How robust are malaria parasite clearance rates as indicators of drug effectiveness and resistance?

Artemisinin combination therapies (ACTs) are currently the first line drugs for treating uncomplicated falciparum malaria, the most deadly of the human malarias. Malaria parasite clearance rates estimated from patients' blood following ACT treatment have been widely adopted as a measure of drug effectiveness and as surveillance tools for detecting the presence of potential artemisinin drug resistance. This metric has not been investigated in detail, nor have its properties or potential shortcomings been identified. Herein, the pharmacology of drug treatment, parasite biology, and human immunity are combined to investigate the dynamics of parasite clearance following ACT treatment. This approach parsimoniously recovers the principal clinical features and dynamics of clearance. Human immunity is the primary determinant of clearance rates unless, or until, artemisinin killing has fallen to near-ineffective levels. Clearance rates are therefore highly insensitive metrics for surveillance that may lead to over-confidence as even quite substantial reductions in drug sensitivity may not be detected as slower clearance rates. Equally serious is the use of clearance rates to quantify the impact of ACT regime changes as this strategy will plausibly miss even very substantial increases in drug effectiveness. In particular, the malaria community may be missing the opportunity to dramatically increase ACT effectiveness through changes in regimen, particularly through a switch to twice-daily regimens and/or increases in artemisinin dosing levels. The malaria community therefore appears over reliant on a single metric of drug effectiveness, parasite clearance rate that has significant and serious shortcomings

Altering Antimalarial Drug Regimens May Dramatically Enhance and Restore Drug Effectiveness.

There is considerable concern that malaria parasites are starting to evolve resistance to the current generation of antimalarial drugs, the artemisinin-based combination therapies (ACTs). We use pharmacological modeling to investigate changes in ACT effectiveness likely to occur if current regimens are extended from 3 to 5 days or, alternatively, given twice daily over 3 days. We show that the pharmacology of artemisinins allows both regimen changes to substantially increase the artemisinin killing rate. Malaria patients rarely contain more than 10(12) parasites, while the standard dosing regimens allow approximately 1 in 10(10) parasites to survive artemisinin treatment. Parasite survival falls dramatically, to around 1 in 10(17) parasites if the dose is extended or split; theoretically, this increase in drug killing appears to be more than sufficient to restore failing ACT efficacy. One of the most widely used dosing regimens, artemether-lumefantrine, already successfully employs a twice-daily dosing regimen, and we argue that twice-daily dosing should be incorporated into all ACT regimen design considerations as a simple and effective way of ensuring the continued long-term effectiveness of ACTs

Stability of Dihydroartemisinin-Piperaquine Tablet Halves during Prolonged Storage under Tropical Conditions

Dihydroartemisinin-piperaquine (DP) is recommended for the treatment of uncomplicated malaria, used in efforts to contain artemisinin resistance, and increasingly considered for mass drug administration. Due to the narrow therapeutic dose range and available tablet strengths, the manufacturers and World Health Organization recommended regimens involve breaking tablets into halves to accurately dose children according to body weight. Use of tablet fractions in programmatic settings under tropical conditions requires a highly stable product, however, the stability of DP tablet fractions is unknown. We aged full and half Eurartesim® DP tablets in a stability chamber at 30°C and 70% humidity level. The active pharmaceutical ingredients dihydroartemisinin and piperaquine remained at ≥95% over the 3 months period of ageing in light and darkness. These findings are reassuring for DP, but highlight the need to assess drug stability under real-life settings during the drug development process, particularly for key drugs of global disease control programmes

The Effects of Pharmacogenetics on Pharmacokinetics\ud of Artemisinin-Based Combinations in Malaria Patients

