The Plasmodium falciparum chloroquine resistance transporter is associated with the ex vivo P. falciparum African parasite response to pyronaridine

The pyronaridine-artesunate combination is one of the most recent oral artemisinin-based therapeutic combinations (ACTs) recommended for the treatment of uncomplicated P. falciparum malaria. The emergence of P. falciparum resistance to artemisinin has recently developed in Southeast Asia. Little data are available on the association between pyronaridine susceptibility and polymorphisms in genes involved in antimalarial drug resistance. The objective of the present study was to investigate the association between ex vivo responses to pyronaridine and the K76T mutation in the pfcrt gene in P. falciparum isolates. The assessment of ex vivo susceptibility to pyronaridine was performed on 296 P. falciparum isolates using a standard 42-h 3H-hypoxanthine uptake inhibition method. The K76T mutation was also investigated. The pyronaridine IC50 (inhibitory concentration 50 %) ranged from 0.55 to 80.0 nM. Ex vivo responses to pyronaridine were significantly associated with the K76T mutation (p-value = 0.020). The reduced susceptibility to pyronaridine, defined as IC50 > 60 nM, was significantly associated with the K76T mutation (p-value = 0.004). Using a Bayesian mixture modelling approach, the pyronaridine IC50 were classified into three components: component A (IC50 median 15.9 nM), component B (IC50 median 34.2 nM) and component C (IC50 median 63.3 nM). The K76T mutation was represented in 46.3 % of the isolates in component A, 47.2 % of the isolates in component B and 73.3 % of the isolates in component C (p-value = 0.021). These results showed the ex vivo reduced susceptibility to pyronaridine, i.e., IC50 > 60 nM, associated with the K76T mutation.

