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Antimalarial activity of azithromycin, artemisinin and dihydroartemisinin in fresh isolates of Plasmodium falciparum in Thailand
Acta Tropica 80 (2001) 39 – 44 www.parasitology-online.com
Antimalarial activity of azithromycin, artemisinin and dihydroartemisinin in fresh isolates of Plasmodium falciparum in Thailand H. Noedl a,b,*, W.H. Wernsdorfer a,b, S. Krudsood b, P. Wilairatana b, H. Kollaritsch a, G. Wiedermann a, S. Looareesuwan b a
Department of Specific Prophylaxis and Tropical Medicine, Institute of Pathophysiology, Uni6ersity of Vienna, Kinderspitalgasse 15, A-1095, Vienna, Austria b Faculty of Tropical Medicine, Bangkok Hospital for Tropical Diseases, Mahidol Uni6ersity, 420 /6 Raj6ithi Road, Bangkok 10400, Thailand Received 29 January 2001; received in revised form 28 March 2001; accepted 17 April 2001
Abstract Antibiotics with antimalarial activity may offer an interesting alternative for the treatment of multidrug-resistant falciparum malaria. Azithromycin, a relatively recent semisynthetic derivative of erythromycin, was tested for its in vitro activity against fresh isolates of Plasmodium falciparum. As the reportedly slow onset of action of azithromycin suggests its combination with fast-acting substances, such as artemisinin-derivatives, dihydroartemisinin (DHA) was tested parallel as a possible combination partner. The effective concentrations found for azithromycin in this study (EC50 =29.3 mmol/l, EC90 =77.1 mmol/l blood medium mixture (BMM)) are comparable to those of other antimalarials in the antibiotics class and are considerably higher than those found for mefloquine or quinine. The absence of an activity correlation between azithromycin and chloroquine, quinine and artemisinin emphasises the independence of azithromycin drug response from the sensitivity to these drugs. A weak activity correlation (zEC90 = 0.352; p= 0.028), which could point to a potential cross-sensitivity but is probably of little clinical importance, was found with mefloquine above the EC50 level. Provided that further clinical trials support the combination of these drugs, DHA may offer an interesting combination partner for azithromycin owing to its rapid onset of action and the comparatively low effective concentrations (EC50 =1.65 nmol/l, EC90 = 7.10 nmol/l BMM). This combination may serve as an interesting alternative for tetracycline and doxycycline, which cannot be used in pregnant women and children, and exhibit phototoxicity. Nevertheless, the relatively high cost of this combination, as well as the controversial reports of the clinical efficacy, may limit the usefulness of azithromycin in malaria therapy and require an adjustment of previously used treatment regimens. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Azithromycin; Dihydroartemisinin; Macrolide antibiotics; Malaria; Plasmodium falciparum; Drug sensitivity; Resistance
* Corresponding author. Fax: + 43-1-403834390. E-mail address: [email protected] (H. Noedl). 0001-706X/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 0 1 ) 0 0 1 4 1 - 3
H. Noedl et al. / Acta Tropica 80 (2001) 39–44
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1. Introduction The spread of multidrug-resistant Plasmodium falciparum in recent years has intensified the search for new antimalarials and antibiotics with antimalarial activity. Among the macrolide antibiotics, erythromycin was the first to be used in the treatment and prophylaxis of malaria. Azithromycin, a relatively recent semisynthetic derivative of erythromycin, differs structurally from erythromycin by the addition of a methylsubstituted nitrogen atom in the lactone ring. This modification improves acid stability and tissue penetration capabilities and also broadens the antibacterial spectrum. It is concentrated intracellularly and in tissues and has a serum half-life of about 2.4 days (Foulds et al., 1990). Its antimalarial and prophylactic activities, however, have only recently been described. In vitro tests with single laboratory-adapted strains of P. falciparum indicate considerably higher effective concentrations for chloroquine-resistant compared with chloroquine-sensitive isolates (Gingras and Jensen, 1992). Recent clinical trials also confirm varying degrees of prophylactic and blood schizontocidal activity (Na-Bangchang et al., 1996; Anderson et al., 1995; Taylor et al., 1999). Its reportedly slow onset of action suggests its combination with fast-acting substances such as artemisinin derivatives. Owing to its excellent activity against P. falciparum, as well as its pharmacological properties, dihydroartemisinin (DHA), the active metabolite of all artemisininderivatives, could be a suitable combination partner for azithromycin. The aim of the study was the establishment of a baseline for the in vitro activity of azithromycin against fresh isolates of P. falciparum in an area with a high prevalence of multidrug resistance, the evaluation of the suitability of DHA as a possible combination partner, and the determination of the relation of azithromycin sensitivity to other currently used antimalarials.
