In vitro sensitivity of Plasmodium falciparum to amodiaquine compared with other major antimalarials in Madagascar

Chloroquine has been used in Madagascar since 1945 and remains the first-line treatment for uncomplicated cases of malaria. Low-grades of resistance type R1 and R2 have been reported. Thus, in vitro tests were performed in order to monitor the drug sensitivity of Plasmodium falciparum from different study sites, with the aim of identifying alternatives to chloroquine. Chloroquine IC50 values ranged from 0.2 nM to 283.4 nM (n = 190, mean IC50 = 52.6 nM; 95% CI = 46.1-59.1 nM). Fifteen isolates (7.9%) were chloroquine-resistant. One mefloquine-resistant isolate was detected (1/139). The test isolates were sensitive to amodiaquine (n = 118), quinine (n = 212), pyrimethamine (n = 86) and cycloguanil (n = 79). The median IC50 for amodiaquine was 12.3 nM (mean IC50 = 15.3 nM, 95% CI = 13.3-17.3 nM). Amodiaquine was 3.4 times as active as chloroquine in vitro and 7 times as active as quinine against P. falciparum. These results indicate that amodiaquine may be a potent alternative to chloroquine in Madagascar. There was positive correlation between tested quinoline-containing drugs activities, which suggests in vitro cross-susceptibility.     

Anti-filarial effects of nine quinoline-containing drugs on adult filariae in vitro

The potencies and efficacies of 9 quinoline-containing anti-malarials including chloroquine, (bis)desethylchloroquine, SN6911, SN12108, amodiaquine, CN-2999-2K, primaquine, quinacrine, and quinine were examined in vitro against adult female Brugia pahangi. Parasite motility and lactate excretion were measured as indicators of drug effects. All of the agents tested showed time-dependent increases in potency over a 24-72-hr incubation period. SN12108 was the most potent at 72 hr, reducing motility by greater than or equal to 50% (IC50) at 1.0 x 10(-7) M. Chloroquine (IC50 2.3 x 10(-6) M), desethylchloroquine (IC50 7.0 x 10(-6) M), quinacrine (IC50 1.9 x 10(-6) M), and quinine (IC50 1.5 x 10(-5) M) were the least potent. All of the drugs caused time-dependent decreases in lactate excretion, except quinine; decreases were found to be dose dependent. A high correlation (r greater than 0.85) was seen between time-dependent effects on motility and lactate excretion. The effects of chloroquine (10 microM) on motility were also examined in female Acanthocheilonema viteae, Dirofilaria immitis, Onchocerca volvulus, and male Onchocerca gutturosa. Dirofilaria immitis was less sensitive to chloroquine than B. pahangi; A. viteae was equally sensitive. Species of Onchocerca were the most sensitive parasites studied. Adult O. gutturosa and O. volvulus were affected by 10 microM chloroquine within 4-6 hr; motility was reduced by 80% within 24 hr. Although the mechanism of anti-filarial activity of the quinoline-containing drugs is not known, their in vitro activity against a variety of adult filariae at clinically relevant concentrations, as well as differential sensitivity seen between the different filariae examined, warrants further study of these compounds.     

Potentiation of antimalarial drug action by chlorpheniramine against multidrug-resistant Plasmodium falciparum in vitro

Chlorpheniramine, a histamine H1 receptor antagonist, was assayed for in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum K1 strain and chloroquine-resistant P. falciparum T9/94 clone, by measuring the 3H-hypoxanthine incorporation. Chlorphenirame inhibited P. falciparum K1 and T9/94 growth with IC50 values of 136.0+/-40.2 microM and 102.0+/-22.6 microM respectively. A combination of antimalarial drug and chlorpheniramine was tested against resistant P. falciparum in vitro. Isobologram analysis showed that chlorpheniramine exerts marked synergistic action on chloroquine against P. falciparum K1 and T9/94. Chlorpheniramine also potentiated antimalarial action of mefloquine, quinine or pyronaridine against both of the resistant strains of P. falciparum. However, chlorpheniramine antagonism with artesunate was obtained in both P. falciparum K1 and T9/94. The results in this study indicate that antihistaminic drugs may be promising candidates for potentiating antimalarial drug action against drug resistant malarial parasites.     

