Targeting ion channels of Plasmodium falciparum-infected human erythrocytes for antimalarial development

Aside from its profound clinical importance, the human malaria parasite, P. falciparum, is intrinsically fascinating to the cell biologist because it resides within the mature red blood cell (RBC). Because the inert RBC cannot otherwise provide sufficient amounts of key substrates to fuel the vigorous parasite metabolism, the parasite must have specialized adaptations for getting these solutes into the RBC and scavenging them from RBC cytosol. Two unusual ion channels have recently been identified within the infected RBC complex; these channels likely function in a sequential diffusive pathway of nutrient acquisition by the intracellular parasite. If so, they present novel opportunities for identification of ion channel inhibitors that may be useful antimalarial compounds.     

Irreversible inhibition of S-adenosylmethionine decarboxylase in Plasmodium falciparum-infected erythrocytes: growth inhibition in vitro

Blocking spermidine and spermine synthesis in Plasmodium falciparum-infected erythrocytes with irreversible inhibitors of S-adenosylmethionine decarboxylase (AdoMet DC; EC 4.1.1.50), prevented the growth of the parasite in vitro. The most potent of these compounds, MDL 73811, inhibited growth of chloroquine-sensitive and -resistant strains of P. falciparum equally, with an IC50 of 2-3 microM. Other structurally related compounds also inhibited parasite proliferation, but to a lesser degree, determined apparently by their potency for inhibition of AdoMet DC. The growth inhibition by MDL 73811 could be alleviated by incubating infected erythrocytes with spermidine and spermine, but not putrescine. Parasites treated with the drug were arrested at the trophozoite stage of the erythrocytic cycle and had putrescine levels which were elevated by about 3- to 4-fold. Treatment of crude extracts of purified parasites with 1 microM MDL 73811 inhibited AdoMet DC activity by greater than 90%. These biochemical changes in P. falciparum-infected cells were consistent with AdoMet DC inhibition being the primary effect of MDL 73811 treatment.     

Adverse neuropsychiatric effects of antimalarial drugs

ABSTRACT

Introduction: Antimalarial drugs are the primary weapon to treat parasite infection, save lives, and curtail further transmission. Accumulating data have indicated that at least some antimalarial drugs may contribute to severe neurological and/or psychiatric side effects which further complicates their use and limits the pool of available medications.

Areas covered: In this review article, we summarize published scientific studies in search of evidence of the neuropsychiatric effects that may be attributed to the commonly used antimalarial drugs administered alone or in combination. Each individual drug was used as a search term in addition to keywords such as neuropsychiatric, adverse events, and neurotoxicity.

Expert opinion: Accumulating data based on published reports over several decades have suggested that among the major commonly used antimalarial drugs, only mefloquine exhibited clear indications of serious neurological and/or psychiatric side effects. A more systematic approach to assess the neuropsychiatric adverse effects of new or repurposed antimalarial drugs on their safety, tolerability and efficacy phases of clinical studies and in post-marketing surveillance, is needed to ensure that these life-saving tools remain available and can be prescribed with appropriate caution and medical judgment.     

A medicinal chemistry perspective on 4-aminoquinoline antimalarial drugs

A broad overview is presented describing the current knowledge and the ongoing research concerning the 4-aminoquinolines (4AQ) as chemotherapeutic antimalarial agents. Included are discussions of mechanism of action, structure activity relationships (SAR), chemistry, metabolism and toxicity and parasite resistance mechanisms. In discussions of SAR, particular emphasis has been given to activity versus chloroquine resistant strains of Plasmodium falciparum. Promising new lead compounds undergoing development are described and an overview of physicochemical properties of chloroquine and amodiaquine analogues is also included.     

Design and synthesis of plasmepsin I and plasmepsin II inhibitors with activity in Plasmodium falciparum-infected cultured human erythrocytes

A series of protease inhibitors targeted at the malarial enzymes plasmepsin I and II, and encompassing a basic hydroxyethylamine transition state isostere scaffold, was prepared. The substituents in the P1' position were varied and the biological activities expressed in K(i)-values ranged from 60 to >2000 nM. A more than 4-fold selectivity for either of the plasmepsins could be achieved. All of the active compounds exhibited high preference for the plasmepsins over cathepsin D, the most closely related human protease. A few active compounds were shown to inhibit parasite growth in cultured infected human erythrocytes. An ED(50) value as low as 1.6 microM was observed for one of the inhibitors despite K(i) values of 115 nM (Plm I) and 121 nM (Plm II).     