2. Material and methods The study was designed as an experimental
laboratory investigation conducted at the Bangkok Hospital for Tropical Diseases (Faculty of Tropical Medicine, Mahidol University) from October 1999 until March 2000. Written informed consent was obtained from all adult participants or from parents or legal guardians of minors. The study protocols were approved by the ethical review board of the Faculty of Tropical Medicine, Mahidol University. A total number of 39 fresh isolates of P. falciparum obtained from patients with microscopically confirmed P. falciparum mono-infections, primarily from the western border regions of Thailand, were successfully tested for their in vitro susceptibility to azithromycin and DHA. Parallel tests were performed with chloroquine, mefloquine, quinine and artemisinin to obtain data for correlation analysis. The in vitro tests for the measurement of the drug sensitivity of P. falciparum followed the WHO standard methodology for the assessment of the inhibition of schizont maturation (WHO, 1990). Microtitre plates were pre-dosed at the Department of Specific Prophylaxis and Tropical Medicine, University of Vienna, with ascending concentrations of azithromycin (0.1–100 mmol/l blood medium mixture (BMM)), DHA (0.3– 300 nmol/l BMM), and artemisinin (3–3000 nmol/l BMM). Distilled water was used as solvent for azithromycin, whereas artemisinin and DHA were dissolved in 50% ethanol, 25% Tween 80 and 25% linoleic acid and further diluted with distilled water before being dispensed into 96-well microtitre plates. Mefloquine (800–51 200 nmol/l blood), quinine (80–5120 nmol/l BMM), and chloroquine (400– 25 600 nmol/l blood) plates were obtained from the WHO, Regional Office for the Western Pacific, Manila, Philippines. Whole blood was collected from every patient by venepuncture and added to RPMI 1640 medium in a 5% dilution, and pre-culture parasitaemia was assessed. Fifty microliters of BMM were added to each well of the culture plates and incubated in candle jars at 37.5 °C for 24 h. The culture results were evaluated microscopically by counting the number of schizonts in each well relative to the control well. In the course of the mathematical analysis of drug response the drug concentrations were log-
H. Noedl et al. / Acta Tropica 80 (2001) 39–44
transformed and the microscopically determined inhibition expressed in probits (Litchfield and Wilcoxon, 1949). A quadratic regression model was found to be best suited for the inhibition characteristics of azithromycin and applied to the transformed data to derive effective concentrations and the minimum inhibitory concentration. Analysis of DHA and artemisinin data was done by standard log-probit analysis. Spearman correlation analysis was employed to determine the activity correlations (z) at a significance level of h =5% (PB 0.05). Two-tailed P-values were used throughout the analyses.
3. Results
3.1. Azithromycin The geometric mean parasite density of the successfully tested blood samples was 18 876 asexual parasites per microlitre blood, and the geometric mean schizont count in the control wells was 58 schizonts per 200 parasites. None of the isolates showed full inhibition at concentrations below 30 mmol/l BMM, and only three (7.7%) isolates were fully inhibited at this concentration. The majority (79.5%), however, had their cut-off point at 100 mmol/l, and five (12.8%) isolates did not show full inhibition even at the highest concentration (100 mmol/l). The overall mean 50% effective concentration (EC50) for all 39 isolates was 29.3 mmol/l (95% confidence intervals (CIs): 24.5 and 35.2 mmol/l), and the corresponding EC90, EC95 and EC99 values were 77.1 mmol/l (95% CIs: 60.6 and 98.0 mmol/l), 98.1 mmol/l (95% CIs: 74.1 and 130.0 mmol/l) and 150.2 mmol/l (95% CIs: 143.0 and 157.7 mmol/l) respectively (Fig. 1). With a value of 5.7, the 2 for the heterogeneity of the quadratic regression (r =0.982) was well within permissible limits. Even though the EC50 and EC90 values for chloroquine covered a wide range, all isolates were considered chloroquine resistant following the WHO criteria. In correlation analysis no activity relation was found between the individual ECs of azithromycin and chloroquine,
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azithromycin and artemisinin and azithromycin and quinine (P\ 0.05). The only significant correlation was found with mefloquine above the EC50 (zEC90 = 0.352; P= 0.028; zEC99 = 0.329; P= 0.041) level (Table 1).
3.2. DHA All isolates were fully inhibited by DHA concentrations of 100 nmol/l BMM. The majority (69%) of the samples had their cut-off point at or below 10 nmol/l. The EC50 for all 39 successfully tested isolates was 1.65 nmol/l (95% CIs: 1.16 and 2.35 nmol/l), and the corresponding EC90, EC95 and EC99 values were 7.10 nmol/l (95% CIs: 4.46 and 11.29 nmol/l), 10.74 nmol/l (95% CIs: 6.39 and 18.04 nmol/l) and 23.33 nmol/l (95% CIs: 12.23 and 44.50 nmol/l) respectively. No association was found between the effective concentrations of DHA and azithromycin (P\0.05). There was an extremely close correlation of DHA with artemisinin (PB 0.001), and both drugs showed almost identical relations to mefloquine, quinine and chloroquine (Table 1). Effective concentrations for artemisinin, mefloquine, quinine, and chloroquine are shown in Table 2.