Low antiplasmodial activity of alkaloids and amides from the stem bark of Zanthoxylum rubescens (Rutaceae)

The stem bark of Zanthoxylum rubescens (syn. Fagara rubescens) is used for treating fevers associated with malaria in the Ivory Coast. Three alkaloids: N-nornitidine, 7,9-dimethoxy-2,3-methylenedioxybenzophenanthridine, and bis[6-5,6-dihydrochelerythrinyl)] ether; and two amides: zanthomamide and lemairamide, were isolated from the stem bark of this plant. These compounds were screened in vitro against the chloroquine-sensitive 3D7 strain and the chloroquine-resistant FCM29 strain of P. falciparum. N-nornitidine was found to be inactive. 7,9-dimethoxy-2,3-methylenedioxybenzophenanthridine, lemairamide and zanthomamide showed weak activity with average IC50 values ranging from 45.6 microM to 149.9 microM. Bis[6-15,6-dihydrochelerythrinyl)] ether was the most active of the tested compounds with mean IC50s of 14.9 +/- 1.4 microM in FCM29 strain and 15.3 +/- 3.4 microM in 3D7 strain (approximately 58 to approximately 1130 times less active than chloroquine respectively). The anti-Plasmodium activities of the tested alkaloids of Z. rubescens were low; and do not encourage the use of this plant as antimalarial.     

Cinanserin is an inhibitor of the 3C-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro

The 3C-like proteinase (3CLpro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for anti-SARS-CoV drugs due to its crucial role in the viral life cycle. In this study, a database containing structural information of more than 8,000 existing drugs was virtually screened by a docking approach to identify potential binding molecules of SARS-CoV 3CLpro. As a target for screening, both a homology model and the crystallographic structure of the binding pocket of the enzyme were used. Cinanserin (SQ 10,643), a well-characterized serotonin antagonist that has undergone preliminary clinical testing in humans in the 1960s, showed a high score in the screening and was chosen for further experimental evaluation. Binding of both cinanserin and its hydrochloride to bacterially expressed 3CLpro of SARS-CoV and the related human coronavirus 229E (HCoV-229E) was demonstrated by surface plasmon resonance technology. The catalytic activity of both enzymes was inhibited with 50% inhibitory concentration (IC50) values of 5 microM, as tested with a fluorogenic substrate. The antiviral activity of cinanserin was further evaluated in tissue culture assays, namely, a replicon system based on HCoV-229E and quantitative test assays with infectious SARS-CoV and HCoV-229E. All assays revealed a strong inhibition of coronavirus replication at nontoxic drug concentrations. The level of virus RNA and infectious particles was reduced by up to 4 log units, with IC50 values ranging from 19 to 34 microM. These findings demonstrate that the old drug cinanserin is an inhibitor of SARS-CoV replication, acting most likely via inhibition of the 3CL proteinase.     

Plasmodium vivax: in vitro susceptibility of blood stages to synthetic trioxolane compounds and the diamidine DB75

Plasmodium vivax is an important human pathogen causing malaria in more temperate climates of the world. Similar to Plasmodium falciparum, the causative agent for malaria tropica, drug resistance is beginning to emerge for this parasite species and this hampers adequate treatment of infection. We have used a short-term ex vivo drug assay to monitor activity of OZ277 (RBx-11160), a fully synthetic anti-malarial peroxide, and the diamidine DB75 against P. vivax. For both compounds as well as the anti-malarial reference compounds artesunate, artemether, and chloroquine, the in vitro IC(50) values were determined in one-cycle hypoxanthine incorporation assays. Results from such assays were found to be very similar compared to IC(50) values obtained from one-cycle P. falciparum hypoxanthine assays. We demonstrate the anti-parasite activity of OZ277 and the reference compounds to be faster than that of DB75. These data warrant clinical testing of OZ277 against P. vivax malaria and support recent data on clinical activity against P. vivax for DB75.     

