Stage-dependent inhibition of chloroquine on Plasmodium falciparum in vitro

Morphological observation of the life cycle of the malaria parasite, Plasmodium falciparum, in highly synchronous cultures after an exposure to therapeutic concentrations of chloroquine in ring, trophozoite and schizont stages, respectively, were carried out in order to determine the influence of chloroquine on the growth of the different stages of the malarial parasites. It was found that chloroquine could not affect merozoite invasion of the erythrocytes; the ring stage was more sensitive to chloroquine than the trophozoite and schizont stages; and chloroquine in therapeutic concentrations prevented only the transformation of rings to trophozoites and could not affect the transformations of trophozoites to schizonts and schizonts to new rings. The determination of the IC50 of chloroquine showed that the IC50 of trophozoites was about 6 times as high as that of rings.     

Inhibition of a protein tyrosine kinase activity in Plasmodium falciparum by chloroquine

The aim of the present study was to establish the importance of phosphorylation events for parasite growth and maturation. Investigations into the cytosolic Plasmodium falciparum protein tyrosine kinase (PTK) activity revealed that there is a stage specific increase in the activity, in the order ring < trophozoite < schizont in both chloroquine sensitive (CQ-S) and chloroquine resistant (CQ-R) strains (p < 0.05). Our data also show that in vivo conversion of the schizont stage to ring stage via release of merozoites is associated with a decrease in PTK activity. Piceatannol, a specific inhibitor of PTK inhibited the activity in both the CQ-S and CQ-R strains of the parasites. The presence of low levels of chloroquine (CQ) inhibited the cytosolic PTK activity in a dose dependent manner (IC50 = 45 mumoles or 23 micrograms/ml) in CQ-S strains. The effect of varying concentration of CQ on the kinetics of peptide phosphorylation reveal that CQ was a competitive inhibitor of PTK with respect to peptide substrate and non-competitive with respect to ATP indicating that CQ inhibits PTK activity by binding with protein substrate binding site. These data thus suggests that maturation of malaria parasite may be due to this cellular PTK and its inhibition by CQ could provide a hypothesis to explain its antimalarial activity and efficacy.     

Sentinel network for monitoring in vitro susceptibility of Plasmodium falciparum to antimalarial drugs in Colombia: a proof of concept

Drug resistance is one of the principal obstacles blocking worldwide malaria control. In Colombia, malaria remains a major public health concern and drug-resistant parasites have been reported. In vitro drug susceptibility assays are a useful tool for monitoring the emergence and spread of drug-resistant Plasmodium falciparum. The present study was conducted as a proof of concept for an antimalarial drug resistance surveillance network based on in vitro susceptibility testing in Colombia. Sentinel laboratories were set up in three malaria endemic areas. The enzyme linked immunosorbent assay-histidine rich protein 2 and schizont maturation methods were used to assess the susceptibility of fresh P. falciparum isolates to six antimalarial drugs. This study demonstrates that an antimalarial drug resistance surveillance network based on in vitro methods is feasible in the field with the participation of a research institute, local health institutions and universities. It could also serve as a model for a regional surveillance network. Preliminary susceptibility results showed widespread chloroquine resistance, which was consistent with previous reports for the Pacific region. However, high susceptibility to dihydroartemisinin and lumefantrine compounds, currently used for treatment in the country, was also reported. The implementation process identified critical points and opportunities for the improvement of network sustainability strategies.     

Time course of in vitro maturation of intra-erythrocytic malaria parasite: a comparison between Plasmodium falciparum and Plasmodium knowlesi

The schizont maturation assay for in vitro drug sensitivity tests has been a standard method employed in the global baseline assessment and monitoring of drug response in Plasmodium falciparum. This test is limited in its application to synchronous plasmodial infections because it evaluates the effect of drug on the maturation of parasite especially from ring to schizont stage and therefore synchronized P. falciparum cultures are required. On the other hand, P. knowlesi, a simian malaria parasite has a unique 24-h periodicity and maintains high natural synchronicity in monkeys. The present report presents the results of a comparative study on the course of in vitro maturation of sorbitol synchronized P. falciparum and naturally synchronous P. knowlesi. Ring stage parasites were incubated in RPMI medium supplemented with 10-15% pooled homologous serum in flat-bottomed 96-well micro plates using a candle jar at 37 degrees C. The results suggest that the ideal time for harvesting the micro-assay plates for in vitro drug sensitivity test for sorbitol-synchronized P. falciparum and naturally synchronous P. knowlesi are from 26 to 30 h and from 22 to 25 h, respectively. The advantages of using P. knowlesi in chemotherapeutic studies are discussed.     