Fig. 1. The log-dose/probit-response quadratic regression of the in vitro drug response of P. falciparum to azithromycin with data points and 95% CIs (azithromycin drug concentration in mM; SMI: schizont maturation inhibition).
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Table 1 In vitro activity correlations between azithromycin (AZI), DHA and other antimalarials at EC50, EC90 and EC99 levels (z: Spearman’s rho, correlation coefficient; P: two-tailed level of significance)
AZI–DHA AZI–chloroquine AZI–quinine AZI–mefloquine AZI–artemisinin DHA–artemisinin DHA–mefloquine DHA–quinine DHA–chloroquine Artemisinin–mefloquine Artemisinin–quinine Artemisinin–chloroquine
n
zEC50
P
zEC90
P
zEC99
P
39 39 39 39 39 39 39 39 39 39 39 39
−0.018 −0.017 0.204 0.296 −0.018 0.853 0.391 0.288 0.366 0.380 0.281 0.470
\0.05 \0.05 \0.05 \0.05 \0.05 B0.001 0.014 \0.05 0.022 0.017 \0.05 0.003
−0.014 −0.072 0.261 0.352 −0.014 0.844 0.422 0.258 0.428 0.486 0.268 0.367
\0.05 \0.05 \0.05 0.028 \0.05 B0.001 0.007 \0.05 0.007 0.002 \0.05 0.022
0.038 0.088 0.244 0.329 0.038 0.729 0.372 0.126 0.380 0.385 0.255 0.222
\0.05 \0.05 \0.05 0.041 \0.05 B0.001 0.020 \0.05 0.017 0.015 \0.05 \0.05
4. Discussion In combination with a fast-acting and chemically unrelated substance such as DHA, azithromycin may offer an interesting alternative for the treatment of falciparum malaria. The fact that no activity correlation between azithromycin and chloroquine was observed suggests that azithromycin is equally active in vitro against chloroquine-resistant and chloroquine-sensitive isolates and that no cross-resistance has to be anticipated. This result contradicts that of a previous study, which described considerably higher effective concentrations for chloroquine-resistant compared to chloroquine-sensitive parasites (Gingras and Jensen, 1992). The apparently conflicting results may, at least in part, be ascribed to the larger number of parasite isolates tested in our study, which is likely to have reduced the influence of individual populations, as well as to differences in laboratory techniques (fresh versus laboratory-adapted isolates; morphology versus radioisotope technique) and statistical evaluation. Similar results for the relation to quinine and artemisinin emphasise the relative independence of azithromycin drug response from the sensitivity of P. falciparum to these drugs. Even though significant, the partial associations between the effective concentrations of azithromycin and mefloquine above the EC50 level must be treated with caution, as they only reflect the flat part of
the dose–response curve and may, therefore, have little clinical impact. The relatively high EC50 and EC90 values reported in this study for azithromycin are comparable to the results previously found with other antimalarials in the antibiotics class. Most of these compounds share a common mode of action and are thought to act upon the 70S ribosomes of the malaria parasite (Blum et al., 1984; Divo et al., 1985). The high ECs may, at least in part, also reflect the slow onset of action of azithromycin, which may lead to higher EC values when tested in a 24 h culture system (Yeo and Rieckmann, 1995). The DHA sensitivity found in the course of this study shows that, even after several years of using artemisinin-derivatives as the first line treatment of uncomplicated falciparum malaria, DHA remains highly effective even in areas with a high prevalence of multidrug resistance, in spite of its close correlation with mefloquine (Noedl et al., 2001). The results are also comparable to recent data from other geographic regions where artemisinin-derivatives have not been used before (Ringwald et al., 1999). The extremely close association of DHA with artemisinin is a natural consequence of the close structural and biochemical relationship between these drugs, and indicates that these findings are probably applicable to most artemisinin-derivatives.
