Most currently available anti-malarial drugs primarily target the disease-causing parasite stages in the human blood system. However, to eradicate malaria, new drugs that block transmission of the parasite between the human host and the mosquito vector, and eliminate the various stages of the parasite during its cycle in the human body, are needed. It is surprising that few studies [
27], to date, have examined drug activity against liver, sexual blood and mosquito stages of the malaria parasite at the same time. In this study, PVB activity was assessed on these different parasite stages. PVB is a MB preparation that complies with the European Pharmacopoeia, contains limited organic impurities and heavy metals of recognized toxicity, and has previously been demonstrated to possess anti-malarial activity on asexual and sexual blood stages. In vitro experiments confirmed PVB anti-malarial potency against the blood stage of 23
P. falciparum strains that were resistant to various anti-malarial drugs [
16]. In combination with mefloquine, quinine and dihydroartemisinin, PVB showed synergistic effects in vitro against 9 well-established
P. falciparum strains [
28,
29]. Ex vivo experiments on 19 samples isolated from patients showed IC
50 for PVB ranging from 0.88 to 40.2 nM with a mean of 5.3 nM [
30]. PVB treatment at 1–10 mg/kg for 5 days significantly reduced or prevented cerebral malaria in mice [
31‐
33]. In humans, several clinical trials conducted in Burkina Faso showed that oral MB was safe and effective in the treatment of uncomplicated falciparum malaria when combined with other anti-malarials [
17,
18,
34‐
36]. Another study reported moderate curative activity with MB monotherapy, illustrating the need for this slow-acting drug to be combined with fast-acting anti-malarials [
37]. Another advantage of MB is that it has gametocytocidal properties and can reduce the transmission of
P. falciparum. In fact, MB interferes in vitro on gametocyte development at all stages and can block transmission through clearance of stage V gametocytes [
38]. Previous clinical observations showed that MB reduced gametocyte carriage rate in treated patients [
18,
39]. So far no published study has analysed the activity of MB on the hepatic stage of
Plasmodium parasites. Here, several established models of malaria liver stages were used to evaluate the effectiveness of PVB. The
P. yoelii murine model has two main interests, it allows: (1) a quick and ‘easy’ in vitro screening of the molecules; and, (2) to draw a parallel between in vitro and in vivo experiments. Murine
Plasmodium species are commonly used to study the liver stage of infection in mice and in vitro, yet it is not clear how well these models reflect
P. falciparum infections in humans. Then, two in vitro models:
P. falciparum and
P. cynomolgi were used to validate the results with
P. yoelii model. The
P. falciparum experiments provide direct data on pharmacodynamic effects against the liver and blood stages of this most important human malaria parasite. The
P. cynomolgi system is being used as a model for
P. vivax and replicates its capability to produce hypnozoites. This
P. cynomolgi model has been used for decades to identify radical curative compounds against
P. vivax [
40,
41]. In all these models, PVB showed no significant activity on
Plasmodium liver stage infection in vitro. Interestingly, MB could partially reduce the parasite burden in the liver of
P. yoelii-infected mice; however this effect was observed only at the highest doses and was not sufficient to prevent transition to a blood stage infection. To date, there is only a limited number of therapeutics capable of eliminating
Plasmodium liver stages [
42,
43]. Whereas PQ, as well as tafenoquine, and atovaquone actively eliminate dividing
Plasmodium liver stages, only PQ has a pronounced effect on the hypnozoites [
22]. However, because of its potential to cause life-threatening acute intravascular haemolysis in individuals with severe G6PD deficiency, PQ cannot be widely used [
44]. Thus, the development of new compounds with hypnozoitocidal activity suitable for mass administration has become a priority in the current drive to eliminate malaria.
In the present study, MB shows low or no activity in vitro in primary hepatocytes while PQ is very effective. Next, in vivo experiments were performed to evaluate the effect of MB on hepatic and blood stages development. In vivo imaging of
Plasmodium growth was used to measure the specific inhibition of parasite hepatic stage development and its consequences on blood stage spreading [
45,
46]. All control mice used showed a strong luminescence signal at 44 h, as well as on day 3 and 4 post-infection while luminescence signal was abrogated in animals treated with PQ (50 mg/kg). In these experiments, while MB was not able to prevent spreading of the parasites into the central circulation (Fig.
4), an inhibitory effect was observed using the high dose of 100 mg/kg, demonstrating once again that to prevent erythrocytic infection, hepatic inhibition has to be close to 100%. These results have to be considered with those obtained by Garavito in his thesis [
47]. Studying the pharmacological effects of MB, he observed a slight delay in the appearance of the erythrocytic infection.
Finally, the activity of MB against
P. yoelii blood stages was confirmed and its transmission blocking activity too. Indeed, parasitaemia was reduced by 80% in mice infected with 10
7 P. yoelii infected red blood cells when MB (50 mg/kg) was applied from day 0 to day 3 (Table
2), leading to the disappearance of gametocytes from the circulation. These results are in agreement with preliminary clinical data showing a complete elimination of asexual stages and gametocytes in the blood stream of MB-treated malaria patients [
18,
39]. Even more interesting, MB considerably decreased the prevalence of infected mosquitoes when the treatment was administrated 2 h before feeding. These observations are in line with results on gametocyte development in the
P. falciparum model [
38].