**4.** *In vivo* **antimalarial drug evaluation**

334 Malaria Parasites

deletion of genes encoding PfHT1, PfCRT and Pgh1 could not be achieved, suggesting that the two latter ones play essential functions in the parasite biology beside being involved in drug-resistance (Sanchez et al., 2010). Such experiments performed using rodent malaria models further indicated that some candidates, apparently dispensable for the asexual development, turned out important for the development of other parasite stages in insects or liver (Martin et al., 2009, Staines et al., 2004). Among all these candidates, PfHT1 is the only malarial transporter that has been validated both chemically and genetically (Staines et al., 2010). Gene deletion studies and D-glucose derivatives used as inhibitors confirmed the essential role of the hexose transporters for the asexual parasite development and other

Advanced antimalarial drug discovery programs revealed four general strategies to discover new drugs (Wu et al., 2011) : 1) To start from specific, validated or not, malaria targets to find new hits (e.g. haem polymerization, enzymes of the pyrimidine synthesis pathway); 2) To synthesize new analogs from known antimalarial pharmacophores (e.g. synthetic endoperoxides (Trioxolanes) or hybrid molecules having two pharmacophores (Trioxaquines)); 3) To start from drugs developed for other diseases whose similar targets are present in malaria parasites (as illustrated by the piggy-back approach undertaken against parasite protein farnesyltransferases using anti-cancer agents targeting protein prenylation developed by pharmaceutical companies); 4) To screen chemicals on whole cellbased assays to identify hits that kill parasite for further optimizations. Most of the current antimalarials were originated from this last phenotypic drug discovery approach. The revolution in molecular biology led to a switch to target-based approaches for drug discovery in pharmaceutical industries. However, this approach failed to deliver the expected results, especially for antimicrobials (Keller et al., 2011, Payne et al., 2007, Sams-Dodd, 2005). In the recent years, the access to large chemical libraries and the improvements of whole cell-based high throughput screening assays led to a renaissance of the phenotypic approach with the forward chemical genetic strategy. Chemical genetics is the study of biological systems using small molecules as tools (O'Connor et al., 2011). Forward chemical genetic uses small molecules to modulate gene-product function leading to a phenotype of interest (parasite killing for example), and the target must be further identified. In contrast, reverse chemical genetic screens specific gene products with libraries of small molecules to identify ligands, which are then tested on cells for phenotypic changes. Over the last few years, reports demonstrated the power of chemical genetic for antimalarial drug discovery. The GlaxoSmithKline (GSK)'s chemical library constituted of nearly 2 million chemical entities was tested upon drug sensitive and multidrug resistant *P. falciparum* strains. More than 8,000 compounds, clustered into 416 molecular frameworks, showed potent antiplasmodial activity. Analysis of historical GSK data suggested that the main target classes affected by these compounds are malaria kinases, proteases and G-protein coupled receptors (Gamo et al., 2010). In a similar study, a library of nearly 310,000 chemicals, designed to cover a large diversity of bioactive compounds, was screened upon drug sensitive and multidrug resistant *P. falciparum* strains (Guiguemde et al., 2010). Amongst hits, 172 were cross-validated by three laboratories using distinct assays. A reverse chemical genetic approach was undertaken with the validated set of compounds using 66 malarial target assays and identified 19 new inhibitors of 4 validated targets. Preliminary

parasite stages (Blume et al., 2010, Slavic et al., 2011).

**3. Renew of phenotypic screening approaches** 

*Plasmodium* species that infect humans are essentially unable to infect non-primate animal models. Historically, *in vivo* evaluation of antimalarial compounds began with the use of avian parasites in birds and simian parasites in monkeys. The discovery by Ignace Vincke and Marcel Lips in 1948 at Keyberg in the then Belgian Congo, of a rodent malaria parasite (*Plasmodium berghei*) which readily infected laboratory mice and rats, greatly facilitated *in vivo* drug screening management. Since then, several other species and subspecies of rodent parasites have been described and employed. With the discovery in the early 90's that the owl monkey (*Aotus trivirgatus*) is susceptible to infection with the human parasites *P. falciparum* and *P. vivax*, the simian models of malaria have regained interest. Infection with *P. falciparum* is now well characterized in both *Aotus* and *Saimiri* monkeys (Collins, 1992), and primate models, because they provide a clear prediction of drug efficacy and pharmacokinetics in humans, are a logical transition to clinical studies. However, there are obvious limitations to their use, and any primary screen dependent upon monkeys appears both wasteful in terms of animal conservation, drug consumption, and ethics. So, the first steps of *in vivo* drug screening typically begins with the use of mouse models of malaria with the rodent parasites *P. berghei*, *P. yoelii*, *P. chabaudi* and *P. vinckei*, that have been validated through the evaluation of several antimalarial drugs.

