Romark Research Laboratory, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK
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Abstract |
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Introduction |
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Following a single oral dose of [14C]nitazoxanide 500 mg in humans, 32% of the radioactivity was recovered in the urine and 66% in the faeces, indicating significant absorption from the gastrointestinal tract. The parent drug, nitazoxanide, is not found in blood, urine or faeces, but is presumably present to some extent in the contents of the intestine. The two major metabolites are produced by hydrolysis and glucurono conjugation; desacetyl-nitazoxanide (tizoxanide) is found in faeces, plasma and urine and tizoxanide glucuronide in plasma, urine and bile. The maximum concentration of tizoxanide in plasma following an oral dose of [14C]nitazoxanide 500 mg was c. 2 mg/L (6.5 µM), and its half-life in plasma was c. 12 h. Minor metabolites include salicyluric acid, tizoxanide sulphate and traces of hydroxytizoxanide in urine. Salicylate is found in faeces.8 Tizoxanide and to some extent nitazoxanide probably account for activity in the intestine, while in other locations tizoxanide may be the most important agent. However, high concentrations of tizoxanide glucuronide are excreted in bile,8 and the action of this compound may be manifested on liver trematodes such as F. hepatica.
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Materials and methods |
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Strains of G. intestinalis (VNB1, VNB2, VNB5, EBC and EBE) were isolated from patientsapos; faecal samples in London.23 The last two strains were used with the agreement of Prof. John P. Ackers [London School of Hygiene and Tropical Medicine (LSHTM)]. The strains were isolated from diagnostic faecal samples from cases of giardiasis returning from the Indian subcontinent, subsequently successfully treated with metronidazole. All of these strains were determined by us to be susceptible to metronidazole in vitro. A further isolate of G. intestinalis (JKH-1) was isolated from a chronic human infection refractory to metronidazole by Dr Tim Paget (University of Hull), and was kindly supplied by him.
Two E. histolytica isolates, HM1:IMSS (Mexico) and NIH200 (Korea), had been held in culture and cryopreservation in the LSHTM laboratory since 1976, when they were supplied to us by the late Dr R. A. Neal (Wellcome Research Laboratories, Beckenham, UK). Isolates IULA: 1092:1 and IULA:0593:2 isolated from amoebic dysentery patients in Venezuela were kindly supplied by Professor John P. Ackers (LSHTM). Other isolates were purchased from the American Type Culture Collection (ATCC). The history and relevant references for the E. histolytica strains used are in Table 1.
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The drugs used in the study were obtained as dry powders. Metronidazole was purchased from SigmaAldrich (Poole, UK), and nitazoxanide, tizoxanide, tizoxanide glucuronide, denitronitazoxanide and denitrotizoxanide were provided by Romark Laboratories, L.C. (Tampa, FL, USA).
In vitro studies
Stabilates were recovered from cryopreservation and cultured using Diamond' TYI-S-33 complete medium.34 Inactivated adult bovine serum was used at 10% and the vitaminTween 80 mixture at 3%. The complete medium was supplemented with bile 0.5 g/L (Sigma) for G. intestinalis.35 Parasites were routinely grown in 12 mL flat-sided plastic test tubes (Nunc) at 35°C. Medium was removed from cultures in the log phase of growth showing a monolayer of trophozoites (observed under the inverted microscope); they were then exposed to 10 mL fresh chilled culture medium at 0°C on an ice/water bath for 10 min and resuspended. For subculturing, 0.20.4 mL of this suspension was transferred to fresh tubes containing culture medium.
For the in vitro drug test the method described by Cedeño & Krogstad25 for E. histolytica was modified as follows. Stocks of metronidazole, dissolved in water, and tizoxanide, nitazoxanide, tizoxanide glucuronide, denitronitazoxanide and denitrotizoxanide, dissolved in DMSO, were prepared and stored at 4°C. Dilutions were made in serum-free culture medium and sterilized through a 0.2 µm filter. Final dilutions were made immediately before the test in complete medium to give twice the top concentration to be used for the in vitro testing. Two hundred microlitres of this was transferred to duplicate drug wells (B1H1, in a flat-bottomed 96-well microtitre test plate) (Corning, New York, USA). Row A, wells 712 and rows BH wells 212 received 100 µL of complete medium. Two-fold serial dilutions were made from B1H1 to B12H12. In row A, wells 112 were drug free, and 200 µL of complete culture medium was dispensed into wells 16 to serve as uninfected controls, while wells 712 were inoculated with 100 µL of parasite suspension as infected controls. Final concentrations of DMSO required in the tests for nitazoxanide and tizoxanide at IC50 values were 0.01% (v/v) or lower, and inactive in the assay systems. DMSO alone began to have inhibitory effects at 0.1% (v/v).