Malaria is a vector-borne infectious disease caused by protozoan parasites of the genus Plasmodium. If not treated appropriately, human P. falciparum malaria can quickly become life-threatening, leading to an estimated 900’000 annual deaths globally. Key interventions to control malaria include prompt diagnosis and effective treatment with artemisinin-based combination therapies (ACTs), use of insecticide treated nets by people at risk, indoor residual spraying with insecticide to control the vector mosquitoes and intermittent preventive treatment for pregnant women (IPTp) and infants (IPTi). Whether antimalarial treatments are effective or not, depends on parasite and host factors. The ability to define resistance leading to treatment failure has been greatly enhanced by our understanding of the underlying molecular mechanisms causing resistance in P. falciparum. However, the potential contribution of host genetic factors, particularly those associated with antimalarial drug metabolism, remains largely unexplored. The same applies for the basic mechanisms involved in the pharmacokinetics of antimalarial drugs and the link between antimalarial drug pharmacokinetics and treatment outcomes. Thus, the purpose of this thesis was to quantify the effects of pharmacogenetics on pharmacokinetics of ACTs. Between 2007 and 2008, three in vivo studies were performed in Cambodia and Tanzania. Patients reporting with fever associated with an infection with Plasmodium falciparum were recruited and treated with ACTs according to the national guidelines in the respective country. In Cambodia, 64 patients were recruited for the treatment with artesunate–mefloquine and 61 for the treatment with dihydroartemisinine–piperaquine. In Tanzania, 150 were treated with artemether–lumefantrine. Blood samples for the pharmacokinetic analysis were taken before treatment and at several time points during and after treatment, e.g. on Days 1, 2 and 7 in all studies and in Cambodia also 1 hour after the first dose and on Day 14. For the analysis of plasma samples collected during our studies, we developed a broad-range liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) assay covering 14 of the currently in-use antimalarial drugs and their metabolites. The assay requires only as little as 200 μl of plasma and is a major improvement over previous methods in terms of convenience, sensitivity, selectivity and throughput. The method was validated according to well-established recommendations. The assay was first used for the analysis of the baseline samples collected in our in vivo studies. In all studies more than half of the patients recruited had still antimalarials in their blood. Theses findings enabled us to get a better assessment of the antimalarials circulating in the local population, and hence of the drug pressure on the parasites in both countries. Single nucleotide polymorphisms (SNPs) in genes encoding enzymes associated with antimalarial drug metabolism, i.e. cytochrome P450 isoenzymes (CYP) and N-acetyltransferase 2 (NAT2), were analyzed. Based on our previous experience, we developed a DNA microarray to affordably generate SNP data. However, after comparison of microarray data and sequencing data, we concluded that the major limit of the microarray technology was lack of robustness which could not be compensated by superior cost-effectiveness. Consequently, the pharmacogenetic profiles of the patients from the three in vivo studies were assessed by direct sequencing of genomic DNA. Whereas for most SNPs allele frequencies were similar in both populations, we found significant inter-ethnic differences in the distribution of genotypes of certain enzymes, namely CYP2D6, CYP3A4/5 and NAT2. Is has been shown that the human CYP3A subfamily plays a dominant role in the metabolic elimination of more drugs than any other biotransformation enzyme. Therefore, our findings might have implications for treatment policies of not only antimalarials and the widely introduced ACTs in particular, but any other drugs metabolized by these enzymes. To quantify the effect of pharmacogenetics on pharmacokinetics of ACTs we developed population pharmacokinetic models. The pharmacokinetic parameters we estimated in our models were in agreement with those from previous studies. In order to account for parts of the inter-individual variability in drug-metabolizing capacity of the liver we included pharmacogenetic data as covariate. For artemether, we found that 9% of the inter-individual variability in clearance could be explained by the genotype of CYP3A5 (reference allele versus variant allele CYP3A5*3). Heterozygous carriers showed a reduction in clearance of 34%. The alterations in clearance were less pronounced for lumefantrine (increase in clearance of 12% in homozygous carriers of variant allele CYP3A4*1B, explaining 2% of the inter-individual variability in clearance) and mefloquine (decrease in clearance of 14% in carriers of homozygous variant allele CYP3A5*5, explaining 1% of the inter-individual variability in clearance). These data might partially provide an explanation for the differences in drug efficacy observed with artemether–lumefantrine combination treatment. In conclusion, we were able to show that there is a correlation between the pharmacogenetic profile of the host and the pharmacokinetics of antimalarial drugs administered in malaria patients. These results suggest that pharmacogenetics could be one of the basic mechanisms involved in the pharmacokinetics of antimalarial drugs. The knowledge gained from this study could facilitate the selection process of first-line treatment for malaria and would allow dosing adaptation based on the pharmacogenetic profile of the population. Such adaptations are needed especially in the most vulnerable groups, including infants, pregnant women, and those with prevalent co-morbidities, where often therapeutic antimalarial drug concentrations over time are not achieve

Pharmacological considerations in the design of anti-malarial drug combination therapies - is matching half-lives enough?

