Efficacy of Proveblue (Methylene Blue) in an Experimental Cerebral Malaria Murine Model Jérome Dormoi, Sébastien Briolant, Camille Desgrouas and Bruno Pradines Antimicrob. Agents Chemother. 2013, 57(7):3412. DOI: 10.1128/AAC.02381-12. Published Ahead of Print 22 April 2013.
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Efficacy of Proveblue (Methylene Blue) in an Experimental Cerebral Malaria Murine Model Jérome Dormoi,a,b Sébastien Briolant,a,b,c,d Camille Desgrouas,e Bruno Pradinesa,b Unité de Parasitologie, Département d’Infectiologie de Terrain, Institut de Recherche Biomédicale des Armées, Marseille, Francea; Aix Marseille Université, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UM 63, CNRS 7278, IRD 198, INSERM 1095, Marseille, Franceb; Direction Interarmées du Service de Santé, Cayenne, French Guiana, Francec; Laboratoire de Parasitologie, Institut Pasteur de la Guyane, Cayenne, French Guiana, Franced; UMR MD3, Institut de Recherche Biomédicale des Armées, Marseille, Francee
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n 2002, the World Health Organization (WHO) recommended that artemisinin-based combination therapy (ACT) should be used for all cases of uncomplicated malaria. Four years later, the WHO added the recommendation that artesunate should be deployed as the first-line treatment for severe malaria in adults and should replace quinine due to its efficacy and better tolerance (1). In 2010, a large-scale trial confirmed artesunate potency in the treatment of severe malaria in children (2). In 2011, the WHO recommended artesunate as the first-line treatment for severe malaria. In recent years, several studies have reported clinical failures or at least extended parasite clearance times in Cambodia (3–5). A large amount of scientific effort is spent on elucidating the mechanisms underlying resistance to antimalarial drugs, with the hope of restoring/improving the efficacy of existing drugs and developing new drugs that can bypass the resistance mechanisms. One strategy that has been used to reduce the prevalence of malaria is drug combinations. The combination protects each drug from the development of resistance and reduces the overall transmission rate of malaria (6). There is an urgent need to discover partners for combination with artemisinin derivatives. Proveblue (international patent no. PCT/FR/2007/001193), which is a methylene blue preparation that complies with the European Pharmacopoeia and contains limited organic impurities and heavy metals of recognized toxicity, was previously demonstrated to possess in vitro antimalarial activity (at a geometric mean 50% inhibitory concentration [IC50] of 3.62 nM) against 23 Plasmodium falciparum strains that were resistant to various other antimalarials (7). No significant association was found between the Proveblue IC50 and polymorphisms in the genes that are involved in quinoline resistance, such as pfcrt, pfmdr1, pfmdr2, pfmrp, and pfnhe-1; furthermore, there was no significant association between the Proveblue IC50 and the copy numbers of pfmdr1 and pfmdr2 (7). Although Proveblue was shown to have in vitro antagonistic effects in combination with chloroquine and additive effects in combination with desethylamodiaquine against nine P. falciparum strains, it exhibited noticeable synergistic effects in combination with mefloquine and quinine and highly synergistic effects in combination with dihydroartemisinin, the active metabolite of artemisinin derivatives (8). The objective of the present work was to evaluate the in vivo
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efficacy of Proveblue alone and in combination with dihydroartemisinin in a murine model of experimental cerebral malaria. Animal models do not exactly reproduce human malaria, but they nevertheless exhibit some similarities to human cerebral malaria, and the Plasmodium berghei ANKA rodent parasite model is generally accepted as a valid model for studying experimental cerebral malaria pathogenesis (9, 10). However, the use of the murine model as an indicator of cerebral malaria has recent detractors (11). Forty female C57Bl6/N mice, 6 to 7 weeks old and weighing 18 to 22 g (Charles River, France), were infected on day 0 (D0) with P. berghei ANKA parasites by intraperitoneal (i.p.) inoculation with 105 parasitized erythrocytes diluted in normal saline from infected donor C57BL/6N mice. All animals were pathogen free and housed under standard conditions, with unlimited access to food and water. All efforts were made to minimize animal suffering. All experiments adhered to French guidelines for animal research and were approved by the ethical committee of the Institut de Recherche Biomédicale des Armées—Antenne de Marseille (number 2007-09). Except for the control group treated by i.p. injection of normal saline solution (8 mice), the mice were treated when parasitemia reached 0.1% by daily i.p. injection with 3 mg/kg dihydroartemisinin (10 mice), 10 mg/kg Proveblue (9 mice), or 3 mg dihydroartemisinin combined with 10 mg/kg Proveblue (9 mice) for 5 days. All the drugs were dissolved in normal saline solution. Parasitemia was determined daily using Giemsa-stained thin blood smears collected from the tail vein as the number of infected red blood cells per 3,000 erythrocytes if ⬎1% and per 10,000 erythrocytes if ⬍1%. The animals were observed daily for clinical signs, neurological symptoms, and weight. Experimental cerebral malaria was diagnosed by clinical signs based on a simplified
Received 29 November 2012 Returned for modification 5 March 2013 Accepted 14 April 2013 Published ahead of print 22 April 2013 Address correspondence to Bruno Pradines,
[email protected]. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.02381-12
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Although 100% of untreated mice infected with Plasmodium berghei died with specific signs of cerebral malaria and 100% of mice treated with 3 mg/kg dihydroartemisinin, the active metabolite of artesunate, which is used as the first-line treatment for severe malaria, also died but showed no specific signs of cerebral malaria, 78% of mice treated with 10 mg/kg Proveblue (methylene blue) and 78% of mice treated with a combination of 3 mg dihydroartemisinin and 10 mg/kg Proveblue survived and showed no specific signs of cerebral malaria or detectable parasites.
