Curdlan sulphate in human severe/cerebral Plasmodium falciparum malaria.

Preclinical studies have shown that curdlan sulphate (CRDS), a sulphated 1-->3-beta-D glucan, inhibits Plasmodium falciparum in vitro and down-modulates the immune response. A direct, non-specific effect on cytoadherence and rosetting may be predicted, as has been described with other sulphated polysaccharides, e.g. heparin. The anticoagulant effect of CRDS is 10-fold lower than heparin. Curdlan sulphate has, therefore, emerged as a candidate for adjunct medication in the treatment of severe/cerebral malaria. Two clinical studies were conducted using CRDS as adjunct medication to conventional therapy (artesunate) in patients with severe and severe/cerebral malaria. Both studies were double-blind and placebo-controlled to evaluate the efficacy and safety of the combination. Curdlan sulphate appeared to reduce the severity of the disease process, e.g. fever clearance time was shortened. Due to the small number of patients, there was no difference in mortality. The two treatment arms in both studies showed similar results for all laboratory parameters. The only adverse event recorded during CRDS treatment was an increase in activated partial thromboplastin time. This can be monitored easily. It seems that the patients who may benefit most are severe/cerebral cases with no organ damage on admission.

Plasmodium falciparum: effects of amantadine, an antiviral, on chloroquine-resistant and -sensitive parasites in vitro and its influence on chloroquine activity.

The lysosomotropic nature of amantadine suggested potential as an antimalarial. Sensitivity tests to amantadine hydrochloride alone and in combination with chloroquine were carried out in 96-well microtitre plates using the tritiated hypoxanthine uptake method to measure parasite growth. Amantadine alone has antimalarial activity. Amantadine is more potent against chloroquine-resistant strains. Combinations of amantadine and chloroquine result in slight synergy in both resistant and sensitive strains.

Malaria pigment and extracellular iron. Possible target for iron chelating agents.

Extracellular iron is necessary for many biochemical reactions involved in Plasmodium falciparum growth and multiplication. The incorporation of radioactive iron taken up by the parasite was found, electrophoretically and via gamma counting, to be mainly associated with the haemozoin only in the presence of the active metabolism of the parasite. The potent antimalarial activity of desferrioxamine, a ferric iron chelating agent, has shown that iron deprivation is inhibitory to the parasite. We propose that the mechanism of action of desferrioxamine in addition to the chelation of iron from the parasitic compartment, chelates iron from the haemozoin crystal resulting in free radical generation and parasite death. The ability of desferrioxamine and not the ferrous iron chelating agent, 2,2'-bipyridyl, to chelate the non-haem iron from the haemozoin structure indicates that the oxidative state of iron associated with the haemozoin structure is ferric in nature.

Structure-activity relationships and inhibitory effects of various purine derivatives on the in vitro growth of Plasmodium falciparum.

The development of novel chemotherapeutic agents has become an urgent task due to the development and rapid spread of drug resistance in Plasmodium falciparum, the protozoan parasite responsible for cerebral malaria. Cyclin-dependent kinases (CDKs) are essential for the regulation of the eukaryotic cell cycle, and several enzymes of this family have been identified in P. falciparum. In recent years, a number of purine-derived kinase inhibitors have been synthesised, some of which display selective activity against CDKs. This report describes a study in which various purine derivatives were screened for in vitro antimalarial activity. The erythrocytic asexual stages of the chloroquine-resistant P. falciparum strain (FCR-3) were cultivated in vitro in the presence of the various purines, and their effect on parasite proliferation was determined by the [3H]hypoxanthine incorporation assay. Our results show considerable variation in the sensitivity of P. falciparum to the different purines, as well as a general independence from their effect on purified starfish CDK1/cyclin B activity, which has been the standard assay used to identify CDK-specific inhibitors. Two subfamilies of purines with moderate to poor activity against CDK1/cyclin B activity showed submicromolar activity against P. falciparum. Structure-activity analysis indicates that certain structural features are associated with increased activity against P. falciparum. These features can be exploited to synthesise compounds with higher activity and specificity towards P. falciparum.