Synthesis and biological evaluation of phenolic Mannich bases of benzaldehyde and (thio)semicarbazone derivatives against the cysteine protease falcipain-2 and a chloroquine resistant strain of Plasmodium falciparum

A targeted series of phenolic Mannich bases of benzaldehyde and (thio)semicarbazone derivatives were synthesized and evaluated in vitro against the malarial cysteine protease falcipain-2 and a chloroquine resistant strain (W2) of Plasmodium falciparum. A novel series of 4-aminoquinoline semicarbazones were the most effective inhibitors of falcipain-2 (most potent inhibitor had IC(50)=0.63microM) while a bisquinoline semicarbazone compound 8f was the most potent antimalarial compound with an IC(50) of 0.07microM against W2. Compound 8f also weakly inhibited falcipain-2, with an IC(50) of 3.16microM, although its principal antiparasitic activity did not appear to be due to inhibition of this enzyme.     

Carbonic anhydrase inhibitors: inhibition of Plasmodium falciparum carbonic anhydrase with aromatic/heterocyclic sulfonamides-in vitro and in vivo studies

A library of aromatic/heterocyclic sulfonamides possessing a large diversity of scaffolds has been assayed for inhibition of the carbonic anhydrase (CA, EC 4.2.1.1) from the malaria parasite Plasmodium falciparum (pfCA). Low micromolar and submicromolar in vitro inhibitors were detected, whereas several compounds showed ex vivo anti-P. falciparum activity, in cell cultures. One derivative, that is, 4-(3,4-dichlorophenylureido)thioureido-benzenesulfonamide was an effective in vitro pfCA inhibitor (K(I) of 0.18 microM), inhibited the ex vivo growth of P. falciparum with an IC(50) of 1 microM, and was also effective as an antimalarial agent in mice infected with P. berghei, an animal model of human malaria infection, with an ID(50) of 10 mg/kg (chloroquine as standard showed an ID(50) of 5 mg/kg). By inhibiting the first step of pyrimidine nucleotide biosyntheses, that is, the CA-mediated carbamoylphosphate biosynthesis, sulfonamide inhibitors of the protozoan CAs may have potential for the development of novel therapies of human malaria.     

Protein tyrosine kinase activity in human malaria parasite Plasmodium falciparum.

Protein tyrosine kinases (PTKs) are believed to be implicated in the parasite growth, maturation and differentiation functions. Protein tyrosine kinase activity was found to be distributed in all the stages of P. falciparum parasite maturation. Membrane bound PTK activity was found to be increased during maturation process (ring stage to trophozoite stage) in chloroquine sensitive strains. In vivo conversion of the schizont stage to ring stage via release of merozoites was associated with a decrease in PTK activity. Chloroquine inhibited the membrane bound PTK activity in a dose dependent manner (IC50 = 45 microM). Kinetic studies show that chloroquine is a competitive inhibitor of PTK with respect to peptide substrate and noncompetitive with respect to ATP indicating that chloroquine inhibits PTK activity by binding with protein substrate binding site. The results suggest that maturation of malaria parasite is related to PTK and inhibition of this activity by chloroquine could provide a hypothesis to explain the mechanism of action of chloroquine.     

In vitro chloroquine resistance modulation study on fresh isolates of Brazilian Plasmodium falciparum: intrinsic antimalarial activity of phenothiazine drugs

Phenothiazine drugs - fluphenazine, chlorpromazine, methotrimeprazine and trifluoperazine - were evaluated as modulating agents against Brazilian chloroquine-resistant fresh isolates of Plasmodium falciparum. Aiming to simulate therapeutic schedules, chloroquine was employed at the concentration used for sensitive falciparum malaria treatment and anti-psychotic therapeutic concentrations of the phenothiazine drugs were adopted in two-fold serial dilutions. The in vitro microtechnique for drug susceptibility was employed. Unlike earlier reported data, the phenothiazine modulating effect was not observed. However, all the drugs demonstrated intrinsic antiplasmodial activity in concentrations lower than those described in the literature. In addition, IC50 estimates have been shown to be inferior to the usual anti-psychotic therapeutic concentrations. Statistical analysis also suggested an increase in the parasitaemia rate or, even, a predominant antiparasitic effect of phenothiazine over chloroquine when used in combination.     