Glutathione reductase inhibitors as potential antimalarial drugs. Effects of nitrosoureas on Plasmodium falciparum in vitro

Malarial parasites are believed to be more susceptible to oxidative stress than their hosts. BCNU(1,3-bis(2-chloroethyl)-1-nitrosourea) and HeCNU(1-(2-chloroethyl)-3-(2-hydroxythyl)-1-nitrosourea), inhibitors of the antioxidant enzyme glutathione reductase, were found to prevent the growth of Plasmodium falciparum in all intraerythrocytic stages. When exposing infected red blood cells to 38 microM BCNU or 62 microM HeCNU for one life cycle of synchronously growing parasites, the parasitemia decreased by 90%. During the formation of new ring forms, the parasites are even more susceptible to these drugs. The treatment with BCNU or HeCNU produced a rapid depletion of GSH in the parasites and their host cells; in addition, protection against lipid peroxidation was impaired in these cells. Possible mechanisms for the antimalarial action of the inhibitors are discussed. Our results suggest that erythrocyte glutathione reductase, an enzyme of known structure, might be considered as a target for the design of antimalarial drugs.     

Abnormal circulatory control in falciparum malaria: the effects of antimalarial drugs

Summary We have studied blood pressure and heart rate responses to standing in 29 previously ambulant adult Thai patients with acute uncomplicated falciparum malaria before and after treatment with quinine or mefloquine.There was significant, symptomatic, and usually profound orthostatic hypotension in 12 patients (41%) before antimalarial treatment. The median maximum fall in systolic pressure was 24 mm Hg, significantly greater than the maximum fall in diastolic pressure 16 mm Hg.Blood pressure fell in two phases: an initial transient and usually asymptomatic fall immediately on standing, and a progressive, usually symptomatic fall, worsening over several minutes without a rise in pulse rate. Orthostatic hypotension was associated with core temperature (r=0.37, P=0.05).Antimalarial treatment accentuated the delayed orthostatic hypotension during malaria, despite (in the case of quinine) a significant reduction in fever. Both antimalarial drugs attenuated the cardioacceleratory response to symptomatic postural hypotension; the mean reduction in heart rate at the time of lowest blood pressure was 22 beats·min^–1.The electrocardiograph ratio of RR intervals at the 30th and 15th beats was reduced significantly in acute malaria, but was not affected further by the drugs. When restudied in convalescence all the patients had normal postural cardiovascular responses.Acute falciparum malaria is associated with impaired circulatory control and the tendency to postural hypotension is worsened significantly by antimalarial treatment with the quinoline antimalarials quinine and mefloquine.     

Membrane effects of various drugs on isolated rat hepatocytes and erythrocytes

The relationship between hepatotoxicity and membrane effects of clinically used drugs on erythrocytes was investigated. The cytotoxicity of various drugs to isolated rat hepatocytes was determined by the leakage of glutamic oxalacetic transaminase (GOT) and ornithine carbamyl transferase (OCT) into the surrounding medium. Inhibition of hypotonic hemolysis of rat erythrocytes was measured as the hemoglobin concentration in the supernatant solution. Drugs such as tricyclic antidepressants, local anesthetics (dibucaine), and bile acids (chenodeoxycholic acid), which have both a membrane stabilizing effect on erythrocytes at low concentrations and a hemolytic effect at higher concentrations, induced enzyme leakage from hepatocytes. The concentration at which hemolysis occurred corresponded to the concentration which caused a marked enzyme leakage from hepatocytes. These phenomena were observed for alkyltrimethylammonium salts (C10 to C16), the order of cytotoxicity to hepatocytes and erythrocytes was C16 greater than C14 greater than C12 greater than C10. Marked enzyme leakage was observed for chenodeoxycholic acid at 1 X 10(-3) M but not for ursodeoxycholic acid. The order of membrane stabilizing and lytic potency of tricyclic antidepressants was chlorimipramine greater than amitriptyline greater than desipramine greater than imipramine. These results suggest that these membrane effects of various drugs on erythrocytes may be useful for screening for hepatocytotoxicity in vitro.     

