artelinic-acid and artemisinin

The appearance of Plasmodium falciparum parasites with decreased in vivo sensitivity but no measurable in vitro resistance to artemisinin has raised the urgent need to characterize the artemisinin resistance phenotype. Changes in the temporary growth arrest (dormancy) profile of parasites may be one aspect of this phenotype. In this study, we investigated the link between dormancy and resistance, using artelinic acid (AL)-resistant parasites. Our results demonstrate that the AL resistance phenotype has (i) decreased sensitivity of mature-stage parasites, (ii) decreased sensitivity of the ring stage to the induction of dormancy, and (iii) a faster recovery from dormancy.

Topics: Animals; Antimalarials; Artemisinins; Cell Culture Techniques; Drug Resistance; Erythrocytes; Genotype; Humans; Inhibitory Concentration 50; Life Cycle Stages; Malaria, Falciparum; Microscopy; Parasitic Sensitivity Tests; Phenotype; Plasmodium falciparum; Recurrence

Emergence of artemisinin resistance in Cambodia highlights the importance of characterizing resistance to this class of drugs. Previously, intermediate levels of resistance in Plasmodium falciparum were generated in vitro for artelinic acid (AL) and artemisinin (QHS). Here we expanded on earlier selection efforts to produce levels of clinically relevant concentrations, and the resulting lines were characterized genotypically and phenotypically. Recrudescence assays determined the ability of resistant and parent lines to recover following exposure to clinically relevant levels of drugs. Interestingly, the parent clone (D6) tolerated up to 1,500 ng/ml QHS, but the resistant parasite, D6.QHS340×3, recovered following exposure to 2,400 ng/ml QHS. Resistant D6, W2, and TM91c235 parasites all exhibited elevated 50% inhibitory concentrations (IC(50)s) to multiple artemisinin drugs, with >3-fold resistance to QHS and AL; however, the degree of resistance obtained with standard methods was remarkably less than expected for parasite lines that recovered from 2,400-ng/ml drug pressure. A novel assay format with radiolabeled hypoxanthine demonstrated a greater degree of resistance in vitro than the standard SYBR green method. Analysis of merozoite number in resistant parasites found D6 and TM91c235 resistant progeny had significantly fewer merozoites than parent strains, whereas W2 resistant progeny had significantly more. Amplification of pfmdr1 increased proportionately to the increased drug levels tolerated by W2 and TM91c235, but not in resistant D6. In summary, we define the artemisinin resistance phenotype as a decrease in susceptibility to artemisinins along with the ability to recover from drug-induced dormancy following supraclinical concentrations of the drug.

Topics: Antimalarials; Artemisinins; Benzothiazoles; Cell Culture Techniques; Diamines; Dose-Response Relationship, Drug; Drug Resistance; Erythrocytes; Gene Dosage; Genotype; Humans; Hypoxanthine; Inhibitory Concentration 50; Malaria, Falciparum; Merozoites; Microscopy; Multidrug Resistance-Associated Proteins; Organic Chemicals; Parasitic Sensitivity Tests; Phenotype; Plasmodium falciparum; Quinolines; Recurrence

Artemisinin and its derivatives are the most rapidly acting and efficacious antimalarial drugs currently available. Although resistance to these drugs has not been documented, there is growing concern about the potential for resistance to develop. In this paper we report the selection of parasite resistance to artelinic acid (AL) and artemisinin (QHS) in vitro and the molecular changes that occurred during the selection. Exposure of three Plasmodium falciparum lines (W2, D6, and TM91C235) to AL resulted in decreases in parasite susceptibilities to AL and QHS, as well as to mefloquine, quinine, halofantrine, and lumefantrine. The changes in parasite susceptibility were accompanied by increases in the copy number, mRNA expression, and protein expression of the pfmdr1 gene in the resistant progenies of W2 and TM91C235 parasites but not in those of D6 parasites. No changes were detected in the coding sequences of the pfmdr1, pfcrt, pfatp6, pftctp, and pfubcth genes or in the expression levels of pfatp6 and pftctp. Our data demonstrate that P. falciparum lines have the capacity to develop resistance to artemisinin derivatives in vitro and that this resistance is achieved by multiple mechanisms, to include amplification and increased expression of pfmdr1, a mechanism that also confers resistance to mefloquine. This observation is of practical importance, because artemisinin drugs are often used in combination with mefloquine for the treatment of malaria.

