lumefantrine and halofantrine

Techniques enabling

Topics: Administration, Oral; Aminoquinolines; Animals; Biological Availability; Clofazimine; Digestion; Drug Compounding; Drug Delivery Systems; Ferrous Compounds; Infant Formula; Lipolysis; Lumefantrine; Metallocenes; Milk; Phenanthrenes; Scattering, Small Angle; Solubility; Spectrum Analysis, Raman; Suspensions; Water; X-Ray Diffraction

Lumefantrine is the mainstay of anti-malarial combination therapy in most endemic countries presently. However, it cannot be used alone owing to its long onset time of action. CDRI 97-78 is a promising trioxane-derivative anti-malarial candidate that is currently being investigated as a substitute for artemisinin derivatives owing to their emerging resistance.. In the present study, a sensitive, simple and rapid high-performance liquid chromatography coupled with positive ion electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the simultaneous determination of lumefantrine and CDRI 97-78's metabolite, 97-63, in rat plasma using halofantrine as an internal standard. Lumefantrine and 97-63 were separated on a Waters Atlantis C18 (4.6×50 mm, 5.0 μm) column under isocratic condition with mobile phase consisting of acetonitrile: methanol (50:50, v/v) and ammonium formate buffer (10 mM, pH 4.5) in the ratio of 95:5 (v/v) at a flow rate of 0.65 mL/min.. The method was accurate and precise within the linearity range 3.9-500 ng/mL for both lumefantrine and 97-63 with a correlation coefficient (r2) of ≥0.998. The intra- and inter-day assay precision ranged from 2.24 to 7.14% and 3.97 to 5.90%, and intra- and inter-day assay accuracy was between 94.93 and 109.51% and 96.87 and 108.38%, respectively, for both the analytes. Upon coadministration of 97-78, the relative bioavailability of lumefantrine significantly decreased to 64.41%.. A highly sensitive, specific and reproducible high-throughput LC-ESI-MS/MS assay was developed and validated to quantify lumefantrine and CDRI 97-78. The method was successfully applied to study the effect of oral co-administration of lumefantrine on the pharmacokinetics of 97-78 in male Sprague-Dawley rats and vice versa. Co-administration of 97-78 significantly decreased the systemic exposure of lumefantrine.

Topics: Animals; Antimalarials; Blood Chemical Analysis; Bridged Bicyclo Compounds, Heterocyclic; Chromatography, High Pressure Liquid; Drug Combinations; Ethanolamines; Fluorenes; Lumefantrine; Male; Phenanthrenes; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

The pharmacokinetic compatibility of short-acting CDRI candidate antimalarial trioxane derivative, 99-411, was tested with long-acting prescription antimalarials, lumefantrine and piperaquine. LC-ESI-MS/MS methods were validated for simultaneous bioanalysis of lumefantrine and 99-411 and of piperaquine and 99-411 combinations. The interaction studies were performed in rats using these validated methods. The total systemic exposure of 99-411 increased when administered with either lumefantrine or piperaquine. However, co-administration of 99-411 significantly decreased the systemic exposure of piperaquine by half-fold while it had no effect on the kinetics of lumefantrine. 99-411, thus, seemed to be a good alternative to artemisinin derivatives for combination treatment with lumefantrine. To explore the reason for increased plasma levels of 99-411, an in situ permeability study was performed by co-perfusing lumefantrine and 99-411. In presence of lumefantrine, the absorption of 99-411 was significantly increased by 1.37 times than when given alone. Lumefantrine did not affect the metabolism of 99-411 when tested in vitro in human liver microsomes. Additionally, ATPase assay suggest that 99-411 was a substrate of human P-gp, thus, indicating the probability of interaction at the absorption level in humans as well.

