Fixed-dose pyronaridine-artesunate combination for treatment of uncomplicated falciparum malaria in pediatric patients in Gabon

BACKGROUND: The development of novel artemisinin-combination therapies suitable for the treatment of pediatric patients suffering from malaria is a research priority. The aim of this study was to investigate a novel fixed-dose pyronaridine-artesunate combination for the treatment of uncomplicated falciparum malaria in Gabonese patients 2-14 years old. METHODS: The study was designed as an open-label dose-escalation study recruiting 60 pediatric patients sequentially in 4 treatment cohorts: study drugs were administered once daily for 3 days, as tablet coformulations (pyronaridine:artesunate ratios of 6:2, 9:3, and 12:4 mg/kg) and as a granule coformulation (pyronaridine:artesunate ratio of 9:3 mg/kg). The primary end points were tolerability, safety, and pharmacokinetics of pyronaridine-artesunate treatment. Efficacy was treated as a secondary outcome measure. RESULTS: The drugs had a good tolerability and safety profile, at all dose levels. Pharmacokinetic analysis revealed a dose-dependent increase in the maximum plasma/blood concentration and the area under the curve, as well as comparable relative bioavailability for the granule coformulation. Polymerase chain reaction-corrected cure rates at day 28 were 100% in per-protocol analysis, at all dose levels. CONCLUSIONS: Pyronaridine-artesunate is a promising novel artemisinin-combination therapy for pediatric patients with uncomplicated Plasmodium falciparum malaria, and the development of both the tablet and the granule coformulations is warranted.     

The Glutamate-Rich Protein (GLURP) of Plasmodium falciparum Is a Target for Antibody-Dependent Monocyte-Mediated Inhibition of Parasite Growth In Vitro

Monocyte-dependent as well as direct inhibitory effects of antimalarial antibodies point toward antigens accessible at the time of merozoite release as targets for biologically active antibodies capable of mediating protection against Plasmodium falciparum. The glutamate-rich protein (GLURP), being an antigen associated with mature schizont-infected erythrocytes, was therefore the object of the present investigation, in which we analyzed whether anti-GLURP antibodies can either interfere directly with merozoite invasion or act indirectly by promoting a monocyte-dependent growth inhibition, antibody-dependent cellular inhibition. GLURP-specific human immunoglobulin G (IgG) antibodies, from pooled IgG of healthy Liberian adults who were clinically immune to malaria, were purified by affinity chromatography on columns containing R0 (N-terminal nonrepetitive region of GLURP) or R2 (C-terminal repetitive region of GLURP) recombinant protein or synthetic peptides as ligands. Analysis of the pattern of reactivity of highly purified anti-GLURP antibodies led to the definition of at least four B-cell epitopes. One epitope was specific for R0, two were specific for R2, and the fourth displayed cross-reactivity between R0 and R2. None of the purified IgG antibodies had direct invasion-inhibitory effects, even at high concentrations. In contrast, when allowed to cooperate with monocytes, all anti-GLURP IgG preparations mediated a strong monocyte-dependent parasite growth inhibition in a dose-dependent manner.Epidemiological surveys performed in areas of intense malaria transmission have consistently shown that individuals who are continuously exposed to repeated malaria infection gradually develop clinical immunity (14, 20, 29). This acquired immunity is strong, although incomplete, and is nonsterilizing (3, 25, 26). Experiments with antibodies purified from the sera of African adults who were clinically immune to malaria and given by passive transfer to susceptible children have established that immunoglobulin G (IgG) is at least a main component of defense against the asexual blood stage of Plasmodium falciparum (5, 9, 11). Recent passive transfer experiments have enabled us to acquire clinically demonstrated protective antibodies from the donor and nonprotective antibodies from the recipients. These sets of antibodies were used to assess the extent to which the in vitro data correlated with the in vivo results for each recipient isolate (5). Results from these in vitro studies suggested that clinically protective antibodies had little direct effect on merozoite invasion, but that they could act in conjunction with blood monocytes to contain parasite multiplication. This mechanism was called antibody-dependent cellular inhibition (ADCI) (5, 17, 19).The assay provides a screen to select molecules which may be targeted by clinically effective antibodies. Further experiments have indicated that antibody-monocyte cooperation in parasite inhibition is mediated not through parasite opsonization but rather through indirect effects. These activities were mediated by soluble monocyte-derived substances whose release was triggered through monocyte interaction with cytophilic antibodies bound to merozoite antigens (7). A critical role for merozoite surface molecules in this mechanism is also supported by the identification of Msp3, a new molecule from the merozoite surface, when an expression library was screened by ADCI (23).Based on the published immunoepidemiological data for the glutamate-rich protein (GLURP) (4, 12, 13, 15) and the report that this molecule is located on the surface of the merozoite (2), we chose to investigate the potential of affinity purified anti-GLURP human IgG in assays of direct parasite inhibition and to compare this with activity in ADCI assays.     