Parasite preparation
Healthy growing cultures in the log phase were selected for in vitro drug testing. Medium was carefully decanted, after gentle centrifugation where necessary, and replaced with fresh complete medium. Parasites were resuspended in 5 mL of medium. Twenty microlitres of suspension were fixed by adding 1 µL of 2% formalin and counts were made using a haemocytometer. The number of organisms per well varied in preliminary experiments with E. histolytica, the most difficult of the organisms to grow consistently, and we found that 50000 organisms/mL were needed in order to obtain optimal reproducibility with the range of strains tested. This is similar to the concentration of 30000/mL found to be optimal by Upcroft et al.4 for Entamoeba, and in contrast to 6000/mL as used by Cedeño & Krogstad25 in their work on strain HK9 of E. histolytica.
Dilutions were made in fresh tubes, with complete medium, to give a density of 100 000 organisms/mL and this was transferred in 100 µL volumes from a sterile vessel, frequently agitated, to each of wells 712 of row A (infected control) and to the test wells of rows B1H1 to B12H12. Each well (test and infected control) finally contained 10000 organisms in a final volume of 200 µL of drugmediumparasite mixture (50 000 organisms/mL) and this system was applied to all three parasites. The final concentration of organisms in the Giardia and Trichomonas cultures is lower than that used by Upcroft et al.4
Incubation
The culture plates were placed in an airtight modular incubator chamber (Billups-Rothenberg, CA, USA) that had been swabbed clean with 70% ethanol and humidified with damp tissue treated with cupric sulphate solution (Sigma) to avoid bacterial and fungal contamination. The chamber was gassed for 5 min with a filter-sterilized gas mixture of 3% O2, 4% CO2 and 93% N2 (a gas phase suitable for microaerophilic organisms and for aerobic tests on T. vaginalis).36 Incubation was at 35°C. After 24 h, plates were viewed on an inverted microscope to monitor parasite growth. Wells were dosed with 5 µL of methyl [3H]thymidine solution (Amersham, UK) to a final concentration of 0.2 µCi/well. Culture plates were returned to the chamber, gassed as above and incubated at 35°C for a further 24 h. Methyl-[3H]thymidine was obtained as 1 mL aqueous solution with a specific activity of 51 Ci/mmol and total activity of 1 mCi. This was transferred to a sterile Universal tube and vial rinsings added in a total volume of 25 mL serum-free culture medium (40 µCi/mL). This was filter-sterilized through a 0.2 µm filter and kept at -20°C until needed.
Assessment of drug activity
After addition of the radiolabelled thymidine and 24 h of incubation, plates were chilled at 4°C in an ice/water-bath. Parasites were harvested with a cell harvester (Skatron Inc., Liev, Norway) on to glass fibre filter paper (ICN Biomedicals Inc., CA, USA).37,38 After drying, the filter discs were punched out into scintillation vials. Three millilitres of scintillation fluid (Ecoscint; National Diagnostics, UK) were added and radioactivity counted (3 min per vial) in a Tricarb liquid scintillation spectrometer (Packard, Meriden, USA). The disintegrations per minute (dpm), representing the incorporation of methyl-[3H]thymidine by surviving parasites, were recorded. Counts from the uninfected control served as 100% inhibition and those from the infected control represented 0% inhibition. The doseresponse was analysed using a non-linear regression (Levenberg-Marquardt algorithm) (XLFit programme #1.02, an add-in for Microsoft Excel). The dpm for each well was converted to percentage inhibition, which was plotted as a function of the logarithm of drug concentration. IC50s were obtained from the sigmoid curves.
All experiments were performed at least three times in duplicate, and the mean values with standard deviations are given in Tables 36. Multiplication factors to convert mg/L to µM are: metronidazole = 5.84; tizoxanide = 3.77; nitazoxanide = 3.254; tizoxanide glucuronide = 2.266; denitronitazoxanide = 3.81; denitrotizoxanide = 4.54.
