Anti-malarial drug formulations and novel delivery systems: a review.

Artemisinin combination therapies have decreased malaria associated morbidity and mortality in several parts of the world. On the other hand, malaria cases have increased in sub-Saharan Africa largely due to falciparum resistance to the most frequently used drugs (chloroquine and sulphadoxine/pyrimethamine (SP) combination). Therapeutic failure has also been attributed in part to adverse effects of anti-malarial drugs and patients' non-compliance due to inconvenient dosing schedules. We consider that formulation and evaluation of novel drug delivery systems is not only less expensive than developing new drugs, but may also improve delivery of anti-malarials at the desired rates. In this review we evaluate the therapeutic efficacy of existing anti-malarial drugs and assess the feasibility of developing novel formulations and delivery systems.

The activity of phosphate-dependent glutaminase from the rat small intestine is modulated by ADP and is dependent on integrity of mitochondria.

The effect of adenine nucleotides and phosphate on rat small intestine phosphate-dependent glutaminase (PDG) activity was investigated in intact mitochondria. Disruption of the integrity of mitochondria by sonication or freeze-thawing resulted in loss of enzyme activity. ADP was the strongest adenine nucleotide activator of the enzyme giving a V(max) that was over 5-fold of that for AMP or ATP. The sigmoid activation curve of PDG by ADP became hyperbolic in presence ATP. ADP also lowered the K(m) for glutamine and increased V(max) and these effects were further enhanced by the presence of ATP. Activation of PDG by phosphate and ADP was not completely additive suggesting some antagonism between the activators. There was no clear relationship between changing ATP/ADP ratios and PDG activity in presence of a constant concentration of phosphate. However, ratios of approximately 1:4 and 4:1 gave the highest and lowest activities, respectively. The pH dependence of PDG activity was affected by phosphate concentration and results suggest that the divalent ion is the activating species.

Potential marker enzymes and metal-metal interactions in Helisoma duryi and Lymnaea natalensis exposed to cadmium.

In this study, we investigate the effects of exposure to cadmium and copper on Lymnaea natalensis and Helisoma duryi. The snails were dosed with Cd2+ or Cu2+ for a period of 96h. Snails dosed with Cd accumulated the metal significantly (P<0.05) in tissues but not in shells. Mortality was observed at approximately 1mg Cd/l of culture water. In tissues and shells of snails dosed with Cd or Cu, synergistic and antagonistic metal-metal interactions involving Cd, Cu, Zn, and Pb were observed and these may affect metal toxicity. Glutamate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase were assayed in whole snail tissue sub-cellular fractions of Cd-dosed snails. Generally, enzyme activity significantly increased at lower concentrations of Cd but decreased at high concentrations of the metal. However, mitochondrial alanine aminotransferase activity progressively declined with increasing Cd concentration. The changes in some of the enzymes' activities suggest biomarker potential.

Phosphate-dependent glutaminase in enterocyte mitochondria and its regulation by ammonium and other ions.

The effects of ammonium and other ions on phosphate dependent glutaminase (PDG) activity in intact rat enterocyte mitochondria were investigated. Sulphate and bicarbonate activated the enzyme in absence and presence of added phosphate. In presence of 10 mM phosphate, ammonium at concentrations <1 mM inhibited the enzyme. This inhibition was reversed by increased concentration of phosphate or sulphate. The inhibition of PDG by ammonium in presence of 10 mM phosphate was biphasic with respect to glutamine concentration, its effect being through a lowering of V(max) at glutamine concentration of

Activities of glutamate dehydrogenase and aspartate and alanine aminotransferases in freshwater snails Helisoma duryi and Lymnaea natalensis exposed to copper.

In this paper we investigate the potential of glutamate dehydrogenase (GDH) and aspartate and alanine aminotransferases (AST and ALT) as biomarkers of water pollution due to copper in the freshwater snails Helisoma duryi and Lymnaea natalensis. Snails were dosed with copper(II) ion concentrations of 0.01, 0.1 and 1 mg kg(-1) breeding water for a period of 96 h, after which those surviving were shelled. The copper content in the breeding water, in whole snail tissue and in the snail shells was determined at the end of the period of exposure. For enzyme determinations, whole snail tissue was first homogenized and fractionated by centrifugation at 500 g to remove the nuclei. The resulting supernatant was then centrifuged at 10,000 g to give a pellet fraction representing the mitochondrial fraction and a supernatant representing the cytosolic fraction. Copper was very toxic to both snail species at concentrations above 0.2 mg l(-1), with only 3% of the Helisoma and 12% of the Lymnaea surviving at concentrations of approximately 1 mg l(-1). The copper content in the shells and tissues of snails rose with increasing copper concentration in the breeding water, and was 2.1- to 4.9-fold in snails exposed to copper ion at a dose of 1 mg kg(-1) water compared with undosed snails. Similarly, the activities of GDH and AST rose by up to 4.7-fold in the homogenate and the mitochondrial and cytosolic fractions with increasing concentrations of copper. These activities, however, fell at copper concentrations of approximately 1 mg l(-1), which coincided with massive death of snails. Mitochondrial ALT disappeared at copper ion concentrations of approximately 0.2 mg l(-1) for Lymnaea and 1 mg l(-1) for Helisoma, possibly indicating mitochondrial degeneration. These results show that GDH, AST and ALT have the potential to be biomarkers of sublethal copper pollution in these two snail species, since their activities were significantly altered by low copper concentrations.

Stabilization of rat liver mitochondrial alanine aminotransferase with ethanol and trehalose.

