Annals of Tropical Medicine & Parasitology
ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
In vitro activity of pyronaridine against African strains of Plasmodium falciparum L. K. Basco & J. Le Bras To cite this article: L. K. Basco & J. Le Bras (1992) In vitro activity of pyronaridine against African strains of Plasmodium falciparum, Annals of Tropical Medicine & Parasitology, 86:5, 447-454, DOI: 10.1080/00034983.1992.11812693 To link to this article: http://dx.doi.org/10.1080/00034983.1992.11812693
Published online: 15 Nov 2016.
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Citing articles: 8 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ypgh19 Download by: [Cornell University Library]
Date: 20 July 2017, At: 05:55
Annals of Tropical Medicine and Parasitology, Vol. 86, No.5,
447~454
(1992)
In vitro activity of pyronaridine against African strains of Plasmodium falciparum BY L. K. BASCO AND]. LE BRAS Centre National de Reference pour Ia Chimiosensibilite du Paludisme, Institut de Midecine et d' Epidemiologic Tropic ales, Laboratoire de Parasitologic, Hopital Bichat-Claude Bernard, 75877 Paris, France Received 13 Apri/1992, Accepted 29 June 1992
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 chloroquineresistant 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.
The emergence and spread of chloroquine resistance in most regions where malaria is endemic pose a serious challenge to the treatment of malaria (Wernsdorfer and Payne, 1991 ). There are only a few alternative drugs which are effective against chloroquine-resistant Plasmodium falciparum, necessitating an urgent search for new drugs. One of the promising candidates for the treatment of chloroquine-resistant malaria is pyronaridine, a drug which was synthesized in China in 1970 (WHO, 1990). Clinical trials of pyronaridine, involving more than 1000 Chinese patients, have shown its high efficacy against P. falciparum and P. vivax, with few side effects (Shao, 1990), and it is currently used for the treatment ofchloroquine-resistant P.falciparum in China. Pyronaridine is an acridine-type Mannich base with a ring system similar to that of mepacrine (Fig. 1). The side chain of the compound is similar to that of amodiaquine and amopyroquine, which are quinoline-type Mannich bases. 0003-4983/92/050447 +08 $08.00/0
The activity of pyronaridine has not been studied against clinical isolates of P.falciparum originating from Central and West Africa. The aim of this study was to assess the in vitro activity of pyronaridine and compare its activity with that of chloroquine, monodesethylamodiaquine (the biologically active metabolite of amodiaquine) and amopyroquine against African strains of P.falciparum.
MATERIALS AND METHODS Parasites The chloroquine-susceptible L-3/Cote d'I voire clone and the chloroquine-resistant FCM 29/ Cameroon clone of P. falciparum were maintained in continuous culture (Trager and Jensen, 1976). Venous blood samples were obtained prior to treatment from 31 malaria-infected travellers returning to France from Central and West Africa between October 1991 and February © 1992 Liverpool School of Tropical Medicine
448
BASCO AND LE BRAS
1992. Giemsa-stained, thin blood smears were examined to determine the parasite density and to verify monoinfection with P. Jalciparum. Blood samples with parasitaemia :2: 0·1% were used to test drug susceptibility. The erythrocytes were washed three times in RPMI 1640. Samples whose parasitaemia exceeded 1·0% were diluted with uninfected erythrocytes to obtain an initial parasitaemia of0·1-1·0%. Drugs Pyronaridine phosphate (56·89% base) was kindly provided by Professor C. Chang of the Institute of Parasitic Diseases, Chinese Academy of Preventive Medicine, Shanghai, China. Chloroquine sulphate was obtained from Specia, Paris, France. Monodesethylamodiaquine dihydrochloride and amopyroquine dihydrochloride were obtained from ParkeDavis, Courbevoie, France. Stock solutions of the test compounds were prepared in sterile distilled water. Two-fold serial dilutions in distilled water were distributed in triplicate in flat-bottom 24-well plates and dried at room temperature. Final concentrations of the drugs were 12 · 5-1600 nmol 1- 1 for chloroquine ( 100--6400 nmol 1- 1 for the highly chloroquine-resistant FCM 29 clone), 5-400 nmol 1- 1 for monodesethylamodiaquine (50--3200 nmol 1- 1 for the clone FCM 29), and 1· 2-160 nmol 1- 1 for amopyroquine and pyronaridine. In vitro Assay The in vitro assay system described by Le Bras and Deloron (1983) was used in this study. The infected erythrocytes were resuspended in RPMI 1640 supplemented with 10% human serum and buffered with 25 mmol 1- 1 of HEPES and 25 mmoll- 1 ofNaHC03 to obtain a haematocrit of2·5%. The suspension (700 J.d/ well) was distributed in 24-well plates predosed with the test compounds and incubated at 37°C in an atmosphere of 5% 0 2, 5% C0 2 and 90% N 2 for 42 hours. Incorporation of [G- 3H]hypoxanthine (specific activity 4· 3 Ci mmoll- 1, 1 11Cijwell; Amersham International, Amersham, U.K.) added 18 hours after initial incubation was used as an index of parasite growth.