Differential effects of chloroquine on the phospholipid metabolism of Plasmodium-infected erythrocytes

The effect of the antimalarial drug chloroquine (CQ) on the phospholipid metabolism in Plasmodium knowlesi-infected simian erythrocytes has been studied by incubating cells with different labeled precursors and various concentrations of CQ. The drug induced considerable modifications of this metabolism but at the same time decreased nucleic acid and protein synthesis as well as the output of 14CO2 from radioactive glucose. Phosphatidylcholine biosynthesis was severely reduced. However, under these conditions, CQ had the early effect of markedly increasing phosphatidylinositol labeling from radioactive inositol, fatty acids, 1-(14C)palmitoyl-lysophosphatidylcholine, but not from glycerol. Synthesis of phosphatidylserine from (14C)serine and of phosphatidylethanolamine from labeled glycerol, ethanolamine, and serine was increased, especially at high CQ concentrations when the whole metabolism of the parasite was severely reduced. These effects reflect a deep differential effect of CQ on the intense phospholipid metabolism of the Plasmodium-infected erythrocytes, which might involve a redirecting of phospholipid metabolism similar to that induced by other cationic amphiphilic drugs, and a compensatory synthesis resulting from the severe blockage of phosphatidylcholine synthesis.     

Clinical pharmacokinetics of antimalarial drugs

For the past 300 years antimalarial dosage regimens have not been based on pharmacokinetic information. However, now that this information is available, it is appropriate to examine current recommendations for prophylaxis and treatment. In healthy subjects, the cinchona alkaloids (quinine and quinidine), primaquine and proguanil (chloroguanide) are all rapidly eliminated with half-lives (t1/2 beta) of between 6 and 12 hours. Hepatic biotransformation accounts for approximately 80, 96 and 50% of their total clearance, respectively. In malaria, the pharmacokinetic properties of quinine and quinidine are significantly altered with a decrease in the apparent volume of distribution (Vd), prolongation of the elimination half-life, and a reduction in systemic clearance (CL) that is proportional to the severity of infection. Red cell concentrations and plasma protein binding are both increased in severe disease. Parenteral quinine or quinidine should be given by slow intravenous infusion rather than by intravenous or intramuscular injection, and a loading dose is necessary in severe infections. Chloroquine (t1/2 beta 6 to 50 days) and mefloquine (t1/2 beta 6.5 to 33 days) have extensive tissue distribution and prolonged activity after a single dose. Both drugs are concentrated in erythrocytes and 55% of chloroquine and 98% of mefloquine in plasma is bound to protein. The pharmacokinetics of chloroquine are complex and, because of the extremely long beta phase, difficult to accurately define. Pyrimethamine (t1/2 35 to 175 hours) has more limited tissue distribution, plasma and erythrocyte concentrations are similar, and 85% of the drug in plasma is bound to plasma proteins. The clearance of quinine, mefloquine and pyrimethamine appears to be higher in children than in adults. Currently, most of the information available on disposition of antimalarial drugs in humans is derived from studies in healthy adult subjects. More information is required on their pharmacokinetics in malaria, pregnancy, and in young children.     

Comparative effects of some antimalarial drugs on isolated cardiac muscle of the guinea pig

The antimalarial drugs, proguanil and pyrimethamine, caused dose-dependent transient negative chronotropic and inotropic effects on spontaneously beating and electrically driven guinea pig atria. In contrast, the quinoline antimalarials (primaquine, chloroquine, quinine, and quinidine) caused a biphasic response—an initial stimulant effect, which could be abolished by reserpine pretreatment, cocaine, or propranolol, followed by a depressant effect of long duration. In electrically driven preparations, all the antimalarial drugs tested caused a dose-dependent decrease in maximal driving frequency and antagonized the positive inotropic effects of isoprenaline and noradrenaline. However, since similar concentrations of the drugs also antagonized calcium-induced responses, this action is unlikely to be due to β-adrenoceptor blockade.     

