In vitro activity of tafenoquine alone and in combination with artemisinin against Plasmodium falciparum

Emergence and spread of drug-resistant falciparum malaria has created an urgent demand for alternative therapeutic agents. This study was conducted to assess the in vitro blood schizontocidal activity of tafenoquine, the most advanced candidate drug of the 8-aminoquinolines, and of its 1:1 combination with artemisinin in fresh isolates of Plasmodium falciparum in an area with multi-drug resistance, measuring the inhibition of schizont maturation. In 43 successfully tested parasite isolates, the mean effective concentrations (ECs) of tafenoquine were 209 nmol/L for the EC50, and 1,414 nmol/L for the EC90. Tafenoquine showed no significant activity relationships with mefloquine, artemisinin, and chloroquine. With quinine, a highly significant activity relationship was observed at the EC50, but not at the EC90. The EC50, and EC90 of the tafenoquine-artemisinin combination were 15.9 nmol/L and 84.3 nmol/L. The combination was synergistic. Tafenoquine appears to be a promising candidate for treating multidrug-resistant falciparum malaria, especially in combination with artemisinin derivatives.     

In vitro activity of chloroquine and quinine in combination with desferrioxamine against Plasmodium falciparum

The activity of chloroquine and quinine, alone and in combination with desferrioxamine (7 μmol/liter), was evaluated in vitro against susceptible and resistant clones of Plasmodium falciparum by a semimicroassay system. The addition of desferrioxamine had no effect on the activity of chloroquine against both clones. Desferrioxamine had no effect on the activity of quinine against the susceptible clone but had slightly enhanced quinine action against the resistant clone. Further development of desferrioxamine as an antimalarial drug may be of limited interest. © 1993 Wiley-Liss, Inc.     

In vitro activity of quinolines against Plasmodium falciparum in Gabon

The assessment of drug sensitivity of Plasmodium falciparum to antimalarial drugs is of vital interest for malaria endemic regions. We conducted a follow-up study to monitor the in vitro activity of the most commonly used quinolines against fresh P. falciparum isolates in Lambaréné, Gabon by measuring schizont maturation inhibition in 2002. Mean 50% effective concentration levels for chloroquine, quinine, and mefloquine were 5.5micromol/l blood, 286nmol/l blood medium mixture (BMM), and 1.1micromol/l blood, respectively. All isolates (n=40) were found to be highly resistant to chloroquine. One isolate was resistant to mefloquine and five isolates were presenting borderline-resistance. All isolates were inhibited by quinine concentrations below the threshold of resistance (n=43).Besides the observation of an increasing number of borderline resistant isolates to mefloquine, an extremly high parasite resistance to chloroquine-still officially the first line antimalarial in Gabon-seems to be of particular concern.     

In vitro activity of artemisone compared with artesunate against Plasmodium falciparum.

Artemisinins show the potential for neurotoxicity in preclinical studies. Artemisone is a leading candidate of second-generation semi-synthetic artemisinin derivatives for antimalarial therapy devoid of neurotoxicity. Artemisone showed 3-5-fold higher in vitro activity (50% effective concentration (EC50) = 0.14 nmol/L, EC90 = 2.55 nmol/L) than artesunate against fresh Plasmodium falciparum isolates from Gabon and a high-activity correlation indicates a shared drug target.     

Potent antimalarial activity of clotrimazole in in vitro cultures of Plasmodium falciparum

The increasing resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands a continuous effort to develop new antimalarial agents. In this quest, the identification of antimalarial effects of drugs already in use for other therapies represents an attractive approach with potentially rapid clinical application. We have found that the extensively used antimycotic drug clotrimazole (CLT) effectively and rapidly inhibited parasite growth in five different strains of P. falciparum, in vitro, irrespective of their chloroquine sensitivity. The concentrations for 50% inhibition (IC(50)), assessed by parasite incorporation of [(3)H]hypoxanthine, were between 0.2 and 1.1 microM. CLT concentrations of 2 microM and above caused a sharp decline in parasitemia, complete inhibition of parasite replication, and destruction of parasites and host cells within a single intraerythrocytic asexual cycle (approximately 48 hr). These concentrations are within the plasma levels known to be attained in humans after oral administration of the drug. The effects were associated with distinct morphological changes. Transient exposure of ring-stage parasites to 2.5 microM CLT for a period of 12 hr caused a delay in development in a fraction of parasites that reverted to normal after drug removal; 24-hr exposure to the same concentration caused total destruction of parasites and parasitized cells. Chloroquine antagonized the effects of CLT whereas mefloquine was synergistic. The present study suggests that CLT holds much promise as an antimalarial agent and that it is suitable for a clinical study in P. falciparum malaria.     

