Activity of pyronaridine and mepacrine against twelve strains of Plasmodium falciparum in vitro

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1 Journal of Antimicrobial Chemotherapy (1996) 37, Activity of pyronaridine and mepacrine against twelve strains of Plasmodium falciparum in vitro E. I. Elueze, S. L. Croft* and D. C. Warhurst Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, London WCIE 7HT, UK, an acndine derivative, has been found effective in China for the treatment of drug-resistant falciparum malaria. The activities of pyronaridine and mepacrine were compared with those of standard antimalarial drugs in vitro against chloroquine-sensitive (CS) and chloroquine-resistant (CR) Plasmodium falciparum isolates to investigate cross resistance. The 50% inhibitory concentrations (IC») against the resistant isolates were 2.8-fold higher than the sensitive isolates for pyronaridine (CS = 7.3 nm; CR = 20.5 nm) and 3.2-fold higher for mepacrine (CS = 13.3 nm; CR = 42.6 nm). These same isolates showed an 11-fold difference in sensitivity to chloroquine with mean IC» values of 21 nm for sensitive and 239 nm for resistant parasites. A significant correlation was observed between parasite sensitivity (IC W ) to pyronaridine and the drugs, mepacrine, amodiaquine and chloroquine. However, the high level of activity seen with pyronaridine, even against the CR isolates, should encourage further field trials with this drug. Introduction The acridine-based compounds have played a prominent role in the history and development of antimalarials., a 9-aminoacridine, was the first synthetic antimalarial blood schizontocide used clinically, but its use was discontinued with the introduction of chloroquine (Wernsdorfer & Payne, 1991). With the emergence of chloroquine-resistant falciparum malaria and reports of parasite resistance to alternative drugs, there has been renewed interest in the antimalarial activity of the acridines (Figgitt el al., 1992) and congeners including acridones (Basco et al., 1994) and dihydroacridinediones (Berman et al., 1994). Two derivatives developed in China, pyronaridine and pyracrine, have shown promising antimalarial activities both in mouse models (Peters & Robinson, 1992) and in clinical trials (Fu & Xiao, 1991). is an azacrine-type Mannich base. While the naphthyridine nucleus resembles acridine, the side chain is similar to those of amopyroquine and amodiaquine (Figure). was synthesised at the Institute of Parasitic Diseases, Shanghai, in Clinical trials began in 1971, and by 1980 it was formally released as a new antimalarial drug in China. Several thousand cases of vivax and falciparum malaria, including cerebral cases, have been treated with pyronaridine with mean parasite clearance time of 2 to 3 days (Fu & Xiao, 1991) and clinical efficacy against Corresponding author. 03O5-7453/96/O3O $12.00/0 511 % 1996 The British Society for Antimicrobial Chemotherapy

2 512 E. I. Elueze et al. CH, NH-CH CH 2 -CH 2 CH 2 -N OCH, C2H5 CH, NH-CH-CHj- CH 2 -CH 2 - N Amodiaquine Figure. Structures of the antimalarials, pyronaridine, mepacrine, amodiaquine and chloroquine. chloroquine-, mefloquine- and multidrug-resistant cases (Chang, Lin-Hua & Jantanavivat, 1992). It is important for future strategy to examine the activity of new antimalarials against different strains of Plasmodium falciparum and to assess their cross resistant patterns with established drugs. There are conflicting reports on cross-resistance. Childs et al. (1988) and Basco & Le Bras (1992) found that pyronaridine was highly active against both chloroquine-sensitive (CS) and chloroquine-resistant (CR) strains of P. falciparum and suggested no cross resistance. However, Schildbach et al. (1990) and Warsame et al. (1991) showed a correlation between chloroquine and pyronaridine responses. In the present study the activity of pyronaridine was compared with the standard antimalarials, chloroquine, mepacrine, amodiaquine, quinine and mefloquine against five CS and seven