Topics: Alleles; Animals; Antimalarials; Artemisinins; Drug Resistance; Gene Amplification; Gene Dosage; Gene Expression; Genes, Protozoan; Humans; In Vitro Techniques; Malaria, Falciparum; Mefloquine; Multidrug Resistance-Associated Proteins; Plasmodium falciparum; RNA, Messenger; RNA, Protozoan

We have produced monoclonal antibodies against artelinic acid and investigated the reactivity with artemisinin drugs and metabolites. Antibody F170-10 is fairly specific for artelinic acid but does bind artemisinin and artemether (3-5% cross-reactivity). Dihydroartemisinin, artesunate, and metabolites of artemisinin showed less reactivity. With this antibody, an inhibition ELISA has been set up to detect artemisinin compounds in urine. In healthy subjects who received a single oral dose of artemisinin, artemether, artesunate or dihydroartemisinin, ELISA reactivity in urine was found. This reactivity in urine paralleled the plasma concentrations of artemether and dihydroartemisinin. The results show that this immunoassay for artelinic acid can be used to detect artemisinin compounds in urine for about 8 hr after intake. With a more sensitive test, this simple method as a urine dipstick may be become useful for drug use and compliance studies in malaria-endemic areas where the artemisinin derivatives are increasingly used.

Topics: Adult; Antibodies, Monoclonal; Antimalarials; Artemisinins; Artesunate; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Humans; Sesquiterpenes

To search for water soluble dihydroartemisinin derivatives with higher efficacy and longer plasma half-life than artesunic or artelinic acid, a series of new stereoisomers of 4-(p-substituted phenyl)-4(R or S)-[10(alpha or beta)-dihydroartemisininoxy]butyric acids were synthesized as new potential antimalarial agents. Two approaches were taken in the design of these new molecules in an attempt to (a) increase the lipophilicity of the molecule and (b) decrease the rate of oxidative dealkylation of the target compounds. The new compounds showed a 2-10-fold increase in in vitro antimalarial activity against D-6 and W-2 clones of Plasmodium falciparum than artemisinin or artelinic acid. R-diastereomers are, in general, more potent than the corresponding S-diastereomers. p-Chlorophenyl and p-bromophenyl derivatives showed in vivo oral antimalarial activity against P. berghei (with 3/8 cured) superior to that of artelinic acid (1/8 cured), whereas p-fluorophenyl and p-methoxyphenyl analogs demonstrated activity only comparable (1/8 cured) to that of artelinic acid at the same dosage level (64 mg/kg twice a day). The in vivo antimalarial activity of these new compounds correlates with their SD50 (50% parasitemia suppression dose). The biological results suggested that an electronic effect, besides the lipophylicity, may play a role in determining the efficacy of this class of compounds.

Topics: Animals; Antimalarials; Artemisinins; Drug Design; Female; Magnetic Resonance Spectroscopy; Malaria; Mice; Molecular Conformation; Molecular Structure; Parasitemia; Plasmodium berghei; Plasmodium falciparum; Sesquiterpenes; Solubility