Design, synthesis, and structure-activity relationships of pyrazolo[3,4-d]pyrimidines: a novel class of potent enterovirus inhibitors

A series of pyrazolo[3,4-d]pyrimidines were synthesized and their antiviral activity was evaluated in a plaque reduction assay. It is very interesting that this class of compounds provide remarkable evidence that they are very specific for human enteroviruses, in particular, coxsackieviruses. Some derivatives proved to be highly effective in inhibiting enterovirus replication at nanomolar concentrations. SAR studies revealed that the phenyl group at the N-1 position and the hydrophobic diarylmethyl group at the piperazine largely influenced the in vitro antienteroviral activity of this new class of potent antiviral agents. It was found that the pyrazolo[3,4-d]pyrimidines with a thiophene substituent, such as compounds 20-24, in general exhibited high activity against coxsackievirus B3 (IC(50) = 0.063-0.089 microM) and moderate activity against enterovirus 71 (IC(50) = 0.32-0.65 microM) with no apparent cytotoxic effect toward RD (rhabdomyosarcoma) cell lines (CC(50) > 25 microM).     

Antimalarial activity of thioacridone compounds related to the acronycine alkaloid

A series of thioacridone compounds that were previously shown to have DNA binding interaction, were screened for antimalarial activity. The new compounds were assessed for in vitro antimalarial activity against a chloroquine sensitive (D10) strain of the malaria parasite Plasmodium falciparum, using a lactate dehydrogenase (PfLDH) assay. In the series, the IC(50) values ranged from 0.4 to 27 microg/ml. 1-(2-Dimethylaminoethylamino)-9(10H)-thioacridone was found to be the most potent against P. falciparum (D10) with an IC(50) value of 0.4 microg/ml. This compound was also evaluated against a South African chloroquine resistant (RSA 11) P. falciparum strain and was found to have an IC(50) value of 1 microg/ml, compared with 0.16 microg/ml for chloroquine. Quantitative structure-activity relationships of this series were also investigated and a multiple linear regression r(2) of 0.58 was found for the best fit equation. The most potent compound, 1-(2-dimethylaminoethylamino)-9(10H)-thioacridone, was docked into the chloroquine binding site of PfLDH and it was found that the slightly lower activity of this compound, compared with chloroquine, is likely due to steric interference within a restricted binding pocket.     

Antimuscarinic effects of chloroquine in rat pancreatic acini

Chloroquine inhibited carbachol-induced amylase release in a dose-dependent fashion in rat pancreatic acini; cholecystokinin- and bombesin-induced secretory responses were almost unchanged by the antimalarial drug. The inhibition of carbachol-induced amylase release by chloroquine was competitive in nature with a Ki of 11.7 microM. Chloroquine also inhibited [3H]N-methylscopolamine binding to acinar muscarinic receptors. The IC50 for chloroquine inhibition of [3H]N-methylscopolamine binding was lower than that for carbachol or the other antimalarial drugs, quinine and quinidine. These results demonstrate that chloroquine is a muscarinic receptor antagonist in the exocrine pancreas.     