Topics: Animals; Antimalarials; Chromatography, High Pressure Liquid; Ethanolamines; Fluorenes; Half-Life; Heterocyclic Compounds; Humans; Lumefantrine; Microsomes, Liver; Phenanthrenes; Quinolines; Rats; Spiro Compounds; Tandem Mass Spectrometry

Malaria's ability to rapidly adapt to new drugs has allowed it to remain one of the most devastating infectious diseases of humans. Understanding and tracking the genetic basis of these adaptations are critical to the success of treatment and intervention strategies. The novel antimalarial resistance locus PF10_0355 (Pfmspdbl2) was previously associated with the parasite response to halofantrine, and functional validation confirmed that overexpression of this gene lowered parasite sensitivity to both halofantrine and the structurally related antimalarials mefloquine and lumefantrine, predominantly through copy number variation. Here we further characterize the role of Pfmspdbl2 in mediating the antimalarial drug response of Plasmodium falciparum. Knockout of Pfmspdbl2 increased parasite sensitivity to halofantrine, mefloquine, and lumefantrine but not to unrelated antimalarials, further suggesting that this gene mediates the parasite response to a specific class of antimalarial drugs. A single nucleotide polymorphism encoding a C591S mutation within Pfmspdbl2 had the strongest association with halofantrine sensitivity and showed a high derived allele frequency among Senegalese parasites. Transgenic parasites expressing the ancestral Pfmspdbl2 allele were more sensitive to halofantrine and structurally related antimalarials than were parasites expressing the derived allele, revealing an allele-specific effect on drug sensitivity in the absence of copy number effects. Finally, growth competition experiments showed that under drug pressure, parasites expressing the derived allele of Pfmspdbl2 outcompeted parasites expressing the ancestral allele within a few generations. Together, these experiments demonstrate that modulation of Pfmspdbl2 affects malaria parasite responses to antimalarial drugs.

Topics: Antimalarials; Base Sequence; DNA Copy Number Variations; Drug Resistance; Ethanolamines; Fluorenes; Gene Dosage; Gene Frequency; Genes, Protozoan; Lumefantrine; Malaria, Falciparum; Mefloquine; Mutation; Parasitic Sensitivity Tests; Phenanthrenes; Plasmodium falciparum; Polymorphism, Single Nucleotide; Protozoan Proteins; Sequence Analysis, DNA

Artemether-lumefantrine (ARM-LUM) has in recent years become the first-line treatment for uncomplicated malaria in many Sub-Saharan African countries. Vigorous monitoring of the therapeutic efficacy of this treatment is needed. This requires high-quality studies following standard protocols; ideally, such studies should incorporate measurement of drug levels in the study patients to exclude the possibility that insufficient drug levels explain an observed treatment failure. Several methods for measuring lumefantrine (LUM) in plasma by HPLC are available; however, several of these methods have some limitations in terms of high costs and limited feasibility arising from large required sample volumes and demanding sample preparation. Therefore, we set out to develop a simpler reversed phase high performance liquid chromatography (RP-HPLC) method based on UV detection for simultaneous measurement of LUM and its major metabolite the desbutyl LUM (DL) in plasma. Halofantrine was used as an internal standard. Liquid-liquid extraction of samples was carried out using hexane-ethyl acetate (70:30, v/v). Chromatographic separation was carried out on a Synergi Polar-RP column (250 mm × 300 mm, particle size 4 μm). The mobile phase consisted of acetonitrile-0.1M ammonium acetate buffer adjusted to pH 4.9 (85:15%, v/v). Absorbance of the compounds was monitored at 335 nm using a reference wavelength of 360 nm. Absolute extraction recovery for LUM and DL were 88% and 90%, respectively. Inter- and intraday coefficients of variation for LUM and DL were ≤ 10%. The lower limits of quantification for LUM and DL were 12.5 and 6.5 ng/ml, respectively. After validation, the methodology was transferred to a local laboratory in Tanga Tanzania and samples from a small subset of malaria patients were analysed for LUM. The method appears to be applicable in settings with limited facilities.