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Table 2 The 50, 90, 95, and 99% ECs of azithromycin, DHA, artemisinin, mefloquine, quinine and chloroquine (n = 39)
Azithromycin (nmol/l BMM) DHA (nmol/l BMM) Artemisinin (nmol/l BMM) Mefloquine (nmol/l blood) Quinine (nmol/l BMM) Chloroquine (nmol/l blood)
EC50
EC90
EC95
EC99
29 300.0 1.65 23.59 583.62 247.02 1720.90
77 100.0 7.10 181.06 1862.09 784.07 4805.34
98 100.0 16.74 322.64 2587.23 1087.80 6429.02
150 200.0 23.33 953.39 4794.37 2010.25 11 098.28
The fact that azithromycin generally shows a slow clinical response in the treatment of falciparum malaria suggests its combination with fastacting substances to reduce the parasitaemia quickly. Our data suggest that DHA may be the most interesting choice. However, quinine, which is similarly unrelated, may be equally effective where artemisinin derivatives are not available. To support this combination, however, additional data will be required from further clinical and in vitro interaction studies, such as the ones previously done with artemisinin and erythromycin (Ye and Van Dyke, 1994). The controversial reports of the clinical efficacy of the combination treatment, as well as its comparatively high cost, may limit the usefulness of azithromycin in the treatment of falciparum malaria to the management of cases in which no other antimalarials can be used (Na-Bangchang et al., 1996; De Vries et al., 1999). The low cure rate with artemether– azithromycin reported in these studies may, at least in part, be attributed to inadequate azithromycin dosage. In conclusion, it may be said that azithromycin may nonetheless serve as an interesting alternative drug to tetracycline and doxycycline, which cannot be used in pregnant women and children and which exhibit phototoxicity. Its independence from the sensitivity to other antimalarials could make it an attractive option for the treatment of multidrug-resistant malaria. Previously published data, as well as our observations, show that its combination with artemisininderivatives, such as DHA, may overcome many of the drawbacks associated with azithromycinmonotherapy.
References Anderson, S.L., Berman, J., Kuschner, R., Wesche, D., Magill, A., Wellde, B., Schneider, I., Dunne, M., Schuster, B.G., 1995. Prophylaxis of Plasmodium falciparum malaria with azithromycin administered to volunteers. Ann. Intern. Med. 123 (10), 771 – 773. Blum, J.J., Yayon, A., Friedman, S., Ginsburg, H., 1984. Effects of mitochondrial protein synthesis inhibitors on the incorporation of isoleucin into Plasmodium falciparum grown in vitro in human erythrocytes. J. Protozool. 31, 475 – 479. De Vries, P.J., Le Ngoc Hung, Le Thi Diem Thuy, Ho Phi Long, Nguyen Van Nam, Trinh Kim Anh, Kager, P.A., 1999. Short course of azithromycin/artesunate against falciparum malaria: no full protection against recrudescence. Trop. Med. Int. Health 4 (5), 407 – 408. Divo, A.A., Geary, T.G., Jensen, J.B., 1985. Oxygen- and time-dependent effects of antibiotics and selected mitochondrial inhibitors on Plasmodium falciparum in vitro. Antimicrob. Agents Chemother. 27, 21 – 27. Foulds, G., Shepard, R.M., Johnson, R.B., 1990. The pharmacokinetics of azithromycin in human serum and tissues. J. Antimicrob. Chemother. 25 (Suppl. A), 73 – 82. Gingras, B.A., Jensen, J.B., 1992. Activity of azithromycin (CP-62,993) and erythromycin against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum in vitro. Am. J. Trop. Med. Hyg. 47 (3), 378 – 382. Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose – effect experiments. J. Pharmac. Exp. Ther. 96, 99 – 113. Na-Bangchang, K., Kanda, T., Tipawangso, P., Thanavibul, A., Suprakob, K., Ibrahim, M., Wattanagoon, Y., Karbwang, J., 1996. Activity of artemether – azithromycin versus artemether – doxycycline in the treatment of multiple drug resistant falciparum malaria. Southeast Asian J. Trop. Med. Public Health 27 (3), 522 – 525. Noedl, H., Wernsdorfer, W.H., Krudsood, S., Wilairatana, P., Viriyavejakul, P., Kollaritsch, H., Wiedermann, G., Looareesuwan, S., 2001. An in vivo – in vitro model for the assessment of clinically relevant antimalarial cross-resistance. Am. J. Trop. Med. Hyg. in press. Ringwald, P., Bickii, J., Basco, L.K., 1999. In vitro activity of dihydroartemisinin against clinical isolates of Plasmodium
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falciparum in Yaounde, Cameroon. Am. J. Trop. Med. Hyg. 61 (2), 187 – 192. Taylor, W.R., Richie, T.L., Fryauff, D.J., Picarima, H., Ohrt, C., Tang, D., Braitman, D., Murphy, G.S., Widjaja, H., Tjitra, E., Ganjar, A., Jones, T.R., Basri, H., Berman, J., 1999. Malaria prophylaxis using azithromycin: a doubleblind, placebo-controlled trial in Irian Jaya, Indonesia. Clin. Infect. Dis. 28 (1), 74 –81. World Health Organisation (WHO), 1990. In vitro micro-test (Mark II) for the assessment of the response of Plasmod-
ium falciparum to chloroquine, mefloquine, quinine, sulfadoxine/pyrimethamine and amodiaquine. WHO document MAP/87.2, Rev. 1. Ye, Z., Van Dyke, K., 1994. Interaction of artemisinin and tetracycline or erythromycin against Plasmodium falciparum in vitro. Parasite 1 (3), 211 – 218. Yeo, A.E., Rieckmann, K.H., 1995. Increased antimalarial activity of azithromycin during prolonged exposure of Plasmodium falciparum in vitro. Int. J. Parasitol. 25 (4), 531 – 532.