Efficacy of artemisinin and mefloquine combinations against Plasmodium falciparum. In vitro simulation of in vivo pharmacokinetics

The activity of artemisinin in combination with mefloquine was tested in vitro against a chloroquine‐sensitive (F32) strain of Plasmodium falciparum . A method of repetitive dosing and extending the culture observation period to 28–30 days was used to mimic the in vivo pharmacokinetic situation. Plasmodium falciparum was exposed to artemisinin from 10⁻⁸ to 10⁻⁵  m , mefloquine from 3×10⁻⁹ to 10⁻⁵  m and their combinations. The exposure time for artemisinin was 3 hours twice daily and for mefloquine 24 hours. The drug‐dosing duration was 3 days.
Neither artemisinin nor mefloquine alone provided radical clearance of P. falciparum , even when maximum concentrations (10⁻⁵  m ) were applied. The antiparasitic activity of artemisinin and mefloquine were significantly higher when dosed alone. Effective concentrations for different degrees of inhibition (EC 50, 90 and 99) of both artemisinin and mefloquine respectively were significantly lower when used in combination. At concentrations normally reached in vivo , this effect was clearly synergistic ( P =0.016)
Our in vitro model of intermittent dosing of artemisinin and mefloquine combinations for 3 days provides significant evidence of positive interaction between the two compounds. Lower combination concentrations around the MIC‐values for the individual compounds showed synergistic effect, and high concentrations showed additive effect. This indicates that such drug combinations may provide radical clearance at concentrations lower than those required for single‐drug treatment.     

Antiplasmodial interactions between artemisinin and triclosan or ketoconazole combinations against blood stages of Plasmodium falciparum in vitro

Emergence of drug-resistant Plasmodium falciparum strains to conventional first-line antimalarial drugs has compelled many countries to reorient their drug policies to adopt artemisinin-based combination therapies (ACTs) for treatment of uncomplicated malaria. This has increased the demand of artemisinin, already a scarce commodity. Synthesis of artemisinin is not yet commercially viable. Extensive use of available ACTs will invariably lead to emergence of resistance to these combinations. Thus, there is need to search for new artemisinin-based synthetic, inexpensive, synergistic combinations to reduce dependence on artemisinin. In vitro cultures of P. falciparum provide an appropriate system for identification of such new combinations. We evaluated interactions of artemisinin with triclosan or ketoconazole against blood stages of P. falciparum by a fixed-ratio isobologram method. Artemisinin shows mild synergistic interaction with triclosan and slight to marked antagonism with ketoconazole in vitro. These antiplasmodial interactions, however, require confirmation using in vivo model systems.     

Activity of azithromycin or erythromycin in combination with antimalarial drugs against multidrug-resistant Plasmodium falciparum in vitro

