Use of radioactive ethanolamine incorporation into phospholipids to assess in vitro antimalarial activity by the semiautomated microdilution technique.

Phospholipid biosynthetic activity is intense in the erythrocytic stage of Plasmodium falciparum because of the parasite's own enzymatic machinery. The incorporation of various labeled phospholipid precursors in comparison with the incorporation of nucleic acid and protein precursors was tested to evaluate P. falciparum growth in vitro. These precursors, namely, [3H]ethanolamine, [3H]hypoxanthine, [3H]palmitate, [14C]serine, [3H]choline, [3H]inositol, and [3H]isoleucine, were all accurate indicators of parasite growth. However, because of its high level of incorporation, [3H]ethanolamine proved to be the best tool for assessing parasite viability. When culture parameters were carefully controlled, [3H]ethanolamine incorporation into phospholipids was proportional to pulse time, precursor concentration, and initial parasitemia and was sensitive to the number of uninfected erythrocytes (hematocrit). It can be used for a wide range of infected erythrocytes, from 2 x 10(4) to 5 x 10(5). The use of [3H]ethanolamine for in vitro antimalarial drug screening is a good alternative to the method of Desjardins et al. (R. E. Desjardins, C. J. Canfield, J. D. Haynes, and J. D. Chulay, Antimicrob. Agents. Chemother. 16:710-718, 1979). The major advantage is that the culture medium can be supplemented with hypoxanthine, which results in better parasite growth. [3H]ethanolamine is also an ideal tool when compounds that interfere with DNA and/or RNA metabolism are to be investigated for their effect on Plasmodium growth.     

Quantitative assessment of antimalarial activities from Malaysian Plasmodium falciparum isolates by modified in vitro microtechnique.

Malaysian, TGR (Thailand), and Gambian (West African) Plasmodium falciparum isolates were cultured in vitro by the candle jar method and were characterized for their susceptibilities to present antimalarial drugs by the modified in vitro microtechnique. Results showed that 93 and 47% of the Malaysian isolates were resistant at 50% inhibitory concentrations of 0.1415 to 0.7737 and 0.1025 to 0.1975 microM, respectively, while the rest were susceptible to choloroquine and cycloguanil at 0.0376 and 0.0306 to 0.0954 microM, respectively. All isolates were susceptible to mefloquine, quinine, and pyrimethamine at 0.0026 to 0.0172, 0.0062 to 0.0854, and 0.0149 to 0.0663 microM, respectively. In contrast, the Gambian isolate was susceptible to multiple drugs at 0.0024 to 0.0282 microM; TGR was resistant to chloroquine at 0.8147 microM but was susceptible to mefloquine, quinine, cycloguanil, and pyrimethamine at 0.0024, 0.0096, 0.0143, and 0.0495 microM, respectively.     

In vitro antimalarial activity of penduline, a bisbenzylisoquinoline from Isopyrum thalictroides.

Two bisbenzylisoquinolines, tetrandrine and penduline, were purified from Isopyrum thalictroides. When tested for antimalarial activity in vitro, penduline was efficient at concentrations fivefold lower than those of tetrandrine. In highly synchronized parasite cultures, penduline mostly interfered between the 8th and the 32nd hours of the parasite cycle.     

Synergistic in vitro antimalarial activity of omeprazole and quinine

Previous studies have shown that the proton pump inhibitor omeprazole has antimalarial activity in vitro. The interactions of omeprazole with commonly used antimalarial drugs were assessed in vitro. Omeprazole and quinine combinations were synergistic; however, chloroquine and omeprazole combinations were antagonistic. Artemisinin drugs had additive antimalarial activities with omeprazole.     

Desbutyl-benflumetol, a novel antimalarial compound: in vitro activity in fresh isolates of Plasmodium falciparum from Thailand.