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Results |
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Six isolates of G. intestinalis (Table 3)
One clearly resistant isolate was three times less susceptible to metronidazole than the mean for the susceptible isolates, and this differential was 13 times for tizoxanide, the active metabolite of nitazoxanide. Tizoxanide was eight times as active against susceptible isolates as metronidazole and twice as active against the resistant isolate. Nitazoxanide results paralleled those of tizoxanide, but for the metabolite tizoxanide glucuronide, which was about one-third as active as metronidazole, there was no detectable difference in activity against the resistant strain (JKH-1). The activity of tizoxanide glucuronide in G. intestinalis VNB1 did not differ from that of denitrotizoxanide (Table 4). Denitrotizoxanide was 48 times less active than tizoxanide, while denitronitazoxanide was 17 times less active than nitazoxanide (see Table 5
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Ten isolates of E. histolytica (see Table 4)
The mean metronidazole IC50 value for the most susceptible isolates of E. histolytica was 18.47 µM, and >30 µM was chosen as the cut-off for resistance. On the basis of literature values this was set at about three times the value for the most susceptible strain.31 Susceptibility to metronidazole showed a seven-fold range from 9.5 to 65.9 µM. On a molar basis, tizoxanide was 1.42 times as active against susceptible isolates as metronidazole, but was 2.44 times as active as that drug against the resistant isolates. Nitazoxanide results paralleled those of tizoxanide, but for tizoxanide glucuronide, which was about half as active as metronidazole on susceptible isolates, there was no detectable difference in activity against the resistant strains. Activity of tizoxanide glucuronide against E. histolytica HMI:IMSS was only marginally higher than that of denitrotizoxanide. Denitrotizoxanide was more than four times less active than tizoxanide, while denitronitazoxanide was 1.9 times less active than nitazoxanide (see Table 5).
Seventeen isolates of T. vaginalis (see Table 6)
The isolates of T. vaginalis used showed a 10-fold range of susceptibility to metronidazole from 0.6 to 60 µM. The cut-off for resistance in our study, following the susceptibility designations of the standard strains, was taken as an IC50 > 20 µM. These strains were still susceptible to tizoxanide. Against metronidazole-resistant isolates, tizoxanide was 4.88 times as active as metronidazole, and 1.39 times as active against susceptible isolates. Nitazoxanide results paralleled those of tizoxanide, but for tizoxanide glucuronide, which was about 0.33 times as active as metronidazole against the susceptible isolates, activity against the resistant strains was 1.93 times higher than metronidazole. Denitrotizoxanide was five times less active against susceptible T. vaginalis PER 014/CGF than tizoxanide, while denitronitazoxanide was 10 times less active than nitazoxanide (Table 4). Nitazoxanide results paralleled those of tizoxanide.
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Discussion |
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In G. intestinalis, tizoxanide was eight times more active than metronidazole against metronidazole-susceptible isolates and twice as active against a resistant isolate. In 10 axenic isolates of E. histolytica tested in the same way, tizoxanide was almost twice as active as metronidazole against more susceptible isolates and was more than twice as active against less susceptible isolates.
In T. vaginalis tizoxanide was 1.5 times as active as metronidazole against susceptible isolates but was nearly five times as active against resistant isolates.
In G. intestinalis, E. histolytica and T. vaginalis the glucuronide metabolite of nitazoxanide was 0.3, 0.63 and 0.33 times as active, respectively, as metronidazole in susceptible strains, and was 1.1, 1.59 and 1.9 times as active as metronidazole against resistant strains. The activity of tizoxanide glucuronide is appreciably less than that of tizoxanide and nitazoxanide, and little difference could be found between the resistant and the susceptible strains (Table 5).
