Brian L. Robinson

Mechanisms of Artemisinin Resistance in the Rodent Malaria Pathogen Plasmodium yoelii

ABSTRACT Artemisinin and its derivatives are important new antimalarials which are now used widely in Southeast Asia. Clinically relevant artemisinin resistance has not yet been reported but is likely to occur. In order to understand how the malaria parasite might become resistant to this drug, we studied artemisinin resistance in the murine malaria parasite Plasmodium yoelii. The artemisinin-resistant strain (ART), which is approximately fourfold less sensitive to artemisinin than the sensitive NS strain, accumulated 43% less radiolabeled drug in vitro (P < 0.01). Within the parasite, the drug appeared to react with the same parasite proteins in both strains. The translationally controlled tumor protein, one of the artemisinin target proteins, did not differ between the strains. No DNA sequence difference was found, but the resistant strain was found to express 2.5-fold-more protein than the sensitive strain (P < 0.01). Thus, the phenotype of artemisinin resistance in P. yoelii appears to be multifactorial.

Anti-malarial efficacy of pyronaridine and artesunate in combination in vitro and in vivo

Pyronaridine is a Mannich base anti-malarial with demonstrated efficacy against drug resistant Plasmodium falciparum, P. vivax, P. ovale and P. malariae. However, resistance to pyronaridine can develop quickly when it is used alone but can be considerably delayed when it is administered with artesunate in rodent malaria models. The aim of this study was to evaluate the efficacy of pyronaridine in combination with artesunate against P. falciparum in vitro and in rodent malaria models in vivo to support its clinical application. Pyronaridine showed consistently high levels of in vitro activity against a panel of six P. falciparum drug-sensitive and resistant strains (Geometric Mean IC50=2.24 nM, 95% CI=1.20-3.27). In vitro interactions between pyronaridine and artesunate showed a slight antagonistic trend, but in vivo compared to pyronaridine and artesunate administered alone, the 3:1 ratio of the combination, reduced the ED90 of artesunate by approximately 15.6-fold in a pyronaridine-resistant P. berghei line and by approximately 200-fold in an artesunate-resistant line of P. berghei. Complete cure rates were achieved with doses of the combination above or equal to 8 mg/kg per day against P. chabaudi AS. These results indicate that the combination had an enhanced effect over monotherapy and lower daily doses of artesunate could be used to obtain a curative effect. The data suggest that the combination of pyronaridine and artesunate should have potential in areas of multi-drug resistant malaria.

PLASMODIUM YOELII: IDENTIFICATION AND PARTIAL CHARACTERIZATION OF AN MDR1 GENE IN AN ARTEMISININ-RESISTANT LINE

The molecular mechanisms by which the malarial parasite has managed to develop resistance to many antimalarial drugs remain to be completely elucidated. Mutations in the pfmdr1 gene of Plasmodium falciparum, as well as an increase in pfmdr1 copy number, have been associated with resistance to the quinoline-containing antimalarial drugs. We investigated the mechanisms of drug resistance in Plasmodium using a collection of P. yoelii lines with different drug resistance profiles. The mdr1 gene of P. yoelii (pymdr1) was identified and characterized. A 2- to 3-fold increase in the pymdr1 gene copy number was observed in the P. yoelii ART line (artemisinin resistant) when compared with the NS parental line. The pymdr1 gene was mapped to a chromosome of 2.1 Mb in all lines analyzed. Reverse transcriptase–polymerase chain reaction and Western blot experiments confirmed the expression of the gene at the RNA and protein levels.

Convenient Access Both to Highly Antimalaria‐Active 10‐Arylaminoartemisinins, and to 10‐Alkyl Ethers Including Artemether, Arteether, and Artelinate

An economical phase‐transfer method is used to prepare 10‐arylaminoartemisinins from DHA and arylamines, and artemether, arteether, and artelinate from the corresponding alcohols. In vivo sc screens against Plasmodium berghei and P. yoelii in mice reveal that the p‐fluorophenylamino derivative 5 g is some 13 and 70 times, respectively, more active than artesunate; this reflects the very high sc activity of 10‐alkylaminoartemisinins. However, through the po route, the compounds are less active than the alkylaminoartemisinins, but still approximately equipotent with artesunate.

Epiartemisinin, a remarkably poor antimalarial: implications for the mode of action.

Epiartemisinin (7) was prepared by the base epimerization of artemisinin (1) and its structure determined by X‐ray analysis. The antimalarial activity of 7 against the chloroquine‐sensitive and resistant strains of Plasmodium berghei and P. yoelii in the mouse was compared with that of the highly effective schizonticide 1 and found to be drastically diminished. It is argued that the mode of action on the intraerythrocytic parasite by 7 is compromised by steric hindrance arising from the α‐disposed Me group. In the initial step, intimate complexation with heme is hindered or biased to favor the formation of a less potent C‐centered radical, the final lethal agent.