At the end of the incubation period, the plates were frozen then thawed to lyse the erythrocytes. The contents of each well were collected onto filter discs with a cell harvester. The discs were dried, and each placed in a scintillation vial containing 1· 5 ml of liquid scintillation cocktail (Optiscint Hisafe; LKB Wallac, U.K.), and counted in a liquid scintillation spectrophotometer (Wallac 1410; Pharmacia, Uppsala, Sweden). The resulting concentration-response data were interpreted by linear regression analysis. The 50% inhibitory concentration (IC 50 ) was defined as the drug concentration giving 50% of the parasite uptake of [G- 3H]hypoxanthine found in drug-free control wells. Resistance to chloroquine and monodesethylamodiaquine was defined as I C 50 > 100 nmol 1- 1 and > 60 nmol 1- 1 respectively (Le Bras et a!., 1991 ). The threshold values for resistance to amopyroquine and pyronaridine have not yet been determined. For each clone of P. falciparum, susceptibility to the test compounds was determined six times. For each fresh clinical isolate of P. falciparum, simultaneous assays based on triplicate determinations were done. Data were expressed in terms of mean IC 50 ± standard deviation for the clones and geometric mean IC 50 and 95% confidence intervals (95% C.I.) for the clinical isolates. Unpaired t-tests were used to compare logarithmic values of IC 50 for chloroquine-susceptible and chloroquine-resistant isolates. Spearman's rankorder correlation test was used to determine the correlation coefficients between different drugs. The significance level of 95% was used in the statistical analysis.
RESULTS The chloroquine-resistant FCM 29 clone (IC 50 : 1250 ± 138 nmoll- 1) was 45 times less susceptible to chloroquine than the chloroquine-susceptible L-3 clone (IC 50 : 28·0± Hnmol 1- 1) (Fig. 2). There were 26-fold, four-fold and 2· 5-fold differences in the responses to monodesethylamodiaquine (IC 50 : 336 ± 27 v. 13·1 ± 3·3 nmol 1- 1), amopyroquine (IC 50 : 34·5±6·4 v. 9·2±
~
~
Cl.
Cl
~
Amodiaquine
"'
.' .,
Chloroquine
'
N
/C2Hs
OH
~
II
~
NH
Amopyroquine
I
\
Pyronaridine
2
OH
r-----1
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ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
In vitro activity of pyronaridine against African strains of Plasmodium falciparum L. K. Basco & J. Le Bras To cite this article: L. K. Basco & J. Le Bras (1992) In vitro activity of pyronaridine against African strains of Plasmodium falciparum, Annals of Tropical Medicine & Parasitology, 86:5, 447-454, DOI: 10.1080/00034983.1992.11812693 To link to this article: http://dx.doi.org/10.1080/00034983.1992.11812693
Published online: 15 Nov 2016.
Submit your article to this journal
View related articles
Citing articles: 8 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ypgh19 Download by: [Cornell University Library]
Date: 20 July 2017, At: 05:55
Annals of Tropical Medicine and Parasitology, Vol. 86, No.5,
447~454
(1992)
In vitro activity of pyronaridine against African strains of Plasmodium falciparum BY L. K. BASCO AND]. LE BRAS Centre National de Reference pour Ia Chimiosensibilite du Paludisme, Institut de Midecine et d' Epidemiologic Tropic ales, Laboratoire de Parasitologic, Hopital Bichat-Claude Bernard, 75877 Paris, France Received 13 Apri/1992, Accepted 29 June 1992
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 chloroquineresistant 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.
The emergence and spread of chloroquine resistance in most regions where malaria is endemic pose a serious challenge to the treatment of malaria (Wernsdorfer and Payne, 1991 ). There are only a few alternative drugs which are effective against chloroquine-resistant Plasmodium falciparum, necessitating an urgent search for new drugs. One of the promising candidates for the treatment of chloroquine-resistant malaria is pyronaridine, a drug which was synthesized in China in 1970 (WHO, 1990). Clinical trials of pyronaridine, involving more than 1000 Chinese patients, have shown its high efficacy against P. falciparum and P. vivax, with few side effects (Shao, 1990), and it is currently used for the treatment ofchloroquine-resistant P.falciparum in China. Pyronaridine is an acridine-type Mannich base with a ring system similar to that of mepacrine (Fig. 1). The side chain of the compound is similar to that of amodiaquine and amopyroquine, which are quinoline-type Mannich bases. 0003-4983/92/050447 +08 $08.00/0
The activity of pyronaridine has not been studied against clinical isolates of P.falciparum originating from Central and West Africa. The aim of this study was to assess the in vitro activity of pyronaridine and compare its activity with that of chloroquine, monodesethylamodiaquine (the biologically active metabolite of amodiaquine) and amopyroquine against African strains of P.falciparum.