In 2002, the World Health Organization (WHO) recommended the use of artemisinin-based combination therapy (ACT) for the treatment of all cases of uncomplicated malaria. The pyronaridine-artesunate combination (Pyramax®) is one of the latest oral ACTs recommended for the treatment of uncomplicated P. falciparum and P. vivax malaria [1]. The combination pyronaridine-artesunate has recently completed phase III trials in humans. The safety and efficacy of this compound were shown in four randomized clinical trials in adults and children in Africa and Asia [2][3][4][5]. Pyronaridine-artesunate showed better efficacy compared with mefloquine-artesunate for the treatment of uncomplicated falciparum malaria in Cambodia and a non-inferior efficacy compared with artemether-lumefantrine in Africa and Southeast Asia. The emergence of P. falciparum resistance to artemisinin and artemisinin derivatives has recently developed in Southeast Asia, manifesting as delayed parasite clearance following treatment with artesunate monotherapy or ACT [6,7]. Resistance has still developed with the most recent ACT in the form of dihydroartemisinin-piperaquine, which demonstrated less than 70 % efficacy [8,9]. In areas where the resistance of artemisinin is emerging, partner drugs are under increasing pressure for the selection of resistance, and new therapeutics are limited. Thus, it is important to use an ACT in which its partner drug shows a different mode of action or mechanism of resistance. The in vitro responses to pyronaridine and piperaquine were differently distributed in a triple normal distribution model for pyronaridine and a quadruple normal distribution model for piperaquine [10]. Significant positive in vitro cross-susceptibility was observed between pyronaridine and piperaquine (coefficient of determination of 0.20-0.23) [11,12]. In vitro and ex vivo responses to piperaquine were not associated with the K76T mutation in the P. falciparum chloroquine resistance transporter gene (pfcrt) [13,14]. Few data are available on the association between pyronaridine susceptibility and polymorphisms in the genes involved in antimalarial drug resistance. A study using 23 P. falciparum strains showed that there was no significant association between in vitro responses to pyronaridine and pfcrt polymorphism [15]. The objective of the present study was to investigate the association between ex vivo responses to pyronaridine and the K76T mutation in the pfcrt gene in 296 P. falciparum African isolates.
In total, 296 P. falciparum isolates were collected between April 2008 and August 2012 from patients hospitalized in France with imported malaria from African malaria-endemic countries (Angola, Benin, Burkina Faso, Cameroon, Central African Republic, Chad, Comoros, Congo, Ivory Coast, Gabon, Gambia, Ghana, Guinea, Madagascar, Mali, Mauritania, Mozambique, Niger, Senegal, Togo, and Zambia). Informed consent was not required for this study because the sampling and testing procedures were conducted according to the French national recommendations for the care and surveillance of malaria. The ex vivo responses to pyronaridine (Shin Poong Pharm Co., Seoul, Korea) and chloroquine (St. Louis, MO, USA) (control for pfcrt polymorphism) were assessed as previously described using a standard 42-h 3H-hypoxanthine uptake inhibition method [10]. Batches of plates were tested and validated using the chloroquine-susceptible 3D7 strain (West Africa) and the chloroquine-resistant W2 strain (Indochina) (MR4, Virginia, USA) in three to six independent experiments. Nucleic acid extraction and pfcrt single-nucleotide polymorphism identification were previously described [14].
The pyronaridine IC 50 values (inhibitory concentration 50 %) ranged from 0.55 to 80.0 nM (Fig. 1). The geometric mean was 20.8 ± 14.6 nM (standard deviation). Ex vivo responses to pyronaridine were significantly associated with the K76T mutation (p-value = 0.020), and similar results were obtained for chloroquine IC 50 (p-value < 0.001). Sixteen isolates (5.4 %) had an IC 50 greater than 60 nM and were considered to display reduced susceptibility to pyronaridine in vitro [10]. The reduced susceptibility to pyronaridine, defined as IC 50 > 60 nM, was significantly associated with the K76T mutation (p-value = 0.004). The odds ratio for reduced susceptibility to pyronaridine associated with the K76T mutation was 4 The results of the present study showed that ex vivo reduced susceptibility to pyronaridine, i.e., IC 50 > 60 nM, was associated with the K76T mutation in the pfcrt gene. These ex vivo results are in contrast with the in vitro results in 23 P. falciparum strains in which the in vitro to pyronaridine were not associated with pfcrt polymorphisms [15]. However, none of the 23 strains showed reduced susceptibility to pyronaridine. In 59 field isolates from Kenya, pyronaridine was more active in vitro against parasites harbouring the wild-type sequence than against those harbouring the K76T mutation (IC 50 of 6 versus 20 nM). However, this difference was not significant (p-value > 0.05) [16]. The odds ratio for the K76T mutation associated with reduced susceptibility to pyronaridine was 4.47, whereas the odds ratio for in vitro resistance to chloroquine was 96.4. The resistance to pyronaridine is certainly multigenic, and additional polymorphisms in other genes could also be involved in this resistance.
In contrast with piperaquine, in which its in vitro responses are not associated with pfcrt polymorphism in isolates from Africa and Asia [13,14,17,18] but rather associated with repeat polymorphisms in an ABC transporter gene, pfmdr6 [19], pyronaridine mechanisms of resistance are different than those involved in piperaquine. Pyronaridine-artesunate could be used in areas where resistance to other ACTs has already emerged.
Pyronaridine-artesunate successfully treats artemisininresistant P. berghei parasites, while artemether-lumefantrine, artesunate-amodiaquine, artesunate-mefloquine and dihydroartemisinin-piperaquine are not effective [20]. Pyronaridine-artesunate showed better efficacy than mefloquine-artesunate for the treatment of uncomplicated falciparum malaria in Cambodia and a non-inferior efficacy compared with that of artemether-lumefantrine in Africa and Southeast Asia [2][3][4]. Pyronaridine-artesunate showed greater than 95 % efficacy when used as an initial falciparum malaria treatment versus the re-treatment of subsequent episodes in a multi-site trial in Mali, Burkina Faso and Guinea [21]. In addition, pyronaridine-artesunate is also affective in the treatment of acute uncomplicated P. vivax malaria [5]. Pyronaridine-artesunate is an alternative artemisinin-based combination treatment for malaria in sub-Saharan Africa.

Ethical approval
According to the French legislation, bio-banking and secondary use for scientific purposes of human clinical samples are possible as long as the corresponding