Azithromycin, an azalide analog of erythromycin was assayed for its in vitro activity against multidrug-resistant Plasmodium falciparum K1 strain by measuring the ³H-hypoxanthine incorporation. Azithromycin caused inhibitory effects on the parasite growth with IC₅₀ and IC₉₀ values of 8.4 ± 1.2 μM and 26.0 ± 0.9 μM, respectively. Erythromycin inhibited growth of P. falciparum with IC₅₀ and IC₉₀ values of 58.2 ± 7.7 μM and 104.0 ± 10.8 μM, respectively. The activity of antimalarial drugs in combination with azithromycin or erythromycin against P. falciparum K1 were compared. Combinations of chloroquine with azithromycin or erythromycin showed synergistic effects against parasite growth in vitro. Combinations of quinine–azithromycin and quinine–erythromycin showed potentiation. Additive effects were observed in mefloquine–azithromycin and mefloquine–erythromycin combinations. Similar results were also produced by pyronaridine in combination with azithromycin or erythromycin. However, artesunate–azithromycin and artesunate–erythromycin combinations had antagonistic effects. The in vitro data suggest that azithromycin and erythromycin will have clinical utility in combination with chloroquine and quinine. The worldwide spread of chloroquine-resistant P. falciparum might inhibit the ability to treat malaria patients with chloroquine–azithromycin and chloroquine–erythromycin in areas of drug-resistant. The best drug combinations against multidrug-resistant P. falciparum are quinine–azithromycin and quinine–erythromycin.     

In vitro activity of pyronaridine against African strains of Plasmodium falciparum.

The in vitro activity of pyronaridine was determined and compared with the activity of monodesethylamodiaquine and amopyroquine against 31 clinical isolates and two clones of Plasmodium falciparum originating from Central and West Africa using a semi-micro drug susceptibility test. Pyronaridine and amopyroquine were 2.5 and four times less active, respectively, against the highly chloroquine-resistant clone, than against the chloroquine-susceptible clone but were equally active against chloroquine-susceptible and chloroquine-resistant clinical isolates. Compared with chloroquine-susceptible isolates, monodesethylamodiaquine was three times less active against chloroquine-resistant parasites. Pyronaridine is highly active against chloroquine-resistant strains of P. falciparum and may be a promising candidate for the treatment of resistant malaria.     

恶性疟原虫氯喹敏感株与抗性株对青蒿素类药物体外敏感性的研究

目的 测定恶性疟原虫氯喹敏感株与抗性株对青蒿素类药物的体外敏感性. 方法 运用体外微量法与酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)测定青蒿琥酯、蒿甲醚及双氢青蒿素等3种青蒿素类抗疟药物对体外培养的恶性疟原虫氯喹敏感株与氯喹抗性株的体外敏感性,并比较两种方法测定的IC50值. 结果 体外微量法测定的3种药物对恶性疟原虫氯喹敏感株的IC50值依次为3.12 nmol/L、4.30 nmol/L、2.18 nmol/L,对恶性疟原虫氯喹抗性株的IC50值依次为4.31nmol/L、3.90 nmol/L、3.17 nmol/L;同时,将体外微量法与ELISA法所获的结果进行相关性分析,两种方法结果基本一致(r2=0.93,P＜0.001). 结论 恶性疟原虫氯喹抗性株对青蒿素类药物无明显的交叉抗性;ELISA法可用于恶性疟原虫对抗疟药物的体外敏感性检测.     

In vitro antiplasmodial activity of Clathria vulpina sponge associated bacteria against Plasmodium falciparum

ObjectiveTo identify the possible antiplasmodial drugs from bacteria associated with marine sponge Clathria vulpina (C. vulpina).MethodsThe C. vulpina samples were collected from Thondi coast and subjected to enumeration and isolation of associated bacteria. Filtered and sterilized extracts (100, 50, 25, 12.5, 6.25 and 3.125 μg/mL) from isolated bacterial strains were screened for antiplasmodial activity against Plasmodium falciparum. Potential extracts were also screened for biochemical constituents.ResultsThirty one bacterial strains were isolated from twelve sponge samples collected from Thondi coast and screened for antiplasmodial assay. The count of bacterial strains were maximum in November 2007 (19×10⁴ CFU/g) and the average count was maximum during the monsoon season (110×10³ CFU/g). The antiplasmodial activity of strain THB15 was highly comparable (IC₅₀ = 20.73 μg/mL) with the positive control chloroquine (IC₅₀ = 19.59 μg/mL) and 21 bacterial strains showed IC₅₀ value of more than 100 μg/mL. Statistical analysis reveals that, significant in vitro antiplasmodial activity (P<0.05) was observed between the concentrations and time of exposure. The chemical injury to erythrocytes showed no morphological changes in erythrocytes by the ethyl acetate extract of bacterial strains after 48 h of incubation. The in vitro antiplasmodial activity might be due to the presence of sugars and alkaloids in the ethyl acetate extracts of bacterial strains.ConclusionThe ethyl acetate extract of THB15 possesses lead compounds for the development of antiplasmodial drugs.     

