Mining for natural product antileishmanials in a fungal extract library.

Leishmaniasis is a Neglected Tropical Disease caused by the insect-vector borne protozoan parasite, Leishmania species. Infection affects millions of the World's poorest, however vaccines are absent and drug therapy limited. Recently, public-private partnerships have developed to identify new modes of controlling leishmaniasis. Most of these collaborative efforts have relied upon the small molecule synthetic compound libraries held by industry, but the number of New Chemical Entities (NCE) identified and entering development as antileishmanials has been very low. In light of this, here we describe a public-private effort to identify natural products with activity against Leishmania mexicana, a causative agent of cutaneous leishmanaisis (CL). Utilising Hypha Discovery's fungal extract library which is rich in small molecule (<500 molecular weight) secondary metabolites, we undertook an iterative phenotypic screening and fractionation approach to identify potent and selective antileishmanial hits. This led to the identification of a novel oxidised bisabolane sesquiterpene which demonstrated activity in an infected cell model and was shown to disrupt multiple processes using a metabolomic approach. In addition, and importantly, this study also sets a precedent for new approaches for CL drug discovery.

Effect of encapsulation on the anti-inflammatory properties of superoxide dismutase after oral administration.

Anti-inflammatory properties of free superoxide dismutase and superoxide dismutase encapsulated into liposomes, with or without ceramides, have been investigated. Two models were investigated: carrageenan paw oedema and pleurisy. Animals were fed by repeated doses, twice daily from day 1 until day 4. Evaluation consisted of measurement of paw oedema volume with determination of prostaglandin E2, thromboxane B2 and 6-keto-prostaglandin F1 alpha levels. Polymorphonuclear oxidative metabolism was evaluated by measurement of superoxide anion production. Levels of superoxide dismutase were determined in cells and pleural exudates. Higher anti-inflammatory effects were obtained after eight administrations of encapsulated forms (0.5 mg/kg) whereas free superoxide dismutase have shown no effects. Ceramides enhanced the results obtained.

Kinetic evaluation of nitric oxide production in pleural exudate after induction of two inflammatory reactions in the rat.

NO generation in the course of two acute, non immune, inflammatory reactions (pleurisy induced by rat isologous serum and carrageenan) was assessed by means of nitrite measurement in pleural exudate from 0.5 to 24 h. NO release varied time-dependently, similarly for the two inflammatory reactions. A first, but transient, peak was reached in 30 min while a second peak, more sustained, began at the fourth hour and was maximum at the tenth. Kinetic evolution of NO release was consistent with activation, in a first step, of a constitutive NO synthase probably from endothelial origin (inhibited by 2-Methyl-2-Thiopseudourea sulfate but not by dexamethasone) and with activation, in a second wave, of inducible NOS from endothelial and exudative cells. NO release was potentiated by administration per os of L-Arginine and seems to be involved in the evolution of acute inflammatory reactions and oxygen metabolite production.

Thermal desorption-gas chromatographic determination of ethane and pentane in breath as potential markers of lipid peroxidation.

Formation of free radicals and lipoperoxidation occur at the onset of cellular damage. These effects are produced during normal metabolism and in pathological states. The peroxidation of polyunsaturated fatty acids, i.e. linoleic acid and linolenic acid, which are both cellular membrane compounds, induces ethane and pentane formation in pulmonary air exhalation. These two volatile hydrocarbons can be considered as potential lipoperoxidation markers. Methodological difficulties limit the use of these gases for assessment of free oxygen radical activity but we have developed and validated a non-invasive technique. A study was performed with ten healthy volunteers.

Increase of singlet oxygen protection of erythrocytes by vitamin E, vitamin C, and beta carotene intakes.

Substantial evidence supports the theory that free radicals, especially oxygen radicals, are involved in the process of aging. The human organisms have two ways to fight them: an enzymatic way with enzymatic intervention like superoxide dismutase, catalase... and a chemical way with the intervention of scavengers such as vitamins, cysteine, methionine, gluthatione... The aim of this work was to determine that an intakes of vitamins association: vitamin E, vitamin C and beta carotene induce an increase of singlet oxygen protection of erythrocytes' subjects. The method was based on the haemolytic effect of singlet oxygen which is generated by irradiation of hematoporphyrine at 365 nm, in 22 p. cent suspension of erythrocytes' subjects. Results show that a supply of beta carotene (15 or 30 mg/day), vitamin E (15 mg/day) and vitamin C (30 mg/day) involves an increase of singlet oxygen protection of erythrocytes of subjects. This protection appears very quickly after 15 days of treatment.

Pharmacokinetics of superoxide dismutase in rats after oral administration.