Differential effects of organic hydroperoxides and hydrogen peroxide on proteolysis in human erythrocytes

The effects of tert-butyl hydroperoxide, cumene hydroperoxide, and hydrogen peroxide on proteolysis in human red blood cells have been examined. The organic hydroperoxides effectively stimulated the rate of protein degradation in red cells and in hemolysate; in contrast, H2O2 addition was without significant effect in either system. tert-Butyl hydroperoxide or cumene hydroperoxide (8 mM) increased the rate of protein degradation in red cells 2.3- and 4-fold, respectively, relative to control as monitored by tyrosine release. In hemolysate, tert-butyl hydroperoxide and cumene hydroperoxide, present at 8 mM, produced a 2- and 3-fold increase in the rate of protein degradation, respectively, as compared to controls. Hydroperoxide-stimulated proteolysis in red cells or in hemolysate was concentration-dependent and reached saturation at 8 mM hydroperoxide. The reaction was linear for 2 h after which a plateau was reached. In contrast to the results observed for the organic hydroperoxides, H2O2 (100 or 200 mM) addition either alone or in the presence of the catalase inhibitor 3-amino-1,2,4-triazole (50-200 mM), failed to stimulate proteolysis. N-Acetylcysteine (20 mM) and dimethylthiourea (50 mM) inhibited the rate of hydroperoxide-stimulated proteolysis in red cells by approximately 50 and approximately 35%, respectively, and in hemolysate by 25 and 40%, respectively. The hydroxyl radical scavengers methyl sulfoxide (50 mM) or dimethylfuran (50 mM), metal ion chelators, or spin traps failed to decrease significantly the rate of organic hydroperoxide stimulated proteolysis. In addition, inhibitors of the calpain/procalpain system in red cell or hemolysate incubations challenged by organic hydroperoxide were without significant effect on the rate of proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of convulsive drugs on photically evoked after-discharge parameters

Since sub-convulsive doses of pentylenetetrazol (Metrazol) had been shown to augment photically evoked after-discharge (AD) patterns both in terms of elicitation threshold and amplitude of spindle bursts, it was questioned whether these observations were indicative of those that might be obtained with other drugs categorized in the general class of convulsive agents. It was found that picrotoxin and strychnine were ineffective in augmenting AD although, consistent with previous observations, Metrazol potentiated the elicitation of AD. Furthermore, Metrazol, when interacted with strychnine or picrotoxin, did not elicit augmented AD patterns. Repeated injections of Metrazol also did not result in additional increments in AD. These results can be related to the modes of action of the three convulsive agents.A major portion of this study was carried out at the Veterans Administration Hospital, Phoenix, Arizona. Additional financial support from the Research Division, Brigham Young University, is gratefully acknowledged.     

Differential effects of serotonergic and catecholaminergic drugs on ingestive behavior

The serotonergic agonists fenfluramine and fluoxetine and the catecholaminergic agonists amphetamine and phenylpropanolamine are well known to cause a reduction in intake in rats. In the studies reported here we investigated the effects of these drugs on the microstructure of licking behavior of the rat ingesting 0.4 M sucrose. The purpose was to examine the similarities in the behavioral effects within and between these two classes of anorectic agents. The serotonergic agonists fenfluramine and fluoxetine caused a reduction in intake primarily by reducing the size of bursts and clusters of licking within the test meal without affecting the duration of the meal, suggesting a reduction in the palatability of the test solution. The catecholamine agonists amphetamine and phenylpropanolamine reduced intake primarily by reducing the number of bursts and clusters without affecting their size, suggesting a fractionation in the organization of the normal pattern of ingestion. The differences between the two serotonin and the two catecholamine agonists on the microstructure of the licking behavior suggest a different effect of the two neurotransmitters on the motor system that controls ingestive behavior. The similarities between the two different agonists within each class suggests a common neurotransmitter mechanism responsible for these two different effects on the behavior of the animals.Supported by NIH Grant DK41563 (JDD).