In addition to artelinic acid, which was demonstrated previously to possess good prophylactic as well as curative antimalarial activity against Plasmodium berghei by transdermal administration, seven artemisinin derivatives in a gel formulation were assessed for their antimalarial activities in this study. Artemisinin, the parent compound of the series, showed moderate prophylactic but poor curative activity. Although methyl artelinate was more active against P. berghei than artelinic acid and sodium artelinate by subcutaneous injection, its transdermal curative and prophylactic activity was only comparable with or weaker than that of artelinic acid. Conversely, both dihydroartemisinin trimethylsilyl ether and dehydrodihydroartemisinin showed weaker antimalarial activity than artelinic acid by the subcutaneous route, yet exhibited comparable activity by transdermal administration. Artemether, a prodrug of dihydroartemisinin, is as effective as the parent dihydroartemisinin, and both compounds were the most potent agents among the compounds studied, with total prophylactic and curative doses of 30 mg/kg and 60 mg/kg, respectively. Complete absorption of dihydroartemisinin appears to occur within 5 min after application. In general, we found that the prophylactic dose is about half that of the curative dose under the protocols used in this study. This novel drug delivery system may be an easy and safe way to administer artemisinin-type antimalarials and also a good alternative dosage form for active compounds with solubility problems.

Topics: Administration, Cutaneous; Animals; Antimalarials; Artemether; Artemisinins; Gels; Malaria; Mice; Plasmodium berghei; Sesquiterpenes; Structure-Activity Relationship

The efficacy of artelinic acid and artemisinin, orally administered at 10 and 50 mg kg-1 day-1, was compared in Plasmodium berghei infected mice. Subsequently, the pharmacokinetics of artelinic acid after intravenous, intramuscular, oral and rectal administration of a 20 mg kg-1 aqueous solution to rabbits were studied in a four-way randomized cross-over experiment. After intravenous administration, artelinic acid concentrations in blood plasma were high (C0: 76 +/- 15 mg L-1), and the drug was rapidly eliminated from the central compartment, showing linear elimination kinetics with an elimination half-life of 15 +/- 3 min. A large inter-subject variation appeared in the absorption rate and the extent of absorption (2-92%) over the 120 min interval after intramuscular administration. Also, a large inter-subject variation in individual rectal bioavailability (17-100%) was shown, which was dependent on the site of absorption in the rectum. The estimated oral bioavailability was low (4.6 +/- 1.7%), probably due to a high first-pass effect and possible decomposition in the acidic gastric environment.

Topics: Absorption; Administration, Oral; Administration, Rectal; Animals; Antimalarials; Artemisinins; Biological Availability; Injections, Intramuscular; Injections, Intravenous; Malaria; Mice; Plasmodium berghei; Sesquiterpenes

The in vitro activities of chloroquine, quinine, mefloquine, halofantrine, artemisinin, arteether, artemether, and artelinate were evaluated against African clones and isolates of Plasmodium falciparum, using an isotopic, semimicro, drug susceptibility test. The chloroquine-resistant FCM 29 clone was 1.6 and 6.2 times more susceptible to artemisinin when compared with the chloroquine-susceptible, mefloquine-, and halofantrine-resistant L-3 and L-16 clones, respectively. Cross-resistance patterns between the standard antimalarial drugs and artemisinin were determined against 36 African isolates of P. falciparum obtained from imported cases of malaria in France. Chloroquine-resistant isolates (n = 21) were significantly more susceptible to artemisinin (50% inhibitory concentration [IC50] 7.67 nM), arteether (IC50 3.88 nM), artemether (IC50 3.71 nM), and artelinate (IC50 3.46 nM), as compared with the 15 chloroquine-susceptible isolates (IC50 11.4, 5.66, 5.14, and 5.04 nM, respectively). Arteether, artemether, and artelinate were equally effective and twice as potent as artemisinin. A significant positive correlation was found between artemisinin and mefloquine (r = 0.424, P = 0.022), artemisinin and halofantrine (r = 0.569, P < 0.001), chloroquine and quinine (r = 0.651, P < 0.001), and mefloquine and halofantrine (r = 0.863, P < 0.001), suggesting in vitro cross-resistance among these drugs. The present in vitro findings require confirmation in clinical studies.