This suggests that the glucuronide may not readily enter the cell to be activated by intracellular reduction. The reductions in tizoxanide activity brought about by removal of the nitro-group and by glucuronidation were remarkably similar within species (Table 5). The measured IC50 concentrations across the species (for denitrotizoxanide, 29.0, 37.0, 32.6 µM; for tizoxanide glucuronide, 33.2, 25.9, 19.6 µM) are remarkably similar considering the differing IC50 values for the parent tizoxanide. These observations support the idea that the glucuronide metabolite may not undergo nitro-reduction in these organisms and indicates that intracellular nitro-group reduction is necessary for activity of tizoxanide. The removal of the nitro-group renders reductive activation impossible, but may also have an impact on other properties of the drug, for example the lipophilic character is increased, and the yellow colour is lost, the latter feature indicating a decrease in the degree of conjugation of the ring system. It is however probable that, as with the 5-nitroimidazoles, the reducibility of the nitro-group is the most important feature involved.40
Nitazoxanide is active against a wider range of organisms than metronidazole. In addition, metronidazole-resistant strains of H. pylori were susceptible.16 Since resistance to metronidazole in G. intestinalis and T. vaginalis apparently depends on decreased ability to activate the drug,41 evidence of some cross-resistance and relative inactivity of denitro-derivatives presented in this paper helps to confirm the probable mode of action. Higher activity than metronidazole seen for nitazoxanide and tizoxanide in relatively metronidazole-refractory strains of all the three organisms studied indicates that resistance mechanisms to metronidazole may be to a variable extent bypassed by nitazoxanide and tizoxanide. This feature may relate to retention of the ability of the metronidazole-resistant organisms to reduce the drug at a less negative redox potential than that required for metronidazole. The redox potential reported for one electron reduction of another 5-nitrothiazole was -390 mV, much less negative than -486 mV seen in the 5-nitroimidazole group of metronidazole.42 The data we have so far on resistant strains are insufficient to draw firm conclusions on the mechanisms of resistance and need further expansion.
Although the effect of tizoxanide and related agents on C. parvum originally seemed to be anomalous, there is now evidence for an unusual cytosolic pyruvate oxidoreductase resembling pyruvate ferredoxin oxidoreductase,43 which may well be involved in tizoxanide nitro-reduction.
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Acknowledgements |
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Notes |
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References |
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2 . Dooley, C. P. & Oapos;Morain, C. A. (1988). Recurrence of hepatic amebiasis after successful treatment with metronidazole. Journal of Clinical Gastroenterology 10, 33942. [ISI][Medline]
3 . Ellis, J. E., Wingfield, J. M., Cole, D., Boreham, P. F. & Lloyd, D. (1993). Oxygen affinities of metronidazole-resistant and -sensitive stocks of Giardia intestinalis. International Journal of Parasitology 23, 359. [ISI][Medline]
4
.
Upcroft, J. A., Campbell, R. W., Benakli, K., Upcroft, P. & Vanelle, P. (1999). Efficacy of new 5-nitroimidazoles against metronidazole-susceptible and -resistant Giardia, Trichomonas, and Entamoeba spp. Antimicrobial Agents and Chemotherapy 43, 736.
5 . Sorvillo, F. & Kerndt, P. (1998). Trichomonas vaginalis and amplification of HIV-1 transmission. Lancet 351, 2134. [ISI][Medline]
6 . Rossignol, J. F. & Cavier, R. (1975). New derivative of 2-benzamido-5-nitro thiazoles. Chemical Abstracts 83, 28216.
7 . Rossignol, J. F. & Stachulski, A. V. (1999). Synthesis and antibacterial activities of tizoxanide, a N-nitrothiazolyl salicylamide, and its O-aryl glucuronide. Journal of Chemical Research S, 445.
8 . Broeckhuysen, J., Stockis, A., Lins, R. L., de Graeve, J. & Rossignol, J. F. (2000). Nitazoxanide: pharmacokinetics and metabolism in man. International Journal of Clinical Pharmacology and Therapeutics 38, 38794. [ISI][Medline]
9
.
Theodos, C. M., Griffiths, J. K., Dapos;Onfro, J., Fairfield, A. & Tzipori, S. (1998). The efficacy of nitazoxanide against Cryptosporidium parvum in cell culture and in animal models. Antimicrobial Agents and Chemotherapy 42, 195965.
10
.
Blagburn, B. L., Drain, K. L., Land, T. M., Kinard, R. G., Moore, P. H., Lindsay, D. S. et al. (1998). Comparative efficacy evaluation of dicationic carbazole compounds, nitazoxanide and paromomycin against Cryptosporidium parvum infections in a neonatal mouse model. Antimicrobial Agents and Chemotherapy 42, 287782.
11
.
Gargala, G., Delaunay, A., Li, X., Brasseur, P., Favennec, L. & Ballet, J. J. (2000). Efficacy of nitazoxanide, tizoxanide and tizoxanide glucuronide against Cryptosporidium parvum development in sporozoite-infected HCT-8 enterocytic cells. Journal of Antimicrobial Chemotherapy 46, 5760.