Rat liver mitochondrial alanine aminotransferase (mALT) is known to be a very unstable enzyme, a property that has hindered efforts to purify it. In this report we examine the possibility of stabilizing mALT with ethanol, trehalose, and protease inhibitors. The presence of ethanol was shown to slow down the inactivation of mALT, increasing its half-life from 1 to 4 h. Trehalose was found to greatly enhance the stability of mALT in a concentration-dependent manner. In the presence of 36.5% trehalose, the half-life of mALT was 85 h. Of the protease inhibitors tested only antipain and chymostatin slowed down the inactivation of mALT but only within the first 24 h following preparation of the crude enzyme. It is concluded that the inclusion of ethanol and trehalose in purification protocols could aid the purification of the enzyme. It is also concluded that the inclusion of protease inhibitors in purification protocols of mALT may not be necessary as its inactivation does not seem to be due to protease activity.

Intramitochondrial localization of alanine aminotransferase in rat-liver mitochondria: comparison with glutaminase and aspartate aminotransferase.

The removal of the outer mitochondrial membrane and hence of constituents of the intermembrane space in rat-liver mitochondria using digitonin showed that phosphate-dependent glutaminase, alanine and aspartate aminotransferase were localized in the mitoplasts. Further fractionation of mitoplasts following their sonication resulted in 90% of glutaminase, 98% of alanine aminotransferase and 48% of aspartate aminotransferase being recovered in the soluble fraction while the remainder of each enzyme was recovered in the sonicated vesicles fraction. These results indicated that glutaminase and alanine aminotransferase were soluble matrix enzymes, the little of each enzyme recovered in the sonicated vesicles fraction being probably due to entrapment in the vesicles. Aspartate aminotransferase had dual localization, in the inner membrane and matrix with the high specific activity in sonicated vesicles confirming its association with the membrane. Activation experiments suggested that the membrane-bound enzyme was localized on the inner side of the inner mitochondrial membrane.

Transamination pathways influencing L-glutamine and L-glutamate oxidation by rat enterocyte mitochondria and the subcellular localization of L-alanine aminotransferase and L-aspartate aminotransferase.

Using analytical subcellular fractionation techniques, 12% of the total L-alanine aminotransferase activity and 26% of the total L-aspartate aminotransferase activity was localized in enterocyte mitochondria. Alanine and aspartate were products from the oxidation of glutamine and glutamate by enterocyte mitochondria. At low concentrations, malate stimulated aspartate synthesis but was inhibitory at higher concentrations. The malate inhibition of aspartate synthesis, which increased in the presence of pyruvate, was accompanied by an increase in alanine synthesis. With glutamine as substrate in the presence of pyruvate and malate, alanine synthesis was increased by 127% on addition of purified L-alanine aminotransferase, in spite of large amounts of glutamate generated. It was concluded that when pyruvate is available the important route for glutamine or glutamate oxidation by transamination was via L-alanine:2-oxoglutarate aminotransferase and not via L-aspartate:2-oxoglutarate aminotransferase. Results suggested that mitochondria may account for 50% of alanine production from glutamine in the enterocyte despite the relatively low activity of L-alanine aminotransferase therein.

The oxidation of glutamine and glutamate in relation to anion transport in enterocyte mitochondria.

The oxidation of L-glutamate and L-glutamine by enterocyte mitochondria was supported by malate. The stimulation of the rate of oxidation of the two amino acids by small amounts of added malate was 93% and 76% respectively. This could not be accounted for by the oxidation of the small amounts of malate added. Amino-oxyacetate added initially inhibited malate-supported oxidation of L-glutamate by 81% and that of L-glutamine by 38%. The inhibition of L-glutamate oxidation was partially reversed by L-glutamine. The dicarboxylate-carrier inhibitor 2-phenylsuccinate inhibited the malate-supported oxidation of both amino acids, but appeared to be slightly stimulatory to L-glutamine oxidation when added initially. The inhibition of L-glutamate oxidation was reversed by L-glutamine. The mitochondrial uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) inhibited malate-supported oxidation of L-glutamate by 78% when added initially. The oxidation of L-glutamine was completely inhibited. However, the uncoupler stimulated the oxidation of both amino acids when added finally. Pyruvate inhibited aspartate synthesis when either of these amino acids was the main substrate, alanine being synthesized. There was no effect on O2 uptake. Mitochondria did not swell in KCl solution, but swelled rapidly in water. Mitochondrial swelling in potassium phosphate and potassium acetate solutions was activated by valinomycin and to a lesser extent by the further addition of FCCP. With potassium malate, swelling was mainly activated by phosphate. The swelling of enterocyte mitochondria in potassium glutamate was slow. In glutamine solution, mitochondrial swelling was greater and appeared to be enhanced by the initial presence of small amounts of phosphate.

Preparation of rat enterocyte mitochondria.

Rat enterocyte mitochondria were prepared with respiratory control ratios of 4 or 5 and occasionally 6. When EGTA was excluded from the mitochondrial incubation medium the calculated P/O ratios were high, especially those based on the first addition of ADP. These ratios were lowered by increasing the EGTA concentration from 1 mM to 2 mM in the mitochondrial preparation medium and including 1 mM-EGTA in the incubation medium. The use of EDTA in the enterocyte isolation medium led to the mitochondria requiring added cytochrome c. Substituting EGTA for EDTA abolished this requirement. The mitochondrial fraction consisted of two components, an upper cream-coloured layer rich in DNA and a lower brown-coloured layer poor in DNA. Both components were capable of oxidative phosphorylation with succinate or the glutamate/malate couple as substrates. The mitochondrial yield was assessed by assaying succinate dehydrogenase activity, and the contamination of the mitochondrial fraction by other cell organelles was assessed by assays for appropriate marker enzymes.