#### **4.1 Rodent models of malaria**

Individual parasite species and strains have been well characterized, including duration of cycle, synchronicity, drug sensitivity and course of infection in genetically defined mouse strains. *P. berghei, P. vinckei* and few strains of *P. yoelii* and *P. chabaudi* cause lethal infections in mice, whereas *P. yoelii, P. chabaudi,* and *P. vinckei petterei* are usually cleared after the initial acute parasitaemia or after a subsequent low-grade chronic parasitaemia.

*P. berghei* - The strains K173, ANKA, NK65 and SP11 are the most frequently used for experimental work. The affinity for immature erythrocytes (reticulocytes) varies according to the strain. ANKA invades preferentially reticulocytes, while NK65 invades indifferently reticulocytes and mature erythrocytes, i.e. normocytes. Both strains are lethal, but ANKA kills the mouse with a low parasitaemia (it rarely exceeds 20%), whereas infection with NK65 induces a continuous increase in parasitaemia until the mouse dies. The erythrocytic cycle is asynchronous and its duration is about 21 h. Schizogony lasts 50 h in hepatocytes. Susceptible mice infected with *P*. *berghei* ANKA die within two weeks after infection with severe neurological symptoms and cerebral microvascular abnormalities. These are common pathologies to both murine and human infections that make of *P. berghei* ANKA a useful experimental model of cerebral malaria. Resistant mice do not show any neurological symptom but die during the third or fourth week post-infection (p.i.) with high parasitaemia.

2002).

or the drug dosage regimen.

is followed daily.

stage of *Plasmodium*.

**4.3.1 Erythrocytic stages of malaria parasites** 

**4.3.2 Other stages of malaria parasites** 

Advances in Antimalarial Drug Evaluation and New Targets for Antimalarials 337

that the enzyme active site is different between rodent plasmodia and *P. falciparum*. This situation has led to question the use of these models in lead optimization (Singh et al.,

Given this, the following criteria must be considered when selecting a mouse model. On the host side, innate factors like peculiarities in pharmacodynamics of the drug; innate immunity; variation between individuals of the same species (strain, age, sex and immune status); environmental factors (temperature and stress, nutrition, intercurrent infections). On the parasite side: variation within a single *Plasmodium* species (variation of sensitivity among different clones, geographic strains, time since isolation of "wild" strain); variation among different *Plasmodium* species; mode and intensity of infection. Also, attention should be paid to drug-dependent factors like the mode of formulation, the route of administration

The most widely used initial test is the "four-day suppressive test", which commonly uses *P. berghei* or *P. chabaudi*. The efficacy of four daily doses of compounds is measured by comparison of mouse survival time and blood parasitaemia on day four p.i. in treated and untreated mice. Compounds identified as being active in those assays can subsequently be progressed through various secondary tests, such as 1) the "dose ranging, full four-day test", in which compounds are tested at a minimum of four different doses to determine effective dose values and get information on oral bioavailability and relative potency of the tested compounds; 2) the "onset/recrudescence" test, in which mice are administered a single dose on day 3 after infection and parasitaemia is monitored daily. Compounds can also be tested for prophylactic activity: the compound is administered prior to infection, and parasitaemia