Anti-malarial drugs are now mainly deployed as combination therapy (CT), primarily as a mechanism to prevent or slow the spread of resistance. This strategy is justified by mathematical arguments that generally assume that drug 'resistance' is a binary all-or-nothing genetic trait. Herein, a pharmacological, rather than a purely genetic, approach is used to investigate resistance and it is argued that this provides additional insight into the design principles of anti-malarial CTs. It is usually suggested that half-lives of constituent drugs in a CT be matched: it appears more important that their post-treatment anti-malarial activity profiles be matched and strategies identified that may achieve this. In particular, the considerable variation in pharmacological parameters noted in both human and parasites populations may compromise this matching and it is, therefore, essential to accurately quantify the population pharmacokinetics of the drugs in the CTs. Increasing drug dosages will likely follow a law of diminishing returns in efficacy, i.e. a certain increase in dose will not necessarily lead to the same percent increase in efficacy. This may allow individual drug dosages to be lowered without proportional decrease in efficacy, reducing any potential toxicity, and allowing the other drug(s) in the CT to compensate for this reduced dosage; this is a dangerous strategy which is discussed further. Finally, pharmacokinetic and pharmacodynamic drug interactions and the role of resistance mechanisms are discussed. This approach generated an idealized target product profile (TPP) for anti-malarial CTs. There is a restricted pipeline of anti-malarial drugs but awareness of pharmacological design principles during the development stages could optimize CT design pre-deployment. This may help prevent changes in drug dosages and/or regimen that have previously occurred post-deployment in most current anti-malarial drugs

Quantifying the pharmacology of antimalarial drug combination therapy.

Most current antimalarial drugs are combinations of an artemisinin plus a 'partner' drug from another class, and are known as artemisinin-based combination therapies (ACTs). They are the frontline drugs in treating human malaria infections. They also have a public-health role as an essential component of recent, comprehensive scale-ups of malaria interventions and containment efforts conceived as part of longer term malaria elimination efforts. Recent reports that resistance has arisen to artemisinins has caused considerable concern. We investigate the likely impact of artemisinin resistance by quantifying the contribution artemisinins make to the overall therapeutic capacity of ACTs. We achieve this using a simple, easily understood, algebraic approach and by more sophisticated pharmacokinetic/pharmacodynamic analyses of drug action; the two approaches gave consistent results. Surprisingly, the artemisinin component typically makes a negligible contribution (≪0.0001%) to the therapeutic capacity of the most widely used ACTs and only starts to make a significant contribution to therapeutic outcome once resistance has started to evolve to the partner drugs. The main threat to antimalarial drug effectiveness and control comes from resistance evolving to the partner drugs. We therefore argue that public health policies be re-focussed to maximise the likely long-term effectiveness of the partner drugs

The effects of pharmacogenetics on pharmacokinetics of artemisinin-based combinations in malaria patients

