry, full blood counts, pharmacokinetic profiles, and electrocardiographic measurements were performed. Fifteen healthy subjects were enrolled. There was no difference in the incidence or severity of adverse events between the three treatment arms. The slight prolongation in QTc (QT interval corrected for heart rate) associated with DHA-piperaquine administration did not increase after administration of DHA-piperaquine + mefloquine. The addition of mefloquine had no significant effect on the pharmacokinetic properties of piperaquine. However, coadministration of mefloquine significantly reduced the exposures to dihydroartemisinin for area under the concentration-time curve (-22.6%; 90% confidence interval [CI], -33.1, -10.4; P = 0.0039) and maximum concentration of drug in serum (-29.0%; 90% CI, -40.6, -15.1; P = 0.0079). Mefloquine can be added safely to dihydroartemisinin-piperaquine in malaria treatment. (This study has been registered at ClinicalTrials.gov under identifier NCT02324738.).

Modeling Prevention of Malaria and Selection of Drug Resistance with Different Dosing Schedules of Dihydroartemisinin-Piperaquine Preventive Therapy during Pregnancy in Uganda.

Dihydroartemisinin-piperaquine (DHA-PQ) is under study for intermittent preventive treatment during pregnancy (IPTp), but it may accelerate selection for drug resistance. Understanding the relationships between piperaquine concentration, prevention of parasitemia, and selection for decreased drug sensitivity can inform control policies and optimization of DHA-PQ dosing. Piperaquine concentrations, measures of parasitemia, and Plasmodium falciparum genotypes associated with decreased aminoquinoline sensitivity in Africa (pfmdr1 86Y, pfcrt 76T) were obtained from pregnant Ugandan women randomized to IPTp with sulfadoxine-pyrimethamine (SP) or DHA-PQ. Joint pharmacokinetic/pharmacodynamic models described relationships between piperaquine concentration and the probability of genotypes of interest using nonlinear mixed effects modeling. An increase in the piperaquine plasma concentration was associated with a log-linear decrease in risk of parasitemia. Our models predicted that higher median piperaquine concentrations would be required to provide 99% protection against mutant infections than against wild-type infections (pfmdr1: N86, 9.6 ng/ml; 86Y, 19.6 ng/ml; pfcrt: K76, 6.5 ng/ml; 76T, 19.6 ng/ml). Comparing monthly, weekly, and daily dosing, daily low-dose DHA-PQ was predicted to result in the fewest infections and the fewest mutant infections per 1,000 pregnancies (predicted mutant infections for pfmdr1 86Y: SP monthly, 607; DHA-PQ monthly, 198; DHA-PQ daily, 1; for pfcrt 76T: SP monthly, 1,564; DHA-PQ monthly, 283; DHA-PQ daily, 1). Our models predict that higher piperaquine concentrations are needed to prevent infections with the pfmdr1/pfcrt mutant compared to those with wild-type parasites and that, despite selection for mutants by DHA-PQ, the overall burden of mutant infections is lower for IPTp with DHA-PQ than for IPTp with SP. (This study has been registered at ClinicalTrials.gov under identifier NCT02282293.).

Improving Methods for Analyzing Antimalarial Drug Efficacy Trials: Molecular Correction Based on Length-Polymorphic Markers msp-1, msp-2, and glurp.

Drug efficacy trials monitor the continued efficacy of front-line drugs against falciparum malaria. Overestimating efficacy results in a country retaining a failing drug as first-line treatment with associated increases in morbidity and mortality, while underestimating drug effectiveness leads to removal of an effective treatment with substantial practical and economic implications. Trials are challenging: they require long durations of follow-up to detect drug failures, and patients are frequently reinfected during that period. Molecular correction based on parasite genotypes distinguishes reinfections from drug failures to ensure the accuracy of failure rate estimates. Several molecular correction "algorithms" have been proposed, but which is most accurate and/or robust remains unknown. We used pharmacological modeling to simulate parasite dynamics and genetic signals that occur in patients enrolled in malaria drug clinical trials. We compared estimates of treatment failure obtained from a selection of proposed molecular correction algorithms against the known "true" failure rate in the model. Our findings are as follows. (i) Molecular correction is essential to avoid substantial overestimates of drug failure rates. (ii) The current WHO-recommended algorithm consistently underestimates the true failure rate. (iii) Newly proposed algorithms produce more accurate failure rate estimates; the most accurate algorithm depends on the choice of drug, trial follow-up length, and transmission intensity. (iv) Long durations of patient follow-up may be counterproductive; large numbers of new infections accumulate and may be misclassified, overestimating drug failure rate. (v) Our model was highly consistent with existing in vivo data. The current WHO-recommended method for molecular correction and analysis of clinical trials should be reevaluated and updated.

Pharmacokinetics/pharmacodynamics of chloroquine and artemisinin-based combination therapy with primaquine.

