Activity of benzothiazoles and chemical derivatives on Plasmodium falciparum.

Malaria is a major health concern particularly in Africa which has about 90% of the worldwide annual clinical cases. The increasing number of drug-resistant Plasmodium falciparum justifies the search for new drugs in this field. Antimalarial activity of 2-substituted 6-nitro- and 6-amino-benzothiazoles and their anthranilic acids has been tested. An in vitro study has been performed on W2 and 3D7 strains of P. falciparum and on clinical isolates from malaria-infected patients. Toxicity has been assessed on THP1 human monocytic cells. For the most active drug candidates, the in vitro study was followed by in vivo assays on P. berghei-infected mice and by in vitro assays in order to determine the stage-dependency and the mechanism of action. Of 39 derivatives tested in vitro, 2 had specific antimalarial properties. Each compound was active on all stages of the parasite, but one was markedly active on mature schizonts, while the other was more active on young schizont forms. Both drugs were also active on mitochondrial membrane potential. In vivo data confirmed efficiency with a sustained decrease of parasitaemia. Products A12 and C7 may be considered as potential antimalarial worthy of further chemical and biological research.

In vitro activity of the beta-carboline alkaloids harmane, harmine, and harmaline toward parasites of the species Leishmania infantum.

Harmane, harmine, and harmaline were investigated for their in vitro antileishmanial activity toward parasites of the species Leishmania infantum. Harmane and Harmine displayed a moderate antiproliferative activity toward human monocytes and exerted a weak antileishmanial activity toward both the promastigote and the amastigote forms of the parasite. Their mechanism of action on the promastigote form of the parasite involved interactions with DNA metabolism leading to an accumulation of parasites in the S-G(2)M phases of the cell-cycle. Harmaline, at the contrary, was deprived from toxicity toward human cells and Leishmania promastigotes, however it exerted a strong antileishmanial activity toward the intracellular amastigote form of the parasite. This property was shown to partly result from the capacity of the molecule to prevent parasite internalization within macrophages by inhibiting Leishmania PKC activity.

[Liposomal amphotericin B in secondary prophylaxis of visceral leishmaniasis in HIV-infected patients: report of five clinical cases]. / L'amphotéricine B liposomale en prophylaxie secondaire chez les patients VIH+ atteints de leishmaniose viscérale: à propos de cinq cas.

Treatment of visceral leishmaniasis in HIV patients encounters inefficacy and relapse due to drug resistance, toxicity and immunodepression. Our goal was to evaluate treatment of these patients by liposomal amphotericin B (L-AmB). Since 1998, five clinical files were exploitable out of 13 patients. Protocols used bolus doses ranging between 2.9 and 4.1 mg/kg dispatched on 5-24 days, followed by maintenance dose ranging from 2.7 to 3.8 mg/kg every 15 days. Attack treatment involved high bolus dose (cumulated doses ranging from 60 to 86 mg/kg at day 30) and allowed favorable clinical and biological results with healing in four patients. Secondary prophylaxis with L-AmB has been efficacious and well tolerated in three patients. Although literature and study results cannot indicate a standard therapeutic care in these patients, an initial treatment by L-AmB at doses higher than marketing-approved doses with a secondary prophylaxis by L-AmB associated with an antiretroviral treatment seem to be major asset in order to obtain healing. Expanding this study to a multicenter trial should allow to better define the frequency and duration of the secondary prophylaxis and to evaluate the risk of therapeutic escape as well as the life-span increase.

Ethnobotanical survey and in vitro antiplasmodial activity of plants used in traditional medicine in Burkina Faso.

In Burkina Faso, most people in particular, in rural areas, use traditional medicine and medicinal plants to treat usual diseases. In the course of new antimalarial compounds, an ethnobotanical survey has been conducted in different regions. Seven plants, often cited by traditional practitioners and not chemically investigated, have been selected for an antiplasmodial screening: Pavetta crassipes (K. Schum), Acanthospermum hispidum (DC), Terminalia macroptera (Guill. et Perr), Cassia siamea (Lam), Ficus sycomorus (L), Fadogia agrestis (Schweinf. Ex Hiern) and Crossopteryx febrifuga (AFZ. Ex G. Don) Benth. Basic, chloroform, methanol, water-methanol and aqueous crude extracts have been prepared and tested on Plasmodium falciparum chloroquine-resistant W2 strain. A significant activity has been observed with alkaloid extract of P. crassipes (IC(50)<4 microg/ml), of A. hispidum, C. febrifuga, and F. agrestis (4

Antiplasmodial activity of alkaloid extracts from Pavetta crassipes (K. Schum) and Acanthospermum hispidum (DC), two plants used in traditional medicine in Burkina Faso.

