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

## **4.3.1 Erythrocytic stages of malaria parasites**

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 is followed daily.

### **4.3.2 Other stages of malaria parasites**

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 stage of *Plasmodium*.

Advances in Antimalarial Drug Evaluation and New Targets for Antimalarials 339

engrafted mouse models have been reported recently. One used the *Fah–/–Rag2–/–Il2rg–/–*  mice, the liver of which was very efficiently repopulated (up to approximately 95% of the murine liver) by human hepatocytes under selection pressure by the drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione) (Bissig et al., 2010). The suitability of this model for antiviral drug testing after successful infection of the chimeric liver by hepatitis B and C viruses was demonstrated, legitimating to believe in future successful infection by human *Plasmodium* species. Also, very recently, researchers at the MIT developed artificial humanised mouse livers engineered by growing human hepatocytes and human liver endothelial cells with mouse fibroblasts in a three-dimensional polymeric scaffold, and implanted them into mice (Chen et al., 2011). The ectopic livers responded to drugs in a way

Obviously those recent experimental models are not yet elaborated enough to meet the requirements for examining the large number of compounds involved in initial drug development. However, they pave the way for more efficient testing of drugs designed for humans. Ultimately, the combination of the immunocompromised mouse model for the liver stage and the model for the blood stage should open access to the complete cycle of the *P. falciparum* development in a small laboratory host, thereby providing an opportunity that should have numerous applications not only in the field of drug testing, pharmacokinetics and toxicology, but also in vaccine development and to investigate some of the biological

Whatever will be the future of these new models, it seems however that exploration of drug potency using the "classical" rodent models might already be improved by considering some points of interest that we would like to list hereafter. 1) Because rodent malaria species can differ significantly in sensitivity to certain classes of compounds, as illustrated above with iron chelators and lipid biosynthesis inhibitors, it may be recommended to test new compounds on different models. 2) Also, the aspect of synergy between drugs should be considered and examination of therapies in conjunction with antimalarials typically given to treat patients with malaria should be favoured. 3) Notions of chronobiology may help in the selection of an experimental model and in the interpretation of chemotherapy experiments. Indeed, specific characteristics such as the duration of the schizogonic cycle, the time of schizogony or the synchronicity/asynchronicity of the chosen *Plasmodium* species and strain may influence its responsiveness to a drug depending on the time of administration and half-life of the drug (Beaute-Lafitte et al., 1994). This can be easily understood in the case of active compounds that preferentially affect a particular period of the parasite life cycle. Drug administration could be planed such that the peak level of the drug in the blood will be reached at the time the sensitive stage of the parasite is present in the circulation. It is assumed that such timing should increase the efficacy of drug treatment and minimize the dose to be injected. 4) Finally, it is remarkable that most of the drug testing performed in laboratories and promoted as potential therapy is carried out before the onset of malaria symptoms, whereas drug administration to treat human malaria is initiated after the onset of symptoms. Primary screening would gain advantage if new compounds were tested also

very similar to the way a human liver does.

and physiological aspects of human malaria.

**4.5 Comments** 

in this configuration.

#### **4.4 Humanized mice models of malaria**

Immunocompromized mice, developed and used in other research contexts, have been used for some years to elaborate new mouse models for human malaria. By grafting them with either human erythrocytes or human hepatocytes, these animals can support, respectively, the asexual blood cycle or the hepatic phase of the human parasite *P. falciparum*. So, drug efficacy and pharmacokinetics can be assessed in an *in vivo* setting against the true parasite target during its blood or hepatic stage development.