CR strains/clones of P. falciparum in vitro. Cultures Materials and methods P. falciparum was maintained in culture by a combination of techniques described by Osisanya, Gould & Warhurst (1981) and Fairlamb, Warhurst & Peters (1985). The P. falciparum strains/clones used in the study included the CS clone T9.96 (Thailand) and CS strains HI (Honduras), FC27 (Papua New Guinea), N (Nigeria) and NF54 (Amsterdam), the CR clone T9.94 (Thailand) and the CR strains Kl (Thailand), LS20 (Zambia), LS21 (India), LS25 (Kenya), LS32 (Nigeria) and LS33 (Ghana). The cultures were maintained at 37 C in a 5% suspension of infected erythrocytes (group A Rh + ) in complete medium (RPMI 1640 supplemented with D-glucose 2 g/l, NaHCO g/l, TES buffer g/l, gentamicin 40 mg/l and 12.5% human A + serum),

3 Antimalarial activity of pyronaridine and mepacrine 513 were flushed for 20 sec with a gas mixture of 3% O 2, 4% CO 2 and 93% N 2, prior to sealing. The culture medium was replaced daily and culture flasks returned to a W-8 Twist Action Shaker (New Brunswick Scientific Co.) set at approximately 370 oscillations per min, maintained at 37 C C. Cultures were passaged when parasitaemia rose above 10%. The state of the parasites and percentage parasitaemia were assessed daily from Giemsa stained thin films. A total of at least 1000 red blood cells or 100 parasites were counted and the parasitaemia expressed as a percentage. Parasite cultures were synchronised by incubating predominantly ring-stage parasitised cells in 15% sorbitol (1:3.5 v/v) for 5 min at 37 C (Vernes et al., 1984). Drugs tetraphosphate was a gift from Professor C. Chang (Institute of Parasitic Diseases, Shanghai, China). Mefloquine hydrochloride was obtained from Dr D. Davidson, Jr. (Walter Reed Army Institute of Research, Washington). diphosphate, amodiaquine dihydrochloride, quinine hydrochloride and mepacrine dihydrochloride were obtained from Sigma. Stock solutions of the test compounds at a concentration of 10 mm were prepared in sterile distilled water except for mefloquine and pyracrine which were initially dissolved in dimethyl sulphoxide (solvent concentration in tests did not exceed 0.02%). Further dilutions of the stock solutions were prepared in culture medium on the day of the experiment. Final drug concentrations used in the study were nm for pyronaridine, pyracrine and amodiaquine, nm for mepacrine and mefloquine, and nm for chloroquine and quinine. In-vitro antimalarial activity The sensitivity of the parasites to the drugs was determined using the 48 h, semi-automated microdilution technique described by O'Neill et al. (1985). Cultures with predominantly ring-stage parasites were used for drug susceptibility tests. Assays were performed using duplicate two-fold serial drug dilutions in 96-well microtitre plates, with each well containing 50 fil of the drug in complete medium. Fifty nl of infected erythrocytes (1 % parasitaemia and haematocrit 5%) in complete medium were added. The total volume of the suspension in each well was 100/^L with a final haematocrit of 2.5%, which has been shown to be an appropriate cell concentration for sensitivity evaluation (Gluzman, Schlesinger & Krogstad, 1987). The plates were kept at 37 C in a modular incubator chamber (Flow Labs), and flushed with the same gas mixture used for parasite culture. After 24 h, 0.2 fid of [ 3 H]-hypoxanthine (Amersham, 17.2 Ci/mmol) was added to each well. After a further 24 h the plates were harvested using a Skatron Cell Harvester to deposit red blood cells on to glass fibre filters (Titertek) and lysed by distilled water. The filters were dried, placed in liquid scintillating fluid (Packard 450) and counted on a liquid scintillation spectrometer (Beckman). Values (dpm) obtained from the scintillation counter were analysed using an Epson QX10 computer programme in which parasite growth inhibition/drug concentration curves were analysed by linear regression giving the ICM and IG» concentrations. Sole reliance on the IC50 is by itself not sufficient since in clinical practice what is desirable is complete inhibition and drug failure is usually due to parasite