In vitro activity of pyronaridine against field isolates and reference clones of Plasmodium falciparum

Pyronaridine, a 9-substituted 1-aza-acridine, was assayed for in vitro activity against clinical and field isolates as well as characterized clones of Plasmodium falciparum. The in vitro antimalarial activity of pyronaridine was compared to activities of standard antimalarials against multidrug-resistant isolates of P. falciparum from eastern and northern Thailand using an assay based on the inhibition of schizont maturation. Isolates from eastern Thailand (n = 30) were susceptible to pyronaridine (IC50 8.40 nM), mefloquine (IC50 6.97 nM), and amodiaquine (IC50 12.7 nM) and resistant to chloroquine (IC50 361 nM), quinine (IC50 388 nM), and pyrimethamine (IC50 11,800 nM). The isolates from northern Thailand (n = 7) showed no statistical difference in susceptibility to pyronaridine (IC50 10.1 nM), amodiaquine (IC50 7.29 nM), and mefloquine (IC50 5.48 nM); however, isolates were significantly more susceptible to chloroquine (IC50 167 nM), quinine (IC50 248 nM), and pyrimethamine (IC50 1,980 nM). These data suggest a lack of cross-resistance between pyronaridine and either chloroquine, quinine, or pyrimethamine. Using the same assay system the in vitro activity of pyronaridine was evaluated against isolates from treatment failures of mefloquine or enpiroline from eastern Thailand. The IC50 values for mefloquine against five recrudescent isolates were significantly higher (IC50 16.4 nM) than the field isolates collected from the same region (IC50 6.97 nM); however, there was no significant difference in the pyronaridine susceptibility between the isolates from the field study (IC50 8.89 nM) and the isolates from the treatment failures (IC50 8.40 nM). These observations suggest a lack of cross-resistance to mefloquine following treatment failure with either mefloquine or enpiroline.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro antiplasmodial activity of marine sponge Stylissa carteri associated bacteria against Plasmodium falciparum

ObjectiveTo identify the possible antiplasmodial drugs from bacteria associated with marine sponge Stylissa carteri (S. carteri).MethodsThe S. carteri samples were collected from Thondi coast and subjected for enumeration and isolation of associated bacteria. Filter sterilized extracts (100, 50, 25, 12.5, 6.25 and 3.125 μ g/mL) from isolated bacterial strains were screened for antiplasmodial activity against Plasmodium falciparum (P. falciparum) and potential extracts were also screened for biochemical constituents.ResultsTwelve samples of S. carteri were collected and subjected for enumeration and isolation of associated bacteria. The count of bacterial strains were maximum in November 2007 (34 × 10⁴ CFU/g) and the average count was maximum during the monsoon season (203 × 10³ CFU/g). Thirty two morphologically different bacterial strains were isolated from S. carteri and the ethyl acetate bacterial extracts were screened for antiplasmodial activity against P. falciparum. The antiplasmodial activity of a strain THB17 (IC₅₀ 20.56 μ g/mL) extract is highly comparable with the positive control chloroquine (IC₅₀ 19.59 μ g/mL) and 13 bacterial extracts which showed IC₅₀ value of more than 100 μ g/mL. Statistical analysis reveals that, significant in vitro antiplasmodial activity (P<0.05) was observed between the concentrations and time of exposure. The chemical injury to erythrocytes showed no morphological changes in erythrocytes by the ethyl acetate extract of bacterial strains after 48 h of incubation. The in vitro antiplasmodial activity might be due to the presence of reducing sugars and alkaloids in the ethyl acetate extracts of bacterial strains.ConclusionsThe ethyl acetate extract of THB17 possesses lead compounds for the development of antiplasmodial drugs.     