### **4.4.1 The** *P. falciparum***-human erythrocyte mouse models**

In 2001, the feasibility of evaluating *in vivo* responses to antimalarial drugs in humanised mice models was demonstrated using the *P. falciparum*-human erythrocyte-BXN model, in which Beige Xid Nude (BXN) mice, genetically deficient in T- and B-cell functions and chemically controlled for response of cells involved in innate defences, were grafted with human erythrocytes infected with *P. falciparum* (Moreno et al., 2001). The mice received human erythrocytes infected with drug (chloroquine/quinine/mefloquine) resistant or sensitive strains of *P. falciparum*, and the blood schizonticidal effects of various antimalarial drugs were assessed. Parasite clearance and parasite reduction rate in the mouse model were shown to parallel those reported in humans infected with *P. falciparum*. Since then, improvements of the *P. falciparum* humanized mouse model have been made using NSG mice with improved genetic deficiency of innate immunity. High reproducibility of human erythrocytes grafting and parasite survival could be achieved, along with the possible adjustment of parasitaemia over a range of 1-40% for several weeks (Arnold et al., 2011). In the near future, a model harbouring a hematopoietic stem cell line capable of producing erythroblasts/cytes should replace current models with the advantage of producing reticulocytes to also support *P. vivax*. To date, we are aware of only one work dealing with drug testing in humanised mouse model of malaria, i.e. the one about trioxaquine selection for drug development (Cosledan et al., 2008). The scarcity of reports exemplifies the serious limitations to the use of such malaria models in drug discovery: 1) in most of the models, parasitaemia remains stable only in a minor subset of mice whereas it is rapidly cleared in the majority of the animals, 2) the use of toxic reagents to minimize the mouse innate defence is susceptible to affect the efficacy of antimalarials or effector cells, 3) management of those experimental models is cumbersome and requires specific facilities.

### **4.4.2 The** *P. falciparum***-human hepatocyte mouse models**

There have been many attempts to develop laboratory models supporting the survival of human hepatocytes, given that *in vivo* only human cells or those of higher primates are receptive to *P. falciparum*. Invasion by and development of *P. falciparum* in human hepatocyte-transplanted animals was demonstrated in 2006, by combining 1) the use of mice with two genetic deficiencies (uPA-SCID mice) affecting hepatocytes and T and B lymphocytes, and 2) the pharmacological control of their non-adaptive defences, which improved the results of xenotransplantation into mice (Morosan et al., 2006). In these mice, *P. falciparum* sporozoites delivered intravenously infected the transplanted human hepatocytes and developed into liver schizonts up to a size that was comparable to that found in humans and chimpanzees. Alternative approaches to elaborate human hepatocyteengrafted mouse models have been reported recently. One used the *Fah–/–Rag2–/–Il2rg–/–*  mice, the liver of which was very efficiently repopulated (up to approximately 95% of the murine liver) by human hepatocytes under selection pressure by the drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione) (Bissig et al., 2010). The suitability of this model for antiviral drug testing after successful infection of the chimeric liver by hepatitis B and C viruses was demonstrated, legitimating to believe in future successful infection by human *Plasmodium* species. Also, very recently, researchers at the MIT developed artificial humanised mouse livers engineered by growing human hepatocytes and human liver endothelial cells with mouse fibroblasts in a three-dimensional polymeric scaffold, and implanted them into mice (Chen et al., 2011). The ectopic livers responded to drugs in a way very similar to the way a human liver does.

#### **4.5 Comments**

338 Malaria Parasites

Immunocompromized mice, developed and used in other research contexts, have been used for some years to elaborate new mouse models for human malaria. By grafting them with either human erythrocytes or human hepatocytes, these animals can support, respectively, the asexual blood cycle or the hepatic phase of the human parasite *P. falciparum*. So, drug efficacy and pharmacokinetics can be assessed in an *in vivo* setting against the true parasite