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Antimalarial activity of azithromycin, artemisinin and dihydroartemisinin in fresh isolates of Plasmodium falciparum in Thailand H. Noedl a,b,*, W.H. Wernsdorfer a,b, S. Krudsood b, P. Wilairatana b, H. Kollaritsch a, G. Wiedermann a, S. Looareesuwan b a
Department of Specific Prophylaxis and Tropical Medicine, Institute of Pathophysiology, Uni6ersity of Vienna, Kinderspitalgasse 15, A-1095, Vienna, Austria b Faculty of Tropical Medicine, Bangkok Hospital for Tropical Diseases, Mahidol Uni6ersity, 420 /6 Raj6ithi Road, Bangkok 10400, Thailand Received 29 January 2001; received in revised form 28 March 2001; accepted 17 April 2001
Abstract Antibiotics with antimalarial activity may offer an interesting alternative for the treatment of multidrug-resistant falciparum malaria. Azithromycin, a relatively recent semisynthetic derivative of erythromycin, was tested for its in vitro activity against fresh isolates of Plasmodium falciparum. As the reportedly slow onset of action of azithromycin suggests its combination with fast-acting substances, such as artemisinin-derivatives, dihydroartemisinin (DHA) was tested parallel as a possible combination partner. The effective concentrations found for azithromycin in this study (EC50 =29.3 mmol/l, EC90 =77.1 mmol/l blood medium mixture (BMM)) are comparable to those of other antimalarials in the antibiotics class and are considerably higher than those found for mefloquine or quinine. The absence of an activity correlation between azithromycin and chloroquine, quinine and artemisinin emphasises the independence of azithromycin drug response from the sensitivity to these drugs. A weak activity correlation (zEC90 = 0.352; p= 0.028), which could point to a potential cross-sensitivity but is probably of little clinical importance, was found with mefloquine above the EC50 level. Provided that further clinical trials support the combination of these drugs, DHA may offer an interesting combination partner for azithromycin owing to its rapid onset of action and the comparatively low effective concentrations (EC50 =1.65 nmol/l, EC90 = 7.10 nmol/l BMM). This combination may serve as an interesting alternative for tetracycline and doxycycline, which cannot be used in pregnant women and children, and exhibit phototoxicity. Nevertheless, the relatively high cost of this combination, as well as the controversial reports of the clinical efficacy, may limit the usefulness of azithromycin in malaria therapy and require an adjustment of previously used treatment regimens. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Azithromycin; Dihydroartemisinin; Macrolide antibiotics; Malaria; Plasmodium falciparum; Drug sensitivity; Resistance
* Corresponding author. Fax: + 43-1-403834390. E-mail address: [email protected] (H. Noedl). 0001-706X/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 0 1 ) 0 0 1 4 1 - 3
H. Noedl et al. / Acta Tropica 80 (2001) 39–44
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1. Introduction The spread of multidrug-resistant Plasmodium falciparum in recent years has intensified the search for new antimalarials and antibiotics with antimalarial activity. Among the macrolide antibiotics, erythromycin was the first to be used in the treatment and prophylaxis of malaria. Azithromycin, a relatively recent semisynthetic derivative of erythromycin, differs structurally from erythromycin by the addition of a methylsubstituted nitrogen atom in the lactone ring. This modification improves acid stability and tissue penetration capabilities and also broadens the antibacterial spectrum. It is concentrated intracellularly and in tissues and has a serum half-life of about 2.4 days (Foulds et al., 1990). Its antimalarial and prophylactic activities, however, have only recently been described. In vitro tests with single laboratory-adapted strains of P. falciparum indicate considerably higher effective concentrations for chloroquine-resistant compared with chloroquine-sensitive isolates (Gingras and Jensen, 1992). Recent clinical trials also confirm varying degrees of prophylactic and blood schizontocidal activity (Na-Bangchang et al., 1996; Anderson et al., 1995; Taylor et al., 1999). Its reportedly slow onset of action suggests its combination with fast-acting substances such as artemisinin derivatives. Owing to its excellent activity against P. falciparum, as well as its pharmacological properties, dihydroartemisinin (DHA), the active metabolite of all artemisininderivatives, could be a suitable combination partner for azithromycin. The aim of the study was the establishment of a baseline for the in vitro activity of azithromycin against fresh isolates of P. falciparum in an area with a high prevalence of multidrug resistance, the evaluation of the suitability of DHA as a possible combination partner, and the determination of the relation of azithromycin sensitivity to other currently used antimalarials.