Several tissue schizontocidal tests (the test of Gregory and Peters (1970) using sporozoites of *P. yoelii nigeriensis*, the test of Rane and Kinnamon with *P. yoelii yoelii*, and the Hill test for causal prophylactic compounds with residual action) have been reported in details (Peters, 1987). Tests that apply to the screening of drugs having gametocidal or sporontocidal action can be found at the same source. A technique such as real time PCR allows quantification of the parasite charge in liver and thus, the inhibitory effect of the molecule tested (Carraz et al., 2006). Recently, the use of a transgenic *P. berghei* parasite expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells in live mice using real-time luminescence imaging was reported (Ploemen et al., 2009). The applicability of real time imaging to assess parasite drug sensitivity in the liver was demonstrated by analysing the effect of primaquine and tafenoquine *in vivo*. The methodology is relatively simple and offers the possibility to analyse liver development in live mice without surgery. It opens up new possibilities for research on *Plasmodium* liver infections and for evaluating the effect of drugs on the liver

**4.3 Current** *in vivo* **antimalarial tests using rodent models of malaria** 

*P. vinckei* - Among four subspecies recognized (*P*. *vinckei vinckei*, *P. vinckei petteri*, *P. vinckei lentum* and *P. vinckei brucechwatti*), *P. v. vinckei* and *P. v. petteri* have been used in some studies for the identification of new antimalarial drugs. Both subspecies preferentially invade normocytes. Schizogony in erythrocytes has a duration time of 24 h and is synchronous. Schizogony in hepatocytes lasts for 60 h or more.

*P. yoelii* – Three subspecies are recognized: *P*. *yoelii yoelii*, *P. yoelii killicki* and *P. yoelii nigeriensis*. *P. yoelii* shows a preference for reticulocytes, but it may also invade normocytes, depending on the virulence of the strain. Duration of the schizogonic cycle is 18 h in erythrocytes. Schizogony is about 45 h long in hepatocytes. The erythrocytic cycle is asynchronous. The parasitaemia of *P. y. yoelii* 17X and 265BY depends mainly on the production of reticulocytes by the host, whereas *P. y. nigeriensis* grows very rapidly by invading all available erythrocytes. Of note, *P. yoelii* is more resistant to chloroquine than are the other species (Beaute-Lafitte et al., 1994). Gametocyte production is constant throughout the infection.

*P. chabaudi* – Two subspecies of *P. chabaudi* have been identified: *P. chabaudi chabaudi* and *P. chabaudi adami*. This species exhibits many similarities to *P. falciparum*, including analogous blood-stage antigens, invasion of reticulocytes and normocytes, suppression of B- and T-cell responses, and parasite sequestration in liver and spleen which induces parasite withdrawal from the peripheral circulation. The schizogonic cycle of *P. chabaudi* is 24 h long in erythrocytes and is synchronous. It runs 54-58 h in liver. In resistant mice, a rapid parasite multiplication during the first week p.i. is followed by parasite elimination by the fourth week p.i., whereas susceptible mice usually die during the second week p.i.

### **4.2 Designing an experimental mouse model**

Many inbred and outbred strains of mice are available to design a mouse model. Considering that every species and every line or clone of a particular *Plasmodium* species exhibits particular characteristics, the resulting infection may vary greatly within the different laboratory strains of mice. As an example, blood-induced infections of the K173 (N) of *P. berghei* follow a fulminating course in many strains of mouse, but are slow in NMRI mice, and infections with *P. y. yoelii* 17X in Swiss mice are lethal, whereas they resolve in CBA/Ca mice. Scientists should use the most appropriate model for their particular research question and take advantage of their particular knowledge about compounds to be tested, especially in terms of molecular and/or biological targets. A rather rich presentation of experimental malaria infections in different mouse strains, comprising some models for cerebral malaria, can be found in (Sanni et al., 2002).

The difference in the degree of infection, lethality and synchronicity between the rodent plasmodia enlarges the number of possible assays for drug evaluation. For example, parasites that generate high parasitaemia and synchronous infections, like *P. chabaudi* and *P. vinckei*, are more appropriate to study compounds exhibiting stage selectivity. Also, it is important to note that the drug sensitivity of a given rodent *Plasmodium* species may not reflect that of the other rodent species. This can be illustrated by *P. berghei* exhibiting less sensitivity than *P. chabaudi* and *P. vinckei* to iron chelators and lipid biosynthesis inhibitors (Peters & Robinson, 1999, Wengelnik et al., 2002). Drug sensitivity may also not mirror that of *P. falciparum*, as shown in the case of cysteine protease inhibitors, owing to the fact