Proveblue Is Effective in Cerebral Malaria
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FIG 1 Survival curve of C57Bl6/N mice infected on day 0 (D0) with P. berghei ANKA parasites and treated daily with 3 mg/kg dihydroartemisinin, 10 mg/kg Proveblue, or 3 mg/kg dihydroartemisinin combined with 10 mg/kg Proveblue for 5 days. Arrows indicate the times of treatment.
SHIRPA protocol (12) with at least two symptoms in at least two of the three different groups: (i) alteration of autonomous functions (piloerection, defecation, urination, and respiration rate); (ii) alteration of muscle tone and strength (grip strength, body tone, limb tone, and abdominal tone); (iii) ataxia, paralysis (mono-, hemi-, para-, or tetraplegia), deviation of the head, convulsions, and coma. The data were analyzed using R software (version 2.10.1). Survival analyses were performed by the Kaplan-Meier log rank test. The comparison of medians between multiple groups was analyzed by the Kruskal-Wallis test. The comparison of medians between two groups was analyzed by the Mann-Whitney test. A difference was considered significant when P values were less than 0.05. In the control group, all the mice died before D10, with specific signs of cerebral malaria and parasitemia of ⬍10.5% (6.7 to 10.5%) (Fig. 1). The observed symptoms for cerebral malaria were consistent with that of previous publications (13, 14). Parasite was detectable by blood smear in six mice (60%) after 2 days of treatment with dihydroartemisinin and in 100% of the mice after 4 days. The mice treated with 3 mg/kg dihydroartemisinin died between D16 and D25, with no specific signs of cerebral malaria and parasitemia between 80.6% and 97.3%. In general, artemisinin derivatives failed to prevent malaria death or cerebral malaria (14). Mice which did not die of cerebral malaria treated by artesunate died of severe anemic malaria (13, 14). In the group of mice treated with 10 mg/kg Proveblue, one mouse (11%) had recurrent parasites at D5 and died at D16 (50.0% parasitemia), and another had recurrent parasites at D11 and died at D25 (91.7% parasitemia), with no specific signs of cerebral malaria. After 2 days of treatment with Proveblue, no parasite was detectable by blood smear. At D45, the surviving mice showed no parasites. In the group of mice treated with 3 mg dihydroartemisinin combined with 10 mg/kg Proveblue, no parasite was detectable by blood smear after 2 days of treatment. One mouse (11%) had recurrent parasites at D5 and died at D15 (70.0% parasitemia), and another had recurrent parasites at D15 and died at D28 (98.0% parasitemia), with no specific signs of cerebral malaria. At D45, the surviving mice had no parasites.
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Treatment with dihydroartemisinin, Proveblue, or a combination of Proveblue and dihydroartemisinin was significantly effective against cerebral malaria (P ⱕ 0.0005). Moreover, all the mice treated with dihydroartemisinin alone died of anemia with a parasitemia of ⱖ80%, not cerebral malaria. Seventy-eight percent of the mice treated with Proveblue survived at a regimen of 10 mg/ kg. This dosage of 10 mg/kg has been shown to be safe in children treated with methylene blue that was noncompliant with the European Pharmacopoeia (15, 16). The combination of Proveblue and dihydroartemisinin was more effective than dihydroartemisinin alone in the experimental cerebral malaria mouse model (P ⫽ 0.005). However, the efficacy of the combination may be due to the efficacy of Proveblue and not to synergism of the two compounds (no significant difference between Proveblue alone and the combination of Proveblue and dihydroartemisinin efficacy; P ⫽ 0.732). Artemisinin should have a synergistic interaction with methylene blue; artemisinin reoxidizes leucomethylene blue, which is produced by the reduction of methylene blue in parasites by the NADPH-flavin reductase system, from methylene blue, and together they oxidize FADH2 (17). However, methylthioninium chloride (Proveblue) is a drug with a short terminal elimination half time of 18.5 h, and dihydroartemisinin has also a short elimination half time (0.5 to 2 h). For future use as antimalarial treatment for uncomplicated malaria, a triple combination with a drug with long elimination half time and a different mode of action, such as piperaquine, should be considered. Some new artemisinin derivatives (artemiside and artemisone) associated with piperaquine prevent malaria death or cerebral malaria in cerebral malaria murine models (14). The present work demonstrates the high efficacy of Proveblue in cerebral malaria in comparison with dihydroartemisinin, the active metabolite of the antimalarial drug recommended by the WHO as the first-line treatment for severe malaria. Another advantage in the use of Proveblue as an antimalarial drug is that methylene blue inhibits the maturation of gametocytes and their transmission (18, 19). These results confirm the therapeutic potential of Proveblue, which is a new methylene blue formulation that contains limited organic impurities and heavy metals of rec-
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ognized toxicity and could be integrated into the pipeline of new antimalarial combination therapies (20).
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ACKNOWLEDGMENTS This work was supported by the Délégation Générale pour l’Armement (grant no PDH-2-NRBC-4-B1-402). We thank Salah Mécheri from the Biology of Host-Parasite Interactions Unit (Pasteur Institute, Paris, France) for providing the Plasmodium berghei ANKA strains and protocols and Philippe Grellier from the National Museum of Natural History (Paris, France) for discussion on protocols. We have no conflicts of interest concerning the work reported in this paper.
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