2. Material and methods The study was designed as an experimental
laboratory investigation conducted at the Bangkok Hospital for Tropical Diseases (Faculty of Tropical Medicine, Mahidol University) from October 1999 until March 2000. Written informed consent was obtained from all adult participants or from parents or legal guardians of minors. The study protocols were approved by the ethical review board of the Faculty of Tropical Medicine, Mahidol University. A total number of 39 fresh isolates of P. falciparum obtained from patients with microscopically confirmed P. falciparum mono-infections, primarily from the western border regions of Thailand, were successfully tested for their in vitro susceptibility to azithromycin and DHA. Parallel tests were performed with chloroquine, mefloquine, quinine and artemisinin to obtain data for correlation analysis. The in vitro tests for the measurement of the drug sensitivity of P. falciparum followed the WHO standard methodology for the assessment of the inhibition of schizont maturation (WHO, 1990). Microtitre plates were pre-dosed at the Department of Specific Prophylaxis and Tropical Medicine, University of Vienna, with ascending concentrations of azithromycin (0.1–100 mmol/l blood medium mixture (BMM)), DHA (0.3– 300 nmol/l BMM), and artemisinin (3–3000 nmol/l BMM). Distilled water was used as solvent for azithromycin, whereas artemisinin and DHA were dissolved in 50% ethanol, 25% Tween 80 and 25% linoleic acid and further diluted with distilled water before being dispensed into 96-well microtitre plates. Mefloquine (800–51 200 nmol/l blood), quinine (80–5120 nmol/l BMM), and chloroquine (400– 25 600 nmol/l blood) plates were obtained from the WHO, Regional Office for the Western Pacific, Manila, Philippines. Whole blood was collected from every patient by venepuncture and added to RPMI 1640 medium in a 5% dilution, and pre-culture parasitaemia was assessed. Fifty microliters of BMM were added to each well of the culture plates and incubated in candle jars at 37.5 °C for 24 h. The culture results were evaluated microscopically by counting the number of schizonts in each well relative to the control well. In the course of the mathematical analysis of drug response the drug concentrations were log-
H. Noedl et al. / Acta Tropica 80 (2001) 39–44
transformed and the microscopically determined inhibition expressed in probits (Litchfield and Wilcoxon, 1949). A quadratic regression model was found to be best suited for the inhibition characteristics of azithromycin and applied to the transformed data to derive effective concentrations and the minimum inhibitory concentration. Analysis of DHA and artemisinin data was done by standard log-probit analysis. Spearman correlation analysis was employed to determine the activity correlations (z) at a significance level of h =5% (PB 0.05). Two-tailed P-values were used throughout the analyses.
3. Results
3.1. Azithromycin The geometric mean parasite density of the successfully tested blood samples was 18 876 asexual parasites per microlitre blood, and the geometric mean schizont count in the control wells was 58 schizonts per 200 parasites. None of the isolates showed full inhibition at concentrations below 30 mmol/l BMM, and only three (7.7%) isolates were fully inhibited at this concentration. The majority (79.5%), however, had their cut-off point at 100 mmol/l, and five (12.8%) isolates did not show full inhibition even at the highest concentration (100 mmol/l). The overall mean 50% effective concentration (EC50) for all 39 isolates was 29.3 mmol/l (95% confidence intervals (CIs): 24.5 and 35.2 mmol/l), and the corresponding EC90, EC95 and EC99 values were 77.1 mmol/l (95% CIs: 60.6 and 98.0 mmol/l), 98.1 mmol/l (95% CIs: 74.1 and 130.0 mmol/l) and 150.2 mmol/l (95% CIs: 143.0 and 157.7 mmol/l) respectively (Fig. 1). With a value of 5.7, the 2 for the heterogeneity of the quadratic regression (r =0.982) was well within permissible limits. Even though the EC50 and EC90 values for chloroquine covered a wide range, all isolates were considered chloroquine resistant following the WHO criteria. In correlation analysis no activity relation was found between the individual ECs of azithromycin and chloroquine,
41
azithromycin and artemisinin and azithromycin and quinine (P\ 0.05). The only significant correlation was found with mefloquine above the EC50 (zEC90 = 0.352; P= 0.028; zEC99 = 0.329; P= 0.041) level (Table 1).
3.2. DHA All isolates were fully inhibited by DHA concentrations of 100 nmol/l BMM. The majority (69%) of the samples had their cut-off point at or below 10 nmol/l. The EC50 for all 39 successfully tested isolates was 1.65 nmol/l (95% CIs: 1.16 and 2.35 nmol/l), and the corresponding EC90, EC95 and EC99 values were 7.10 nmol/l (95% CIs: 4.46 and 11.29 nmol/l), 10.74 nmol/l (95% CIs: 6.39 and 18.04 nmol/l) and 23.33 nmol/l (95% CIs: 12.23 and 44.50 nmol/l) respectively. No association was found between the effective concentrations of DHA and azithromycin (P\0.05). There was an extremely close correlation of DHA with artemisinin (PB 0.001), and both drugs showed almost identical relations to mefloquine, quinine and chloroquine (Table 1). Effective concentrations for artemisinin, mefloquine, quinine, and chloroquine are shown in Table 2.
Fig. 1. The log-dose/probit-response quadratic regression of the in vitro drug response of P. falciparum to azithromycin with data points and 95% CIs (azithromycin drug concentration in mM; SMI: schizont maturation inhibition).
