IC50 values and in vitro potentiation tests.
values obtained for artemisinin, artemether, arteflene, Mn-TPPS, and Mn-TMPS on P. falciparum
FcB1 strain are reported in Table . These results indicate that, on the chloroquine-resistant P. falciparum
FcB1-Columbia strain, artemether is 3.5-fold as efficient as the parent drug artemisinin (IC50
, 0.1 nM, compared to 0.35 nM after 32 h of incubation) and close to 500-fold as efficient as arteflene (IC50
, 49 nM). The IC50
values of Mn-TPPS and Mn-TMPS were also determined and were found in the same range as previously reported (4
). In fact, these metalloporphyrins are both devoid of significant antimalarial activity when used alone (IC50
, 276 μM and 257 μM for Mn-TPPS and Mn-TMPS, respectively).
TABLE 1 IC50 values of endoperoxides and manganese metalloporphyrins tested independently against the chloroquine-resistant P. falciparum strainFcB1-Columbia
Artemether is a widely used derivative of artemisinin. It is effective against multidrug-resistant P. falciparum
), and several studies in Asia have suggested that artemether is more effective than quinine in reducing the number of fatal issues in severe malaria (16
). However, decreasing the efficient doses is a desirable goal in order to decrease the risk of resistance and also from an economic point of view. The potentiation of artemether by Mn-TPPS and Mn-TMPS is reported in Fig. A and B, respectively. A significant synergistic effect was observed in the presence of Mn-TPPS (Fig. A), the efficiency of artemether being multiplied by a factor of 3.5 or 5.5, after 32 or 72 h of incubation, respectively. This result is consistent with that obtained for the potentiation of artemisinin by Mn-TPPS on the same parasite strain (activity × 3.6 after 72 h) (4
). On the other hand, Mn-TMPS also increased the efficiency of artemether after 32 h of incubation but was only additive after 72 h (Fig. B). An additive effect of Mn-TMPS associated with artemisinin after 72 h was also previously reported (4
), but presently we have no rational explanation for the difference in effect of Mn-TMPS with respect to the incubation time.
Potentiation of artemether (A and B) and arteflene (C and D) by Mn-TPPS (A and C) and Mn-TMPS (B and D) on P. falciparum FcB1 strain after 32 h (■) or 72 h (●) of incubation.
Arteflene is a potent, long-lasting antimalarial drug, which is also active on chloroquine- and/or pyrimethamine-resistant strains (19
). A clinical study of arteflene in the treatment of patients with mild malaria showed good efficacy and no adverse effects (35
). However, other studies showed that the same dose was not sufficient to cure children with P. falciparum
). The potentiation of arteflene by simple and cheap molecules is of particular interest as is its combination with other antimalarial drugs.
The potentiation of arteflene by Mn-TPPS and Mn-TMPS is reported in Fig. C and D, respectively, after 32 and 72 h of incubation time. Both resulting isobolograms are close to the diagonal, therefore indicating that an additive effect was obtained, with the activity of arteflene mainly unchanged in the presence of a synthetic metalloporphyrin. When radiolabeled arteflene is incubated with P. falciparum
-infected erythrocytes, this drug alkylates some parasite proteins (2
). Furthermore, the in vitro activation of arteflene by a reduced-heme model produces drug-derived fragments which are able to react with nucleophilic residues of proteins to generate covalent adducts. It is therefore unlikely that arteflene did not react with Mn-TPPS or react to produce inert derivatives. The more reasonable hypothesis is that the in vivo activation of arteflene by sulfonated metalloporphyrin complexes probably occurs away from the parasite target of arteflene.
In vivo potentiation by Mn-TPPS.
values of artemisinin and artemether on the P. vinckei petteri
-infected mice were 3 ± 2 mg/kg of body weight and 0.3 ± 0.2 mg/kg of body weight, respectively, which is consistent with values previously reported by Jaquet et al. (19
) and Posner et al. (28
) (drugs injected subcutaneously on Plasmodium berghei
In the 4-day test, the ED50
values of Mn-TPPS were found to be 25 ± 10 mg/kg of body weight per day. Since Mn-TPPS was shown to enhance in vitro the antimalarial activity of both artemisinin and artemether when incubated with the P. falciparum
FcB1 strain and is devoid of toxicity in several cell lines (e.g., HeLa human fibroblast [4
] and MT-4 lymphocyte [37
]), we decided to treat a group of healthy mice with Mn-TPPS for 4 days. At a dose of 40 mg/kg of body weight per day, (close to twice as high as ED50
), the treated mice were still alive, with normal behavior, after a 2-month period. No visible effect on organs was noticed during autopsy. These data confirmed the absence of toxicity of Mn-TPPS in mice.