In the course of the search for new antimalarial compounds, a study of plants traditionally used against malaria in Burkina Faso was made. An ethnobotanical study permitted the identification of plants currently used by the traditional healers and herbalists. Two plants among them were selected for further study: Pavetta crassipes (K. Schum) and Acanthospermum hispidum (DC). Alkaloid extracts of these plants were tested in vitro against two reference clones of Plasmodium falciparum: the W2 chloroquine-resistant and the D6 chloroquine-sensitive strains. Significant inhibitory activity was observed with Pavetta crassipes (IC(50)=1.23 microg/ml) and A. hispidum (IC(50)=5.02 microg/ml). Antiplasmodial activity was also evaluated against six Plasmodium falciparum isolates from children between 4 and 10 years old. The IC(50) values for the alkaloid extracts were in the range 25-670 ng/ml. These results indicated that P. falciparum wild strains were more sensitive to the alkaloid extracts than strains maintained in continuous culture. Moreover, the alkaloid extracts exhibit good in vitro antimalarial activity and weak cytotoxicity against three human cell lines (THP1, normal melanocytes, HTB-66). Isolation and structural determination are now necessary in order to precisely determine the active compounds.

Antimalarial activity of extracts and alkaloids isolated from six plants used in traditional medicine in Mali and Sao Tome.

Methanol and chloroform extracts were prepared from various parts of four plants collected in Mali: Guiera senegalensis (Gmel.) Combretaceae, Feretia apodanthera (Del.) Rubiaceae, Combretum micranthum (Don.) Combretaceae, Securidaca longepedunculata (Fres.) Polygalaceae and two plants -collected in Sao Tome: Pycnanthus angolensis (Welw.) Myristicaceae and Morinda citrifolia (Benth.) Rubiaceae were assessed for their in vitro antimalarial activity and their cytotoxic effects on human monocytes (THP1 cells) by flow cytometry. The methanol extract of leaves of Feretia apodanthera and the chloroform extract of roots of Guiera senegalensis exhibited a pronounced antimalarial activity. Two alkaloids isolated from the active extract of Guiera senegalensis, harman and tetrahydroharman, showed antimalarial activity (IC(50) lower than 4 microg/mL) and displayed low toxicity against THP1. Moreover, the decrease of THP1 cells in S phase of the cell cycle, after treatment with harman and tetrahydroharman, was probably due to an inhibition of total protein synthesis.

Synergistic in vitro antimalarial activity of plant extracts used as traditional herbal remedies in Mali.

In Mali, where malaria is endemic, plants are extensively used for treating periodic fevers and malaria. According to the advice of traditional medicine, plants are often mixed during the preparation of febrifugal decoctions. In previous studies, we demonstrated the potent in vitro antimalarial activity of extracts isolated from four plants commonly used in traditional remedies: Mitragyna inermis (Willd.) O. Kuntze, Rubiaceae, Nauclea latifolia (Sm.), Rubiaceae, Guiera senegalensis (Gmel.), Combretaceae, and Feretia apodanthera (Del.), Rubiaceae. In the present work, we evaluate the potent in vitro synergistic antimalarial interaction between these extracts, using standard isobologram analysis. Then, we evaluate their cytotoxicity on human monocytes and their mutagenic activity on an in vitro system of two beta-carboline alkaloids isolated from Guiera senegalensis (harman and tetrahydroharman). Three combinations demonstrate a strong, synergistic, inhibitory effect on in vitro plasmodial development and are devoid of cytotoxicity towards human cells. These results justify their use in association in traditional medicine. Moreover, tetrahydroharman, isolated from G. senegalensis, presents interesting antimalarial activity, no cytotoxicity and is not genotoxic in the Salmonella Ames test with and without metabolic activation.