4 514 E. I. Elueze et at. Table I. Antimalarial activity of chloroquine, pyronaridine, mepacrine, amodiaquine, mefloquine and quinine. Mean IC50 values (standard deviations) in nmol/l Strain/clone Amodiaquine Mefloquine Quinine T9.96 HI FC27 N NF54 T9.94L KJ LS20 LS21 LS25 LS32 LS (4) 14.4(5) 40.7(12) 20.2(10) 12.9(3) 31.5(11) 214.0(88) (2) 286.5(129) (42) (53) 284.0(13) 3.7(2) 4.4 (3) 14.5(8) 12.0(10) 1.9(0.1) 14.2(7) 8.2 (8) 11.0(11) 47.8 (23) 35.2 (4) 21.6(16) 5.4 (2) 13.0(6) 9.1(5) 18.3(8) 17.9(1) 8.3 (0.5) 17.8(6) 39.9(18) 24.8 (6) 66.4 (36) 67.2(14) 52.9(10) 29.1 (2) 6.4 (4) 8.5(1) 22.8(19) 16.3 (6) 10.1 (0.1) 18.3(10) 11.2(5) 19.0(4) 35.8(19) 31.2(8) 24.8 (6) 12.0(3) 115.0(72) 27.2 (6) 169.0(60) 28.1 (9) 43.9 (4) 17.5(2) 22.8 (2) 3.5(0.3) 15.2(15) 60.9 (27) 31.6(19) 14.5(12) 650.0(171) 122.0(46) 418.0(149) 159.5(14) 132.0(10) 310.0(80) 461.8(172) 285.0(14) (59) (26) (390) 490.0(113) survival at higher drug levels (Wernsdorfer & Payne, 1991). Each susceptibility test for a strain/clone was repeated at least twice. In the analysis, resistance to chloroquine was defined as IC*, ^ 160 nm (de Francisco Serpa et al., 1988; WHO, 1990). Unpaired /-tests were used to compare the IC50 and IG9 values of the test compounds for the CS and CR strains/clones. The Pearson correlation coefficients between the different drugs weje also determined. Results The IC50 and IG» values for pyronaridine, mepacrine, chloroquine, amodiaquine, quinine and mefloquine against all the strains/clones are shown in Tables I and II respectively. Parasite resistance to chloroquine, amodiaquine, mefloquine and quinine was defined using the cut-off values (minimum inhibitory concentrations) defined by Payne (1990), which are comparable to 99% inhibitory concentrations obtained from Table II. Antimalarial activity of chloroquine, pyronaridine, mepaenne, amodiaquine, mefloquine and quinine. Mean IC» values (standard deviations) in nmol/l Strain/clone T9.96 HI FC27 N NF54 T9.94L JC1 LS20 LS21 LS25 LS32 LS (13) 61.5(39) 96.3 (42) 60.5(12) 43.0(1) 196.0(75) (292) 699.0(251) (223) 1166(1.4) 924.0(141) 1325(388) 11.1(6) 15.9(10) 61.5(48) 38.8(13) 10.0(3) 63.1 (30) 20.9 (20) 65.6(60) 144.0(64) 117.0(61) 88.1 (11) 27.5 (20) 46.3(33) 23.9 (6) 104.0(90) 41.8(7) 21.4(5) 38.4(14) 95.3(38) 65.6 (6) (93) (205) 124.0(18) 101.0(0.7) Amodiaquine 32.0(16) 24.1 (0.2) 74.5 (38) 54.7 (40) 26.8 (0.4) 51.1(22) 26.2(10) 53.8(5) 80.7(31) 171.0(101) 69.1 (11) 39.4(12) Mefloquine 650.0(216) 281.0(4) (4) 118.0(29) (10) 163.0(64) 109.8(34) 48.3(3) (22) 174.0(64) 177.0(84) 66.7 (28) Quinine 2461 (1045) 1253(759) 2985(1038) (32) (243) (520) (17) (381) (511) 4132(1141) (130) (98)

5 Antimalarial activity of pyronaridine and mepacrine 515 the hypoxanthine incorporation test. No established threshold resistance values for pyronaridine and mepacrine have been defined. and mepacrine and mepacrine showed high antimalarial activity against both the CS and CR strains/clones with overall mean ICJO values of 15 and 30.4 nm respectively. The mean \C X value of chloroquine for all strains/clones was 148 nm. The ICM concentrations were 2.8-fold higher for pyronaridine (CS = 7.3 nm; CR = 20.5 nm), and 3.2-fold higher for mepacrine (CS = 13.3 nm; CR = 42.6) against isolates defined as CR than those defined as CS. -resistant parasites were 11 times less sensitive to chloroquine, with mean IC50 values of 21 nm for CS and 239 nm for CR. The IC50 and IG>9 values of chloroquine, mepacrine and pyronaridine compared between the CS and CR parasites showed a significant difference only for chloroquine (P = 0.003) and mepacrine (P = 0.01). The ratios of mean IC50 chloroquine/pyronaridine values for the CS and CR parasites respectively were 3.7 and The values for chloroquine/ mepacrine ratios were 1.7 and 7.1 respectively. These results indicate that, although pyronaridine and mepacrine were less active