Transport and pharmacodynamics of albitiazolium, an antimalarial drug candidate.

BACKGROUND AND PURPOSE: Choline analogues, a new type of antimalarials, exert potent in vitro and in vivo antimalarial activity. This has given rise to albitiazolium, which is currently in phase II clinical trials to cure severe malaria. Here we dissected its mechanism of action step by step from choline entry into the infected erythrocyte to its effect on phosphatidylcholine (PC) biosynthesis. EXPERIMENTAL APPROACH: We biochemically unravelled the transport and enzymatic steps that mediate de novo synthesis of PC and elucidated how albitiazolium enters the intracellular parasites and affects the PC biosynthesis. KEY RESULTS: Choline entry into Plasmodium falciparum-infected erythrocytes is achieved both by the remnant erythrocyte choline carrier and by parasite-induced new permeability pathways (NPP), while parasite entry involves a poly-specific cation transporter. Albitiazolium specifically prevented choline incorporation into its end-product PC, and its antimalarial activity was strongly antagonized by choline. Albitiazolium entered the infected erythrocyte mainly via a furosemide-sensitive NPP and was transported into the parasite by a poly-specific cation carrier. Albitiazolium competitively inhibited choline entry via the parasite-derived cation transporter and also, at a much higher concentration, affected each of the three enzymes conducting de novo synthesis of PC. CONCLUSIONS AND IMPLICATIONS: Inhibition of choline entry into the parasite appears to be the primary mechanism by which albitiazolium exerts its potent antimalarial effect. However, the pharmacological response to albitiazolium involves molecular interactions with different steps of the de novo PC biosynthesis pathway, which would help to delay the development of resistance to this drug.

The antimalarial ferroquine: from bench to clinic.

Ferroquine (FQ, SSR97193) is currently the most advanced organo-metallic drug candidate and about to complete phase II clinical trials as a treatment for uncomplicated malaria. This ferrocene-containing compound is active against both chloroquine-susceptible and chloroquine-resistant Plasmodium falciparum and P. vivax strains and/or isolates. This article focuses on the discovery of FQ, its antimalarial activity, the hypothesis of its mode of action, the current absence of resistance in vitro and recent clinical trials.

Free radical depolymerization of hyaluronan by Maillard reaction products: role in liquefaction of aging vitreous.

The degradation of hyaluronan was followed by viscosimetry and by HPLC in order to study the possible role of Maillard products (lysine-glucose) on the alteration of the vitreous gel in aging and diabetes. Lysine-glucose generated Maillard products produced a decrease of viscosity and of the number average molecular weight (Mn) of hyaluronan during a 1 h incubation at 37 degrees C. This effect was comparable to that produced by 1 U/ml of testicular hyaluronidase but was weaker than the effect of a Fenton-type reagent (Udenfriend's reagent). The polydispersity of hyaluronan incubated with Maillard products appeared higher than with hyaluronidase suggesting a more random reaction. Antioxydant enzymes (SOD, catalase), the iron chelators (desferrioxamine, transferrin) and the free radical scavengers (uric acid, carnosine) inhibited the degradation by Maillard products confirming its free radical nature and the intervention of trace metals. Maillard products have been detected in diabetic vitreous and may play a role in its accelerated modifications (liquefaction) in diabetes as compared to normal aging.

Biochemical and pharmacological properties of SR 27388, a dual antioxidant and PAF receptor antagonist.