In vitro antileishmanial activity of three saponins isolated from ivy, alpha-hederin, beta-hederin and hederacolchiside A(1), in association with pentamidine and amphotericin B.

The in vitro antileishmanial activity of three saponins isolated from ivy, alpha-hederin, beta-hederin and hederacolchiside A(1), was investigated on parasites of the species Leishmania mexicana, in their promastigote and amastigote forms compared with their toxicity versus human monocytes. The results showed that saponins exhibited a strong antiproliferative activity on all stages of development of the parasite but demonstrated a strong toxicity versus human cells. Association of subtoxic concentrations of saponins with antileishmanial drugs such as pentamidine and amphotericin B demonstrated that saponins could enhance the efficiency of conventional drugs on both the promastigote and the amastigote stages of development of the parasite. The results demonstrated moreover that the action of saponins on promastigote membrane was cumulative with those of amphotericin B.

[Long-term treatment of Alzheimer's disease: followup of a cohort of 255 patients treated with lacrine for four years]. / Traitement de la maladie d'Alzheimer au long cours: à propos d'une cohorte de 255 patients traités quatre ans par tacrine.

We describe the follow-up of a cohort of 255 Alzheimer's disease (AD) patients (81 males, 174 females) treated by tacrine during 4 years. We performed the survey of hepatic, cholinergic and general tolérance. Drug efficacy was measured by MMS examination on weeks 0, 18, 30, 52, 104, 156 and 208. A total of 190 patients (74.5 percent) were dropped out of this study, 75 (29 percent) for adverse events. We found 85 hepatic (33 percent), 79 cholinergic (31 percent), 31 (12 percent) neuropsychiatric and 72 general (28 percent) side effects. In term of drug efficacy we observed a global decline of 2.5 MMS points during the first year and 2 MMS points between W52 and W156. Tacrine's symptomatic efficacy, defined as the number of patients improved or stabilized at W30, was present in 50 patients (46 percent) among the 109 patients reaching W30. The intensity of symptomatic efficacy was expressed by a 2.7 MMS points increase in 37 patients improved on W30. The long term effects of Tacrine, measured by the MMS score at one year, showed a positive impact as the MMS was 2.5 points above the expected score in non treated AD patients. This study raises the practical problem of optimal cholinesterase inhibitors use in AD and the theoretical question of long term action of cholinesterase inhibitors on cerebral lesions of AD.

No evidence of oxidant events in amphotericin B cytotoxicity versus L. infantum promastigotes.

Amphotericin B is used for the treatment of systemic mycoses and visceral leishmaniasis. The objective of our study was to evaluate the impact of catalase, ascorbic acid and ketoconazole on the amphotericin B toxicity towards Leishmania promastigotes membrane by two flow cytometric tests, the membrane potential assay using a cationic dye, [DiOC5(3)], and the membrane permeability test using propidium iodide. The collapse of membrane potential appeared at amphotericin B concentrations weaker than those assessed by the membrane permeability test. The binding of amphotericin B to membrane sterol was not modified by catalase or ascorbic acid whereas amphotericin B-induced growth inhibition could be modulated by these products. The permeabilizing effect of amphotericin B on parasite membrane was strongly reduced in the presence of ketoconazole. These results confirmed the pore hypothesis of amphotericin B action and suggested that flow cytometric methods constituted a valuable alternative to conventional methods for assessing the effect of drugs on cellular membrane and evaluating parasite susceptibility to polyene antibiotics.

Toxicity and genotoxicity of antimalarial alkaloid rich extracts derived from Mitragyna inermis O. Kuntze and Nauclea latifolia.

The toxicity and the genotoxicity of antimalarial alkaloid rich extracts derived from two plants used in traditional medicine in Mali (Mitragyna inermis (Willd.) O. Kuntze Rubiaceae and Nauclea latifolia (Sm.) Rubiaceae) were evaluated on in vitro and in vivo systems. The results demonstrated that an alkaloid rich extract derived from M. inermis induced a strong inhibition of protein synthesis in mammalian cells but did not exhibit mutagenic or genotoxic activity. An alkaloid rich extract derived from N. latifolia could interact in vitro with DNA of bacteria and mammalian cells, leading to G2-M cell cycle arrest and heritable DNA-damage, as well as inducing in vivo single-strand breaks in liver, kidney and blood cells.

