C mice infected with L. (L.) amazonensis. First, the efficacy of GV and TPM 6 in a 1 gel was compared to a control group that received placebo. As seen in figure 3A, treatment with TPM 6 gel led to a get 256373-96-3 significant decrease in the parasite burdens at site of infection, from 16107 (control group) to 16104 (TPM 6 treated group), whereas, no parasites were found at lesion site in the GV treated group. In a dose-effect assay, GV was tested in a gel either at 0.1, 0.5 or 1 . Five animals per group were treated twice a day for 20 days, as above described. As shown in Figure 3B, the number of parasites within the lesion decreased when gel concentration were increased, although a linear dose-response has been not observed. The number of parasites in the control group (2.26107) was higher than that observed in the groups treated with GV gel at 0.1 (2.26106), 0.5 (2.626105), or 1 (parasites were not detected). Statistical analysis showed a significant reduction in parasite numbers only in 1 GV treated group when compared with the control group (p,0.05).this study, 10 novel TPM were evaluated against promastigotes and amastigotes from 3 species of Leishmania, recognized worldwide as major etiological agents of CL. The most effective compounds proved to be GV and TPM 6 for all the Leishmania species tested. Overall, there was no significant difference in the efficacy of the same compound against the promastigotes of three different species of Leishmania. Table 3. Cytotoxicity, anti-leishmanial in vitro activity and selectivity index (SI) of TPM 1, TPM 2, TPM 6, TPM 9 and GV against L. (L.) amazonensis and L. (V.) braziliensis on intracellular amastigotes assay.TPMCytotoxicity IC50 (mM)L. (L.) amazonensisIC50 (mM) 0.76 (0.53; 0.99) 1.59 (1.25; 1.93) 0.10 (0.08; 0.11) 0.34 (0.29; 0.39) 0.17 (0.16; 0,18) 23.71 20.68 41.60 5.97 SI 10.L.(V.) braziliensisIC50 (mM) 0.52 (0.23; 0.81) 1.53 (1.07; 1.99) 0.10 (0.09; 0.11) 0.17 (0.08; 0.26) n.d. n.d. 41.35 41.60 6.20 SI 15.TPM8.21 (7.46; 8.96)TPM9.49 (8.68; 10.30)TPM4.16 (3.18; 5.14)TPM7.03 (6.07; 7.99)GV4.03 (3.36; 4.70)DiscussionGiven the worldwide prevalence of Leishmania infection in countries that have low budgets for health care, finding a safe and inexpensive treatment for leishmaniasis is still an unmet need. InIC50 values correspond to mean and 95 CI of results obtained from triplicates; n.d., not determined; data obtained from linear regression on MiniTabH 15.1 software; mean value of parasite growth inhibition observed for control drug (0.2 mg/ml AmB) was 98 for L. (V.) braziliensis and 99.5 for L. (L.) amazonensis. doi:10.1371/journal.pone.Docosahexaenoyl ethanolamide biological activity 0051864.tTriphenylmethane Activity against LeishmaniasisFigure 3. In vivo efficacy of GV and TPM6 topical treatment in L (L.) amazonensis-infected BALB/c mice. Female BALB/c mice were infected with L (L.) amazonensis at the base of the tail; 6 weeks after inoculation. A) Lesions were covered with 50 ml of a gel formulation containing either 1 GV or 1 TPM 6, twice a day, for 20 days. Animals from control group were treated with the gel formulation without GV or TPM 6 (placebo). The treatment efficacy was evaluated through of the parasite quantification at the site of infection. B) Dose-effect study of GV. The GV gel formulation was applied topically at 0.1, 0.5 or 1.0 twice a day, for 20 days. Animals from control group were treated with the gel formulation without GV (placebo). In both experiments, parasite numbers recovered from lesions were evaluated by.C mice infected with L. (L.) amazonensis. First, the efficacy of GV and TPM 6 in a 1 gel was compared to a control group that received placebo. As seen in figure 3A, treatment with TPM 6 gel led to a significant decrease in the parasite burdens at site of infection, from 16107 (control group) to 16104 (TPM 6 treated group), whereas, no parasites were found at lesion site in the GV treated group. In a dose-effect assay, GV was tested in a gel either at 0.1, 0.5 or 1 . Five animals per group were treated twice a day for 20 days, as above described. As shown in Figure 3B, the number of parasites within the lesion decreased when gel concentration were increased, although a linear dose-response has been not observed. The number of parasites in the control group (2.26107) was higher than that observed in the groups treated with GV gel at 0.1 (2.26106), 0.5 (2.626105), or 1 (parasites were not detected). Statistical analysis showed a significant reduction in parasite numbers only in 1 GV treated group when compared with the control group (p,0.05).this study, 10 novel TPM were evaluated against promastigotes and amastigotes from 3 species of Leishmania, recognized worldwide as major etiological agents of CL. The most effective compounds proved to be GV and TPM 6 for all the Leishmania species tested. Overall, there was no significant difference in the efficacy of the same compound against the promastigotes of three different species of Leishmania. Table 3. Cytotoxicity, anti-leishmanial in vitro activity and selectivity index (SI) of TPM 1, TPM 2, TPM 6, TPM 9 and GV against L. (L.) amazonensis and L. (V.) braziliensis on intracellular amastigotes assay.TPMCytotoxicity IC50 (mM)L. (L.) amazonensisIC50 (mM) 0.76 (0.53; 0.99) 1.59 (1.25; 1.93) 0.10 (0.08; 0.11) 0.34 (0.29; 0.39) 0.17 (0.16; 0,18) 23.71 20.68 41.60 5.97 SI 10.L.(V.) braziliensisIC50 (mM) 0.52 (0.23; 0.81) 1.53 (1.07; 1.99) 0.10 (0.09; 0.11) 0.17 (0.08; 0.26) n.d. n.d. 41.35 41.60 6.20 SI 15.TPM8.21 (7.46; 8.96)TPM9.49 (8.68; 10.30)TPM4.16 (3.18; 5.14)TPM7.03 (6.07; 7.99)GV4.03 (3.36; 4.70)DiscussionGiven the worldwide prevalence of Leishmania infection in countries that have low budgets for health care, finding a safe and inexpensive treatment for leishmaniasis is still an unmet need. InIC50 values correspond to mean and 95 CI of results obtained from triplicates; n.d., not determined; data obtained from linear regression on MiniTabH 15.1 software; mean value of parasite growth inhibition observed for control drug (0.2 mg/ml AmB) was 98 for L. (V.) braziliensis and 99.5 for L. (L.) amazonensis. doi:10.1371/journal.pone.0051864.tTriphenylmethane Activity against LeishmaniasisFigure 3. In vivo efficacy of GV and TPM6 topical treatment in L (L.) amazonensis-infected BALB/c mice. Female BALB/c mice were infected with L (L.) amazonensis at the base of the tail; 6 weeks after inoculation. A) Lesions were covered with 50 ml of a gel formulation containing either 1 GV or 1 TPM 6, twice a day, for 20 days. Animals from control group were treated with the gel formulation without GV or TPM 6 (placebo). The treatment efficacy was evaluated through of the parasite quantification at the site of infection. B) Dose-effect study of GV. The GV gel formulation was applied topically at 0.1, 0.5 or 1.0 twice a day, for 20 days. Animals from control group were treated with the gel formulation without GV (placebo). In both experiments, parasite numbers recovered from lesions were evaluated by.