Desbutyl-benflumetol (DBB) is a novel antimalarial compound closely related to benflumetol (lumefantrine), of which it is a putative metabolite. The in vitro response of Plasmodium falciparum to DBB was studied in Mae Hong Son and Mae Sot, in northwest Thailand, in 1997 and 1998. In total, 155 fresh isolates were successfully tested using the World Health Organization standard in vitro microtest system (Mark II). The mean 50% effective concentration (EC(50)) and 90% effective concentration of DBB were 6.36 and 31.09 nmol/liter, respectively. The comparison of the activity of DBB and benflumetol yielded a highly significant potency ratio of 4.52, corresponding to a more than four times higher efficacy of DBB. A considerable potency difference was found between isolates from Mae Hong Son and those from Mae Sot, reflecting lesser sensitivity in the area with marked resistance to mefloquine and quinine. This observation is also supported by a highly significant activity correlation with benflumetol (P < 0.001) and to a similar degree with mefloquine (P < 0.001), reflecting a close relationship of DBB with the class II aryl amino alcohol blood schizontocides. A less distinct association was also found with artemisinin, which was significant only at the EC(50) level, and there was no correlation at all with chloroquine. DBB is a promising antimalarial compound that merits further investigation in order to define its practical therapeutic potential.     

In vitro antimalarial activity and drug interactions of fenofibric acid

Plasmodium falciparum has developed resistance to most available treatments, underscoring the need for novel antimalarial drugs. Fibrates are lipid-modifying agents used to reduce morbidity and mortality associated with cardiovascular disease. They may have antimalarial activity through modulation of P-glycoprotein and ATP-binding cassette subfamily A member (ABC-1)-mediated nutrient transport and/or via a putative peroxisome proliferator-activated receptor alpha-like protein. We therefore examined in vitro antimalarial activities of fibrates and their interactions with chloroquine and dihydroartemisinin in chloroquine-sensitive (3D7) and chloroquine-resistant (W2mef) strains of P. falciparum using the conventional isotopic assay microtechnique. A bioassay was used to assess inhibition activities of human plasma after therapeutic fenofibrate doses. Fenofibric acid, the main metabolite of fenofibrate, was the most potent of the fibrates tested, with mean 50% inhibitory concentrations of 152 nM and 1,120 nM for chloroquine-sensitive and -resistant strains, respectively. No synergistic interaction between fibrates and chloroquine or dihydroartemisinin was observed. Plasma fenofibric acid concentrations, quantified by high-performance liquid chromatography in seven healthy volunteers after treatment (mean, 15.3 mg/liter, or 48 μM), inhibited P. falciparum. BLAST analysis revealed the likely presence of an ABC-1 transporter homolog in P. falciparum. Our findings demonstrate that fenofibric acid has activity similar to the activities of conventional antimalarial drugs at concentrations well below those achieved after therapeutic doses. It may inhibit P. falciparum growth by inhibiting intracellular lipid transport.     

In vitro activity of a novel broad-spectrum antifungal, E1210, tested against Candida spp. as determined by CLSI broth microdilution method

The in vitro activity of the novel antifungal agent E1210 and four comparators (caspofungin, fluconazole, posaconazole, and voriconazole) was determined against 90 clinical isolates of Candida using Clinical and Laboratory Standards Institute methods. The collection was composed of 21 Candida albicans, 20 C. glabrata, 25 C. parapsilosis, and 24 C. tropicals, and also included 21 fluconazole-resistant and 15 caspofungin-resistant strains. E1210 was highly active against all the species tested and was more potent than all comparators. The MIC₉₀ results (μg/mL) for E1210, caspofungin, fluconazole, posaconazole, and voriconazole, respectively, were as follows by species: C. albicans (0.06, 4, ≥64, 0.5, 0.5), C. glabrata (0.06, 2, 32, 1, 1), C. parapsilosis (0.06, 4, 16, 0.12, 0.25), and C. tropicalis (0.06, 4, ≥64, 0.5, 2). E1210 was also the most active agent against fluconazole-resistant strains of C. albicans (MIC range, 0.015–0.12 μg/mL), C. glabrata (0.06 μg/mL), C. parapsilosis (MIC range, 0.06–0.05 μg/mL), and C. tropicalis (MIC range, 0.008–0.06 μg/mL), and was the most potent agent tested against caspofungin-resistant strains of C. albicans (MIC range, 0.008–0.12 μg/mL), C. glabrata (MIC range, 0.03–0.06 μg/mL), and C. tropicalis (MIC range, 0.015–0.06 μg/mL).     