against CR than CS parasites, the difference in activity is much less than that for chloroquine., amodiaquine, mefloquine and quinine Of the twelve P. falciparum parasite strains/clones studied, five were sensitive to chloroquine and seven resistant (IG» > 160 nm). The 'LS' parasites (Table I) were CR isolates from patients who presented at the Hospital for Tropical Diseases, London, between 1988 and One CR strain (LS25) was clearly resistant to amodiaquine (IG» > 80 nm), and another one (LS21) showed a borderline value (80.7 nm). All the parasites studied were susceptible to mefloquine under the current WHO definition (IC99 < 1280 nm). Quinine was also effective against all the parasites, although the 99% inhibitory concentrations of two CR strains (LS25 and LS32) were close to the discriminatory value for quinine (5120 nm). Correlations The relationships between the sensitivity of the parasites to pyronaridine, mepacrine or chloroquine with the other test compounds were analysed (Table III). Using the IC» values there was a positive significant correlation between the response of the parasites to pyronaridine and to the following drugs: chloroquine (r = 0.63; P = 0.03), mepacrine (r = 0.87; P < 0.01) and amodiaquine (r = 0.95; P < 0.01). The pyronaridine IC*, values showed a significant correlation with those of mepacrine (r = 0.85; P < 0.01), amodiaquine (r = 0.77; P < 0.01) and quinine (r = 0.71; P = 0.0\). This significant correlation was paralleled by the IC50 and IG» responses to mepacrine and amodiaquine. There was no correlation between the sensitivity of the strains to chloroquine and to mefloquine (IC50 P = 0.33; IG» P = 0.07) or between the sensitivity to mefloquine and quinine (IC» P = 0.4; IG» P = 0.6).

6 516 E. I. Elueze et al. TaWe III. Relationship of response (IC» and IC99) of 12 strains/clones of P. falciparum to pyronaridine, mepacrine, chloroquine and other test compounds Drug Drug r P r P Significant. chloroquine amodiaquine quinine mefloquine chloroquine amodiaquine quinine mefloquine amodiaquine quinine mefloquine Discussion 0.03* * * 0.02* * * A high level of activity of pyronaridine, with a mean IC50 value of 15.0 nm (S.E.M. ± 4.0), was observed against all strains of P. falciparum in vitro in this study. Alin, Bjorkman & Ashton (1990) and Childs et al. (1988), using the WHO in-vitro microtest technique, also reported values between 10 and 20 nm, and Basco & Le Bras (1992), using the 3 H-hypoxanthine uptake technique, reported IC50 values between 6 and 10 nm. However, there are conflicting reports on cross resistance between pyronaridine and chloroquine. Studies in China (Chang et al., 1992) and on isolates from Central and West Africa (Basco & Le Bras, 1992) showed no correlation between resistance to the two drugs, while studies on isolates from Thailand (Schildbach et al., 1990) and Somalia (Warsame et al., 1991) showed significant correlation. In the present study we found a positive correlation between the sensitivity of P. falciparum parasites to chloroquine and pyronaridine at the ICJO level but not at the IC*> level for isolates from a wide range of geographical locations. also showed high activity against both CS and CR parasites. Activity was significantly correlated to the activities of pyronaridine and chloroquine. has the same alkyl amino alkyl side chain as chloroquine and the nucleus is close in structure to that of pyronaridine (Figure). The response to amodiaquine (which has a similar quinoline nucleus to chloroquine, and a Mannich base side chain like that of pyronaridine Figure) also was correlated to that of pyronaridine and chloroquine. These results suggest that the structures of both the nucleus and side chains of these antimalarials are important in activity and that both may be important in the evolution of cross resistance, a proposition supported by the observations of Kilimali, Mkufya & Kilama (1989). Peters & Robinson (1992) reported that the activities of a variety of Mannich base antimalarials were difficult to predict against lines of rodent Plasmodium that were resistant to chloroquine or to other Mannich bases. The relative structure-specificity of cross-resistance in P. falciparum in vitro contrasts with the rodent malaria system where a highly pyronaridine-resistant line was cross-resistant to

