Christian Bories

Miltefosine Induces Apoptosis-Like Death in Leishmania donovani Promastigotes

ABSTRACT Miltefosine (hexadecylphosphocholine [HePC]) has proved to be a potent oral treatment for human visceral leishmaniasis due to Leishmania donovani. The molecular mechanisms that contribute to the antileishmanial activity of HePC are still unknown. We report that in wild-type promastigotes of Leishmania donovani HePC is able to induce a cell death process with numerous cytoplasmic, nuclear, and membrane features of metazoan apoptosis, including cell shrinkage, DNA fragmentation into oligonucleosome-sized fragments, and phosphatidylserine exposure. None of these changes were detected in an HePC-resistant clone treated with the same drug concentration. Therefore, HePC does not appear to kill L. donovani promastigotes by a direct toxic mechanism but, rather, kills the promastigotes by an indirect one. Pretreatment of wild-type promastigotes with two broad caspase inhibitors, z-Val-Ala-dl-Asp(methoxy)-fluoromethylketone and Boc-Asp(methoxy)-fluoromethylketone, as well as a broad protease inhibitor, calpain inhibitor I, prior to drug exposure interfered with DNA fragmentation but did not prevent cell shrinkage or phosphatidylserine externalization. These data suggest that at least part of the apoptotic machinery operating in wild-type promastigotes involves proteases. Identification of the death-signaling pathways activated in HePC-sensitive parasites appears to be essential for a better understanding of the molecular mechanisms of action and resistance in these parasites.

Antileishmanial and trypanocidal activity of Brazilian Cerrado plants

The side effects and the emerging resistance to the available drugs against leishmaniasis and trypanosomiasis led to the urgent need for new therapeutic agents against these diseases. Thirty one extracts of thirteen medicinal plants from the Brazilian Cerrado were therefore evaluated in vitro for their antiprotozoal activity against promastigotes of Leishmania donovani, and amastigotes of Trypanosoma cruzi. Among the selected plants, Casearia sylvestris var. lingua was the most active against both L. donovani and T. cruzi. Fifteen extracts were active against promastigotes of L. donovani with concentrations inhibiting 50% of parasite growth (IC50) between 0.1-10 microg/ml, particularly those of Annona crassiflora (Annonaceae), Himatanthus obovatus (Apocynaceae), Guarea kunthiana (Meliaceae), Cupania vernalis (Sapindaceae), and Serjania lethalis (Sapindaceae). With regard to amastigotes of T. cruzi, extracts of A. crassiflora, Duguetia furfuracea (Annonaceae), and C. sylvestris var. lingua were active with IC50 values between 0.3-10 microg/ml. Bioassay fractionations of the more active extracts are under progress to identify the active antiparasite compounds.

Efficacy of Orally Administered 2-Substituted Quinolines in Experimental Murine Cutaneous and Visceral Leishmaniases

ABSTRACT We report in this study the in vivo efficacy of nine 2-substituted quinolines on the Leishmania amazonensis cutaneous infection murine model and on the Leishmania infantum and Leishmania donovani visceral infection murine models. In the case of the L. amazonensis model, quinolines were administered orally at 25 mg/kg twice daily for 15 days. Quinolines 1, 2, 3, and 7 reduced by 80 to 90% the parasite burdens in the lesion, whereas N-methylglucamine antimoniate (Glucantime), administered by subcutaneous injections at 100 mg [28 mg Sb(V)] per kg of body weight daily, reduced the parasite burdens by 98%. In visceral leishmaniasis due to L. infantum, mice treated orally at 25 mg/kg daily for 10 days with quinolines 1, 4, 5, and 6 showed a significant reduction of parasite burdens in the liver and spleen. These quinolines were significantly more effective than meglumine antimoniate to reduce the parasite burden in both the liver and spleen. Also, the oral in vivo activity of three quinolines (quinolines 4, 5, and 2-n-propylquinoline) were determined against L. donovani (LV 9) at 12.5 and 25 mg/kg for 10 days. Their activity was compared with that of miltefosine at 7.5 mg/kg. Miltefosine, 2-n-propylquinoline, and quinoline 5 at 12.5 mg/kg significantly reduced the parasite burdens in the liver by 72, 66, and 61%, respectively. From the present study, quinoline 5 is the most promising compound against both cutaneous and visceral leishmaniasis. The double antileishmanial and antiviral activities of these compounds suggest that this series could be a potential treatment for coinfection of Leishmania-human immunodeficiency virus.