Hydrocortisone and tumor necrosis factor in severe community-acquired pneumonia. A randomized controlled study.

BACKGROUND: Community-acquired pneumonia is a major cause of death in third world countries. Antimicrobial therapy may have little impact on the natural history of patients with severe pneumonia. We hypothesized that the intrapulmonary production of tumor necrosis factor-alpha (TNF-alpha) may be responsible for the progressive lung injury and shock commonly seen in patients with severe pneumonia after commencing antibiotic therapy. AIM: To investigate the effects of a single bolus of hydrocortisone on the clinical course and serum TNF-alpha levels of patients with severe community-acquired pneumonia. DESIGN: Randomized placebo-controlled study. SETTING: Multidisciplinary ICU of a tertiary care teaching hospital. PATIENTS AND METHODS: Patients with three or more British Thoracic Society criteria of severe pneumonia were studied. Patients were randomized to receive either a single dose of hydrocortisone (10 mg/kg) or placebo 30 min prior to commencing antibiotic therapy. Patients were treated with cefotaxime and other antibiotics as clinically indicated. Blood for TNF-alpha was taken at the time of hospital admission and repeated 2, 6, and 12 h after starting antibiotic therapy. RESULTS: Thirty patients were studied: 16 received placebo and 14 received hydrocortisone. The patients who received placebo tended to be sicker than the patients who received hydrocortisone. The baseline TNF-alpha value was 989 +/- 374 pg/ml in the placebo group and 827 +/- 394 pg/ml in the hydrocortisone group. In both groups of patients, the TNF-alpha levels did not change significantly with time. There was no correlation between the TNF-alpha levels and the APACHE II score, lung injury score, or outcome. The only variable that predicted outcome was the APACHE II score. CONCLUSION: Bactericidal antibiotics do not increase serum TNF-alpha levels in patients with severe pneumonia. Hydrocortisone given prior to antibiotic treatment had no effect on the serum TNF-alpha levels or the clinical course of patients with severe community-acquired pneumonia.

Effect of pH on in vitro potency of amantadine against Plasmodium falciparum.

Amantadine is a monoprotic weak base that inhibits intraerythrocytic growth in in vitro cultures of Plasmodium falciparum, specifically chloroquine-resistant strains. Changes in the external PH of the medium are expected to result in a shift in the relative proportion of ionized and unionized species of amantadine by virtue of the weak base characteristic of the drug, influencing passage of the drug through the membrane. The ability of amantadine to alkalinize the food vacuole was determined using the accumulation of acridine orange as a vacuolar probe. Drug sensitivity following alteration of the pH gradient was assessed using the hypoxanthine method. Amantadine was able to alkalinize the food vacuole in the millimolar range; however, since its antimalarial activity is in the micromolar range, alkalinization of the food vacuole is not the primary action of the drug. The pH of the medium profoundly influenced susceptibility to chloroquine; the log of the 50% inhibitory concentration (IC50) values were linearly dependent on the external pH in both chloroquine-resistant and chloroquine-sensitive strains. Log IC50 values of amantadine exhibited a linear dependence on external pH in the chloroquine-sensitive strain, but in the chloroquine-resistant strain, a nonlinear parabolic function was found with the minimum IC50 at pH 7.03. Ammonium chloride did not interfere with the antimalarial activity of amantadine. The presence of the amine group on the hydrocarbon cage is essential for the activity of amantadine in Plasmodium falciparum. These results suggest factors in addition to pH gradient are involved in the effect of amantadine, possibly interactions with membrane phospholipids.

Comparison of the pharmacokinetic profiles of soluble aspirin and solid paracetamol tablets in fed and fasted volunteers.