Determination of susceptibilities of 26 Leptospira sp. serovars to 24 antimicrobial agents by a broth microdilution technique

The MICs of 24 antimicrobials for 26 Leptospira spp. serovars were determined using a broth microdilution technique. The MICs at which 90% of isolates tested were inhibited (MIC(90)s) of cefepime, imipenem-cilastatin, erythromycin, clarithromycin, and telithromycin were all /=3.13 microg/ml. Many antimicrobials have excellent in vitro activity against Leptospira.     

Plants as sources of antimalarial drugs: in vitro antimalarial activities of some quassinoids.

Fourteen quassinoids, obtained from simaroubaceous plants, were tested for in vitro antimalarial activity. All of these inhibited the incorporation of [3H]hypoxanthine into Plasmodium falciparum in vitro at concentrations below 0.41 microgram ml-1. The two most potent quassinoids, bruceantin and simalikalactone D, showed 50% inhibitory concentration values of 0.0008 and 0.0009 microgram ml-1, respectively. The results are compared with the antiamoebic, antileukemic, and cytotoxic activities of these compounds reported in the literature.     

In vitro activity of a novel broad-spectrum antifungal, E1210, tested against Aspergillus spp. determined by CLSI and EUCAST broth microdilution methods

E1210 is a first-in-class broad-spectrum antifungal that suppresses hyphal growth by inhibiting fungal glycophosphatidylinositol (GPI) biosynthesis. In the present study, we extend these findings by examining the activity of E1210 and comparator antifungal agents against Aspergillus spp. by using the methods of the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) to test wild-type (WT) as well as amphotericin B (AMB)-resistant (-R) and azole-R strains (as determined by CLSI methods). Seventy-eight clinical isolates of Aspergillus were tested including 20 isolates of Aspergillus flavus species complex (SC), 22 of A. fumigatus SC, 13 of A. niger SC, and 23 of A. terreus SC. The collection included 15 AMB-R (MIC, ≥ 2 μg/ml) isolates of A. terreus SC and 10 itraconazole-R (MIC, ≥ 4 μg/ml) isolates of A. fumigatus SC (7 isolates), A. niger SC (2 isolates), and A. terreus SC (1 isolate). Comparator antifungal agents included anidulafungin, caspofungin, amphotericin B, itraconazole, posaconzole, and voriconazole. Both CLSI and EUCAST methods were highly concordant for E1210 and all comparators. The essential agreement (EA; ± 2 log(2) dilution steps) was 100% for all comparisons with the exception of posaconazole versus A. terreus SC (EA = 91.3%). The minimum effective concentration (MEC)/MIC(90) values (μg/ml) for E1210, anidulafungin, caspofungin, itraconazole, posaconazole, and voriconazole, respectively, were as follows for each species: for A. flavus SC, 0.03, ≤ 0.008, 0.12, 1, 1, and 1; for A. fumigatus SC, 0.06, 0.015, 0.12, >8, 1, and 4; for A. niger SC, 0.015, 0.03, 0.12, 4, 1, and 2; and for A. terreus SC, 0.06, 0.015, 0.12, 1, 0.5, and 1. E1210 was very active against AMB-R strains of A. terreus SC (MEC range, 0.015 to 0.06 μg/ml) and itraconazole-R strains of A. fumigatus SC (MEC range, 0.03 to 0.12 μg/ml), A. niger SC (MEC, 0.008 μg/ml), and A. terreus SC (MEC, 0.015 μg/ml). In conclusion, E1210 was a very potent and broad-spectrum antifungal agent regardless of in vitro method applied, with excellent activity against AMB-R and itraconazole-R strains of Aspergillus spp.     

Potent in vitro antimalarial activity of metacycloprodigiosin isolated from Streptomyces spectabilis BCC 4785

Bioassay-guided fractionation of the extract from the fermentation broth of Streptomyces spectabilis BCC 4785 led to the isolation of three principle antimalarial agents, metacycloprodigiosin, bafilomycin A(1), and spectinabilin. Metacycloprodigiosin exhibited potent in vitro activity against Plasmodium falciparum K1, with a 50% inhibitory concentration of 0.0050 +/- 0.0010 microg/ml, while its cytotoxicity was much weaker.     