Malaria is a vector-borne infectious disease caused by protozoan parasites of the genus Plasmodium. If not treated appropriately, human P. falciparum malaria can quickly become life-threatening, leading to an estimated 900’000 annual deaths globally. Key interventions to control malaria include prompt diagnosis and effective treatment with artemisinin-based combination therapies (ACTs), use of insecticide treated nets by people at risk, indoor residual spraying with insecticide to control the vector mosquitoes and intermittent preventive treatment for pregnant women (IPTp) and infants (IPTi). Whether antimalarial treatments are effective or not, depends on parasite and host factors. The ability to define resistance leading to treatment failure has been greatly enhanced by our understanding of the underlying molecular mechanisms causing resistance in P. falciparum. However, the potential contribution of host genetic factors, particularly those associated with antimalarial drug metabolism, remains largely unexplored. The same applies for the basic mechanisms involved in the pharmacokinetics of antimalarial drugs and the link between antimalarial drug pharmacokinetics and treatment outcomes. Thus, the purpose of this thesis was to quantify the effects of pharmacogenetics on pharmacokinetics of ACTs. Between 2007 and 2008, three in vivo studies were performed in Cambodia and Tanzania. Patients reporting with fever associated with an infection with Plasmodium falciparum were recruited and treated with ACTs according to the national guidelines in the respective country. In Cambodia, 64 patients were recruited for the treatment with artesunate–mefloquine and 61 for the treatment with dihydroartemisinine–piperaquine. In Tanzania, 150 were treated with artemether–lumefantrine. Blood samples for the pharmacokinetic analysis were taken before treatment and at several time points during and after treatment, e.g. on Days 1, 2 and 7 in all studies and in Cambodia also 1 hour after the first dose and on Day 14. For the analysis of plasma samples collected during our studies, we developed a broad-range liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) assay covering 14 of the currently in-use antimalarial drugs and their metabolites. The assay requires only as little as 200 μl of plasma and is a major improvement over previous methods in terms of convenience, sensitivity, selectivity and throughput. The method was validated according to well-established recommendations. The assay was first used for the analysis of the baseline samples collected in our in vivo studies. In all studies more than half of the patients recruited had still antimalarials in their blood. Theses findings enabled us to get a better assessment of the antimalarials circulating in the local population, and hence of the drug pressure on the parasites in both countries. Single nucleotide polymorphisms (SNPs) in genes encoding enzymes associated with antimalarial drug metabolism, i.e. cytochrome P450 isoenzymes (CYP) and N-acetyltransferase 2 (NAT2), were analyzed. Based on our previous experience, we developed a DNA microarray to affordably generate SNP data. However, after comparison of microarray data and sequencing data, we concluded that the major limit of the microarray technology was lack of robustness which could not be compensated by superior cost-effectiveness. Consequently, the pharmacogenetic profiles of the patients from the three in vivo studies were assessed by direct sequencing of genomic DNA. Whereas for most SNPs allele frequencies were similar in both populations, we found significant inter-ethnic differences in the distribution of genotypes of certain enzymes, namely CYP2D6, CYP3A4/5 and NAT2. Is has been shown that the human CYP3A subfamily plays a dominant role in the metabolic elimination of more drugs than any other biotransformation enzyme. Therefore, our findings might have implications for treatment policies of not only antimalarials and the widely introduced ACTs in particular, but any other drugs metabolized by these enzymes. To quantify the effect of pharmacogenetics on pharmacokinetics of ACTs we developed population pharmacokinetic models. The pharmacokinetic parameters we estimated in our models were in agreement with those from previous studies. In order to account for parts of the inter-individual variability in drug-metabolizing capacity of the liver we included pharmacogenetic data as covariate. For artemether, we found that 9% of the inter-individual variability in clearance could be explained by the genotype of CYP3A5 (reference allele versus variant allele CYP3A5*3). Heterozygous carriers showed a reduction in clearance of 34%. The alterations in clearance were less pronounced for lumefantrine (increase in clearance of 12% in homozygous carriers of variant allele CYP3A4*1B, explaining 2% of the inter-individual variability in clearance) and mefloquine (decrease in clearance of 14% in carriers of homozygous variant allele CYP3A5*5, explaining 1% of the inter-individual variability in clearance). These data might partially provide an explanation for the differences in drug efficacy observed with artemether–lumefantrine combination treatment. In conclusion, we were able to show that there is a correlation between the pharmacogenetic profile of the host and the pharmacokinetics of antimalarial drugs administered in malaria patients. These results suggest that pharmacogenetics could be one of the basic mechanisms involved in the pharmacokinetics of antimalarial drugs. The knowledge gained from this study could facilitate the selection process of first-line treatment for malaria and would allow dosing adaptation based on the pharmacogenetic profile of the population. Such adaptations are needed especially in the most vulnerable groups, including infants, pregnant women, and those with prevalent co-morbidities, where often therapeutic antimalarial drug concentrations over time are not achieved

Age‑shifting in malaria incidence as a result of induced immunological deficit: a simulation study

Effective population-level interventions against Plasmodium falciparum malaria lead to age-shifts, delayed morbidity or rebounds in morbidity and mortality whenever they are deployed in ways that do not permanently interrupt transmission. When long-term intervention programmes target specific age-groups of human hosts, the age-specific morbidity rates ultimately adjust to new steady-states, but it is very difficult to study these rates and the temporal dynamics leading up to them empirically because the changes occur over very long time periods. This study investigates the age and magnitude of age- and time- shifting of incidence induced by either pre-erythrocytic vaccination (PEV) programmes or seasonal malaria chemo-prevention (SMC), using an ensemble of individual-based stochastic simulation models of P. falciparum dynamics. The models made various assumptions about immunity decay, transmission heterogeneity and were parameterized with data on both age-specific infection and disease incidence at different levels of exposure, on the durations of different stages of the parasite life-cycle and on human demography. Effects of transmission intensity, and of levels of access to malaria treatment were considered. While both PEV and SMC programmes are predicted to have overall strongly positive health effects, a shift of morbidity into older children is predicted to be induced by either programme if transmission levels remain static and not reduced by other interventions. Predicted shifting of burden continue into the second decade of the programme. Even if long-term surveillance is maintained it will be difficult to avoid mis-attribution of such long-term changes in age-specific morbidity patterns to other factors. Conversely, short-lived transient changes in incidence measured soon after introduction of a new intervention may give over-positive views of future impacts. Complementary intervention strategies could be designed to specifically protect those age-groups at risk from burden shift