In 2001, the feasibility of evaluating *in vivo* responses to antimalarial drugs in humanised mice models was demonstrated using the *P. falciparum*-human erythrocyte-BXN model, in which Beige Xid Nude (BXN) mice, genetically deficient in T- and B-cell functions and chemically controlled for response of cells involved in innate defences, were grafted with human erythrocytes infected with *P. falciparum* (Moreno et al., 2001). The mice received human erythrocytes infected with drug (chloroquine/quinine/mefloquine) resistant or sensitive strains of *P. falciparum*, and the blood schizonticidal effects of various antimalarial drugs were assessed. Parasite clearance and parasite reduction rate in the mouse model were shown to parallel those reported in humans infected with *P. falciparum*. Since then, improvements of the *P. falciparum* humanized mouse model have been made using NSG mice with improved genetic deficiency of innate immunity. High reproducibility of human erythrocytes grafting and parasite survival could be achieved, along with the possible adjustment of parasitaemia over a range of 1-40% for several weeks (Arnold et al., 2011). In the near future, a model harbouring a hematopoietic stem cell line capable of producing erythroblasts/cytes should replace current models with the advantage of producing reticulocytes to also support *P. vivax*. To date, we are aware of only one work dealing with drug testing in humanised mouse model of malaria, i.e. the one about trioxaquine selection for drug development (Cosledan et al., 2008). The scarcity of reports exemplifies the serious limitations to the use of such malaria models in drug discovery: 1) in most of the models, parasitaemia remains stable only in a minor subset of mice whereas it is rapidly cleared in the majority of the animals, 2) the use of toxic reagents to minimize the mouse innate defence is susceptible to affect the efficacy of antimalarials or effector cells, 3) management of those experimental models is

There have been many attempts to develop laboratory models supporting the survival of human hepatocytes, given that *in vivo* only human cells or those of higher primates are receptive to *P. falciparum*. Invasion by and development of *P. falciparum* in human hepatocyte-transplanted animals was demonstrated in 2006, by combining 1) the use of mice with two genetic deficiencies (uPA-SCID mice) affecting hepatocytes and T and B lymphocytes, and 2) the pharmacological control of their non-adaptive defences, which improved the results of xenotransplantation into mice (Morosan et al., 2006). In these mice, *P. falciparum* sporozoites delivered intravenously infected the transplanted human hepatocytes and developed into liver schizonts up to a size that was comparable to that found in humans and chimpanzees. Alternative approaches to elaborate human hepatocyte-

**4.4 Humanized mice models of malaria** 

target during its blood or hepatic stage development.

cumbersome and requires specific facilities.

**4.4.2 The** *P. falciparum***-human hepatocyte mouse models** 

**4.4.1 The** *P. falciparum***-human erythrocyte mouse models** 

Obviously those recent experimental models are not yet elaborated enough to meet the requirements for examining the large number of compounds involved in initial drug development. However, they pave the way for more efficient testing of drugs designed for humans. Ultimately, the combination of the immunocompromised mouse model for the liver stage and the model for the blood stage should open access to the complete cycle of the *P. falciparum* development in a small laboratory host, thereby providing an opportunity that should have numerous applications not only in the field of drug testing, pharmacokinetics and toxicology, but also in vaccine development and to investigate some of the biological and physiological aspects of human malaria.

Whatever will be the future of these new models, it seems however that exploration of drug potency using the "classical" rodent models might already be improved by considering some points of interest that we would like to list hereafter. 1) Because rodent malaria species can differ significantly in sensitivity to certain classes of compounds, as illustrated above with iron chelators and lipid biosynthesis inhibitors, it may be recommended to test new compounds on different models. 2) Also, the aspect of synergy between drugs should be considered and examination of therapies in conjunction with antimalarials typically given to treat patients with malaria should be favoured. 3) Notions of chronobiology may help in the selection of an experimental model and in the interpretation of chemotherapy experiments. Indeed, specific characteristics such as the duration of the schizogonic cycle, the time of schizogony or the synchronicity/asynchronicity of the chosen *Plasmodium* species and strain may influence its responsiveness to a drug depending on the time of administration and half-life of the drug (Beaute-Lafitte et al., 1994). This can be easily understood in the case of active compounds that preferentially affect a particular period of the parasite life cycle. Drug administration could be planed such that the peak level of the drug in the blood will be reached at the time the sensitive stage of the parasite is present in the circulation. It is assumed that such timing should increase the efficacy of drug treatment and minimize the dose to be injected. 4) Finally, it is remarkable that most of the drug testing performed in laboratories and promoted as potential therapy is carried out before the onset of malaria symptoms, whereas drug administration to treat human malaria is initiated after the onset of symptoms. Primary screening would gain advantage if new compounds were tested also in this configuration.

Advances in Antimalarial Drug Evaluation and New Targets for Antimalarials 341

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