BACKGROUND: Activation of hypnozoites of vivax malaria causes multiple clinical relapses, which contribute to the Plasmodium vivax burden and continuing transmission. Artemisinin-based combination therapy (ACT) is effective against blood-stage P. vivax but requires co-administration with primaquine to achieve radical cure. The therapeutic efficacy of primaquine depends on the generation of a therapeutically active metabolite via cytochrome P450 2D6 (CYP2D6). Impaired CYP2D6 metabolism has been associated with primaquine treatment failure. This study investigated the association between impaired CYP2D6 genotypes, drug-exposure to the long-acting ACT component (schizonticidal drugs) and tolerance and efficacy. METHODS: Adult patients with acute vivax malaria were enrolled in a recently completed trial and treated with artesunate-mefloquine, chloroquine or artemether-lumefantrine. All received concomitant primaquine (0.5 mg/kg/day for 7-9 days). The association between efficacy and safety and drug exposure was explored using area-under-the-curve (AUC) and half-life (t1/2) estimates obtained by non-compartmental analysis of the long half-life drugs. Parasite recurrences by day 63 were categorized as related relapses or re-infections/unrelated hypnozoite activation by genotyping three microsatellite loci and two polymorphic loci of merozoite surface antigen-1. The CYP2D6 genotype was identified with Taqman assays by real-time PCR to 9 polymorphisms (8 SNPs and one deletion). Impaired CYP2D6 activity was inferred using the Activity Score System. RESULTS: Most recurrences in the ASMQ (67%), CQ (80%) and AL (85%) groups were considered related relapses. Eight of nine (88.9%) of the patients with impaired CYP2D6 activity relapsed with related parasite compared to 18/25 (72%) with normal activity (RR = 1.23, 0.88; 1.72, p = 0.40). There were no associations between the measured PK parameters and recurrence. Patients with longer chloroquine half-lives had more pruritus (RR = 1.09, 1.03; 1.14, p = 0.001). Higher CQ AUCs were associated with reduced falls in haemoglobin by day 14 (Coef - 0.02, - 0.005; - 0.03, p = 0.01). All regimens were well tolerated. CONCLUSION: Genotyping of P. vivax showed that activation of related (homologous) hypnozoites was the most frequent cause of recurrence. The high proportion of the impaired CYP2D6 activity among patients with recurrent infections suggests that slow primaquine metabolism might influence related relapse rates in Brazil among patients receiving primaquine for radical cure, although confirmatory studies are needed. There was no association between drug exposure of the long-acting ACT component (schizonticidal drugs) and risk of related relapse. ACT was well tolerated. These results provide further re-assurance about the safety and efficacy of ACT when combined with short course primaquine to treat uncomplicated malaria vivax in Brazil. Trial registration RBR-79s56s ( http://www.ensaiosclinicos.gov.br/rg/RBR-79s56s/ ).

A Microbial Transformation Model for Simulating Mammal Metabolism of Artemisinin.

Artemisinin (ART) is a highly effective antimalarial agent isolated from the traditional Chinese herb Qinghao. Metabolism of ART and its derivatives in the body is one of the most pressing issues for pharmaceutical scientists. Herein, an efficient in vitro microorganism model for simulation of metabolism of ART in vivo was developed employing Cunninghamella elegans. Metabolites in the microbial transformation system and plasma of mice pre-administrated ART orally were analyzed by ultra-performance liquid chromatography (UPLC)-electrospray ionization (ESI)-quadrupole time-of-flight (Q-TOF)-mass spectrometry (MSE) combined with UNIFI software. Thirty-two metabolites were identified in vitro and 23 were identified in vivo. After comparison, 16 products were found to be common to both models including monohydroxylated ART, dihydroxylated ART, deoxyartemisinin, hydroxylated deoxyartemisinin, hydroxylated dihydroartemisinin (DHA), and hydroxylated deoxy-DHA. These results revealed that C. elegans CICC 40250 functioned as an appropriate model to mimic ART metabolism in vivo. Moreover, an overall description of metabolites of ART from C. elegans CICC 40250 has been provided. Notably, DHA was detected and identified as a metabolite of ART in mouse plasma for the first time.

Population Pharmacokinetics of Artemether, Dihydroartemisinin, and Lumefantrine in Rwandese Pregnant Women Treated for Uncomplicated Plasmodium falciparum Malaria.