Multiple-dose kinetics in healthy volunteers and in vitro antimalarial activity of proguanil plus dapsone

The multiple-dose kinetics of a daily dose of proguanil (200 mg) coadministered with dapsone (10 mg) was investigated in 6 healthy adult male volunteers. The kinetics of dapsone (DDS), monoacetyldapsone (MADDS), proguanil (PROG) and its active metabolite cycloguanil (CYCLO) were derived from plasma drug concentrations after the last maintenance dose. The following kinetic parameters (mean values) were estimated for DDS and PROG, respectively: maximum concentration (Cmax) = 285 and 151 ng/ml, minimum concentration (Cmin) = 125 and 31 ng/ml, elimination half-life (t1/2) = 23.3 and 18.3 h, plasma clearance (Cl) = 0.032 and 1.27 l/h/kg and apparent volume of distribution (Vss) = 1.05 and 33.32 l/kg. The Cmax, Cmin and t1/2 of CYCLO were 56 ng/ml, 17 ng/ml and 15.0 h, respectively. The antimalarial activity of the proguanil/dapsone combination was assessed in vitro by measuring the inhibition of re-invasion of two Plasmodium falciparum isolates grown in the presence of volunteers' sera. Both FC-27 [chloroquine (CQ)- and pyrimethamine (PYR)-sensitive] and K1 (CQ- and PYR-resistant) isolates were completely inhibited by the drug combination at steady-state concentrations. These findings suggest that the drug regimen may be effective against drug-resistant falciparum malaria.     

Nonisotopic, semiautomated plasmodium falciparum bioassay for measurement of antimalarial drug levels in serum or plasma

A simple, nonisotopic, semiautomated bioassay for the measurement of antimalarial drug levels in plasma or serum based on the quantitation of histidine-rich protein II in malaria culture is presented. The assay requires only small sample volumes and was found to be highly sensitive and reproducible. The results closely paralleled those obtained with isotopic bioassays (R = 0.988, P < 0.001) and high-performance liquid chromatography-electrochemical detection (R = 0.978, P < 0.001).     

Interactions of DB75, a novel antimalarial agent, with other antimalarial drugs in vitro

Pafuramidine is a novel orally active antimalarial. To identify a combination partner, we measured the in vitro antimalarial activities of the active metabolite, DB75, with amodiaquine, artemisinin, atovaquone, azithromycin, chloroquine, clindamycin, mefloquine, piperaquine, pyronaridine, tafenoquine, and tetracycline. None of the drugs tested demonstrated antagonistic or synergistic activity in combination with pafuramidine.     

Iron chelators as antimalarial agents: in vitro activity of dicatecholate against Plasmodium falciparum

The present study was undertaken to explore the antimalarial effect of a series of dicatecholate iron chelators. They may be made more or less lipophilic by increasing or reducing the length of the R substituent on the nitrogen. In vitro activity against the W2 and 3D7 clones of Plasmodium falciparum, toxicity on Vero cells and toxicity on uninfected erythrocytes by measure of the released haemoglobin were assessed for each compound. These findings were compared with the ability of iron(III), iron(II) and ferritin to reverse the inhibitory effect of catecholates. This study shows that increased lipid solubility of catecholate iron chelators does not lead to improved antimalarial activity. However, their activity is well correlated with their interaction with iron and with their toxicity against Vero cells. This study demonstrates a potent antimalarial effect of FR160 (R = C9H19) on five different strains of P. falciparum in vitro. FR160 inhibited parasite growth with an IC50 between 0.8 and 1.5 micro M. The effects of FR160 on mammalian cells were minimal compared with those obtained with malaria parasites. FR160 acted on parasites at considerably higher rates than desferrioxamine, and at all stages of parasite growth. The drug was more effective at the late trophozoite and young schizont stages, although FR160 affected rings and schizonts as well. Ascorbic acid, a free radical scavenger, reduced the activities of FR160 and artesunate. FR160 might induce formation of free radicals, which could explain why FR160 antagonized the effects of artesunate and dihydroartemisinin.     