The kinetic behaviour of bovine erythrocyte Cu-Zn SOD was investigated in Sprague Dawley male rats after subcutaneous and oral administrations of doses ranging from 0 center dot 5 to 20 mg kg-1. Studies have been carried out with SOD and SOD encapsulated into liposomes containing or not containing ceramides. The maximum concentration (Cmax) in blood cell pellets ranged from 8 center dot 65 to 11 center dot 03 U/mg haemoglobin (Hb) after subcutaneous injection, and from 4 center dot 48 to 8 center dot 23 U/mg Hb after oral administration. The maximum concentrations were reached in 5 h (t max) for the two routes. Comparison between the areas under the curves (AUCs) obtained after subcutaneous and oral administration allowed the calculation of relative bioavailability (F'). The maximum bioavailability after oral administration was 14% for free SOD, 22% for SOD encapsulated into liposomes, and 57% when ceramides were added to liposomes. Poor SOD bioavailability was enhanced by liposome encapsulation, and ceramide addition seemed to be beneficial for oral encapsulated SOD administration.

Influence of beta carotene, vitamin E, and vitamin C on endogenous antioxidant defenses in erythrocytes.

OBJECTIVE: To evaluate the in vivo radical scavenger activity of vitamin E, vitamin C, and beta carotene on erythrocyte membranes. DESIGN: A prospective, open trial without placebo. SETTING: Department of Clinical Pharmacy. PATIENTS: Ten healthy volunteers being supplemented with beta carotene, vitamin E, and vitamin C. MEASUREMENTS: Erythrocytes were incubated in water bath with 2,2' azobis (2 amidinopropane) hydrochloride (AAPH). AAPH decomposes spontaneously at 37 degrees C to generate free radicals inducing membrane cellular damage and hemolysis. The absorbance was measured at 405 nm at 0, 30, and 60 min, and then every 20 minutes for four hours. The time for 50 percent of maximal hemolysis (T50%), which expresses the radical scavenger activity of erythrocytes, was determined. RESULTS: The physiologic T50% value determined in 52 healthy volunteers is 117 +/- 12 min. Patients receiving these supplements have a higher value of T50% (143.2 +/- 11.6 min at 30 d and 145.7 +/- 10.5 min at 60 d) than the physiologic value (p < 0.001). CONCLUSIONS: These data suggest that vitamin C, vitamin E, and beta carotene stimulate the radical scavenger activity of erythrocyte membranes after 30 days.

Plasma concentrations of beta-carotene, vitamin A and vitamin E after beta-carotene and vitamin E intake.

We have studied the metabolism (absorption) of beta-carotene and vitamin E by assigning eleven volunteers to receive daily two capsules of OENOBIOL, each containing 15 mg of beta-carotene and 15 mg of vitamin E, over 60 days. The beta-carotene, vitamin E and vitamin A plasma levels were then determined using new methods developed in our laboratory. After two months, the actively treated group's median beta-carotene and vitamin E levels were significantly higher than those of a control group. However, no significant change between treated and control groups in the mean of vitamin A (retinol) plasma levels were observed. Treatment with beta-carotene, a vitamin A precursor, does not significantly modify the vitamin A levels. This conclusion had already been observed and it is assumed that a plasma level of beta-carotene equal or higher than 0.3 mg/L reflects a nutritional intake of provitamins sufficient to support homeostasis of retinol (Brubacher et al., 1982).

In vivo inhibition of nitric oxide synthase by bisisothiouronium and bisguanidinium salts.

The ability of two S,S'-(alkane-1,omega-diyl) bisisothiouronium dibromides, three N,N'-(alkane-1, omega-diyl) bis guanidinium dinitrates and N,N'-bis (3-guanidinopropyl)piperazine dinitrate to inhibit constitutive (i.e. endothelial and neuronal forms) and inducible forms of nitric oxide synthases has been evaluated in vivo. These compounds, synthesized by two of us (J. C. L. and C. S.), have been tested in vivo; they were administered simultaneously with an irritant (carrageenan lambda) into the pleural cavity. The amount of nitrites collected 0.5 and 7 hours after this injection can be considered as an indicator of nitric oxide (NO) production. According to previous data, the first harvesting time can be related to activation of constitutive NO synthases and the second to activation of inducible NO synthases. These substances significantly inhibited nitrite production as did 2-methyl-2-thiopseudourea sulphate, previously described as a potent inhibitor of NO synthases and considered as the reference compound. The inhibiting effect varied according to the chemical structure of the compounds. Results were significantly different from controls at 0.5 h only with the S,S'-(octane-1,8-diyl) bisisothiouronium dibromide and the S,S'-(nonane-1,9-diyl) bisisothiouronium dibromide at the highest concentration, N,N'-(heptane-1,7-diyl) bisguanidinium dinitrate and N,N'-bis (3-guanidinopropyl)piperazine dinitrate. At 7 h, all the results were significantly different from controls, with a major effect observed with N,N'-(heptane-1,7-diyl) bisguanidinium dinitrate. The most active substances exerted similar effects to the reference substance.

Correlation between alimentary mycotoxin contamination and specific diseases.

Several pathological cases including primitive hepatomas, Reye's syndrome, alimentary toxic aleukaemia, were encountered in two different Tunisian Sahel hospitals. Contamination of some nutriments of the patients by mycotoxins (aflatoxins, trichothecenes, ochratoxin A, citrinin) are most likely involved in the origin of these diseases.