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Am. J. Trop. Med. Hyg., 71(3), 2004, pp. 294-296
Copyright © 2004 by The American Society of Tropical Medicine and Hygiene

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SHORT REPORT: LACK OF PREDICTION OF AMODIAQUINE EFFICACY IN TREATING PLASMODIUM FALCIPARUM MALARIA BY IN VITRO TESTS

AGNÉS AUBOUY, JUSTICE MAYOMBO, ANNICK KEUNDJIAN, MOHAMED BAKARY, JACQUES LE BRAS, AND PHILIPPE DELORON
Centre International de Recherches Médicales de Franceville, Unité de Parasitologie Médicale, Franceville, Gabon; Unité de Parasitologie, Institut de Médecine Tropicale du Service de Santé des Armées, le Pharo, Marseille Armées, France; Hôpital de Bakoumba, Bakoumba, Gabon; Hôpital Bichat-Claude Bernard, Laboratoire de Parasitologie, Paris, France; Institut de Recherche pour le Développement, Unité de Recherche 010 Mother and Child Health in the Tropics, Paris, France

 

ABSTRACT

Amodiaquine (AQ) is currently a major candidate for new antimalarial combinations, although in vivo and in vitro tests have been rarely simultaneously investigated. The efficacy of AQ was assessed at the dose of 30 mg/kg in treating Plasmodium falciparum malaria attacks in 74 children from southeast Gabon, and the in vitro activity of monodesethylamodiaquine (MdAQ), the main metabolite of AQ, was measured against P. falciparum parasites isolated from these children. Treatment failures were observed in 40.5% of the children, while 5.4% of the isolates showed in vitro resistance to MdAQ. No relationship was observed between in vivo and in vitro susceptibility. The in vitro activities of MdAQ and chloroquine were correlated. The reasons for such disparities between in vivo and in vitro AQ activities are discussed and the issue of the validity of in vitro tests to measure AQ efficacy is raised.

In vivo and in vitro tests have been in use for more than 30 years for assessing the activity of drugs against Plasmodium falciparum.1 Although it has been clearly shown that the in vitro activity of chloroquine (CQ) was highly correlated with CQ treatment efficacy,2 this is not the case with numerous other antimalarials. Amodiaquine (AQ) is one of the cost-efficient alternatives to resistant parasites in Africa. However, very few studies investigated AQ activity, simultaneously using both in vivo and in vitro tests, and the relationship between both methods remains poorly documented. Therefore, we compared results of both methods for AQ and analyzed the reasons for the disparities.

The study was part of a larger treatment efficacy assay in children (<10 years old) comparing sulfadoxine-pyrimethamine versus AQ described elsewhere.3 Briefly, AQ treatment efficacy (30 mg/kg over a three-day period given under supervision, Camoquin®; Parke Davis, Dakar, Senegal) was assessed by the revised World Health Organization (WHO) in vivo protocol for areas of intense transmission,4 extended to 28 days of follow-up. A total of 125 children were enrolled in this study, of whom 118 completed the 28-day follow-up. Merozoite surface protein 1 (msp 1) and msp 2 genotyping was used to distinguish recurrence of malaria due to recrudescence from those due to reinfection. In vivo responses were classified according to the WHO scheme4 for an adequate clinical and parasitologic response, early treatment failure, late clinical failure, or parasitologic failure. Plasma samples were collected at day 3 for measurement by high-performance liquid chromatography of the levels of AQ and monodesethylamodiaquine (MdAQ), the main metabolite of AQ. The study was reviewed and approved by the Center International de Recherches Médicales de Franceville ethical committee, and informed consent was obtained from all parents or guardians.

The in vitro drug sensitivity assay was assessed by the isotopic microtest with MdAQ and CQ, as described.5 The 50% inhibitory concentration (IC50) value was defined as the drug concentration corresponding to 50% of the uptake of 3H-hypoxanthine in the drug-free control wells. The calculation was based on linear regression analysis of the logarithm of concentrations plotted against the percentage of growth inhibition. According to previously published data,2,6 the threshold IC50 values for CQ and MdAQ in vitro resistance were fixed at 100 nM (according to a direct comparison of in vivo and in vitro data) and 60 nM (more arbitrarily), respectively. Data were analyzed with the Statview software (SAS Institute Inc, Cary, NC).