Efficacy and Pharmacokinetics of Intravenous Nanocapsule Formulations of Halofantrine in Plasmodium berghei-Infected Mice

ABSTRACT The efficacy and pharmacokinetics of a new parenteral formulation of halofantrine were studied in mice infected with Plasmodium berghei. The formulation consisted of nanocapsules with an oily core, prepared from either poly(d,l-lactide) (PLA) homopolymer or PLA that was surface modified with grafted polyethylene glycol chains. They were compared with a previously described intravenous halofantrine preparation. No toxic effects were observed with halofantrine in form of nanocapsules after intravenous administration for doses of up to 100 mg/kg, whereas the solubilized form in polyethylene glycol-dimethylacetamide was toxic at this dose. The halofantrine-loaded nanocapsules showed activity that was similar to or better than that of the solution in the 4-day test and as a single dose in severely infected mice, with only minimal differences between the two nanocapsule formulations. Halofantrine pharmacokinetics were determined in parallel with parasite development in severely infected mice. Nanocapsules increased the area under the curve for halofantrine in plasma more than sixfold compared with the solution throughout the experimental period of 70 h. Furthermore, nanocapsules induced a significantly faster control of parasite development than the solution in the first 48 h posttreatment. While the parasitemia fell more rapidly with PLA nanocapsules, the effect was more sustained with the surface-modified ones. This is consistent with surface-modified nanocapsules remaining longer in the circulation. These results suggest that nanocapsule formulations could provide a more favorable halofantrine profile in the plasma and reduce the intravenous dose necessary and therefore the toxicity, thus suggesting the use of halofantrine by a parenteral route in severe malaria.

Noninvasive Monitoring of the Health of Pan troglodytes schweinfurthii in the Kibale National Park, Uganda

We assessed the health status of chimpanzees (Pan troglodytes schweinfurthii) of the Kanyawara group in the Kibale National Park in Western Uganda via noninvasive methods. We conducted visual veterinary inspection, parasitological and urine analysis in association with behavioral observations, causing minimal disturbance or stress to individually recognized chimpanzees. We applied multiple parasitological techniques to 252 stool samples to compare their efficacy in detecting parasitic infection and to increase the power of detecting a wide range of parasites at a more sensitive level. We examined 76 urine samples via a quick detection method to evaluate multiple parameters of urine that indicate organ dysfunction. Results of the different analyses are mutually supportive and provided useful information for monitoring bodily condition and diseases. The multifaceted health evaluation system is a beneficial tool for monitoring long-term and short-term changes in health status due to environmental stress, seasonal dietary change, and disease in wild chimpanzee populations. Use of this method to detect changes in health, when employed together with behavioral observations, may also provide important insights into the potential effects of self-medicative behaviors.

Leishmania Resistance to Miltefosine Associated with Genetic Marker

To the Editor: During 2000–2010, serial Leishmania isolates obtained from an HIV-infected patient who was not responding to treatment showed a gradual decrease in in vitro miltefosine susceptibility. We performed L. donovani miltefosine transporter (Ldmt) gene analysis to identify an association between miltefosine resistance of reference L. donovani lines and variability in miltefosine response of L. infantum isolates. A new single-nucleotide polymorphism (SNP), L832F, was identified, which might be a marker of miltefosine resistance in leishmaniasis. The patient, a 46-year-old woman, had lived in France since 1994 but regularly returned to Algeria, her country of birth. HIV-1 infection was diagnosed in 1991. Antiretroviral therapy was initiated in 1993, leading to undetectable viral load and a CD4+ T-cell count of 185 cells/mm3 (reference >450/mm3). Concurrent conditions were thoracic herpes zoster in 1996, hairy leukoplakia of tongue, oropharyngeal candidiasis, and chronic renal failure of unknown cause since 2000. Visceral leishmaniasis was diagnosed in 1998 by culture of a bone marrow smear, which showed intracellular amastigotes. Use of meglumine antimonate (Glucantime; Sanofi, Paris, France), a drug of choice for the treatment of leishmaniasis, was contraindicated because of pancreatitis in the patient and in vitro isolate susceptibility variation; therefore, induction therapy consisted of liposomal amphotericin B (AmpB [AmBisome; Astellas Pharma US, Deerfield, IL, USA]) at a dose of 3 mg/kg/d for 5 consecutive days, then 1× week for 5 weeks (total dose 30 mg/kg) during 1998–2000 (Table). The same medication was administered for relapses at 4 mg/kg/d for 5 days, then 4 mg/kg 1× week for 5 weeks (total dose 40 mg/kg) during 2001–2010. Given the adverse effects of AmpB and the availability of oral miltefosine (Impavido; AEterna Zentaris Inc., Quebec City, Quebec, Canada), the latter drug was used for maintenance treatment during 2001–2007 at 50 mg 2×/d. Leishmaniasis was monitored by leukocytoconcentration and culture of blood samples on Novy-Nicolle-McNeal medium. Table Comparions of IC50 for AmpB and miltefosine against promastigotes and axenic amastigotes and distribution of LdMT SNPs in Leishmania infantum isolates and reference strains* When signs of biological and clinical relapse appeared, bone marrow was aspirated for parasite detection. After culture of the aspirate and isoenzyme determination, the strain was identified as L. infantum, zymodeme MON-24. Eleven relapses were documented; all were confirmed by positive direct examination of bone marrow or blood, but cultures of only 7 samples yielded positive results (Table). The susceptibility of 4 cryopreserved isolates (S1, S3, S4, and S6; Table) to AmpB and to miltefosine was studied in the in vitro promastigote and axenic amastigote form by determining the concentrations inhibiting parasite growth by 50% (1,2). The 50% inhibitory concentration (IC50) was determined in parallel for the following reference L. donovani lines: a wild-type L. donovani LV9 (MHOM/ET/67/HU3) line (LV9 WT), a wild-type L. donovani DD8 (MHOM/IN/80/DD8) line (DD8 WT), a laboratory miltefosine-resistant line obtained from LV9 WT (LV9 miltefosine-R, resistant to 90 μmol/L miltefosine), and the laboratory AmB-resistant line obtained from DD8 WT (DD8 AmB-R, resistant to 1.4 μmol/L AmB) on promastigote and axenic amastigote forms (3,4). The AmB susceptibility of the isolates did not change notably over time; IC50 values ranged from 0.09 µmol/L to 0.24 µmol/L, regardless of parasite form, similar to those of wild-type reference strains (Table). In contrast, the IC50 values of miltefosine increased greatly over time, from 5.00 µmol/L to 50.10 μmol/L. During the 6 years of follow-up with miltefosine maintenance therapy, the susceptibility of the isolate (S6) obtained 6 months after miltefosine treatment withdrawal in 2008 was 6-fold higher than that of the first isolate (S1) obtained in 2000. The L. donovani miltefosine transporter protein (LdMT) promotes miltefosine translocation (5), and LdMT inactivation in L. donovani promastigotes leads to miltefosine resistance at the promastigote and amastigote stages (6). In 2003 and 2006 studies, several mutations were linked to the inability of parasites to take up miltefosine and to miltefosine resistance (5,7). In a 2009 study, the weak expression of LdMT and its β subunit LdROS3 in L. braziliensis isolates was linked to diminished sensitivity (8). We sequenced the entire Ldmt gene (3,294 bp) in the reference strains and the clinical isolates for SNP analysis (5,7). Only 1 new SNP, L832F, was found in the miltefosine-resistant reference strain (LV9 miltefosine-R) and in clinical isolate S6. The L832 wild-type allele was found in isolate S1 and in the miltefosine-sensitive reference lines (LV9, DD8, and DD8 AmpB-R), whereas both alleles were found in isolates S3 and S4, with a decrease in the wild-type allele (Table). The last isolate, which was obtained 3 years after miltefosine withdrawal and could not be subcultured, had reverted to the wild-type allele (L832). These results point to a relation between the 832F allele and diminished susceptibility to miltefosine. Analysis of this case of miltefosine resistance in a patient co-infected with Leishmania sp. and HIV strongly suggests that an SNP (L832F) in the Ldmt gene could represent a molecular marker of miltefosine resistance in L. infantum and L. donovani.

