TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1999) 93,651-652

idine diluted in PBS (experimental group) or an equivalent volume of PBS alone (control group). This dose is equivalent to that given to humans over 3 days (CHEN et al., 1992). Giemsa-stained peripheral blood Assessment pyronaridine activity in smears were observed daily until 14 days after sporozoite of inoculation. vivo and in vitro against the hepatic In vitro, pyronaridine was hepatotoxic at the concen- stages of malaria in laboratory mice trations of 100 phi, 10 pM, and 1 pM, as evidenced by morphological features of cell death as well as cell detachment. No hepatotoxic effect was observed at Leonardo K asco’, Pasc ’ gwald’,Jean-Fran- 100 nM, 10 nM, and 1 nM in the hepatocyte culture. Fois Franeti&’ and Do&t%Maziel2 ‘Institutde However, there was only a slight decrease (P > 0.05) in Recherche pour le Développement (IRD) - Laboratoire de the number of hepatic schizonts in pyronaridine-treated Recherche sur le Paludisnte, Laboratoire Associé Franco- culture at 100 nM, as compared with untreated culture, phone 302, Organisation de Coordination pour la lutte contre while there was no schizontocidal effect at 10 n~ and les Endémies en Afrique Centrale (OCEAC), B. P. 288, 1 n~ (Figure). Yaoundé, Cameroon; ’INSERM Unité 51 1, Immunobiolo- In vivo, 5 days post-infection, 4 of 5 control mice were gie Cellulaire et Moléculaire des bzfections Parasitaires, found to be parasitized, whereas the 5 mice treated with Centre Hospitalo- Universitaire Pitié-SalpêtGre, 91 Boule- pyronaridine were free of blood-stage parasites (Table). vard de l’Hôpital, 75013 Paris, France From day 6 to day 12, all untreated controls were found to be parasitized, and all treated mice were still negative. Our experiments in vitro and in vivo suggest that Keywords: malaria, Plasmodium yoelii, mice, sporozoites, liver pyronaridine has a blood-schizontocidal, but not hepa- stages, drug resistance, pyronaridine tic-schizontocidal, action against P. yoelii. Pyronaridine is known to exert a potent blood-scliizontocidal action. Chloroquine-resistant Plasmodiziiiz falciparunz malaria The growth in vitro of the human malaria parasites P. is now widespread in many parts ofthe African continent, faleiparum, P. ovale, and P. malariae is idhibited at where a large majority of malaria infections occur. One of (50 nM during a 48-72-h incubation (CHILDS et al., the promising new drugs that may replace chloroquine 1988; RINGWALDet al., 1996, 1997; PRADINES et al., for the first-line treatment of uncomplicated falciparum malaria in Afiica is pyronaridine. This synthetic deriva- tive of the acridine-type Mannich bases was developed in China and was found to possess high blood-schizonto- 100 cidal activity against rodent, simian, and human malaria 2., parasites (CHEN et al., 1992). Subsequent clinical stud- Y ies have confirmed the safety, tolerance, and efficacy of u7 pyronaridine to treat chloroquine-resistant P. faleiparum 80 malaria infections (LOOAREESUWANet al., 1996; RING- f U WALD etal., 1996). Although its potent action against the .-E asexual blood stages of various malaria species has been 60 .I2 demonstrated, the possible activity of pyronaridine L.