The median (range) IC50 values were 146.5 (3–629) nM and 20 (2–257) nM for CQ and MdAQ, respectively. The IC50 values for 75.6% and 5.4% of the isolates were greater than the threshold of in vitro resistance to CQ and MdAQ, respectively. The IC50 values for both drugs showed a correlation with each other (Figure 1Go) (r = 0.45, P < 0.0001, by linear regression analysis). Discrepancies between in vivo and in vitro results were observed in 31 of the 74 cases successfully studied by both methods: 29 isolates were susceptible in vitro (IC50 ≤ 60 nM) to MdAQ, but led to treatment failures, while 2 of the 4 isolates with in vitro resistance to MdAQ led to treatment successes. The role of the factors that may influence treatment outcome was investigated (Table 1Go). Subjects with in vivo failures were younger (P = 0.04, by Mann-Whitney U test). Day 3 plasma concentrations of MdAQ, or MdAQ plus AQ did not differ in children in which AQ treatment outcome was a success or a failure. Similarly, these concentrations did not correlate with IC50 values for CQ or MdAQ.



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  		    FIGURE 1. Correlation between in vitro responses to monodesethylamodiaquine (MdAQ) and chloroquine (CQ) among 72 Plasmodium falciparum Gabonese isolates from Bakoumba, Gabon, 2000. Resistance thresholds are shown at 60 nM for MdAQ and 100 nM for CQ. Two samples, one amodiaquine (AQ) resistant and one AQ sensitive, were not studied with CQ. IC50 = 50% inhibitory concentration.

 

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TABLE 1
Malaria attack characteristics, in vitro activity of monodesethylamodiaquine (MdAQ) and chloroquine (CQ), and post-treatment plasma drug levels according to amodiaquine (AQ) treatment outcome Bakoumba, Gabon, 2000*
 
In this study, 5.4% of the isolates showed in vitro resistance to MdAQ, while a treatment failure to AQ occurred in 40.5% of the children. No clear relationship between treatment response and the in vitro IC50 of AQ was detected. Similarly, Brasseur and others7 reported a two-fold higher rate of resistance in vivo than in vitro. However, the in vitro tests were carried out with AQ instead of MdAQ. Conversely, Trenholme and others,8 Ringwald and others,9 and Basco and others10 reported lower treatment failure rates compared with the rate of in vitro resistance. In endemic areas, the higher prevalence rate of in vitro resistance may be related to the immune response, adding its effect to that of the drug in clearing the parasite infection. Although differences in the methodology of in vivo and in vitro tests do not allow an accurate comparison of these studies to our results, there was no correlation between resistance in vivo and in vitro in most of these studies.

The accuracy of the threshold for MdAQ in vitro resistance may be questioned. The threshold for CQ was established 25 years ago2,6 by direct comparison of data from treatment efficacy of the standard therapeutic regimen of CQ and in vitro tests results. In the case of MdAQ, as well as most other antimalarial drugs, this threshold was calculated from the mean IC50 of a number of isolates plus two standard errors.6 These determinations were done in areas where the level and the prevalence rate of AQ resistance were then low. Nevertheless, in our study, MdAQ IC50 values were similarly low in isolates from both adequate responses and therapeutic failures.

Drug intake before enrollment can modify the outcome of in vivo as well as in vitro tests results. An inadequate treatment regimen of AQ may also constitute a possible factor of discordance with in vitro results. The regimen of 25 mg/kg was first used and is still commonly used, but the higher efficiency of a 35 mg/kg regimen has been reported.11,12 No other comparative study was conducted to determine the best regimen, but it was shown that increasing the doses of CQ from 25 to 35, 40, or 50 mg/kg only delayed the appearance of recrudescences.13

Our larger study reporting AQ versus SP efficacy for malaria treatment in Gabonese children3 showed that treatment failures were characterized by reduced MdAQ, and/or MdAQ plus AQ plasma concentrations. The metabolism of AQ via MdAQ depends on cytochrome P(450) 2C8 enzyme activity14 and large interindividual variation in metabolizing CYP2C8 substrates has been reported.15 Genetic variants of CYP2C8 have also been reported16 with various frequencies among different ethnic groups, and some variants being associated with altered activity.17 Therefore, the large interindividual variations in pharmacokinetics of AQ may contribute to in vivo and in vitro discrepancies.