The aim of this study was to investigate the absorption of popular preparations of two common analgesics--soluble aspirin and solid paracetamol tablets. An open, randomised, crossover study design was used to compare the pharmacokinetic parameters of soluble aspirin and solid paracetamol tablets in 16 healthy, male volunteers from the University of the Witwatersrand, South Africa, in both fed and fasted states. Plasma concentrations of paracetamol, aspirin and salicylic acid were measured. It was found that the rate of absorption was significantly faster for soluble aspirin than for solid paracetamol, regardless of fed or fasting state, considering time to maximum concentration (p < 0.01), time to first quantifiable concentrations (p < 0.05) and absorption rate (p < 0.01). Absorption rate was significantly affected by food for both soluble aspirin (p = 0.028) and for solid paracetamol (p = 0.0003). Time to maximum concentration was not significantly affected by food for soluble aspirin (p = 0.17) but significantly lengthened for solid paracetamol (p = 0.0003). The extent of absorption was affected by food in terms of maximum concentration for both drugs (p = 0.0001), with a reduction of 49% in the fed state for solid paracetamol compared to 18% for soluble aspirin, the difference between the drugs being statistically significant (p = 0.0024). The overall bioavailability of soluble aspirin was unaffected by food and the bioavailability of salicylic acid was increased in the fed state, whereas that of solid paracetamol was lowered in the fed state. Greater inter-individual variation was seen in paracetamol concentrations compared with aspirin or salicylic acid levels. In conclusion, these results show that the absorption of soluble aspirin is largely unaffected by food, whereas, in the same volunteers, the absorption of solid paracetamol tablets is greatly affected. In some volunteers, maximum plasma concentrations of paracetamol following food did not reach levels previously reported to be required for effective analgesia, and this may have implications for pain relief in some individuals. The practice in some individuals of taking aspirin tablets after food to minimise potential gastric disturbance should not affect the level of analgesia.

In vitro drug interaction between amantadine and classical antimalarial drugs in Plasmodium falciparum infections.

The interactions of amantadine with classical antimalarial drugs were evaluated against a chloriquine-resistant and a chloroquine-sensitive strain of Plasmodium falciparum in vitro. Amantadine potentiated the effect of chloroquine and quinine in both strains; it also potentiated the effect of mefloquine, halofantrine and primaquine in the chloroquine-resistant strain but had no effect in the chloroquine-sensitive strain. Amantadine had no effect on the response to pyrimethamine of either strain. Amantadine does not interfere with the activity of these compounds and may possibly enhance it.

The effect of curdlan sulphate on development in vitro of Plasmodium falciparum.

Sulphated glycoconjugates have been reported to inhibit malarial merozoite invasion and interfere with rosetting and adhesion. Curdlan sulphate, a sulphated glycoconjugate with a favourable toxicity profile, exhibits antimalarial activity in vitro. The aim of this study was to characterize the antimalarial activity of curdlan and investigate its effect on adhesion. The antimalarial activity of curdlan at different points in the intraerythrocytic developmental cycle was investigated using morphological observation and radiolabelled hypoxanthine uptake as indices of parasite growth. Effects on adhesion were investigated using a platelet model. Curdlan suphate had no effect on the ability of the parasite to develop through the intraerythrocytic cycle. Inhibition of invasion was dependent on the drug being present at the time of invasion. Curdlan did not interfere with the ability of the parasite to adhere to the C36 receptor in the platelet model. In conclusion, the low toxicity of curdlan and its marked anti-invasion activity on merozoites make curdlan a potential auxiliary treatment for severe malaria.

The effects of herbal oxytocics on the isolated "stripped" myometrium model.