against the hepatic stages has not been documented. I4 We performed experiments in vitro and in vivo to evaluate 40 whether pyronaridine also acts against the liver stages of 8 malaria parasites. ... Experiments in vitro were performed using C57/B16 ...8 20 mouse hepatocytes infected with sporozoites of P. yoelii Y yoelii. In brief, hepatocytes were isolated by collagenase z3 perfusion as previously described (MAZIERet al., 1986) and seeded in 8-chamber plastic Lab-Teck slides (Nalge O Nunc International,O4 Naperville, IL, USA) at a concen- 1WnM lOnM 1nM tration of 8 X 1 cells per well, in Williams Medium E Pyronandine concenuarion (Bio Whittaker, Walkersville, ML, USA), supplemented with 10% fetal calf serum, 100 units/mL of penicillin Figure. Activiry in vitro ofpyronaridine against pre-erythrocytic and 10 mg/mL of streptomycin. Sporozoites were ob- stages of P. yoelii. Reduction in the number of hepatic schizonts tained by dissection of salivary glands of Anopheles was estimated by counting schizonts in 48-h cultures. Results are expressed as mean f standard deviation of treated cultures stephensi infected with malaria parasites, crushed in a compared to untreated cultures (number of schizonts = 77.75 glass grinder and diluted in culture media. Pyronaridine f was first diluted in phosphate-buffered saline (PBS) for a 142. 1O-mM stock solution and then diluted from 1 n~ to phi 100 in culture medium and added with sporozoites Table. Activity of pyronaridine in vivo against P. (35 OOO/well) on hepatocyte cultures (24 h old). After yoelii mice 3 h, cultures were washed and the medium was replaced in with fresh medium or’diluted drug. Forty-eight hours Number of parasitized mice after infection, cultures were fixed with cold methanol and parasites labelled with an immunofluorescent assay. Schizonts were counted and culture was observed with a Mouse group Day5 Day6 Day 12 fluorescence microscope. Untreated controls q5 515 515 Experiments in vivo were carried out in C57/B16 Pyronaridine-treated 015 015 015 mice. One hour after intravenous injection of 5000 P. Treated and control group mice (n = 5 in each group) were yoelii yoelii sporozoites, mice were injected subcuta- X injected intravenously with 5 lo3 P. yoelii sporozoites in neously with 30 mg/kg (0.6 mg per mouse) of pyronar- phosphate-buffered salme (PBS). Treatment, given 1 h after infection, was with 30 mg/kg of pyronaridine (experimental Address for correspondence: Leonardo Basco, OCEAC, B. P. group) diluent only (control group). The delay the O0 or of 288, Yaoundé, Cameroon; phone 4-237 23 61, fax +237 23 appearance of blood parasites was detennined from Giemsa- O0 61, e-mail [email protected] stained blood smears taken daily. “I i - I Fonds Documentaire QRSTOM LEONARDO K. BASCO ETAL.

1998). In the present study, no growth inhibition was of pyronaridine for the treatment of amte uncomplicated observed in the infected hepatocytes at lOOnM and falciparum malaria in Thailand. Americaiz Jountal of Tropical 10 nM of pyronaridine. The results of higher drug Medicine aitd Hygiene,S., 54, 205-209. concentrations were uninterpretable owing to drug Mazier, D., Mellouk, Beaudoin, R. I., Texier, B., Druilhe, P., toxicity on hepatic cells. Thus, the suppressive effect of Hockmeyer, W., Trosper, P., Paul, C., Charoenvit, Y., Young, J., Miltgen, F., Chedid, L., Chigot, J. P., Galley, B., pyronaridine observed in vivo in our study was most Brandicourt, O. & Gentilini, M. (1986). Effect of antibodies , likely to have been due to its blood-schizontocidal action. to recombinant and synthetic peptides on Phntodium falci- Currently available antimalarial agents and antibiotics parum sporozoites in vitro. Science, 231, 156-159. used in antimalarial chemotherapy that possess tissue- Radines, B., Tall, A., Parzy, D., Spiegel, A., Fusai, T., Hienne, schizontocidal activity include primaquine, doxycycline, R., Trape, J. F. & Doury, J. C. (1998). In-vitro activity of and proguanil (WERNSDORFER,1997). Like other deri- pyronaridine and amodiaquine against African isolates (Se- vatives belonging to the Mannich bases (amodiaquine, negal) of Plasinodiunzfalciparuin in comparison with standard amopyroquin) and 4-aminoquinolines, pyronaridine did antimalarial agents. JoumalofAnti?iaic~obialChemotherapy, 42, 333-339. not exert any effect on the hepatic stages of malaria Ringwald, P., Bickii, J. &Basco, L. (1996). Randomised mal of parasites. Nonetheless, because of its established blood- pyronaridine versus chloroquine for acute uncomplicated schizontocidal activity, even against chloroquine-resis- falciparum malaria in Africa. Lancet, 347, 24-28. tant malarial infections, pyronaridine is a promising drug Ringwald, P., Bickii, J., Same-Ekobo, A. &Basco, L. K. (1997). for antimalarial chemotherapy. Pyronaridine for the treatment of Plarmodiuin ovale and P.