H. Noedl et al. / Acta Tropica 80 (2001) 39–44
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Table 1 In vitro activity correlations between azithromycin (AZI), DHA and other antimalarials at EC50, EC90 and EC99 levels (z: Spearman’s rho, correlation coefficient; P: two-tailed level of significance)
AZI–DHA AZI–chloroquine AZI–quinine AZI–mefloquine AZI–artemisinin DHA–artemisinin DHA–mefloquine DHA–quinine DHA–chloroquine Artemisinin–mefloquine Artemisinin–quinine Artemisinin–chloroquine
n
zEC50
P
zEC90
P
zEC99
P
39 39 39 39 39 39 39 39 39 39 39 39
−0.018 −0.017 0.204 0.296 −0.018 0.853 0.391 0.288 0.366 0.380 0.281 0.470
\0.05 \0.05 \0.05 \0.05 \0.05 B0.001 0.014 \0.05 0.022 0.017 \0.05 0.003
−0.014 −0.072 0.261 0.352 −0.014 0.844 0.422 0.258 0.428 0.486 0.268 0.367
\0.05 \0.05 \0.05 0.028 \0.05 B0.001 0.007 \0.05 0.007 0.002 \0.05 0.022
0.038 0.088 0.244 0.329 0.038 0.729 0.372 0.126 0.380 0.385 0.255 0.222
\0.05 \0.05 \0.05 0.041 \0.05 B0.001 0.020 \0.05 0.017 0.015 \0.05 \0.05
4. Discussion In combination with a fast-acting and chemically unrelated substance such as DHA, azithromycin may offer an interesting alternative for the treatment of falciparum malaria. The fact that no activity correlation between azithromycin and chloroquine was observed suggests that azithromycin is equally active in vitro against chloroquine-resistant and chloroquine-sensitive isolates and that no cross-resistance has to be anticipated. This result contradicts that of a previous study, which described considerably higher effective concentrations for chloroquine-resistant compared to chloroquine-sensitive parasites (Gingras and Jensen, 1992). The apparently conflicting results may, at least in part, be ascribed to the larger number of parasite isolates tested in our study, which is likely to have reduced the influence of individual populations, as well as to differences in laboratory techniques (fresh versus laboratory-adapted isolates; morphology versus radioisotope technique) and statistical evaluation. Similar results for the relation to quinine and artemisinin emphasise the relative independence of azithromycin drug response from the sensitivity of P. falciparum to these drugs. Even though significant, the partial associations between the effective concentrations of azithromycin and mefloquine above the EC50 level must be treated with caution, as they only reflect the flat part of
the dose–response curve and may, therefore, have little clinical impact. The relatively high EC50 and EC90 values reported in this study for azithromycin are comparable to the results previously found with other antimalarials in the antibiotics class. Most of these compounds share a common mode of action and are thought to act upon the 70S ribosomes of the malaria parasite (Blum et al., 1984; Divo et al., 1985). The high ECs may, at least in part, also reflect the slow onset of action of azithromycin, which may lead to higher EC values when tested in a 24 h culture system (Yeo and Rieckmann, 1995). The DHA sensitivity found in the course of this study shows that, even after several years of using artemisinin-derivatives as the first line treatment of uncomplicated falciparum malaria, DHA remains highly effective even in areas with a high prevalence of multidrug resistance, in spite of its close correlation with mefloquine (Noedl et al., 2001). The results are also comparable to recent data from other geographic regions where artemisinin-derivatives have not been used before (Ringwald et al., 1999). The extremely close association of DHA with artemisinin is a natural consequence of the close structural and biochemical relationship between these drugs, and indicates that these findings are probably applicable to most artemisinin-derivatives.
H. Noedl et al. / Acta Tropica 80 (2001) 39–44
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Table 2 The 50, 90, 95, and 99% ECs of azithromycin, DHA, artemisinin, mefloquine, quinine and chloroquine (n = 39)
Azithromycin (nmol/l BMM) DHA (nmol/l BMM) Artemisinin (nmol/l BMM) Mefloquine (nmol/l blood) Quinine (nmol/l BMM) Chloroquine (nmol/l blood)
EC50
EC90
EC95
EC99
29 300.0 1.65 23.59 583.62 247.02 1720.90
77 100.0 7.10 181.06 1862.09 784.07 4805.34
98 100.0 16.74 322.64 2587.23 1087.80 6429.02
150 200.0 23.33 953.39 4794.37 2010.25 11 098.28
The fact that azithromycin generally shows a slow clinical response in the treatment of falciparum malaria suggests its combination with fastacting substances to reduce the parasitaemia quickly. Our data suggest that DHA may be the most interesting choice. However, quinine, which is similarly unrelated, may be equally effective where artemisinin derivatives are not available. To support this combination, however, additional data will be required from further clinical and in vitro interaction studies, such as the ones previously done with artemisinin and erythromycin (Ye and Van Dyke, 1994). The controversial reports of the clinical efficacy of the combination treatment, as well as its comparatively high cost, may limit the usefulness of azithromycin in the treatment of falciparum malaria to the management of cases in which no other antimalarials can be used (Na-Bangchang et al., 1996; De Vries et al., 1999). The low cure rate with artemether– azithromycin reported in these studies may, at least in part, be attributed to inadequate azithromycin dosage. In conclusion, it may be said that azithromycin may nonetheless serve as an interesting alternative drug to tetracycline and doxycycline, which cannot be used in pregnant women and children and which exhibit phototoxicity. Its independence from the sensitivity to other antimalarials could make it an attractive option for the treatment of multidrug-resistant malaria. Previously published data, as well as our observations, show that its combination with artemisininderivatives, such as DHA, may overcome many of the drawbacks associated with azithromycinmonotherapy.