Topics: Acetates; Artemether; Artemisinins; Calibration; Chemistry Techniques, Analytical; Chromatography; Chromatography, High Pressure Liquid; Ethanolamines; Fluorenes; Hexanes; Kinetics; Lumefantrine; Models, Chemical; Phenanthrenes; Regression Analysis; Reproducibility of Results; Spectrophotometry, Ultraviolet; Ultraviolet Rays

The Plasmodium falciparum parasite's ability to adapt to environmental pressures, such as the human immune system and antimalarial drugs, makes malaria an enduring burden to public health. Understanding the genetic basis of these adaptations is critical to intervening successfully against malaria. To that end, we created a high-density genotyping array that assays over 17,000 single nucleotide polymorphisms (∼ 1 SNP/kb), and applied it to 57 culture-adapted parasites from three continents. We characterized genome-wide genetic diversity within and between populations and identified numerous loci with signals of natural selection, suggesting their role in recent adaptation. In addition, we performed a genome-wide association study (GWAS), searching for loci correlated with resistance to thirteen antimalarials; we detected both known and novel resistance loci, including a new halofantrine resistance locus, PF10_0355. Through functional testing we demonstrated that PF10_0355 overexpression decreases sensitivity to halofantrine, mefloquine, and lumefantrine, but not to structurally unrelated antimalarials, and that increased gene copy number mediates resistance. Our GWAS and follow-on functional validation demonstrate the potential of genome-wide studies to elucidate functionally important loci in the malaria parasite genome.

Topics: Antimalarials; Drug Resistance; Ethanolamines; Fluorenes; Gene Dosage; Gene Expression; Genetic Association Studies; Genetic Loci; Genetic Variation; Genotype; Haplotypes; Linkage Disequilibrium; Lumefantrine; Malaria, Falciparum; Mefloquine; Phenanthrenes; Plasmodium falciparum; Polymorphism, Single Nucleotide; Selection, Genetic

Resistance to the amino alcohol quinine has been associated with polymorphisms in pfnhe, a sodium hydrogen exchanger. We investigated the role of this gene in quinine resistance in vitro in isolates from Kenya. We analyzed pfnhe whole-gene polymorphisms, using capillary sequencing, and pfcrt at codon 76 (pfcrt-76) and pfmdr1 at codon 86 (pfmdr1-86), using PCR-enzyme restriction methodology, in 29 isolates from Kilifi, Kenya, for association with the in vitro activities of quinine and 2 amino alcohols, mefloquine and halofantrine. In vitro activity was assessed as the drug concentration that inhibits 50% of parasite growth (IC50). The median IC50s of quinine, halofantrine, and mefloquine were 92, 22, and 18 nM, respectively. The presence of 2 DNNND repeats in microsatellite ms4760 of pfnhe was associated with reduced susceptibility to quinine (60 versus 227 nM for 1 and 2 repeats, respectively; P<0.05), while 3 repeats were associated with restoration of susceptibility. The decrease in susceptibility conferred by the 2 DNNND repeats was more pronounced in parasites harboring the pfmdr1-86 mutation. No association was found between susceptibility to quinine and the pfcrt-76 mutation or between susceptibility to mefloquine or halofantrine and the pfnhe gene and the pfcrt-76 and pfmdr1-86 mutations. Using previously published data on the in vitro activities of chloroquine, lumefantrine, piperaquine, and dihydroartemisinin, we investigated the association of their activities with pfnhe polymorphism. With the exception of a modulation of the activity of lumefantrine by a mutation at position 1437, pfnhe did not modulate their activities. Two DNNND repeats combined with the pfmdr1-86 mutation could be used as an indicator of reduced susceptibility to quinine.