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.     

Monitoring the drug-sensitivity of Plasmodium falciparum in coastal towns in Madagascar by use of in vitro chemosensitivity and mutation detection tests

The dissemination of mutant and resistant strains of Plasmodium falciparum makes a considerable contribution to the spread of drug-resistant malaria. Populations around harbours and airports could be particularly exposed to Plasmodium isolates introduced with imported cases of malaria. The use of chloroquine as well as the use of and sulfadoxine/pyrimethamine is currently an effective method for treating uncomplicated cases of malaria in Madagascar. As part of a monitoring programme, in vitro methods were used to assess the sensitivity of P. falciparum isolates in two coastal towns in Madagascar: Mahajanga on the west coast and Toamasina on the east coast. All of the isolates from both sites were sensitive to amodiaquine, quinine, pyrimethamine and cycloguanil. All of the isolates from Mahajanga were sensitive to chloroquine (n = 25; mean IC50 = 22.6 nM, 95% confidence interval: 16.8-28.7 nM), whereas three of the isolates from Toamasina were resistant to chloroquine (n = 18; mean IC50 = 66.3 nM; 95% confidence interval: 42.6-90 nM). The frequency of the Pfcrt Thr-76 and the dhfr Asn-108 mutations was estimated by PCR/RFLP. The 43 P. falciparum isolates examined, including the three in vitro chloroquine-resistant isolates from Toamasina were all wild-type (Lys-76). Phenotyping and genotyping studies suggested that the prevalence of chloroquine- and pyrimethamine-resistant isolates and of mutant strains of P. falciparum is very low. These results showed that in vitro test and genotyping of resistance markers approaches could be successfully used to monitor the emergence of drug-resistant malaria and to try to alleviate the lack of medical teams able to carry out in vivo test. The possible hazard/risk associated with imported cases of malaria is discussed.     

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.     

Malaria chemoprophylaxis in travellers to east Africa: a comparative prospective study of chloroquine plus proguanil with chloroquine plus sulfadoxine-pyrimethamine

As malaria caused by Plasmodium falciparum has become resistant to chloroquine alternative drug regimens need to be developed. The prophylactic efficacy against malaria and the side effects of chloroquine phosphate 500 mg weekly with proguanil hydrochloride 200 mg daily was compared with the efficacy of chloroquine 500 mg weekly with sulfadoxine 500 mg-pyrimethamine 25 mg weekly in a randomised study of Scandinavian travellers to Kenya and Tanzania during 1984-5. A total of 767 subjects (416 male and 351 female; 384 taking chloroquine phosphate with proguanil hydrochloride and 383 taking chloroquine with sulfadoxine-pyrimethamine) completed a diary on the breakthrough of malaria and the side effects of treatment while taking the drugs. They were also asked to make thick blood films when symptoms like those of malaria occurred, which were sent to and analysed in Denmark. Four subjects taking chloroquine with proguanil hydrochloride and three taking chloroquine with sulfadoxine-pyrimethamine developed falciparum malaria, which was verified microscopically. Side effects were reported by 36 subjects taking chloroquine phosphate with proguanil hydrochloride and 55 taking the other regimen (p=0·043). The side effects of both regimens were generally mild, but the combination of chloroquine phosphate with proguanil hydrochloride is recommended because it results in fewer side effects.As breakthroughs of malaria occurred at the earliest after seven weeks self treatment should not be recommended for travellers staying only a short time. Thick blood films are useful for diagnosis of suspected cases of malaria, can be prepared by non-specialists in Africa, and can be analysed successfully after long delays.     

Plasmodium falciparum: drug-resistant malaria complicating leukemias and lymphomas in children.