Flow cytometric detection of Leishmania parasites in human monocyte-derived macrophages: application to antileishmanial-drug testing.

A flow cytometric technique was developed for detection of amastigotes of the protozoan Leishmania infantum in human nonadherent monocyte-derived macrophages. The cells were fixed and permeabilized with paraformaldehyde-ethanol, and intracellular amastigotes were labeled with Leishmania lipophosphoglycan-specific monoclonal antibody. Results showed that flow cytometry provided accurate quantification of the infection rates in human macrophages compared to the rates obtained by the conventional microscopic technique, with the advantage that a large number of cells could be analyzed rapidly. The results demonstrated, moreover, that labeling of intracellular amastigotes could reliably be used to evaluate the antileishmanial activities of conventional drugs such as meglumine antimoniate, amphotericin B, pentamidine, and allopurinol. They also established that various Leishmania species (L. mexicana, L. donovani) could be detected by this technique in other host-cell models such as mouse peritoneal macrophages and suggested that the flow cytometric method could be a valid alternative to the conventional method.

New pyrazolo[3,4-b]pyrazines: synthesis and biological activity.

Some new pyrazolo[3,4-b]pyrazines and related heterocycles were synthesized and evaluated for their antifungal and antiparasitic activities. The key intermediate, 6-amino-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine-5-carbonitrile (3) was obtained in a one-pot synthesis via the reaction of 5-amino-3-methyl-4-nitroso-1-phenylpyrazole 2 with malononitrile.

Antileishmanial activity of three saponins isolated from ivy, alpha-hederin, beta-hederin and hederacolchiside A1, as compared to their action on mammalian cells cultured in vitro.

The in vitro antileishmanial activity of three saponins isolated from ivy, alpha-hederin, beta-hederin and hederacolchiside A1, was investigated on Leishmania infantum. The assessment of possible targets (membrane integrity, membrane potential, DNA synthesis and protein content) was performed in both Leishmania promastigotes and human monocytes (THP1 cells). Results observed in Leishmania showed that the saponins exhibited a strong antiproliferative activity on all stages of development of the parasite by altering membrane integrity and potential: hederacolchiside A1 appeared to be the most active compound against both promastigotes and amastigotes. Results observed in THP1 cells demonstrated that the saponins exerted also a potent antiproliferative activity against human monocytes, by producing a significant DNA synthesis inhibition. The ratio between antileishmanial activity on amastigotes and toxicity to human cells suggested that the saponins could be considered as possible antileishmanial drugs.

Structure and antiprotozoal activity of triterpenoid saponins from Glinus oppositifolius.

Two new triterpenoid saponins, glinosides A and B, isolated from the aerial parts of Glinus oppositifolius, have been characterized by 1D, 2D, NMR and high-resolution mass spectral (HRMS) techniques. Their structures were established respectively as 16-O-(beta-arabinopyranosyl)-3-oxo-12,16 beta,21 beta,22-tetrahydroxyhopane for glinoside A and 16-O-(beta-arabinopyranosyl)-3-oxo-12,16 beta,22-trihydroxyhopane for glinoside B. Results presented evidence that fractions had a better antiplasmodial activity (IC50 = 31.80 micrograms/ml) than pure glinoside A (IC50 = 42.30 micrograms/ml).

Functional derivatives of 5-benzo[1, 3]dioxol-5-yl-1-methyl-1H-imidazole-2-carbaldehyde and evaluation of leishmanicidal activity.

New imidazole derivatives were obtained after subjecting 5-benzo[1, 3]dioxol-5-yl-1-methyl-1H-imidazole-2-carbaldehyde to a Knoevenagel reaction. Some of these compounds exhibit significant in vitro leishmanicidal activity.

Antimalarial activity of four plants used in traditional medicine in Mali.