Topics: Animals; Antimalarials; Artemether; Artemisinins; Chloroquine; Drug Resistance; Mefloquine; Phenanthrenes; Plasmodium falciparum; Quinine; Sesquiterpenes

1. In this communication, induction of hydrogen peroxide production by the semisynthetic antimalarial drugs of the artemisinin class (beta-arteether, beta-artelinic acid and dihydroartemisinin) in rat liver microsomes, is reported. 2. Endogenous, NADPH-dependent, production of hydrogen peroxide in rat liver microsomes was enhanced in the presence of arteether and artelinic acid, but not in the presence of dihydroartemisinin. 3. NADPH-dependent metabolism of arteether and artelinic acid was closely coupled to the drug-induced production of hydrogen peroxide. 4. The redox cycle of cytochrome P-450 was presented, which describes satisfactorily both the endogenous and the drug-assisted hydrogen peroxide production in rat liver microsomes; also, the rate-limiting step of the cycle was identified.

Topics: Animals; Antimalarials; Artemisinins; Hydrogen Peroxide; Kinetics; Male; Microsomes, Liver; NADP; Rats; Rats, Inbred Strains; Sesquiterpenes

alpha-Artelinic acid [8], a potent, stable, and water-soluble antimalarial agent, has been synthesized from artemisinin [1], the sesquiterpene lactone endoperoxide isolated from Artemisia annua. The blood schizontocidal antimalarial activity of alpha-artelinic acid evaluated against Plasmodium knowlesi is also reported.

Topics: Animals; Antimalarials; Artemisinins; Macaca mulatta; Malaria; Plasmodium knowlesi; Sesquiterpenes

Sodium artelinate, a new water-soluble and relatively stable derivative of artemisinin, and its parent compound were tested for their antimalarial action. Experiments were done in vitro with synchronous cultures of Plasmodium berghei. The inhibition of growth by different concentrations of sodium artelinate and artemisinin was determined using flow cytometry. In vivo testing was done by subcutaneous injection of each drug in mice infected with P. berghei. Sodium artelinate, being stable in aqueous solution, was also administered orally to infected mice by its addition to their drinking water. Comparison of the parent compound and the derivative showed that sodium artelinate was slightly less active than artemisinin both in culture and in vivo. However, after oral administration of sodium artelinate, parasites were cleared from the blood with one-half to one-tenth of the dose used in the experiments with subcutaneous injection. The number of mice which were cured by oral administration of sodium artelinate was greater than after subcutaneous injection, even with a total oral dose lower than the injected dose.

Topics: Administration, Oral; Animals; Antimalarials; Artemisinins; DNA; Drugs, Chinese Herbal; Flow Cytometry; Injections, Subcutaneous; Malaria; Mice; Plasmodium berghei; Sesquiterpenes

A reversed-phase high-performance liquid chromatographic method is described for the analysis of the new antimalarial drug artelinic acid in blood plasma. The influence of mobile phase composition, pH and type of mobile phase modifier on the retention of artelinic acid on the reversed-phase column is reported. Linear calibration curves were obtained in the range 0-500 ng/ml artelinic acid. Intra-assay and inter-assay variability in the analysis of plasma samples spiked with the drug were less than or equal to 15%. Plasma samples of the drug were found to be unstable when stored at -20 degrees C, the concentration of the drug decreasing by over 50% within three days. Plasma samples stored at -70 degrees C remained stable for at least two weeks. Initial pharmacokinetic studies in the rat showed that following intravenous administration, plasma concentrations of artelinic acid declined mono-exponentially. The relatively short elimination half-life (17 +/- 5 min) of artelinic acid is consistent with what is known for qinghaosu and its derivatives.

Topics: Animals; Antimalarials; Artemisinins; Chemical Phenomena; Chemistry; Chromatography, High Pressure Liquid; Humans; Hydrogen-Ion Concentration; Male; Mass Spectrometry; Rats; Rats, Inbred Strains; Sesquiterpenes; Spectrophotometry, Ultraviolet