Phytochemical analysis and in vitro antiviral activities of the essential oils of seven Lebanon species

The chemical composition of the essential oils of Laurus nobilis, Juniperus oxycedrus ssp. oxycedrus, Thuja orientalis, Cupressus sempervirens ssp. pyramidalis, Pistacia palaestina, Salvia officinalis, and Satureja thymbra was determined by GC/MS analysis. Essential oils have been evaluated for their inhibitory activity against SARS-CoV and HSV-1 replication in vitro by visually scoring of the virus-induced cytopathogenic effect post-infection. L. nobilis oil exerted an interesting activity against SARS-CoV with an IC(50) value of 120 microg/ml and a selectivity index (SI) of 4.16. This oil was characterized by the presence of beta-ocimene, 1,8-cineole, alpha-pinene, and beta-pinene as the main constituents. J. oxycedrus ssp. oxycedrus oil, in which alpha-pinene and beta-myrcene were the major constituents, revealed antiviral activity against HSV-1 with an IC(50) value of 200 microg/ml and a SI of 5.     

Organic phenyl arsonic acid compounds with potent antileukemic activity

A series of 12 organic arsonic acid compounds has been synthesized and evaluated against human B-lineage (NALM-6) and T-lineage (MOLT-3) acute lymphoblastic leukemia (ALL) cell lines. The lead compounds 2-trichloromethyl-4-[4'-(4"-phenylazo)phenylarsonic acid]aminoquinazoline (compound 19, PHI-P518; IC(50)=1.1+/-0.5 microM against NALM-6 and 2.0+/-0.8 microM against MOLT-3) and 2-methylthio-4-(2'-phenylarsonic acid)aminopyrimidine (compound 15, PHI-P381; IC(50)=1.5+/-0.3 microM against NALM-6 and 2.3+/-0.5 microM against MOLT-3) exhibited potent antileukemic activity at low micromolar concentrations.     

Design, synthesis, and preliminary in vitro and in silico antiviral activity of [4,7]phenantrolines and 1-oxo-1,4-dihydro-[4,7]phenantrolines against single-stranded positive-sense RNA genome viruses

Following the antiviral screening of a wide series of new angular and linear N-tricyclic systems both in silico and in vitro, the [4,7]phenantroline nucleus emerged as a new ring system endowed with activity against viruses containing single-stranded, positive-sense RNA genomes (ssRNA+). Here, we report our new pathway to the synthesis of this nucleus and of several related derivatives, as well as the results of both cell-based antiviral assays and molecular dynamics simulations. In the antiviral screening, several compounds (9 and 16-20) showed to be fairly active against BVDV, CVB-2, and Polio 1 (EC50, 6-25 microM). According to molecular dynamics simulations, compounds (15) and (17) emerged for its potency against the HCV NS5B, with a calculated IC50 of 11-12 microM.     

Effect of chloroquine phosphate and toxic concentrations of lead acetate on Ca2+-ATPase activity in isolates and clones of Plasmodium Falciparum

The basal activity of Ca2+-ATPase in two isolates (NL56, UNC) and two clones (D6, W2) of P.falciparum was assessed. The effects of various concentrations of chloroquine phosphate and toxic concentrations of lead acetate were also evaluated in the clones and strains of P.falciparum. The Ca2+-ATPase activity was measured by monitoring the rate of release of inorganic phosphate from the gamma-position of ATP on spectrophotometer at 820nm wavelength. The various concentrations of chloroquine (3, 6, 9, 12, 18μg/ml) and lead acetate (5, 10, 20, 30, 40μg/ml) on Ca2+-ATPase activity were measured respectively. Chloroquine phosphate inhibited Ca2+-ATPase activity in both the isolates and the cloned strains of P.falciparum in concentration dependent manner. Median Inhibitory concentration of chloroquine (MIC50) estimated from the plot of activity against chloroquine concentration was found to be 2.6mg/ml at pH 7.4 for both the isolates and cloned strains examined. Lead acetate at concentrations 5-20μg/ml inhibited Ca2+-ATPase activity in concentration dependent manner in clone W2 (Chloroquine resistant strain) while the same range of concentrations of lead acetate stimulated the activity of the enzyme in clone D6 (Chloroquine sensitive strain).The inhibitory effect of lead acetate on the enzyme in clone D6 was observed at concentrations above 20μg/ml. The result also suggests that lead ions could modulate and moderate calcium ion homeostasis in P. falciparum via its effect on Ca2+-ATPase activity. Also sufficient influx of lead ions into P. falciparum may transform the biochemical or bioenergetics nature of chloroquine sensitive strain of P. falciparum (D6) to that similar to chloroquine resistant strain (W2). In conclusion, inhibition of Ca2+-ATPase activity of P.falciparum may be part of the mechanism of action of chloroquine in its use as chemotherapy for malaria. The study implies that populations simultaneously exposed to lead pollution and malaria infection may experience failure in chloroquine therapy.     

