Activity of Chitosan and Its Derivatives against Leishmania major and Leishmania mexicana In Vitro.

There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million people worldwide annually. Chitosan, a biodegradable cationic polysaccharide, has previously been reported to have antimicrobial, antileishmanial, and immunostimulatory activities. We investigated the in vitro activity of chitosan and several of its derivatives and showed that the pH of the culture medium plays a critical role in antileishmanial activity of chitosan against both extracellular promastigotes and intracellular amastigotes of Leishmania major and Leishmania mexicana Chitosan and its derivatives were approximately 7 to 20 times more active at pH 6.5 than at pH 7.5, with high-molecular-weight chitosan being the most potent. High-molecular-weight chitosan stimulated the production of nitric oxide and reactive oxygen species by uninfected and Leishmania-infected macrophages in a time- and dose-dependent manner at pH 6.5. Despite the in vitro activation of bone marrow macrophages by chitosan to produce nitric oxide and reactive oxygen species, we showed that the antileishmanial activity of chitosan was not mediated by these metabolites. Finally, we showed that rhodamine-labeled chitosan is taken up by pinocytosis and accumulates in the parasitophorous vacuole of Leishmania-infected macrophages.

Synthesis, Profiling, and in Vivo Evaluation of Cyclopeptides Containing N-Methyl Amino Acids as Antiplasmodial Agents.

Malaria is a major tropical disease where important needs are to mitigate symptoms and to prevent the establishment of infection. Cyclopeptides containing N-methyl amino acids with in vitro activity against erythrocytic forms as well as liver stage are presented. The synthesis, parasitological characterization, physicochemical properties, in vivo evaluation, and mice pharmacokinetics are described.

Synthesis of a Microcystis aeruginosa predicted metabolite with antimalarial activity.

The synthesis of a Microcystis aeruginosa predicted metabolite analog of aerucyclamide B was performed. This hexacyclopeptide was obtained from three heterocyclic building blocks by a convergent macrocycle-assembly methodology. The compound exhibited good in vitro antiplasmodial activity (IC(50): 0.18 µM, K1, cholorquine resistant strain).

Synthesis and antiprotozoal evaluation of new N4-(benzyl)spermidyl-linked bis(1,3,5-thiadiazinane-2-thiones).

The synthesis and in-vitro antiprotozoal evaluation of novel N(4)-(benzyl)spermidyl-linked bis(1,3,5-thiadiazinane-2-thione) (bis-THTT) derivatives from N(4)-(benzyl)spermidine is disclosed. Several of the new bis-THTT have in-vitro activities against L. donovani and T. cruzi that are comparable or superior to those of currently employed protozoocidal agents.

American tegumentary leishmaniasis: Is antimonial treatment outcome related to parasite drug susceptibility?

BACKGROUND: Antimonials are the first drug of choice for the treatment of American tegumentary leishmaniasis (ATL); however, their efficacy is not predictable, and this may be linked to parasite drug resistance. We aimed to characterize the in vitro antimony susceptibility of clinical isolates of Peruvian patients with ATL who were treated with sodium stibogluconate and to correlate this in vitro phenotype with different treatment outcomes. METHODS: Thirty-seven clinical isolates were obtained from patients with known disease and treatment histories. These isolates were typed, and the susceptibility of intracellular amastigotes to pentavalent (SbV) and trivalent (SbIII) antimonials was determined. RESULTS: We observed 29 SbV-resistant isolates among 4 species of subgenus Viannia, most of which exhibited primary resistance; isolates resistant only to SbIII; and 3 combinations of in vitro phenotypes: (1) parasites sensitive to both drugs, (2) parasites resistant to both drugs, and (3) parasites resistant to SbV only (the majority of isolates fell into this category). There was no correlation between in vitro susceptibility to both antimonials and the clinical outcome of therapy. CONCLUSION: Antimony insensitivity might occur in a stepwise fashion (first to SbV and then to SbIII). Our data question the definition of true parasite resistance to antimonials. Further studies of treatment efficacy should apply standardized protocols and definitions and should also consider host factors.

Alkyl-linked bis-THTT derivatives as potent in vitro trypanocidal agents.

The effect of several alkyl-linked bis tetrahydro-(2H)-1,3,5-thiadiazine-2-thione (bis-THTT) on Leishmania donovani, Trypanosoma brucei rhodesiense, and Plasmodium falciparum is reported. Most of the compounds exhibited a potent activity against the three parasitic strains but the best in vitro activity profiles were found against T. b. rhodesiense with IC(50) values ranging between 0.3 and 4 microM for the most active compounds.

The sensitivity of clinical isolates of Leishmania from Peru and Nepal to miltefosine.

Clinical isolates of Leishmania, from visceral leishmaniasis (VL) cases in Nepal and from cutaneous leishmaniasis (CL) cases in Peru, were cultured using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to type species and strain. Promastigotes from 38 isolates, within eight passages from isolation, were used to infect mouse peritoneal macrophage cultures in vitro, and the amastigote sensitivity to miltefosine was determined. The concentration required to kill 50% of intracellular amastigotes from Nepalese VL isolates, all typed as Leishmania (L.) donovani (N = 24) from both Sbv responders and nonresponders, ranged from 8.7 to 0.04 microg/mL. In contrast, the concentration required to kill 50% intracellular amastigotes from isolates from Peru, typed as L.(V.) braziliensis (N = 8), was > 30 to 8.4 microg/mL, L.(V.) guyanensis (N = 2) > 30 to 1.9 microg/mL, L.(L.) mexicana (N = 1) > 30 microg/mL, and L. (V.) lainsoni (N = 4) was 3.4 to 1.9 microg/mL. This demonstrates a notable difference in the intrinsic sensitivity of Leishmania species to miltefosine in vitro. If this model can be correlated to therapeutic outcome, it may have implications for the interpretation of clinical trials.

Inhibitors of Leishmania mexicana CRK3 cyclin-dependent kinase: chemical library screen and antileishmanial activity.

The CRK3 cyclin-dependent kinase of Leishmania has been shown by genetic manipulation of the parasite to be essential for proliferation. We present data which demonstrate that chemical inhibition of CRK3 impairs the parasite's viability within macrophages, thus further validating CRK3 as a potential drug target. A microtiter plate-based histone H1 kinase assay was developed to screen CRK3 against a chemical library enriched for protein kinase inhibitors. Twenty-seven potent CRK3 inhibitors were discovered and screened against Leishmania donovani amastigotes in vitro. Sixteen of the CRK3 inhibitors displayed antileishmanial activity, with a 50% effective dose (ED50) of less than 10 microM. These compounds fell into four chemical classes: the 2,6,9-trisubstituted purines, including the C-2-alkynylated purines; the indirubins; the paullones; and derivatives of the nonspecific kinase inhibitor staurosporine. The paullones and staurosporine derivatives were toxic to macrophages. The 2,6,9-trisubstituted purines inhibited CRK3 in vitro, with 50% inhibitory concentrations ranging from high nanomolar to low micromolar concentrations. The most potent inhibitors of CRK3 (compounds 98/516 and 97/344) belonged to the indirubin class; the 50% inhibitory concentrations for these inhibitors were 16 and 47 nM, respectively, and the ED50s for these inhibitors were 5.8 and 7.6 microM, respectively. In culture, the indirubins caused growth arrest, a change in DNA content, and aberrant cell types, all consistent with the intracellular inhibition of a cyclin-dependent kinase and disruption of cell cycle control. Thus, use of chemical inhibitors supports genetic studies to confirm CRK3 as a validated drug target in Leishmania and provides pharmacophores for further drug development.