Mitragyna inermis (De Willd.) O. Kuntze Rubiaceae, Nauclea latifolia (Sm.) Rubiaceae, Glinus oppositofolius (Linn) Molluginaceae and Trichilia roka (Forsk.) Chiv. Meliaceae were investigated for their in vitro antimalarial activity. Leaves, roots and stem barks were submitted to aqueous, hydromethano and chloroform extractions and antimalarial activity was evaluated by microscopic and flow cytometric analysis. The results present evidence that the alkaloids contained in chloroform extracts and ursolic acid, purified from the hydromethanol extract of M. inermis induced a significant decrease of parasite proliferation. However, aqueous extracts, traditionally used for medication did not show high antimalarial activity. Statistical comparison between microscopic and cytometric analysis demonstrated the validity of this new technique for the screening of active antimalarial compounds isolated from plants.

[Balance sheet of tolerance-efficacy on the use of tacrine in 100 cases of Alzheimer's disease]. / Bilan tolérance-efficacité d'un an d'utilisation de la tacrine dans 100 cas de maladie d'Alzheimer.

We report our first 100 cases of Alzheimer's (AD) patients treated with tacrine (Cognex) for a period of one year. At the beginning of treatment the mean Mini-Mental-Status (MMS) score was 15.1. To date 71 patients are still under treatment (12 for more than 12 months). Forty-three instances of side-effects were observed, of which 31 involved hepatic side-effects with an increase in ALAT > IN (normal value) (6 cases > 3N), the mean date of appearance was 10.4 +/- 6.8 weeks, there were 16 cholinergic side-effects (nausea, vomiting, diarrhoea), plus 4 neurologic and 2 cutaneous side-effects. These side-effects led to the arrest of the treatment in 19 cases (16 for hepatic toxicity). Treatment was reattempted after interruption in 13 cases; successfully in 3 instances only. The measure of tacrine efficacy was based on 52 MMS score re-evaluations in week 18: there was an increase of the MMS score in 22 cases (3.3 points +/- 2.5), a stabilisation in 11 cases and a decrease in 19 cases (3.3 points +/- 2.2.). In week 30, the MMS scores (35 patients) increased in 9 cases (3.6 points +/- 2.4), stabilized in 5 cases and decreased in 21 cases (3.9 points +/- 3.3). At week 52, only 28 per cent of the patients were considered as either improved or stabilized. We conclude that there is a necessity for close follow-up of tacrine-treated patients, and that globally at 8 months there is an improvement or a stabilization in 40 per cent of patients and long term (at one year) a stabilizing effect on AD patients.

Leishmania infantum promastigotes: flow cytometry as a possible tool for assessing the effects of drugs on cellular functions.

The capacity of flow-cytometric techniques to detect drug-specific biochemical targets and side effects in Leishmania infantum promastigotes was estimated by assessing the effects of three antileishmanial drugs (pentamidine, allopurinol, and amphotericin B) on parasite metabolism. Cell cycle and total protein content were estimated by staining cells with propidium iodide and fluorescein isothiocyanate, nonprotein thiols were stained by mercury orange, and membrane potential was measured by the accumulation of 3,3'-dipenthyloxacarbocyanine iodide inside the cell. Results showed that dynamic studies in parasites treated with subtoxic concentrations of drugs allowed the detection of drug-specific targets: pentamidine primarily affected nonprotein thiol contents and DNA synthesis, allopurinol primarily affected intracellular protein contents, and amphotericin B primarily affected membrane potential. Moreover, the assessment of cellular functions in parasites treated with increasing concentrations of drugs certified the capacity of these techniques to establish dose-response curves and to permit the detection of side effects.

Assessment of amphotericin B susceptibility in Leishmania infantum promastigotes by flow cytometric membrane potential assay.

Flow cytometry was used for measuring the effects of amphotericin B on the membrane of Leishmania infantum strains. The technique was adapted from the rapid flow cytometric membrane potential assay developed by Ordonez and Wehman (Cytometry 22:154-157, 1995) for evaluating antibiotic-susceptibility of Candida species. The study consisted of measuring membrane potential changes induced by amphotericin B in 3 initial strains and 12 laboratory-generated variants adapted to grow with amphotericin B. Results showed that, after 3 h of incubation, amphotericin B induced a dose-related decrease of membrane potential that reached its maximal level at the same concentrations that inhibited parasite growth. These results suggest that the flow cytometric membrane potential assay could be used to assess the susceptibility of Leishmania promastigotes to amphotericin B.