Decoctions of Agapanthus africanus and Clivia miniata are used as oxytocic agents in South African traditional herbal medicine. Aqueous extracts of A. africanus and C. miniata leaves have been shown to possess similar uterotonic activities in the isolated whole uterus preparation. The uterus however, comprises a myometrial and an endometrial layer and the activity of both oxytocin and the prostaglandins differs in these layers. The aim of this study was to determine the uterotonic activity of the herbal remedies in an endometrium-free preparation (i.e. "stripped" myometrium) and, if active, whether this effect could be related to prostaglandin synthesis or to interaction with specific receptors. The effects of the herbal extracts were tested on the isolated "stripped" rat myometrium preparation. Both herbal extracts caused a direct contractile response by the isolated tissue. Pretreatment of the myometrium with either plant extract augmented the initial response to acetylcholine. Preincubation with atropine inhibited the response to cumulative dosage of Agapanthus extract but had no effect on the response to Clivia. Indomethacin administration did not affect the response of the myometrium to cumulative dosage of acetylcholine, oxytocin or Clivia extract but inhibited the response to Agapanthus extract. These results clearly indicate that the Agapanthus and Clivia herbal extracts exhibited uterotonic activity in this model. The study illustrates that the "stripped" myometrium model has successfully differentiated between the mechanisms of action of two herbal oxytocics compared to the whole uterus preparation where their uterotonic activity was thought to be similar.

Preliminary screening of plants used in South Africa as traditional herbal remedies during pregnancy and labour.

Agapanthus africanus, Pentanisia prunelloides and Gunnera perpensa are indigenous plants which are used in traditional herbal remedies during pregnancy and childbirth. A crude decoction of each plant was tested on isolated rat uterus and ileum for pharmacological activity. Agapanthus africanus and Pentanisia prunelloides exhibited direct smooth muscle activity on the uterus and ileum preparations, while Gunnera perpensa showed direct smooth muscle activity on the uterus only. All the plant extracts potentiated the initial response of the uterus to oxytocin but Pentanisia prunelloides reduced the maximum response of the uterus to oxytocin. The Agapanthus africanus and Pentanisia prunelloides extracts potentiated the initial response of the ileum to acetylcholine but all the plant extracts inhibited the maximum response of the ileum to acetycholine.

Pharmacological effects of Agapanthus africanus on the isolated rat uterus.

The Agapanthus africanus plant is used by South African traditional healers as a phytomedicine in herbal remedies to treat pregnancy-related ailments and to augment labour. It has already been shown that an aqueous extract of A. africanus causes smooth muscle contractions in the isolated uterus and ileum preparations. In the present study, the effects of an aqueous extract of A. africanus leaves was examined on receptor systems involved in contraction of the uterine smooth muscle in order to determine the mechanism of its pharmacological effect relevant to its ethnic use to augment labour. The extract was tested on the isolated rat uterus preparation. The aqueous extract of A. africanus leaves was found to exhibit agonist activity on uterine muscarinic receptors and to promote the synthesis of prostaglandins in the oestrogenized rat uterus. Some pharmacological justification for the ethnic use of A. africanus as a herbal oxytocic in prolonged labour has been provided.

Continuous growth of Candida utilis under periodic change of growth-limiting substrate.

A periodic change of limitation by glucose and ammonia was effected during continuous cultivation of Candida utilis. Values of feed parameters providing periodic nitrogen limitation were established. Cell biomass yield, macromolecular composition and parameters of individual cells (cell mass, budding percentage and cell-wall polysaccharide per surface square unit) were examined. The cyclic regime was found to result in culture synchrony. Parameters obtained on the basis of cell counts displayed a high sensitivity to changes in limitation where as the remaining parameters were less sensible.

Effects of potassium ethylxanthogenate and 2,3-dimercaptopropane sulphonate sodium on the pentobarbital pharmacokinetics and metabolism in male mice.