In humans, the metabolism of AQ results in two metabolites other than MdAQ: bidesethylamodiaquine (BdAQ) and hydroxydesethylamodiaquine (HdAQ). The in vitro antimalarial activity of HdAQ is 1% that of AQ against CQ-sensitive strains.18,19 In addition, BdAQ was shown to have a very low in vitro activity and to achieve very low blood levels.20 However, Mount and others showed that the in vitro activity of BdAQ was 10–30% that of AQ,19 suggesting that in vitro tests performed with MdAQ may not adequately reproduce the complex in vivo situation achieved following treatment with AQ. Amodiaquine was shown to be 1–3 times more effective in vitro than MdAQ,18,19 suggesting that remaining levels of AQ after metabolism should be considered when measuring blood concentrations of the drug. In our study, the addition of AQ concentrations to MdAQ did not alter the results or improve the in vivo-in vitro data agreement. This also suggests that it may be worthwhile to conduct in vitro testing not only with either AQ or MdAQ, but also with a mixture of these two drugs.

High-level transmission areas are characterized by large number of infective clones, as we previously showed at the site of this study.3 Such a factor may increase the complexity of in vitro tests, and raise questions about their significance. A resistant clone that is poorly represented in the blood may not have a sufficient effect to substantially increase the IC50, but may later cause an in vivo failure. Furthermore, at the time of sampling, selected parasites may be sequestered and absent from the peripheral circulation.21,22

New strategies are urgently needed to address the question of the waning lifetime of antimalarial treatments. Methods to evaluate treatment efficacy are of utmost importance because they help to develop treatment recommendations. Our data demonstrate that treatment failure may not necessarily be due to resistance, and that in the case of AQ and probably many other antimalarials, in vitro testing cannot replace in vivo testing and should instead used as an early warning tool. Our study raises the problem of the validity of in vitro tests to measure AQ efficacy, and stresses the necessity to validate an in vitro threshold of resistance and to validate its predictive value.

Received January 23, 2004. Accepted for publication April 9, 2004.

Acknowledgments: We are grateful to the children who participated in the study, as well as to their mothers and guardians. We also thank J. Bourgeais (Le Site de la Société d’Exploitation du Parc de la Lékédi) for logistical support in Bakoumba.

Financial support: This work was supported by the French Ministry of Research (VIHPAL grant) and by the Fondation pour la Recherche Médicale. Agnes Aubouy was the recipient of a fellowship from the French Ministry of Research.

Authors’ addresses: Agnes Aubouy and Philippe Deloron, Faculté de Pharmacie, Institut de Recherche pour le Développement, Unité de Recherche 010, 4 Avenue de l’Observatoire, 75006 Paris, France, Telephone: 33-1-53-73-96-22. Justice Mayombo, Center International de Recherches Médicales de Franceville, BP 769 Franceville, Gabon, Telephone: 241-677-096. Annick Keundjian, Unité de Parasitologie, Institut de Médecine Tropicale du Service de Santé des Armées, le Pharo, BP46, 13998 Marseille Armées, France, Telephone: 33-4-91-50116. Mohamed Bakary, Hôpital de Bakoumba, BP52, Bakoumba, Gabon, Telephone: 241-661-101. Jacques Le Bras, Hôpital Bichat-Claude Bernard, 75877 Paris, France, Telephone: 33-1-40-25-78-99.

Reprint requests: Philippe Deloron, Institut de Recherche pour le Développement, Unité de Recherche 010, Faculté de Pharmacie, 4 Avenue de l’Observatoire, 75006 Paris, France, Telephone: 33-1-53-73-96-22, Fax: 33-1-53-73-96-17, E-mail: Philippe.Deloron{at}ird.fr.

 

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