12 . Didier, E., Maddry, J., Kwong, C., Green, L., Snowden, K. & Shadduck, J. (1998). Screening of compounds for antimicrosporidial activity in vitro. Folia Parasitologica 45, 12939. [ISI][Medline]
13 . Cavier, R. & Rossignol, J.-F. (1982). Etude de diverses associations dapos;anthelminthiques chez la souris. Revue de Medicine Veterinaire 133, 77983.
14 . Euzeby, J., Prom Tep, S. & Rossignol, J. F. (1980). Experimentation des proprietes anthelminthiques de la nitazoxanide chez le chien, le chat, et les ovins. Revue de Medicine Veterinaire 131, 68796.
15 . Dubreuil, L., Houcke, I., Mouton, Y. & Rossignol, J. F. (1996). In vitro evaluation of activities of nitazoxanide and tizoxanide against anaerobes and aerobic organisms. Antimicrobial Agents and Chemotherapy 40, 226670. [Abstract]
16
.
Mégraud, F., Occhialini, A. & Rossignol, J. F. (1998). Nitazoxanide, a potential drug to eradicate Helicobacter pylori with no cross resistance to metronidazole. Antimicrobial Agents and Chemotherapy 42, 283640.
17 . Doumbo, O., Rossignol, J.-F., Prichard, E., Traore, H., Dembele, M., Diakite, M. et al. (1997). Nitazoxanide in the treatment of cryptosporidial diarrhoea and other intestinal parasitic infections associated with acquired immunodeficiency syndrome in tropical Africa. American Journal of Tropical Medicine and Hygiene 56, 6379. [ISI][Medline]
18 . Rossignol, J. F., Ayoub, A. & Ayers, M. S. (2001). Treatment of diarrhea caused by Cryptosporidium parvum: a prospective randomized, double-blind, placebo-controlled study of nitazoxanide. Journal of Infectious Diseases 184, 1036. [ISI][Medline]
19
.
Bicart-See, A., Massip, P., Linas, M. D. & Datry, A. (2000). Successful treatment with nitazoxanide of Enterocytozoon bieneusi microsporidiosis in a patient with AIDS. Antimicrobial Agents and Chemotherapy 44, 1678.
20 . Romero Cabello, R., Guerrero, L. R., Munoz Garcia, M. R. & Geyne Cruz, A. (1997). Nitazoxanide for the treatment of intestinal protozoan and helminthic infections in Mexico. Transactions of the Royal Society of Tropical Medicine and Hygiene 91, 7013. [ISI][Medline]
21 . Rossignol, J. F. & Maisonneuve, H. (1984). Nitazoxanide in the treatment of Taenia saginata and Hymenolepis nana. American Journal of Tropical Medicine and Hygiene 33, 5112. [ISI][Medline]
22 . Rossignol, J. F., Abaza, H. & Friedman, H. (1998). Successful treatment of heavily infected human fascioliasis with nitazoxanide: a case report. Transactions of the Royal Society of Tropical Medicine and Hygiene 92, 1034. [ISI][Medline]
23 . Bhatia, V. N. & Warhurst, D. C. (1981). Hatching and subsequent cultivation of cysts of Giardia intestinalis in Diamond' medium. Journal of Tropical Medicine and Hygiene 84, 45. [ISI][Medline]
24 . Deschiens, R. (1937). Considerations sur lapos;action pathogene dapos;une souche dapos;amibes dysenterique (Souche C. Dobell DKB). Bulletine de la Societe de Pathologie Exotique 30, 5624.