SR 27388 (N-(2-dimethylaminoethyl)-N-(3-pyridinylmethyl[4-(3,5-di(tert- butyl)-4-hydroxylphenyl)thiazol-2-yl]amine) is a potent and competitive antagonist of the binding of [3H]PAF to its receptor on rabbit platelets exhibiting an equilibrium inhibition constant for PAF binding of 10.5 +/- 1.2 nM (n = 3). SR 27388 potently inhibited PAF-induced aggregation of rabbit platelets in vitro (IC50 = 65 +/- 12 nM) (n = 4). In this respect, SR 27388 was as potent as the triazolothienodiazepine WEB-2086 against PAF-induced aggregation of rabbit platelets and had no effect on the action of other platelet aggregating agents. SR 27388 prevented in a dose-dependent manner the formation of thiobarbituric acid reactive substances during membrane peroxidation (IC50 = 0.7 microM) and inhibited reduction of the stable 1,1-diphenyl-2-picrylhydrazyl radical, indicating that the antioxidant potency of SR 27388 was due to an efficient radical scavenging activity. SR 27388 displayed marked in vitro inhibition of zymosan-induced oxidative burst in human monuclear cells (IC50 = 3 microM). In vivo, SR 27388 protected mice from 100 micrograms/kg PAF-induced death with an ED50 value of 500 micrograms/kg, when given i.v., 5 min before PAF challenge or p.o. (ED50 = 800 micrograms/kg) when given 1 h before PAF administration. Similarly, i.v. or oral doses of SR 27388 afforded in mice complete protection against endotoxin-induced lethality (ED50 values were 250 micrograms/kg and 1.3 mg/kg, respectively). Neither BHT, vitamin E nor catechin exhibited significant protection against PAF- or endotoxin-induced death. In ovalbumin-presensitized rabbits, SR 27388 premixed with the allergen inhibited in a dose-dependent manner allergen-induced oedema formation in the skin (ED50 = 0.1 mumol/site). After an i.v. administration of 10 mg/kg, SR 27388 significantly protected mice against alloxan-induced diabetes. These results show that SR 27388 is a potent and orally active dual PAF receptor antagonist and antioxidant.

The organo-specific external NADH dehydrogenase of mammal heart mitochondria has an artefactual origin.

The existence of an organo-specific (heart) external NADH dehydrogenase located on the outer face of the inner mitochondrial membrane has been recently proposed. We have studied the respiration on external NADH in rat and beef heart mitochondrial fractions: (i) by using different mitochondrial isolation procedures on the rat, we observed that the higher the criteria of quality toward classical substrate respiration of mitochondrial fractions, the lower the external NADH-linked respiration; (ii) by using an especially loosely fitting glass-Teflon homogenizer, we obtained rat heart mitochondrial fractions practically free from external NADH linked respiration and with the highest respiratory control ratio on glutamate plus malate respiration. In rat and beef heart mitochondrial fractions containing an external NADH respiration: (i) ethoxyformic anhydride used previously to distinguish internal and external NADH oxidation was shown not to be specific; (ii) external NADH-linked respiration (although associated to the normally functioning respiratory chain as was shown by the effects of classic respiratory inhibitors) did not lead to ADP phosphorylation while glutamate plus malate did; (iii) respiratory activity on glutamate plus malate and external NADH was totally additive and the oxidation corresponded to two separate cytochrome oxidase pools, indicating a total functional separation between the two respiratory systems; (iv) NAD+ addition stimulated states 3 and 4 glutamate plus malate respiration to the same extent, indicating the presence of an appreciable number of internal dehydrogenases accessible to external cofactors. These results show that external NADH-linked dehydrogenase activity, which is usually detectable in mammal heart mitochondrial fractions, is of artefactual origin.

Long-chain-substituted uric acid and 5,6-diaminouracil derivatives as novel agents against free radical processes: synthesis and in vitro activity.

A new series of N-alkylated uric acids (2,6,8-purinetrione) and 5,6-diaminouracils (5,6-diamino-2,4-pyrimidinedione) were synthesized, and their activities against free radicals were evaluated. Long-chain derivatives of both series exhibited a large inhibitory activity against oxygen radical induced lipid peroxidation in bovine heart mitochondria (IC50 lower than 1 microM), compared to the reference antioxidants trolox C or alpha-tocopherol. This activity appeared related to (i) the ability of these compounds to reduce the stable radical 1,1-diphenyl-2-picrylhydrazyl and (ii) their lipophilicity estimated by log P determination. In order to study the scavenging mechanisms of diaminouracils and urate derivatives against lipid radicals, they were also tested against the azo-initiated peroxidation of either methyl linoleate in organic solvents or a liposomal suspension of dilinoleoylphosphatidylcholine. Urate derivatives reacted moderately with lipid radicals and were slowly consumed, significantly affecting the propagation of the peroxidation. Diaminouracils strongly reduced the propagation rate. They were quickly consumed and were able to deactivate about 1 mol of lipid radical per mole of compound in organic solvent. Dodecyl urates and decyl- and dodecyldiaminouracils were chosen for further in vitro investigation and in vivo evaluation.

