pfmdr1 Amplification Is Related to Increased Plasmodium falciparum In Vitro Sensitivity to the Bisquinoline Piperaquine

Abstract

The 4-aminoquinoline bisquinoline piperaquine is an important partner drug in one of the presently recommended artemisinin combination therapies. Recent clinical trials have confirmed its high efficacy in combination with dihydroartemisinin. Resistance to piperaquine alone has, however, been documented. Amplification in copy number of the Plasmodium falciparum multidrug resistance locus on chromosome 5, containing the pfmdr1 gene, has been shown to confer resistance to structurally unrelated antimalarials. Through the determination of the 50% inhibitory concentrations (IC 50 s) and IC 90 s for piperaquine and chloroquine in a set of 46 adapted P. falciparum cultures originating from the Thai-Burmese border, we have characterized the regions around the pfmdr1 gene and identified a significant association between the presence of pfmdr1 duplications and enhanced sensitivity to piperaquine (P ‫؍‬ 0.005 for IC 50 and P ‫؍‬ 0.002 for IC 90 ) and chloroquine, reaching statistical significance at IC 90 s (P ‫؍‬ 0.026). These results substantiate the potential importance of pfmdr1 copy number amplifications in the efficacy of the combination therapy piperaquine-dihydroartemisinin. It supports the rational use of 4-aminoquinolines and artemisininbased compounds, as they independently select for mutually incompatible combinations of mutations.A rtemisinin combination therapy (ACT) is instrumental in the global decrease of Plasmodium falciparum malaria in recent years. However, P. falciparum resistance to several partner drugs, namely, artesunate-mefloquine (25), artemether-lumefantrine (26, 31), and artesunate-amodiaquine (15), has been documented. These early signs have been associated with decreased parasite responses to the slowly eliminated partner drugs, a phenomenon believed to be accelerated in circumstances of high transmission due to the exposure of reinfecting parasites to subtherapeutic concentrations (14).A more recent ACT, dihydroartemisinin-piperaquine (DHA-PPQ), has been widely used in Southeast Asia and is now ready to be launched in sub-Saharan Africa. PPQ is a bisquinoline, structurally a 4-aminoquinoline-based antimalarial like chloroquine (CQ). Piperaquine is not a recent newcomer, having been extensively used in monotherapy regimens in the southern regions of China in the 1970s and 1980s, as a response to the rise of CQ resistance. Later, it was adopted by the national Vietnamese malaria control program in several formulations (9). The most recent development of PPQ-based ACT is commercially known as Artekin (Hollekyn Pharmaceuticals, China) or Eurartesim (SigmaTau, Italy), each tablet containing 40 mg dihydroartemisinin plus 320 mg piperaquine phosphate.DHA-PPQ has shown excellent efficacy in recent clinical trials in Africa (1,4,17), making it a promising fixed-dose formulation for malaria treatment on the continent. Nevertheless, the PPQ long elimination half-life of Ͼ4 weeks after the standard 3-day course (2.25 mg/kg [of body weight] DHA and 18 mg/kg of piperaquine phosphate per day) raises concer...

In Vitro Sensitivities of Plasmodium falciparum Isolates from the China-Myanmar Border to Piperaquine and Association with Polymorphisms in Candidate Genes

“…Microarray analysis showed deamplification of the pfmdr1 allele and amplification of an adjacent region on chromosome 5. Analysis of parasite samples from the Thai-Myanmar border area confirmed the role of pfmdr1 amplification with enhanced sensitivity to PPQ (32). pfmdr1 amplification is normally associated with resistance to amino-alcoholic drugs such as MQ (6).…”

“…In order to elucidate the genetic basis of PPQ resistance, Eastman et al used an in vitro scheme to select PPQ-resistant lines from Dd2 parasite and identified deamplification of a region in chromosome 5 encompassing pfmdr1 and amplification of an adjacent region associated with the selection, suggesting a potential link between pfmdr1 copy number and PPQ sensitivity (31). To corroborate this genetic study, Veiga et al determined the in vitro response to PPQ in parasite isolates from western Thailand, where pfmdr1 copy number has been increased due to MQ selection and found that pfmdr1 amplification was significantly associated with enhanced sensitivity to PPQ (32). It is worth noting that most of these in vitro drug studies used parasite isolates that were not exposed or rarely exposed to PPQ.…”

“…It is suspected that cross-resistance might occur between PPQ and other quinoline drugs because of structural similarities of these drugs and possibly shared modes of ac- tion (46). Studies showing correlation between in vitro susceptibilities of parasite isolates to CQ and PPQ (20,22,24,25,32) and demonstration of elevated resistance to PPQ in well-characterized laboratory CQ-resistant parasite lines (25,26) suggest a role of the major CQ-resistant marker pfcrt in PPQ resistance. However, it is noteworthy that the difference between the PPQ responses in CQsensitive and -resistant strains which led to the suggestion of cross-resistance between CQ and PPQ resistance was insignificant and only at 3-to 4-fold (Table 2).…”

Evidence of Plasmodium falciparum Malaria Multidrug Resistance to Artemisinin and Piperaquine in Western Cambodia: Dihydroartemisinin-Piperaquine Open-Label Multicenter Clinical Assessment

“…Resistance has developed sequentially in response to the sequential use of antimalarial drugs; artemisinin resistance is the most recent addition. The rapid development of DHA-PP treatment failure in western Cambodia appears to be highly correlated with the presence of K13 mutations and the increased sensitivity of P. falciparum isolates to mefloquine (36,37). Interestingly, DHA-PP treatment failures have not been reported in other countries of the Greater Mekong Subregion, such as Myanmar and Vietnam, despite high prevalence rates of K13 mutant alleles in these countries and many years of intense use (18,19,38,39).…”

Temporal and Seasonal Changes of Genetic Polymorphisms Associated with Altered Drug Susceptibility to Chloroquine, Lumefantrine, and Quinine in Guinea-Bissau between 2003 and 2012

“…The pfmdr1 86 ϩ 184 ϩ 1246 NFD haplotype has been selected for by AL (7,15,16). Furthermore, an increased pfmdr1 copy number has been linked to reduced susceptibility to artemisinin derivatives, lumefantrine, piperaquine, and mefloquine and to artesunate plus mefloquine treatment failure (17)(18)(19)(20). In addition to these well-characterized resistance-associated SNPs, pfmdr1 polymorphisms N86Y, S1034C, N1042D, and D1246Y have been suggested to modulate levels of CQ and quinine resistance (21,22).…”