*P. vinckei* - Among four subspecies recognized (*P*. *vinckei vinckei*, *P. vinckei petteri*, *P. vinckei lentum* and *P. vinckei brucechwatti*), *P. v. vinckei* and *P. v. petteri* have been used in some studies for the identification of new antimalarial drugs. Both subspecies preferentially invade normocytes. Schizogony in erythrocytes has a duration time of 24 h and is

*P. yoelii* – Three subspecies are recognized: *P*. *yoelii yoelii*, *P. yoelii killicki* and *P. yoelii nigeriensis*. *P. yoelii* shows a preference for reticulocytes, but it may also invade normocytes, depending on the virulence of the strain. Duration of the schizogonic cycle is 18 h in erythrocytes. Schizogony is about 45 h long in hepatocytes. The erythrocytic cycle is asynchronous. The parasitaemia of *P. y. yoelii* 17X and 265BY depends mainly on the production of reticulocytes by the host, whereas *P. y. nigeriensis* grows very rapidly by invading all available erythrocytes. Of note, *P. yoelii* is more resistant to chloroquine than are the other species (Beaute-Lafitte et al., 1994). Gametocyte production is constant throughout

*P. chabaudi* – Two subspecies of *P. chabaudi* have been identified: *P. chabaudi chabaudi* and *P. chabaudi adami*. This species exhibits many similarities to *P. falciparum*, including analogous blood-stage antigens, invasion of reticulocytes and normocytes, suppression of B- and T-cell responses, and parasite sequestration in liver and spleen which induces parasite withdrawal from the peripheral circulation. The schizogonic cycle of *P. chabaudi* is 24 h long in erythrocytes and is synchronous. It runs 54-58 h in liver. In resistant mice, a rapid parasite multiplication during the first week p.i. is followed by parasite elimination by the fourth

Many inbred and outbred strains of mice are available to design a mouse model. Considering that every species and every line or clone of a particular *Plasmodium* species exhibits particular characteristics, the resulting infection may vary greatly within the different laboratory strains of mice. As an example, blood-induced infections of the K173 (N) of *P. berghei* follow a fulminating course in many strains of mouse, but are slow in NMRI mice, and infections with *P. y. yoelii* 17X in Swiss mice are lethal, whereas they resolve in CBA/Ca mice. Scientists should use the most appropriate model for their particular research question and take advantage of their particular knowledge about compounds to be tested, especially in terms of molecular and/or biological targets. A rather rich presentation of experimental malaria infections in different mouse strains, comprising some models for

The difference in the degree of infection, lethality and synchronicity between the rodent plasmodia enlarges the number of possible assays for drug evaluation. For example, parasites that generate high parasitaemia and synchronous infections, like *P. chabaudi* and *P. vinckei*, are more appropriate to study compounds exhibiting stage selectivity. Also, it is important to note that the drug sensitivity of a given rodent *Plasmodium* species may not reflect that of the other rodent species. This can be illustrated by *P. berghei* exhibiting less sensitivity than *P. chabaudi* and *P. vinckei* to iron chelators and lipid biosynthesis inhibitors (Peters & Robinson, 1999, Wengelnik et al., 2002). Drug sensitivity may also not mirror that of *P. falciparum*, as shown in the case of cysteine protease inhibitors, owing to the fact

week p.i., whereas susceptible mice usually die during the second week p.i.

**4.2 Designing an experimental mouse model** 

cerebral malaria, can be found in (Sanni et al., 2002).

synchronous. Schizogony in hepatocytes lasts for 60 h or more.

the infection.

that the enzyme active site is different between rodent plasmodia and *P. falciparum*. This situation has led to question the use of these models in lead optimization (Singh et al., 2002).

Given this, the following criteria must be considered when selecting a mouse model. On the host side, innate factors like peculiarities in pharmacodynamics of the drug; innate immunity; variation between individuals of the same species (strain, age, sex and immune status); environmental factors (temperature and stress, nutrition, intercurrent infections). On the parasite side: variation within a single *Plasmodium* species (variation of sensitivity among different clones, geographic strains, time since isolation of "wild" strain); variation among different *Plasmodium* species; mode and intensity of infection. Also, attention should be paid to drug-dependent factors like the mode of formulation, the route of administration or the drug dosage regimen.