Assay of phenylethanolamine N-methyltransferase activity using high-performance liquid chromatography with ultraviolet absorbance detection.

A simple and rapid method for measuring phenylethanolamine N-methyltransferase (PNMT) activity by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection is described. This assay requires a partially purified PNMT preparation derived from bovine adrenals, with noradrenaline and S-adenosyl-L-methionine (SAM) as co-substrates. After incubation, the reaction is stopped by addition of acid and the reaction mixture is analysed directly by HPLC. The enzymatically formed S-adenosyl-L-homocysteine (SAH) is detected at 258 nm and determined. Under optimum conditions, the stability of SAH allowed automation of the HPLC detection. This assay was validated by the determination of the kinetic properties of PNMT. Km values for noradrenaline and SAM defined in this assay (16 and 5.7 microM, respectively) are consistent with previously published values. This assay is simple enough to be used for large series of measurements of PNMT activity testing new methyl acceptors, potential inhibitors or PNMT activity in adrenal medulla.

Inhibition of the iron-catalysed formation of hydroxyl radicals by nitrosouracil derivatives: protection of mitochondrial membranes against lipid peroxidation.

A new series of metal ligands containing the 1,3-dimethyl-6-amino-5- nitrosouracil moiety has been synthesized and they have been studied as potential inhibitors of iron-dependent lipid peroxidation. For this purpose, these new derivatives have been tested in the Fenton induced deoxyribose degradation assay, which allows a quantitative measurement of their inhibitory effect towards hydroxyl radical generation. When iron(II) is complexed by these ligands, a strong inhibition of deoxyribose degradation is observed, especially in the case of tris-[2-(1,3-dimethyl-5-nitrosouracil-6-yl)aminoethyl] amine (5). This inhibitory effect is clearly related to a specific complexation of iron(II) and is not due to the direct scavenging of hydroxyl radical by the ligand. Inhibition of the iron mediated Fenton reaction presumably results from inactivation of the reactivity of the metal center towards hydrogen peroxide. These derivatives, as well as long alkyl chain substituted nitrosouracils were evaluated in the protection of biological membranes against lipid peroxidation (induced by iron(II)/dihydroxyfumaric acid and determined with the 2-thiobarbituric acid test). Ligand 5 inhibited lipid peroxidation at a rate similar to Desferal (desferrioxamine B) and slightly higher than bathophenanthroline sulphonate (BPS), which are respectively good iron(III) and iron(II) chelators. When covalently bound with a long alkyl chain, the increase of lipophilic character of the ligand allows its location near the mitochondrial membrane, where lipid peroxidation occurs. Lower concentrations (IC50 = 4 microM) are then necessary to inhibit lipid peroxidation. This IC50 concentration should be compared to those obtained for Trolox (IC50 = 3 microM) or the 21-aminosteroid U74500A (IC50 = 1 microM) described previously.

Flux-dependent increase in the stoichiometry of charge translocation by mitochondrial ATPase/ATP synthase induced by almitrine.

After studying the effects of almitrine, a new kind of ATPase/ATP synthase inhibitor, on two kinds of isolated mammalian mitochondrion, we have observed that: (1) Almitrine inhibits oligomycin-sensitive ATPase; it decreases the ATP/O value of oxidative phosphorylations without any change in the magnitude of delta mu H+. (2) Almitrine increases the mechanistic H+/ATP stoichiometry of ATPase as shown by measuring either (i) the extent of potassium acetate and of potassium phosphate accumulation sustained by ATP utilisation, or (ii) the electrical charge/ATP (K+/ATP) ratio at steady-state of ATPase activity. (3) Rat liver mitochondria are at least 10-times more sensitive to almitrine than beef heart mitochondria. (4) The change in H+/ATP stoichiometry induced by almitrine depends on the magnitude of the flux through ATPase. The inhibitory effect of almitrine on ATPase/ATP synthase complex, as a consequence of such an H+/ATP stoichiometry change, is discussed.