Toxicity and Antileishmanial Activity of a New Stable Lipid Suspension of Amphotericin B

ABSTRACT The aim of the present study was to evaluate the toxicity and the activity of a new lipid complex formulation of amphotericin B (AMB) (LC-AMB; dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, and AMB) that can be produced by a simple process. Like other lipid formulations, this new complex reduced both the hemolytic activity of AMB (the concentration causing 50% hemolysis of human erythrocytes, >100 μg/ml) and its toxicity toward murine peritoneal macrophages (50% inhibitory concentration, >100 μg/ml at 24 h). The in vivo toxicity of the new formulation (50% lethal dose,> 200 mg/kg of body weight for CD1 mice) was similar to those of other commercial lipid formulations of AMB. The complex was the most effective formulation against the DD8 strain of Leishmania donovani. It was unable to reverse the resistance of an AMB-resistant L. donovani strain. In vivo LC-AMB was less efficient than AmBisome against L. donovani.

Antiprotozoal activity of aporphine alkaloids isolated from Unonopsis buchtienii (Annonaceae).

On a preliminary screening, substantial leishmanicidal activity was observed for the petroleum ether and alkaloidal extracts of the stem bark of Unonopsis buchtienii, the alkaloids and sterols isolated from these were studied. Of the alkaloids, liriodenine exhibited the highest activity against Leishmania major and L. donovani (IC100 = 3.12 µg/mL). On the other hand, O‐methylmoschatoline and the petroleum ether extract without alkaloids showed an interesting in vitro activity against Trypanosoma brucei with an IC100 of 6.25 µg/mL. The highest cytotoxic activities were found with the petroleum ether extracts without alkaloids and with all alkaloids isolated (IC50 < 9 µg/mL for Vero cell line). Copyright

Leishmania major and HIV co-infection in Burkina Faso.

The incidence of cutaneous leishmaniasis (CL) has increased in Ouagadougou, Burkina Faso since 1996. A study was carried out from September to November 2000 to determine the impact of HIV on this outbreak. Of 74 CL patients, 10 were co-infected with HIV. The percentage of CL in patients positive for HIV was slightly higher than the percentage of HIV patients in Ouagadougou. However, the study showed that HIV infection did not increase the risk of CL infection.

Nosocomial Dermatitis Caused by Dermanyssus gallinae

The mite Dermanyssus gallinae may cause pruritic dermatitis in humans. We describe a case of nosocomial infestation with D. gallinae from an abandoned pigeon nest suspended on the front wall of the Hôpital Henri Mondor near a window. Close surveillance and regular destruction of pigeon nests could prevent these incidents of infection in humans.