7 Antlmalarial activity of pyronaridlne and mepacrine 517 chloroquine, piperaquine, amopyroquine, bispyroquine, mepacrine and quinine, but was highly sensitive to sulphadoxine and pyrimethamine (Shao, 1990). Amodiaquine was active against most of the strains (mean ICso 18.0 nm; S.E.M. ± 2.7). Previous studies have established that amodiaquine is usually more active than chjoroquine against CR parasites in vitro but, as also observed in this study, amodiaquine frequently shows cross resistance with chloroquine (Childs et al., 1991; White, 1992). Activity of amodiaquine in vitro does not always correlate with sensitivity in vivo. In man, amodiaquine is rapidly transformed to the primary metabolite desethylamodiaquine, which is less active against CR P. falciparum than the parent compound. The susceptibility of the 'LS' isolates to quinine and mefloquine is reassuring since these drugs are used routinely either alone or in combination to treat cases of malaria at the Hospital for Tropical Diseases, London. A positive correlation was noted between the parasite response to quinine and chloroquine confirming the established 'chloroquine/quinine' relationship (Peters, 1987; Brasseur et al., 1988). In addition, there was a positive correlation between the parasite response to quinine and pyronaridine. The inverse relationship in the sensitivity of parasites to mefloquine and chloroquine observed in this study has also been reported by other workers (Hassan et al., 1992; Sowunmi et al., 1992). Despite the positive correlation between the sensitivity of the falciparum strains to chloroquine and pyronaridine, the high antimalarial activity of pyronaridine is evident from this study. If pyronaridine is used for the treatment and control of chloroquineresistant malaria, it will be important to monitor continuously the response of parasite populations to this drug and other standard antimalarials. Such field studies will also help determine the appropriate threshold values for parasite resistance to pyronaridine. Acknowledgement E. I. Elueze was sponsored by the Association of Commonwealth Universities Commission, UK. References Alin, M. H., Bjorkman, A. & Ashton, M. (1990). In vitro activity of artemisinin, its derivatives, and pyronaridine against different strains of Plasmodium falciparum. Transactions of the Royal Society of Tropical Medicine and Hygiene 84, Basco, L. K. & Le Bras, J. (1992). In vitro activity of pyronaridine against African strains of Plasmodium falciparum. Annals of Tropical Medicine and Parasitology 86, Basco, L. K, Mitaku, S., Skaltsounis, A.-L., Ravelomanantsoa, N., Tillequin, F., Koch, M. et al. (1994). In vitro activities of furoquinoline and acridone alkaloids against Plasmodium falciparum. Antimicrobial Agents and Chemotherapy 38, Berman, J., Brown, L., Miller, R., Andersen, S. L., McGreevy, P., Schuster, B. G. et al. (1994). Antimalarial activity of WR , a Dihydroacridinedione. Antimicrobial Agents and Chemotherapy 38, Brasseur, P., Kouamouo, J., Brandicourt, O., Moyou-Somo, R. & Druilhe, P. (1988). Patterns of in vitro resistance to chloroquine, quinine and mefloquine of Plasmodium falciparum in Cameroon, American Journal of Tropical Medicine and Hygiene 39, Chang, C, Lin-Hua, T. & Jantanavivat, C. (1992). Studies on the new antimalarial compound: pyronaridine. Transactions of the Royal Society of Tropical Medicine and Hygiene 86, Childs, G. E., Boudreau, E. F., Wimonwattratee, T., Pang, L. & Milhous, W. K. (1991). In vitro and clinical correlates of mefloquine resistance of Plasmodium falciparum in Eastern Thailand. American Journal of Tropical Medicine and Hygiene 44,

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