**1. Introduction**

Parasitic infections due to the protozoa *Plasmodium* are responsible for malaria, a severe disease that still caused about 225 million cases and 781,000 human deaths in 2009, despite the efforts developed during the last decade to fight this disease (Alonso et al., 2011). The international funding allocated to antimalarial strategies has increased regularly since 2003 from about 0.3 billions to 1.7 billion dollars in 2009 (Collier, 2009), allowing many countries to undertake or strengthen effective fights against the parasite, the disease and the vectors. Nonetheless, more than half of the world population still lives in area where there is a risk of malaria transmission. The difficulty in fighting malaria is that five species of *Plasmodium,*  namely *P. ovale*, *P. malariae*, *P. vivax*, *P. falciparum* and *P. knowlesi* (until recently considered as a nonhuman primate parasite) transmitted by over 30 species of *Anopheles* female mosquitoes are known to cause human malaria. The most virulent, *P. falciparum*, is responsible for severe clinical malaria and death. Furthermore, an increasing prevalence of resistance of vectors to insecticides, and of parasites to the standard antimalarial drugs has been observed for decades.

Today, the chemotherapeutic arsenal for malaria treatment is limited to three main families of compounds: quinolines, antifolates and artemisinin derivatives. Recommended chemotherapy is based on combinations of existing drugs with artemisinin derivatives (artemisinin combination therapies or ACT), the only antimalarial drug having no clear resistance recorded but for which alarming reports of tolerance in the field indicate it could be just a question of time (Noedl et al., 2008). From 2000 to 2008, the use of ACT combined with vector control allowed to reduce considerably the number of cases of malaria in a dozen African countries, so that nowadays, about 50 % of the total cases of malaria in Africa are found in mainly five countries (Enserink, 2010). However, no new class of antimalarials has been introduced into clinical practice since 1996 due to the intrinsic difficulties in discovering and developing new antimicrobials. A recent review of the global antimalarial drug development (Figure 1), including drugs at various clinical stage development and those expected to enter in phase I studies, showed that the pipeline is rather strong but novelty in terms of drug targets that is required to circumvent resistance is relatively low (Olliaro & Wells, 2009). This situation and the complexity in developing efficient vaccines require an urgent need for new drugs with original mechanisms of actions.

Advances in Antimalarial Drug Evaluation and New Targets for Antimalarials 323

humans during a bite. Different antimalarial bioassays have been developed based on the *in vitro* inhibition of parasite growth or, more recently, on the inhibition of potential parasite

The intraerythrocytic cycle, being responsible for the symptoms of the disease, is still the main parasite stage against which drugs are tested. Initially, drug screenings were limited to the use of animal malaria models (rodent, chicken or monkey). The development of the continuous culture of *P. falciparum* on human erythrocytes (Trager & Jensen, 1976) was a critical advance, allowing drug evaluation on well established laboratory strains and on fresh isolates from patients. Typically, parasites are maintained on leucocyte-free erythrocytes at 2- 5% haematocrit, in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered Roswell Park Memorial Institute (RPMI) medium and supplemented with 5-10% human serum, at 37 °C, under a reduced percentage of oxygen. Hypoxanthine can be added to the culture medium to stimulate parasite growth and to sustain high parasitemia, and it is recommended for the culture of fresh isolates. Standardized protocols have been proposed e.g. (in *Methods in Malaria Research*, http://www.mr4.org/Publications/tabid/326/Default.aspx). *Plasmodium vivax* is the most widespread species and, except in equatorial Africa, is responsible for the most prevalent malaria infection of humans, causing 70 to 80 million clinical cases per year. Although *P. vivax* infections are rarely fatal, they remain an important cause of morbidity, particularly in Asia Pacific region. Compared to *P. falciparum*, *P. vivax* can be considered as a neglected disease. Resistance of *P. vivax* to the antimalarial drug chloroquine, the reference drug for treating *P. vivax* infection, has been reported since the 90's. It is therefore of importance to develop tools for monitoring drug resistance and developing new drugs. In contrast to *P. falciparum*, establishment of continuous cultured lines of *P. vivax* has not yet been achieved limiting drug evaluation, particularly high throughput screening. *P. vivax* does not easily grow in culture, requiring removal of leucocytes and enrichment of the growth media. Parasite growth can only be performed for short periods, but maintaining cultures up to 4 weeks can be obtained by supplying reticulocytes from normal blood (Udomsangpetch et al., 2008). Drug assays developed for *P. falciparum* are transposable to fresh and cryopreserved *P. vivax* isolates (Kosaisavee et al., 2006). It is generally assumed that drugs active against *P. falciparum* blood stages will be also active against *P. vivax* blood stages; this has been shown in clinical studies for dihydroartemisinin–piperaquine and for artesunate–pyronaridine (Olliaro & Wells, 2009). Such assumption and the technical constraints to study *P. vivax* explain the limited interest for this species. For the other human malaria parasites, *P. knowlesi* has been adapted to longterm culture on monkey erythrocytes (Kocken et al., 2002) but no continuous cultivation of

Standard protocols of drug and resistance evaluation on *P. falciparum* are recommended by the World Health Organisation to facilitate comparison of data. They generally involve evaluation by using Giemsa-stained smears and counting parasitemia or parasite stage distribution in treated and non-treated cultures. These assays require minimal equipment and can be easily applied in the field. However they are time-consuming thereby preventing rapid, large-scale screening of molecules. Several methods have been developed for

screening large numbers of compounds in 96-well plates, or even in 384-well plates.

targets, allowing the screening of chemical compounds.

**2.2 Bioassays against the erythrocytic stage** 

*P. ovale* and *P. malariae* has been set up.


Fig. 1. Global antimalarial drug development pipeline (February 2009), after (Olliaro & Wells, 2009). Artemisinin (Art) derivatives or drugs containing the trioxane ring of artemisinin are illustrated in blue; aminoquinolines and structurally related compounds as well as aryl alcohols are in green; antibiotics are in orange; others drugs having different targets or mechanisms of action are in brown-red.