Desbutyl-lumefantrine is a metabolite of lumefantrine with potent in vitro antimalarial activity that may influence artemether-lumefantrine treatment outcome

Desbutyl-lumefantrine (DBL) is a metabolite of lumefantrine. Preliminary data from Plasmodium falciparum field isolates show greater antimalarial potency than, and synergy with, the parent compound and synergy with artemisinin. In the present study, the in vitro activity and interactions of DBL were assessed from tritium-labeled hypoxanthine uptake in cultures of the laboratory-adapted strains 3D7 (chloroquine sensitive) and W2mef (chloroquine resistant). The geometric mean 50% inhibitory concentrations (IC(50)s) for DBL against 3D7 and W2mef were 9.0 nM (95% confidence interval, 5.7 to 14.4 nM) and 9.5 nM (95% confidence interval, 7.5 to 11.9 nM), respectively, and those for lumefantrine were 65.2 nM (95% confidence interval, 42.3 to 100.8 nM) and 55.5 nM (95% confidence interval, 40.6 to 75.7 nM), respectively. An isobolographic analysis of DBL and lumefantrine combinations showed no interaction in either laboratory-adapted strain but mild synergy between DBL and dihydroartemisinin (sums of the fractional inhibitory concentrations of 0.92 [95% confidence interval, 0.87 to 0.98] and 0.94 [95% confidence interval, 0.90 to 0.99] for 3D7 and W2mef, respectively). Using a validated ultra-high-performance liquid chromatography-tandem mass spectrometry assay and 94 day 7 samples from a previously reported intervention trial, the mean plasma DBL was 31.9 nM (range, 1.3 to 123.1 nM). Mean plasma DBL concentrations were lower in children who failed artemether-lumefantrine treatment than in those with an adequate clinical and parasitological response (ACPR) (P = 0.053 versus P > 0.22 for plasma lumefantrine and the plasma lumefantrine-to-DBL ratio, respectively). DBL is more potent than the parent compound and mildly synergistic with dihydroartemisinin. These properties and the relationship between day 7 plasma concentrations and the ACPR suggest that it could be a useful alternative to lumefantrine as a part of artemisinin combination therapy.     

Chemical Determinants of antimalarial activity of reversed siderophores.

Reversed siderophores (RSFs) are artificial hydroxamate-based iron chelators designed after the natural siderophore ferrichrome. The modular molecular design of RSF derivatives allowed the synthesis of various congeners with controlled iron-binding capacities and partition coefficients. These two physicochemical properties were assessed by a novel fluorescent method and were found to be the major determinants of RSF permeation across erythrocyte membranes and scavenging of compartmentalized iron. The partition coefficient apparently conferred upon RSFs two major features: (i) the ability to rapidly access iron pools of in vitro-grown Plasmodium falciparum at all developmental stages and to mobilize intracellular iron and transfer it to the medium and (ii) the ability to suppress parasite growth at all developmental stages. These features of RSFs were assessed by quantitative determination of the structure-activity relationships of the biological activities and partition coefficients spanning a wide range of values. The most effective RSF containing the aromatic group of phenylalanine (RSFm2phe) showed 50% inhibitory concentration of 0.60 +/- 0.03 nmol/ml in a 48-h test and a 2-h onset of inhibition of ring development at 5 nmol/ml. The lipophilic compound RSFm2phe and the lipophilic and esterase-cleavable compound RSFm2pee inhibited parasite growth at all developmental stages whether inhibition was assessed in a continuous mode or after discontinuing drug administration. The antimalarial effects of RSFm2phe and cleavable RSFm2pee were potentiated in the presence of desferrioxamine (DFO) at concentrations at which DFO alone had no effect on parasite growth. These studies provide experimental evidence indicating that the effective and persistent antimalarial actions of RSFs are associated with drug access to infected cells and scavenging of iron from intracellular parasites. Moreover, the optimal antimalarial actions of RSFs are apparently also determined by improved accessibility to critical iron pools or by specific interactions with critical parasite targets.     