The effects of potassium ethylxanthogenate (PEX) and 2,3-Dimercaptopropane sulphonate sodium (Unithiol) on the pentobarbital (40 mg/kg, body weight i.v.) (PB) sleeping time, pharmacokinetics, and metabolism, were studied in comparative experiments on male albino mice. It was established that the pretreatment of animals with PEX (80 mg/kg of body weight s.c.) potentiated the PB sleeping time. Unithiol in an equimolar dose (105.2 mg/kg of body weight s.c.) had no effect. The pharmacokinetics of PB could be fitted to be a biexponential equation of the type Cp(t) = A.e-alpha t + B.e-beta t. PEX caused an almost two-fold decrease in the elimination rate constant of plasma PB, which led to a higher half-time and a lower total clearance compared with the controls. The curve for the plasma PB metabolites in the PEX-pretreated mice was significantly lower than of the controls. A higher PB level and decreased rate of elimination in PEX-pretreated animals was observed also in the: liver, lungs, kidney, and brain. A conclusion was drawn that the potentiating effect of PEX on the PB sleeping time is mainly due to inhibition of PB liver metabolism. It was suggested that the differences in the biological effects of the two thiol compounds are due to the differences in their chemical structures: PEX possesses a C = S group but Unithiol lacks this group.

On the prenatal noxious effects of trypan blue and of a related azo dye.

Since 1948 trypan blue has been a well-known and extensively used experimental teratogen, belonging to the group of azo dyes. Chemically, trypan blue consists of a biphenyl molecule (0-tolidine or benzidine) combined by means of azo linkages with two molecules of a substituted naphthalene. Between 1987-89 the effect of the replacement of the biphenyl molecule by a molecule of p,p'-diaminobenzanilide upon the prenatal noxious action of trypan blue has been controlled. Investigations were carried out on three species: chick embryos, albino rats and albino mice. In the species used, the replacement annihilates the teratogenic properties of the dye, with the persistence of some embryotoxic effects. On the other hand, the control of o-tolidine and of p,p'-diaminobenzanilide revealed that no one had teratogenic properties (only some embryotoxic effect, more marked in the case of o-tolidine). It results that the teratogenic action of trypan blue cannot be attributed to the o-tolidine molecule proper but to an effect which results (in a for the moment unknown manner) from its combination with the other parts of the dye molecule.

The combined effect of iron chelators and classical antimalarials on the in-vitro growth of Plasmodium falciparum.

The emergence of drug resistant malaria has prompted an intensified search for new antimalarials or combinations of such drugs. Iron chelating agents may represent a new approach to antimalarial treatment and could possibly be used in combination with classical antimalarials. Plasmodium falciparum (FCR-3) strain used at a 1% haematocrit, was subjected to various combinations of the classic antimalarials (chloroquine, pyrimethamine and quinine) and iron chelating agents (desferrioxamine and 2,2'-bipyridyl) in vitro. Tritiated hypoxanthine incorporation was used to determine the growth of the malarial parasites. The iron chelating agents and classic antimalarials when tested alone were found to inhibit the growth of the late stages of the parasite. The combination of the classic antimalarials and iron chelating agents resulted in additive effects on the in-vitro growth of P. falciparum.

The pharmacological action of Rhoicissus tridentata on isolated rat uterus and ileum.

Decoctions and infusions of Rhoicissus tridentata subsp cuneifolia (Vitaceae) roots and lignotubers are widely used in South African traditional herbal remedies during pregnancy. The pharmacological action of an aqueous extract of R. tridentata subsp cuneifolia roots was investigated using isolated rat uterus and ileum. The extract directly stimulated concentration dependent contractions of the uterus and ileum. Pretreatment with the muscarinic antagonist atropine (40 nM) and the cyclooxygenase inhibitor indomethacin (5 microM) both blocked the contractile response to the Rhoicissus extract suggesting that muscarinic receptors and cyclooxygenase metabolites could be involved in the contractile response to the extract. The serotonergic antagonist methysergide (1 microM) and the alpha-adrenoceptor antagonist prazosin (2 microM) had no effect on the direct action of the extract. This infers that the contractile response to the plant extract is independent of serotonin receptors and alpha-adrenoceptors.