Topics: Amino Acid Sequence; Animals; Antimalarials; Drug Resistance; Humans; Kenya; Mefloquine; Membrane Transport Proteins; Molecular Sequence Data; Multidrug Resistance-Associated Proteins; Parasitic Sensitivity Tests; Phenanthrenes; Plasmodium falciparum; Polymorphism, Genetic; Protozoan Proteins; Quinine; Sequence Analysis, DNA; Sodium-Hydrogen Exchangers

A simple, sensitive and rapid method for the analysis of lumefantrine in rat plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed. Detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The method included a chromatographic run of 5 min using a C(18) analytical column and the calibration curve was linear over the concentration range of 2-500 ng/mL with a correlation coefficient (r) of 0.996 or better. The intra- and inter-day assay precision ranged from 1.5 to 7.5% and 5.5 to 7.7%, respectively, and intra- and inter-day assay accuracy was between 91.3-109.7% and 97.0-104.7%, respectively. The method was successfully applied for the pharmacokinetic study in rats.

Topics: Animals; Antimalarials; Calibration; Chromatography, High Pressure Liquid; Ethanolamines; Fluorenes; Indicators and Reagents; Lumefantrine; Male; Phenanthrenes; Rats; Rats, Sprague-Dawley; Reference Standards; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization

The global dissemination of drug-resistant Plasmodium falciparum is spurring intense efforts to implement artemisinin (ART)-based combination therapies for malaria, including mefloquine (MFQ)-artesunate and lumefantrine (LUM)-artemether. Clinical studies have identified an association between an increased risk of MFQ, MFQ-artesunate, and LUM-artemether treatment failures and pfmdr1 gene amplification. To directly address the contribution that pfmdr1 copy number makes to drug resistance, we genetically disrupted 1 of the 2 pfmdr1 copies in the drug-resistant FCB line, which resulted in reduced pfmdr1 mRNA and protein expression. These knockdown clones manifested a 3-fold decrease in MFQ IC(50) values, compared with that for the FCB line, verifying the role played by pfmdr1 expression levels in mediating resistance to MFQ. These clones also showed increased susceptibility to LUM, halofantrine, quinine, and ART. No change was observed for chloroquine. These results highlight the importance of pfmdr1 copy number in determining P. falciparum susceptibility to multiple agents currently being used to combat malaria caused by multidrug-resistant parasites.

Topics: Animals; Antimalarials; Artemisinins; ATP-Binding Cassette Transporters; DNA, Protozoan; Drug Resistance, Multiple; Ethanolamines; Fluorenes; Genes, MDR; Inhibitory Concentration 50; Lumefantrine; Malaria, Falciparum; Mefloquine; Parasitic Sensitivity Tests; Phenanthrenes; Plasmodium falciparum; Polymerase Chain Reaction; Protozoan Proteins; Quinine; Sesquiterpenes

The effects of antimalarial drugs halofantrine and lumefantrine on the fluoresence anisotropy of diphenylhexatriene (DPH)-containing phospholipid vesicles have been examined. Lumefantrine increases DPH anisotropy, indicating a condensing effect on bilayers of dipalmitoylphosphatidyl choline (DPPC), dioleoylphosphatidylcholine (DOPC), egg lecithin and mouse erythrocyte membranes (including membranes isolated from plasmodial-infected mice). Its condensing effect is more pronounced in bilayers of lower microviscosity. In contrast, increases or decreases in DPH anisotropy are observed with halofantrine, depending on the nature of the lipid. Decreases in anisotropy, which reflect a perturbing effect, are observed in bilayers of high microviscosity (for example, gel state of DPPC bilayers). Increases in anisotropy are observed in bilayers of low microviscosity (such as DOPC and egg lecithin bilayers). The perturbing effect of halofantrine is further confirmed by the increases in permeability of calcein-containing DPPC vesicles in the presence of the drug. However the perturbative effects of halofantrine are observed to the same magnitude in uninfected and plasmodial-infected erythrocyte membranes, and may not be relevant to the antimalarial action of the drug. In contrast, the condensing effect of lumefantrine is significantly greater in infected erythrocyte membranes and may contribute to its antimalarial action.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Antimalarials; Erythrocyte Membrane; Ethanolamines; Fluorenes; Lipid Bilayers; Lumefantrine; Male; Mice; Phenanthrenes; Phosphatidylcholines