The present communication deals with drug-resistant Plasmodium falciparum malaria complicating hematologic malignancies (leukemias, n = 24, and lymphomas, n = 7) in children. Of 50 cases of hematologic malignancies, 31 patients were microscopically diagnosed as having P. falciparum infection (MP +). Initially, all the patients were treated with chloroquine. The results of primary treatment showed chloroquine resistance in 16 (51. 62%) cases. Of these 16 chloroquine-resistant cases, 13 were secondarily treated with a combination of pyrimethamine plus sulfamethopyrazine. The results of secondary treatment also revealed resistance to pyrimethamine plus sulfamethopyrazine in 6 of 13 (46. 10%) cases. The 6 pyrimethamine plus sulfamethopyrazine-resistant P. falciparum cases were finally cured by quinine therapy, against which no resistance was encountered. Conversely, in the control group comprising 38 cases of P. falciparum without malignancy, the incidence of chloroquine resistance was found in only 9 cases, which is rather low (23.70%). Of these 7 chloroquine-resistant cases, 5 were found to be sensitive to pyrimethamine plus sulfamethopyrazine treatment, while the 2 nonresponders were finally cured with quinine. The overall results of this study show a high prevalence of chloroquine resistance among clinical cases of falciparum malaria (25/69; 30.6%). Among the nonresponders (n = 20) 40% of cases were also resistant to the pyrimethamine plus sulfamethopyrazine combination. There was no resistance to quinine.     

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.     

Persistence of low levels of Plasmodium falciparum resistance to chloroquine in the autochthonous population of the Central African Republic.

In Central African Republic, the first cases of resistant P. falciparum to chloroquine occurred in 1983 in non immune expatriate residents on regular chemoprophylaxis. From 1984 to 1991, 950 in vivo tests with a seven days observation period were performed in semi-immune autochtonous children living in seven towns of the country. Chloroquine treatments were given at 25 mg/kg over 3 days period to children with P. falciparum parasitaemia > 500 (634 simplified methods) or > 1000/mm3 (316 WHO standard field tests), usually asymptomatic. Until 1988, the surveys show an absence or a low frequency of chloroquine resistance (usually below 10%) according to the town; since 1989, the resistance has been present in all towns but everywhere with a frequency under 20%. None cases of R III level resistance was observed and all children became or remained asymptomatic at day 7. Until further surveys demonstrate a decreased efficacity of chloroquine, it is advised that chloroquine be used at 25 mg/kg over 3 days period as the treatment of choice in uncomplicated acute malaria in the Central African Republic.     

Plasmodium vivax Drug Resistance Genes; Pvmdr1 and Pvcrt-o Polymorphisms in Relation to Chloroquine Sensitivity from a Malaria Endemic Area of Thailand

The aim of the study was to explore the possible molecular markers of chloroquine resistance in Plasmodium vivax isolates in Thailand. A total of 30 P. vivax isolates were collected from a malaria endemic area along the Thai-Myanmar border in Mae Sot district of Thailand. Dried blood spot samples were collected for analysis of Pvmdr1 and Pvcrt-o polymorphisms. Blood samples (100 μl) were collected by finger-prick for in vitro chloroquine susceptibility testing by schizont maturation inhibition assay. Based on the cut-off IC₅₀ of 100 nM, 19 (63.3%) isolates were classified as chloroquine resistant P. vivax isolates. Seven non-synonymous mutations and 2 synonymous were identified in Pvmdr1 gene. Y976F and F1076L mutations were detected in 7 (23.3%) and 16 isolates (53.3%), respectively. Analysis of Pvcrt-o gene revealed that all isolates were wild-type. Our results suggest that chloroquine resistance gene is now spreading in this area. Monitoring of chloroquine resistant molecular markers provide a useful tool for future control of P. vivax malaria.     

Synthesis of ring-substituted 4-aminoquinolines and evaluation of their antimalarial activities

A simple two-step synthesis method was used to make 51 B-ring-substituted 4-hydroxyquinolines allowing analysis of the effect of ring substitutions on inhibition of growth of chloroquine sensitive and resistant strains of Plasmodium falciparum, the dominant cause of malaria morbidity. Substituted quinoline rings other than the 7-chloroquinoline ring found in chloroquine were found to have significant activity against the drug-resistant strain of P. falciparum W2.     

Stage-dependent effects of chloroquine on Plasmodium falciparum in vitro

The erythrocytic developmental cycle of Plasmodium falciparum can be conveniently divided into the ring, trophozoite, and schizont stages based on morphology and metabolism. Using highly synchronous cultures of P. falciparum, considerable variation was demonstrated among these stages in sensitivity to chloroquine. The effects of timed, sequential exposure to several clinically relevant concentrations of chloroquine were monitored by three techniques: morphological analysis, changes in the rate of glucose consumption, and changes in the incorporation of 3H-hypoxanthine into parasite nucleic acids. All three techniques gave essentially identical results. The trophozoite and schizont stages were considerably more sensitive to the drug than ring-stage parasites. Chloroquine sensitivity decreased as nuclear division neared completion. The increase in chloroquine sensitivity was coincident with a marked rise in the rate of glucose consumption and nucleic acid synthesis. The rate of nucleic acid synthesis decreased as schizogony progressed while glucose consumption continued at high rates during this process. The degree of chloroquine sensitivity was not highly correlated with either metabolic activity.     

pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum

Efforts to control malaria worldwide have been hindered by the development and expansion of parasite populations resistant to many first-line antimalarial compounds. Two of the best-characterized determinants of drug resistance in the human malaria parasite Plasmodium falciparum are pfmdr1 and pfcrt, although the mechanisms by which resistance is mediated by these genes is still not clear. In order to determine whether mutations in pfmdr1 associated with chloroquine resistance affect the capacity of the parasite to persist when drug pressure is removed, we conducted competition experiments between P. falciparum strains in which the endogenous pfmdr1 locus was modified by allelic exchange. In the absence of selective pressure, the component of chloroquine resistance attributable to mutations at codons 1034, 1042 and 1246 in the pfmdr1 gene also gave rise to a substantial fitness cost in the intraerythrocytic asexual stage of the parasite. The loss of fitness incurred by these mutations was calculated to be 25% with respect to an otherwise genetically identical strain in which wild-type polymorphisms had been substituted at these three codons. At least part of the fitness loss may be attributed to a diminished merozoite viability. These in vitro results support recent in vivo observations that in several countries where chloroquine use has been suspended because of widespread resistance, sensitive strains are re-emerging.     

Plasmodium falciparum: in vitro drug interaction between chloroquine and enantiomers of amlodipine

Both enantiomers of amlodipine, whose calcium antagonist action resides almost exclusively in the R(-) enantiomer, reversed chloroquine resistance in Plasmodium falciparum in vitro. R(-) enantiomer was slightly more effective than the S(+) enantiomer in potentiating chloroquine action against chloroquine-resistant strains of parasites. No potentiating effect was observed in chloroquine-sensitive parasites. Both enantiomers entered rapidly into parasitized erythrocytes to the same extent. Reversal of chloroquine resistance by the enantiomers of amlodipine was related to dose-dependent increase in the accumulation of chloroquine inside the erythrocytes parasitized by resistant parasites. These results suggest that the potentiating effect on chloroquine is independent of calcium metabolism of malaria parasites.     

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.     

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.     

Review of pyronaridine anti-malarial properties and product characteristics

ABSTRACT: Pyronaridine was synthesized in 1970 at the Institute of Chinese Parasitic Disease and has been used in China for over 30 years for the treatment of malaria. Pyronaridine has high potency against Plasmodium falciparum, including chloroquine-resistant strains. Studies in various animal models have shown pyronaridine to be effective against strains resistant to other anti-malarials, including chloroquine. Resistance to pyronaridine appears to emerge slowly and is further retarded when pyronaridine is used in combination with other antimalarials, in particular, artesunate. Pyronaridine toxicity is generally less than that of chloroquine, though evidence of embryotoxicity in rodents suggests use with caution in pregnancy. Clinical pharmacokinetic data for pyronaridine indicates an elimination T1/2 of 13.2 and 9.6 days, respectively, in adults and children with acute uncomplicated falciparum and vivax malaria in artemisinin-combination therapy. Clinical data for mono or combined pyronaridine therapy show excellent anti-malarial effects against P. falciparum and studies of combination therapy also show promise against Plasmodium vivax. Pyronaridine has been developed as a fixed dose combination therapy, in a 3:1 ratio, with artesunate for the treatment of acute uncomplicated P. falciparum malaria and blood stage P. vivax malaria with the name of Pyramax(R) and has received Positive Opinion by European Medicines Agency under the Article 58 procedure.     

3H-azidopine photoaffinity labeling of high molecular weight proteins in chloroquine resistant falciparum malaria

Using 3H-azidopine, we have succeeded in labeling proteins from chloroquine resistant (CR) human falciparum malaria parasites in the molecular weight range of 155-170 kd. Vinblastine does not compete, but azidopine blocks the labeling using 3H-azidopine. Relatively little or no labeling of the 155-170 kd protein is seen in the chloroquine sensitive strain using 3H-azidopine. Further competition can be seen with nicardipine and reserpine (71%) respectively and verapamil (61%), chloroquine (48%), quinacrine (56%), trifluoperazine (32%) and chlorpromazine (33%). We speculate that this may be the glycoprotein responsible for the resistance to chloroquine in falciparum malaria.