Solid phase synthesis and antiprotozoal evaluation of di- and trisubstituted 5'-carboxamidoadenosine analogues

The rapid increase of resistance to drugs commonly used in the treatment of tropical diseases such as malaria and African sleeping sickness calls for the prompt development of new safe and efficacious drugs. The pathogenic protozoan parasites lack the capability of synthesising purines de novo and they take up preformed purines from their host through various transmembrane transporters. Adenosine derivatives constitute a class of potential therapeutics due to their selective internalisation by these transporters. Automated solid-phase synthesis can speed up the process of lead finding and we pursued the solid-phase synthesis of di- and trisubstituted 5'-carboxamidoadenosine derivatives by using a safety-catch approach. While efforts with Kenner's sulfonamide linker remained fruitless, successful application of the hydrazide safety-catch linker allowed the construction of two representative combinatorial libraries. Their antiprotozoal evaluation identified two compounds with promising activity: N(6)-benzyl-5'-N-phenylcarboxamidoadenosine with an IC(50) value of 0.91 microM against Trypanosoma brucei rhodesiense and N(6)-diphenylethyl-5'-phenylcarboxamidoadenosine with an IC(50) value of 1.8 microM against chloroquine resistant Plasmodium falciparum.     

Clinical efficacy of chloroquine versus artemether-lumefantrine for Plasmodium vivax treatment in Thailand

Chloroquine remains the drug of choice for the treatment of vivax malaria in Thailand. Mixed infections of falciparum and vivax malaria are also common in South-East Asia. Laboratory confirmation of malaria species is not generally available. This study aimed to find alternative regimens for treating both malaria species by using falciparum antimalarial drugs. From June 2004 to May 2005, 98 patients with Plasmodium vivax were randomly treated with either artemether-lumefantrine (n = 47) or chloroquine (n = 51). Both treatments were followed by 15 mg of primaquine over 14 days. Adverse events and clinical and parasitological outcomes were recorded and revealed similar in both groups. The cure rate was 97.4% for the artemether-lumefantrine treated group and 100% for the chloroquine treated group. We concluded that the combination of artemether-lumefantrine and primaquine was well tolerated, as effective as chloroquine and primaquine, and can be an alternative regimen for treatment of vivax malaria especially in the event that a mixed infection of falciparum and vivax malaria could not be ruled out.     

Plasmodium falciparum: in vitro interactions of artemisinin with amodiaquine, pyronaridine, and chloroquine

In the scenario of drug-resistant Plasmodium falciparum malaria combination therapy represents an effective approach. Artemisinin and its derivatives are of special interest because they represent the most effective group of compounds against multidrug-resistant malaria with a rapid onset of action and a short half-life. Interactions of artemisinin with amodiaquine, pyronaridine, and chloroquine were therefore investigated against three strains of P. falciparum using a 48-h in vitro culture assay. Two of the strains were chloroquine sensitive and one was partially chloroquine resistant. Observed effective concentrations (O) of the combined compounds at different concentration ratios were calculated for different degrees of inhibition (EC50, EC90, EC99) and compared to expected calculated effective concentrations (E) using a probit method. Synergism with mean O/E EC90 values of 0.25 and 0.8 were found with the combination of artemisinin and the two Mannich bases, amodiaquine and pyronaridine, respectively, whereas chloroquine showed addition with a mean value of 1.2. Although both amodiaquine and chloroquine are 4-aminoquinolines, their interaction with artemisinin appears to be different. The combination of artemisinin with amodiaquine represents an important option for the treatment of falciparum malaria.