MATERIALS AND METHODS Parasites The chloroquine-susceptible L-3/Cote d'I voire clone and the chloroquine-resistant FCM 29/ Cameroon clone of P. falciparum were maintained in continuous culture (Trager and Jensen, 1976). Venous blood samples were obtained prior to treatment from 31 malaria-infected travellers returning to France from Central and West Africa between October 1991 and February © 1992 Liverpool School of Tropical Medicine
448
BASCO AND LE BRAS
1992. Giemsa-stained, thin blood smears were examined to determine the parasite density and to verify monoinfection with P. Jalciparum. Blood samples with parasitaemia :2: 0·1% were used to test drug susceptibility. The erythrocytes were washed three times in RPMI 1640. Samples whose parasitaemia exceeded 1·0% were diluted with uninfected erythrocytes to obtain an initial parasitaemia of0·1-1·0%. Drugs Pyronaridine phosphate (56·89% base) was kindly provided by Professor C. Chang of the Institute of Parasitic Diseases, Chinese Academy of Preventive Medicine, Shanghai, China. Chloroquine sulphate was obtained from Specia, Paris, France. Monodesethylamodiaquine dihydrochloride and amopyroquine dihydrochloride were obtained from ParkeDavis, Courbevoie, France. Stock solutions of the test compounds were prepared in sterile distilled water. Two-fold serial dilutions in distilled water were distributed in triplicate in flat-bottom 24-well plates and dried at room temperature. Final concentrations of the drugs were 12 · 5-1600 nmol 1- 1 for chloroquine ( 100--6400 nmol 1- 1 for the highly chloroquine-resistant FCM 29 clone), 5-400 nmol 1- 1 for monodesethylamodiaquine (50--3200 nmol 1- 1 for the clone FCM 29), and 1· 2-160 nmol 1- 1 for amopyroquine and pyronaridine. In vitro Assay The in vitro assay system described by Le Bras and Deloron (1983) was used in this study. The infected erythrocytes were resuspended in RPMI 1640 supplemented with 10% human serum and buffered with 25 mmol 1- 1 of HEPES and 25 mmoll- 1 ofNaHC03 to obtain a haematocrit of2·5%. The suspension (700 J.d/ well) was distributed in 24-well plates predosed with the test compounds and incubated at 37°C in an atmosphere of 5% 0 2, 5% C0 2 and 90% N 2 for 42 hours. Incorporation of [G- 3H]hypoxanthine (specific activity 4· 3 Ci mmoll- 1, 1 11Cijwell; Amersham International, Amersham, U.K.) added 18 hours after initial incubation was used as an index of parasite growth.
At the end of the incubation period, the plates were frozen then thawed to lyse the erythrocytes. The contents of each well were collected onto filter discs with a cell harvester. The discs were dried, and each placed in a scintillation vial containing 1· 5 ml of liquid scintillation cocktail (Optiscint Hisafe; LKB Wallac, U.K.), and counted in a liquid scintillation spectrophotometer (Wallac 1410; Pharmacia, Uppsala, Sweden). The resulting concentration-response data were interpreted by linear regression analysis. The 50% inhibitory concentration (IC 50 ) was defined as the drug concentration giving 50% of the parasite uptake of [G- 3H]hypoxanthine found in drug-free control wells. Resistance to chloroquine and monodesethylamodiaquine was defined as I C 50 > 100 nmol 1- 1 and > 60 nmol 1- 1 respectively (Le Bras et a!., 1991 ). The threshold values for resistance to amopyroquine and pyronaridine have not yet been determined. For each clone of P. falciparum, susceptibility to the test compounds was determined six times. For each fresh clinical isolate of P. falciparum, simultaneous assays based on triplicate determinations were done. Data were expressed in terms of mean IC 50 ± standard deviation for the clones and geometric mean IC 50 and 95% confidence intervals (95% C.I.) for the clinical isolates. Unpaired t-tests were used to compare logarithmic values of IC 50 for chloroquine-susceptible and chloroquine-resistant isolates. Spearman's rankorder correlation test was used to determine the correlation coefficients between different drugs. The significance level of 95% was used in the statistical analysis.
RESULTS The chloroquine-resistant FCM 29 clone (IC 50 : 1250 ± 138 nmoll- 1) was 45 times less susceptible to chloroquine than the chloroquine-susceptible L-3 clone (IC 50 : 28·0± Hnmol 1- 1) (Fig. 2). There were 26-fold, four-fold and 2· 5-fold differences in the responses to monodesethylamodiaquine (IC 50 : 336 ± 27 v. 13·1 ± 3·3 nmol 1- 1), amopyroquine (IC 50 : 34·5±6·4 v. 9·2±
~
~
Cl.
Cl
~
Amodiaquine
"'
.' .,
Chloroquine
'
N
/C2Hs
OH
~
II
~
NH
Amopyroquine
I
\
Pyronaridine
2
OH
r-----1
2
\ CH -CH
CH,-