25 . Cedeño, J. R. & Krogstad, D. J. (1983). Susceptibility testing of Entamoeba histolytica. Journal of Infectious Diseases 148, 10905. [ISI][Medline]
26 . Diamond, L. S., Mattern, C. F. & Bartgis, I. L. (1972). Viruses of Entamoeba histolytica: identification of transmissible virus-like agents. Journal of Virology 9, 32641. [ISI][Medline]
27 . Clark, C. G. & Diamond, L. S. (1997). Intraspecific variation and phylogenetic relationships in the genus Entamoeba as revealed by riboprinting. Journal of Eukaryotic Microbiology 44, 14254. [ISI][Medline]
28 . Urdaneta, H., Rondon, M., Munoz, M. & Hernandez, M. (1995). Isolation and axenization of two Entamoeba histolytica strains. G E N 49, 238. [Medline]
29 . Sargeaunt, P. G., Williams, J. E. & Neal, R. A. (1980). A comparative study of Entamoeba histolytica (NIH 200, HK9 etc) E. histolytica-likeapos; and other morphologically identical amoebae using isoenzyme electrophoresis. Transactions of the Royal Society of Tropical Medicine and Hygiene 74, 46974. [ISI][Medline]
30 . Diamond, L. S., Phillips, B. P. & Bartgis, I. L. (1974). A comparison of the virulence of nine strains of axenically cultivated E. histolytica in the hamster liver. Archivos de Investigaciones Medica (Mexico) 5, Suppl. 2, 4238.
31 . Aguirre-Cruz, M. L., Valadez-Salazar, A. & Munoz, O. (1990). In vitro sensitivity of Entamoeba histolytica to metronidazole. Archivos de Investigaciones Medica (Mexico) 21, Suppl. 1, 236.
32 . Wright, C. W., Oapos;Neill, M. J., Phillipson, J. D. & Warhurst, D. C. (1988). Use of microdilution to assess in vitro antiamoebic activities of Brucea javanica fruits, Simarouba amara stem, and a number of quassinoids. Antimicrobial Agents and Chemotherapy 32, 17259. [ISI][Medline]
33 . Müller, M., Lossick, J. G. & Gorrell, T. E. (1988). In vitro susceptibility of Trichomonas vaginalis to metronidazole and treatment outcome in vaginal trichomoniasis. Sexually Transmitted Diseases 15, 1724. [ISI][Medline]
34 . Diamond, L. S., Harlow, D. R. & Cunnick, A. C. (1978). A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Transactions of the Royal Society of Tropical Medicine and Hygiene 72, 4312. [ISI][Medline]
35 . Keister, D. B. (1983). Axenic culture of Giardia lamblia in TYIS-33 medium supplemented with bile. Transactions of the Royal Society of Tropical Medicine and Hygiene 77, 4878. [ISI][Medline]
36
.
Meri, T., Jokiranta, T. S., Suhonen, L. & Meri, S. (2000). Resistance of Trichomonas vaginalis to metronidazole: report of the first three cases from Finland and optimization of in vitro susceptibility testing under various oxygen concentrations. Journal of Clinical Microbiology 38, 7637.
37 . Ekong, R. M., Kirby, G. C., Patel, G., Phillipson, J. D. & Warhurst, D. C. (1990). Comparison of the in vitro activities of quassinoids with activity against Plasmodium falciparum, anisomycin and some other inhibitors of eukaryotic protein synthesis. Biochemical Pharmacology 40, 297301. [ISI][Medline]
38 . Oapos;Neill, M. J., Bray, D. H., Boardman, P., Phillipson, J. D. & Warhurst, D. C. (1985). Plants as sources of antimalarial drugs. Part 1. In vitro test method for the evaluation of crude extracts from plants. Planta Medica 1985, 3948.
39
.
Upcroft, P. & Upcroft, J. A. (2001). Drug susceptibility testing of anaerobic protozoa. Antimicrobial Agents and Chemotherapy 45, 18104.
40 . Edwards, D. I. (1993). Nitroimidazole drugsaction and resistance mechanisms. I. Mechanisms of action. Journal of Antimicrobial Chemotherapy 31, 920. [ISI][Medline]
41
.
Upcroft, P. & Upcroft, J. A. (2001). Drug targets and mechanisms of resistance in the anaerobic protozoa. Clinical Microbiology Reviews 14, 15064.
42 . Wardman, P. (1985). Some reactions and properties of nitro radical-anions important in biology and medicine. Environmental Health Perspectives 64, 30920. [ISI][Medline]
43
.
Rotte, C., Stejskal, F., Zhu, G., Keithly, J. S. & Martin, W. (2001). Pyruvate:NADP oxidoreductase from the mitochondrion of Euglena gracilis and from the Apicomplexan Cryptosporidium parvum: a biochemical relic linking pyruvate metabolism in mitochondriate and amitochondriate protists. Molecular Biology and Evolution 18, 71020.
Received 25 January 2001; returned 19 April 2001; revised 21 August 2001; accepted 24 September 2001