8 malariae infections. Antinticmbial Agents and Clzewzotherapy, References 41,2317-2319. In: Chen, C., Tang, L. H. & Jantanavivat, C. (1992). Studies on a Wemsdorfer, W. H. (1997). Antimalarial drugs. Handbook new antimalarial compound pyronaridine. Traitsactiom of the of Malaria btfection in the Tropics, Carosi, G. & Castelli, F. Royal Society of Tropical Medicine and Hygiene, 86,7- 10. (editors). Organizzazione per la Cooperazione Sanitaria Childs, G. E., Hausler, B., Milhous, W., Chen, C., Wimonwat- Internazionale, Bologna, Italy, Quademi di cooperazione trawatee, T., Pooyindee, N. & Boudreau, E. F. (1988). In sanitaria, no. 15, pp. 151-198. vim activity of pyronaridine against field isolates and refer- ence clones of Plasmodium falciparuna. American Jo~malof Tropical Medicine and Hyp.ene, 38, 24-29. Looareesuwan, S., Kyle, D. E., Viravan, C., Vanijanonta, S., Received 2 July 1999; revised 21 July 1999; accepted for Wilairatana, P. & Wemsdorfer, W. H. (1996). Clinical study publication 27Jdy 1999

I BookReview I assessment’ is of interest, as its Australian setting means I I that stakeholder participation is taken for granted; this is refreshing in view of how often it is neglected in the Water Resources: Health, Environment and developing world, and so is Birley’s re-christening of Development. B. H. Kay (editor). London: E & F N ‘Health opportunities assessment’ in the following Spon: 1999. xviii + 250pp. Price E55.00. ISBN 0-419- chapter. 22290-1. Where things get more interesting is in the case- studies; there are 3 chapters on Australia, 2 on the Coming 25 years after Stanley and Alpers’ classic book Tennessee Valley Authority (an old example, but the on man-made lakes and human health, this book can be update is worth reading), 1 from Thailand, a fascinating seen in some senses as a sequel. The former book had an historical chapter on fish culture and malaria in Indone- Australian editor, and so does this, although it is not sia, and the editor adds a final overview of urban vector- unduly antipodean; Australians have a significant experi- borne disease problems. ence to share in this area, and their 6 chapters (out of 15) One recurring theme is the unpredictability of many of do not seem too many. The area has been expanded th? health impacts and political forces which can now be significantly through the WHO/FAOEJNEP Panel of documented with hindsight. No one foresaw that Murray Experts on Environmental Modification for Vector Valley encephalitis could be transported over long dis- Control (PEEM), and this book arose from a PEEM- tances by viraemic birds, that wastewater-irrigated wet- sponsored conference in Brisbane. lands would produce 30 times more mosquitoes, or that The era of building large dams is over now, except in. villagers downstream of a dam would complain that the China, which has half the world’s big dams. Elsewhere, low level of dissolved oxygen would make the water maintenance is the chief concern. The book tells us much unsuitable for bathing or washing. In the circumstances, about maintenance, but precious little about dams in it is salutary that the case-studies mention a number of China, which in the circumstances is a shortcoming. One ways to monitor some of the health impacts, such as the would also have wished to see more about the research use of sentinel flocks of chickens to provide early warning work sponsored by the West African Rice Development of arbovirus epidemics. As the authors of the Thai case- Association, which is mentioned only once. study conclude, ‘it would seem rather ambitious to The editor’s avowed aim was to ‘acquaint the reader forecast the health impact of water resources develop- with a variety of topics without going into too much ment’. It is nevertheless worth the attempt, and this book detail’, and indeed the book is something ofa dilettante’s will be of interest to anyone who tries to do so. delight (although some of the introductory chapters on overall principles and the policies of intemational agen- Sandy Cairncross cies are so general that it is hard to find the interest in WELL Resource Centre them). PEEM’s manuals are more useful to practitioners London School of Hygiene and Tropical Medicine than these, but the guidelines and checklists do help. Keppel Street The chapter on ‘Environmental and health impact London WClE 7HT, UK I 1

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