The effect of artemisinin combined with standard antimalarials against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum in vitro

The activity of artemisinin (qinghaosu) in combination with some commonly-used antimalarial drugs was tested in vitro against a chloroquine-sensitive (NF54) and a chloroquine-resistant (K1) strain of Plasmodium falciparum. Both mefloquine and tetracycline showed marked potentiative synergism with artemisinin against both strains, whilst primaquine showed potentiation against K1. Combinations of artemisinin with pyrimethamine and with chloroquine were antagonistic. The results confirm observations on rodent malaria in vivo and indicate that the drug interactions seen were direct, through actions on the parasite, and not merely effects on drug distribution and metabolism in the mouse host.     

In vitro blood schizonticidal activity of sera containing mefloquine‐quinine against Plasmodium falciparum

Serum samples collected at intervals from healthy volunteers, after the administration of 3 drug regimens (quinine (QN) 600 mg, mefloquine (MQ) 750 mg, and MQ 750 mg plus QN 600 mg) were investigated for their blood schizonticidal activities against K₁ strain Plasmodium falciparum in vitro . Superiority of activity was shown in the sera collected after the combination regimen. In the diluted sera of the QN regimen, a complete inhibitory effect was observed for only 24 hours, whereas the effect was sustained for 72 hours in the sera collected after MQ in either regimen (MQ alone or MQ/QN). The pattern of minimum inhibitory concentrations (MICs) of QNEq of the sera from QN alone was constant throughout a 24‐hour period, with significantly higher concentrations than that from the combination regimen (118–150 vs 21.25–73.5 μg/l). In sera collected after the combination regimen, however, the MIC gradually decreased from 0.5 until 2.5 and 4 h, and thereafter gradually returned to the same levels again during a period of 6–24 hours. The MICs of MQEq when given as MQ alone or in combination appeared constant, with a significantly higher value in the former regimen (24.4–26.8 vs 17–19.2 μg/l).     

In vitro activity of dihydroartemisinin against clinical isolates of Plasmodium falciparum in Yaounde, Cameroon.

The in vitro activities of dihydroartemisinin (the biologically active metabolite of artemisinin derivatives), chloroquine, monodesethylamodiaquine (the biologically active metabolite of amodiaquine), quinine, mefloquine, halofantrine, and pyrimethamine were assessed in 65 African isolates of Plasmodium falciparum from Yaounde, Cameroon using an isotopic microtest. The 50% inhibitory concentration (IC50) values for dihydroartemisinin were within a narrow range from 0.25 to 4.56 nM, with a geometric mean of 1.11 nM (95% confidence interval = 0.96-1.28 nM). Dihydroartemisinin was equally active (P > 0.05) against the chloroquine-sensitive isolates (geometric mean IC50 = 1.25 nM, 95% confidence interval = 0.99-1.57 nM) and the chloroquine-resistant isolates (geometric mean IC50 = 0.979 nM, 95% confidence interval = 0.816-1.18 nM). A significant positive correlation was observed between the responses to dihydroartemisinin and mefloquine (r = 0.662) or halofantrine (r = 0.284), suggesting in vitro cross-resistance. There was no correlation between the responses to dihydroartemisinin and other antimalarial drugs.     

Analogues of N-benzyloxydihydrotriazines: in vitro antimalarial activity against Plasmodium falciparum

The antimalarial activities of a series of chlorophenyloxyalkoxy and chlorophenalkoxy N-substituted diamino-dihydrotriazines were determined in vitro against three strains of Plasmodium falciparum (Malayan Camp, Vietnam Smith, FCB) with diverse levels of resistance to chloroquine, pyrimethamine, and cycloguanil. Parasite viability was assayed by the inhibition of the uptake of radiolabelled hypoxanthine. Most of the ID-50S of these compounds were less than 1.0 ng ml-1. Consistent differences in sensitivities to these compounds were observed and appeared to be strain related. The Malayan Camp was the most sensitive and Vietnam Smith was the least sensitive. These differences appeared to be related primarily to an inherent sensitivity of a particular strain to the series of analogues examined rather than to a pattern of cross-resistance to chloroquine, pyrimethamine, or cycloguanil.