超声造影伽马拟合分析技术对肝癌微循环血流灌注的定量研究

目的探讨新型超声造影定量分析软件SW-UCS-1评价原发性肝癌微循环灌注的应用价值。方法选择由手术病理证实的原发性肝癌患者35例,于术前一周内进行超声造影检查。使用具有伽马拟合分析技术的超声造影分析软件对动态造影数据进行脱机分析,并引入拟合优度检验指数（GOF）来评价拟合曲线的可靠性。分别选择肿瘤区、肝实质浅部和深部3个感兴趣区（ROI）绘制时间-强度曲线,并得出各项定量参数：强度增量（a1）、下降曲率（a2）、上升支斜率（a3）、峰值强度（PI）、达峰时间（TTP）和造影剂到达时间（AT）。结果所得拟合曲线的GOF值均高于0.90。与肝实质相比,肿瘤区的时间-强度曲线上升快,下降也快,峰值强度大,达峰时间早,上述参数均有统计学差异（P〈0.05）;不同深度肝实质组间的同一定量参数,其差异有统计学意义（P〈0.05）。结论伽马拟合定量分析软件较为真实客观地反映原发性肝癌及肝实质间不同的灌注特点,为肝肿瘤的诊断和血流动力学研究提供了依据。     

Reproducibility of a semiautomated acetylene rebreathing technique for measuring cardiac output in humans at rest

The specific aims of the present study were to determine: (1) the day-to-day reproducibility of a semiautomated acetylene rebreathing technique for measuring cardiac output under resting conditions; (2) the reproducibility of this technique among subjects differing in gender and age; and (3) the number of trials within a session necessary to maximize the day-to-day reproducibility of the technique. To address these aims, cardiac output was measured in 21 healthy men (n = 8) and women (n = 13) between the ages of 25 and 71 years in the supine posture on two separate days. Mean levels of cardiac output at rest were similar on day 1 vs. day 2 in the overall group. Cardiac output measured on day 1 was highly correlated (r = 0·98, P<0·001) with cardiac output measured on day 2. The day 1 to day 2 mean difference in cardiac output for the individual subjects was <4%. The mean levels of heart rate and stroke volume also were similar between day 1 and day 2. The relation between cardiac output measured on day 1 vs. day 2 in the gender and age subgroups was similar to that observed in the overall group. The mean absolute difference among the three rebreathing trials within a day was 360 ml min^?1 in the overall group, with a coefficient of variation of 7%. The variability between rebreathing trials measured on day 1 vs. day 2 in the gender and age subgroups was similar to that observed in the overall group. The reliability of cardiac output measured on different days was excellent with a single rebreathing trial (r = 0.93) and improved significantly up to three trials (r = 0.98). In conclusion, the findings of the present study indicate that the acetylene rebreathing technique can be a highly reproducible method for measuring cardiac output under resting conditions. The reproducibility is consistently strong in healthy humans of varying age and in both genders, and is enhanced by the use of multiple trials.     

In vitro antimalarial activity of nucleic acid precursor analogues in the simian malaria Plasmodium knowlesi

Analogues of nucleic acid precursors were screened for antimalarial activity in Plasmodium knowlesi by using an in vitro culture system. Activity was assessed by the degree of inhibition of incorporation of l-[methyl-(14)C]methionine into protein and of [8-(14)C]adenosine and [6-(14)C]orotic acid into ribonucleic acid and deoxyribonucleic acid. The incorporation of adenosine or orotic acid was effectively inhibited by many of the compounds, including 3' analogues of purine nucleosides, many of the 6-position analogues of purine bases and nucleosides, and 5-position analogues of orotic acid. Only a few compounds inhibited methionine incorporation into protein, and in each instance adenosine or orotic acid incorporation also was inhibited. Some compounds inhibited adenosine or orotic acid incorporation into both ribonucleic acid and deoxyribonucleic, whereas other analogues inhibited incorporation into one nucleic acid only. The qualitative and quantitative differences suggest that this experimental system may be appropriate for investigation of metabolic pathways of the malaria parasite, as well as for demonstration of antimalarial activity of candidate antimalaria drugs.