The control-mouse group was inoculated with Plasmodium spp. and received no treatment but only physiological serum during the 4-day test. The mean parasitemia of untreated control mice on day 4 was found to vary from 32% to 63% (mean, 46%). No death occurred during the treatment. All but one (97%) of the nontreated control mice died between days 5 and 14.
The results obtained for the potentiation of the drug by Mn-TPPS on P. vinckei petteri
were analyzed by using the Dunnett statistical test. For α = 0.05 and in bilateral situations, the groups of mice treated by the peroxide drug alone or by the peroxide associated with Mn-TPPS have ED50
values statistically different from those obtained with the untreated control group (11
The results obtained for the potentiation of artemisinin and artemether by Mn-TPPS on P. vinckei petteri are reported in Fig. and , respectively. They are expressed as percentages of parasitemia inhibition with respect to the control group. The inhibition of parasitemia of the control group (line 1) is therefore 0%. In Fig. , lines 2 to 4 and 5 to 7 report parasitemia inhibition in the presence of increasing doses of artemisinin alone and Mn-TPPS alone, respectively. Lines 8 to 10 report parasitemia inhibition in the presence of 1 mg of artemisinin per kg of body weight per day and increasing doses of Mn-TPPS (2, 10, and 20 mg/kg/day).
Parasitemia inhibition in mice treated with artemisinin (art) associated with Mn-TPPS. Hatched zones represent the expected inhibition if both drugs were additive.
Parasitemia inhibition in mice treated with artemether (artm) associated with Mn-TPPS. Hatched zones represent the expected inhibition if both drugs were additive.
The potentiation was calculated as [inh (A + MnP) − (inh A + inh MnP)]/(inh A + inh MnP), where inh A is the parasitemia inhibition value due to the applied dose of artemisinin when used alone, inh MnP is the parasitemia inhibition value due to the applied dose of Mn-TPPS when used alone, and inh (A + MnP) is the parasitemia inhibition value due to artemisinin and Mn-TPPS used together.
When the subinhibitory dosage of 1 mg of artemisinin per kg of body weight per day was associated with 10 or 20 mg of Mn-TPPS per kg of body weight per day, the inhibition of parasitemia was 54% ± 7% and 55% ± 10%, respectively (lines 9 and 10 in Fig. ), significantly higher than that of mice treated with artemisinin used alone (15% ± 3% [Fig. , line 3]) or the inhibition of parasitemia expected in the case of an additive effect (15% + 22% = 37%). The potentiations reached in these cases, 46 and 49% (Fig. , lines 9 and 10), were two similar values. With 1 mg of artemisinin per kg of body weight per day associated with 2 mg of Mn-TPPS per kg of body weight per day, the potentiation was 25% (line 8). The decreased parasitemia of mice treated with artemisinin associated with Mn-TPPS is consistent with the in vitro potentiation of artemisinin by the metalloporphyrin on the FcB1 strain.
The potentiation study of artemether reported in Fig. is rather puzzling. When 0.25 mg of artemether per kg of body weight per day was associated with 2 or 10 mg of Mn-TPPS per kg body weight per day, in the hypothesis of an additive effect of these two drugs, 84 or 91%, respectively, of parasitemia inhibition was expected. In fact, only 42% ± 8% and 45% ± 10% of parasitemia inhibition was observed in these cases (Figure , lines 8 and 10).
This reduced efficiency of artemether in the presence of the metalloporphyrin did not correlate with the potentiation observed in vitro (Fig. B). The living mice are obviously more complex than the in vitro parasite culture medium, and artemether was found to be more fragile than artemisinin, especially in acidic conditions (34
). It is also probable that artemether can be activated by the metalloporphyrin outside the parasite, far from the target heme or proteins, leading to a loss of “useful” artemether and then to a decrease of efficiency when artemether and Mn-TPPS are associated. In fact, different studies have shown that the results of antimalarial assays obtained in vitro with drug associations are difficult to extrapolate from one strain to another and from in vitro to in vivo (12
). For example, on one hand, the association of artemisinin and mefloquine has been found to be additive in vitro on K1 and NF-54 strains (19
). On the other hand, this association exhibits a synergistic effect in vivo on P. berghei
) and is widely used against multidrug-resistant Plasmodium
spp. in humans (17
). Furthermore, it was recently reported that rats might be better animal models than mice for parasitical studies, due to the numerous differences between the immunity systems of the latter and that of humans (6
The main result of the present in vivo potentiation study is that the artemisinin efficiency can be increased by ca. 50% in the presence of a nontoxic and cheap manganese porphyrin complex, which is by itself devoid of any antiplasmoidal activity. In addition, these results confirm that the activation of artemisinin by heme or a heme model in the present case is a key step in the mechanism of action of this antimalarial trioxane.