The artemisinin-based combination therapy artemether-lumefantrine is commonly used in pregnant malaria patients. However, the effect of pregnancy-related changes on exposure is unclear, and pregnancy has been associated with decreased efficacy in previous studies. This study aimed to characterize the population pharmacokinetics of artemether, its active metabolite dihydroartemisinin, and lumefantrine in 22 Rwandese pregnant women in their second (n = 11) or third (n = 11) trimester with uncomplicated Plasmodium falciparum malaria. These patients were enrolled from Rwamagana district hospital and received the standard fixed oral dose combination of 80 mg of artemether and 480 mg of lumefantrine twice daily for 3 days. Venous plasma concentrations were quantified for all three analytes using liquid chromatography coupled with tandem mass spectroscopy, and data were analyzed using nonlinear mixed-effects modeling. Lumefantrine pharmacokinetics was described by a flexible but highly variable absorption, with a mean absorption time of 4.04 h, followed by a biphasic disposition model. The median area under the concentration-time curve from 0 h to infinity (AUC0-∞) for lumefantrine was 641 h · mg/liter. Model-based simulations indicated that 11.7% of the study population did not attain the target day 7 plasma concentration (280 ng/ml), a threshold associated with increased risk of recrudescence. The pharmacokinetics of artemether was time dependent, and the autoinduction of its clearance was described using an enzyme turnover model. The turnover half-life was predicted to be 30.4 h. The typical oral clearance, which started at 467 liters/h, increased 1.43-fold at the end of treatment. Simulations suggested that lumefantrine pharmacokinetic target attainment appeared to be reassuring in Rwandese pregnant women, particularly compared to target attainment in Southeast Asia. Larger cohorts will be required to confirm this finding.

Efficacy and Tolerability Outcomes of a Phase II, Randomized, Open-Label, Multicenter Study of a New Water-Dispersible Pediatric Formulation of Dihydroartemisinin-Piperaquine for the Treatment of Uncomplicated Plasmodium falciparum Malaria in African Infants.

Artemisinin combination therapies are considered the mainstay of malaria treatment, but pediatric-friendly formulations for the treatment of infants are scarce. We sought to evaluate the efficacy and safety of a new dispersible-tablet formulation of dihydroartemisinin/piperaquine phosphate (DHA/PQP) in comparison to the marketed tablet (Eurartesim) in the treatment of infants with uncomplicated Plasmodium falciparum malaria. Reported here are the results of a large phase II, randomized, open-label, multicenter trial conducted in African infants (6 to 12 months of age) from Mozambique, Burkina Faso, The Gambia, the Democratic Republic of the Congo, and Tanzania. Primary efficacy endpoint was the PCR-corrected adequate clinical and parasitological response (ACPR) at day 28. Analysis was performed for the intention-to-treat (ITT) and per-protocol (PP) populations. A total of 201 patients received the dispersible-tablet formulation, and 99 received the conventional one administered as crushed tablets. At day 28, the PCR-corrected ACPRs were 86.9% (ITT) and 98.3% (PP) in the dispersible-tablet group and 84.9% (ITT) and 100% (PP) in the crushed-tablet group. At day 42, these values were 85.9% (ITT) and 96.5% (PP) in the dispersible-tablet group and 82.8% (ITT) and 96.4% (PP) in the crushed-tablet group. The comparison between survival curves for time to new infections showed no statistically significant differences (P = 0.409). The safety and tolerability profile for the two groups was similar in terms of type and frequency of adverse events and was consistent with that expected in African infants with malaria. A standard 3-day treatment with the new dispersible DHA/PQP formulation is as efficacious as the currently used tablet in African infants and has a comparable safety profile. (This trial was registered at ClinicalTrials.gov under registration no. NCT01992900.).

Pharmacokinetics and Safety Profile of Artesunate-Amodiaquine Coadministered with Antiretroviral Therapy in Malaria-Uninfected HIV-Positive Malawian Adults.

There are limited data on the pharmacokinetic and safety profiles of artesunate-amodiaquine in human immnunodeficiency virus-infected (HIV+) individuals receiving antiretroviral therapy. In a two-step intensive sampling pharmacokinetic trial, we compared the area under the concentration-time curve from 0 to 28 days (AUC0-28) of an active metabolite of amodiaquine, desethylamodiaquine, and treatment-emergent adverse events between antiretroviral therapy-naive HIV+ adults and those taking nevirapine and ritonavir-boosted lopinavir-based antiretroviral therapy. In step 1, malaria-uninfected adults (n = 6/arm) received half the standard adult treatment regimen of artesunate-amodiaquine. In step 2, another cohort (n = 25/arm) received the full regimen. In step 1, there were no safety signals or significant differences in desethylamodiaquine AUC0-28 among participants in the ritonavir-boosted lopinavir, nevirapine, and antiretroviral therapy-naive arms. In step 2, compared with those in the antiretroviral therapy-naive arm, participants in the ritonavir-boosted lopinavir arm had 51% lower desethylamodiaquine AUC0-28, with the following geometric means (95% confidence intervals [CIs]): 23,822 (17,458 to 32,506) versus 48,617 (40,787 to 57,950) ng · h/ml (P < 0.001). No significant differences in AUC0-28 were observed between nevirapine and antiretroviral therapy-naive arms. Treatment-emergent transaminitis was higher in the nevirapine (20% [5/25]) than the antiretroviral therapy-naive (0.0% [0/25]) arm (risk difference, 20% [95% CI, 4.3 to 35.7]; P = 0.018). The ritonavir-boosted lopinavir antiretroviral regimen was associated with reduced desethylamodiaquine exposure, which may compromise artesunate-amodiaquine's efficacy. Coadministration of nevirapine and artesunate-amodiaquine may be associated with hepatoxicity.