References Anderson, S.L., Berman, J., Kuschner, R., Wesche, D., Magill, A., Wellde, B., Schneider, I., Dunne, M., Schuster, B.G., 1995. Prophylaxis of Plasmodium falciparum malaria with azithromycin administered to volunteers. Ann. Intern. Med. 123 (10), 771 – 773. Blum, J.J., Yayon, A., Friedman, S., Ginsburg, H., 1984. Effects of mitochondrial protein synthesis inhibitors on the incorporation of isoleucin into Plasmodium falciparum grown in vitro in human erythrocytes. J. Protozool. 31, 475 – 479. De Vries, P.J., Le Ngoc Hung, Le Thi Diem Thuy, Ho Phi Long, Nguyen Van Nam, Trinh Kim Anh, Kager, P.A., 1999. Short course of azithromycin/artesunate against falciparum malaria: no full protection against recrudescence. Trop. Med. Int. Health 4 (5), 407 – 408. Divo, A.A., Geary, T.G., Jensen, J.B., 1985. Oxygen- and time-dependent effects of antibiotics and selected mitochondrial inhibitors on Plasmodium falciparum in vitro. Antimicrob. Agents Chemother. 27, 21 – 27. Foulds, G., Shepard, R.M., Johnson, R.B., 1990. The pharmacokinetics of azithromycin in human serum and tissues. J. Antimicrob. Chemother. 25 (Suppl. A), 73 – 82. Gingras, B.A., Jensen, J.B., 1992. Activity of azithromycin (CP-62,993) and erythromycin against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum in vitro. Am. J. Trop. Med. Hyg. 47 (3), 378 – 382. Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose – effect experiments. J. Pharmac. Exp. Ther. 96, 99 – 113. Na-Bangchang, K., Kanda, T., Tipawangso, P., Thanavibul, A., Suprakob, K., Ibrahim, M., Wattanagoon, Y., Karbwang, J., 1996. Activity of artemether – azithromycin versus artemether – doxycycline in the treatment of multiple drug resistant falciparum malaria. Southeast Asian J. Trop. Med. Public Health 27 (3), 522 – 525. Noedl, H., Wernsdorfer, W.H., Krudsood, S., Wilairatana, P., Viriyavejakul, P., Kollaritsch, H., Wiedermann, G., Looareesuwan, S., 2001. An in vivo – in vitro model for the assessment of clinically relevant antimalarial cross-resistance. Am. J. Trop. Med. Hyg. in press. Ringwald, P., Bickii, J., Basco, L.K., 1999. In vitro activity of dihydroartemisinin against clinical isolates of Plasmodium
44
H. Noedl et al. / Acta Tropica 80 (2001) 39–44
falciparum in Yaounde, Cameroon. Am. J. Trop. Med. Hyg. 61 (2), 187 – 192. Taylor, W.R., Richie, T.L., Fryauff, D.J., Picarima, H., Ohrt, C., Tang, D., Braitman, D., Murphy, G.S., Widjaja, H., Tjitra, E., Ganjar, A., Jones, T.R., Basri, H., Berman, J., 1999. Malaria prophylaxis using azithromycin: a doubleblind, placebo-controlled trial in Irian Jaya, Indonesia. Clin. Infect. Dis. 28 (1), 74 –81. World Health Organisation (WHO), 1990. In vitro micro-test (Mark II) for the assessment of the response of Plasmod-
ium falciparum to chloroquine, mefloquine, quinine, sulfadoxine/pyrimethamine and amodiaquine. WHO document MAP/87.2, Rev. 1. Ye, Z., Van Dyke, K., 1994. Interaction of artemisinin and tetracycline or erythromycin against Plasmodium falciparum in vitro. Parasite 1 (3), 211 – 218. Yeo, A.E., Rieckmann, K.H., 1995. Increased antimalarial activity of azithromycin during prolonged exposure of Plasmodium falciparum in vitro. Int. J. Parasitol. 25 (4), 531 – 532.
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