Quasi-compulsory treatment of drug dependent offenders: an international literature review

This paper reports on a review of the literature on the quasi-compulsory treatment (QCT) of drug dependent offenders in five languages; English, German, French, Italian and Dutch. The findings of this review on previous reviews and on the availability, process, and outcomes of QCT are summarized. The review found that previous, anglophone reviews have tended to present positive outcomes from QCT, but that there are some problems with this research. QCT is increasingly available internationally, but may be applied at different stages of the criminal justice process, and to different types of offender. Research on the process of QCT is comparatively rare. The available research does suggest problems of system integration between criminal justice and treatment agencies in implementing QCT. The research in languages other than English shows a wider range of outcomes (including negative effects) for QCT than was found in the English literature. We conclude that the international literature shows that QCT does not inevitably produce worse outcome than voluntary treatment, but that we need more multimethod, multisite studies of QCT in order to inform policy and practice, which is currently being made in the absence of reliable evidence in many countries.     

Human malaria in immunocompromised mice: an in vivo model to study defense mechanisms against Plasmodium falciparum

We have recently described that sustained Plasmodium falciparum growth could be obtained in immunodeficient mice. We now report the potential of this new mouse model by assaying the effect of the passive transfer of antibodies (Abs) which in humans have had a well-established effect.Our results show that the total African adult hyperimmune immunoglobulin Gs (HI-IgGs) strongly reduce P. falciparum parasitemia similarly to that reported in humans, but only when mice are concomitantly reconstituted with human monocytes (HuMNs). In contrast, neither HI-IgGs nor HuMNs alone had any direct effect upon parasitemia. We assessed the in vivo effect of epitope-specific human Abs affinity-purified on peptides derived either from the ring erythrocyte surface antigen (RESA) or the merozoite surface protein 3 (MSP3). The inoculation of low concentrations of anti-synthetic peptide from MSP3, but not of anti-RESA Abs, consistently suppressed P. falciparum in the presence of HuMNs. Parasitemia decrease was stronger and faster than that observed using HI-IgGs and as fast as that induced by chloroquine. Our observations demonstrate that this mouse model is of great value to evaluate the protective effect of different Abs with distinct specificity in the same animal, a step hardly accessible and therefore never performed before in humans.     

Immunization of Saimiri sciureus monkeys with Plasmodium falciparum merozoite surface protein-3 and glutamate-rich protein suggests that protection is related to antibody levels

The immunogenicity and protective efficacy of various antigen-adjuvant formulations derived either from the merozoite-surface protein-3 (MSP-3) or the glutamate-rich protein (GLURP) of Plasmodium falciparum were evaluated in Saimiri sciureus monkeys. These proteins were selected for immunogenicity studies based primarily on their capacity of inducing an antibody-dependent cellular inhibition effect on parasite growth. Some of the S. sciureus monkeys immunized with MSP-3(212-380)-AS02 or GLURP(27-500)-alum were able to fully or partially control parasitaemia upon an experimental P. falciparum [Falciparum Uganda Palo Alto (FUP-SP) strain] blood-stage infection, and this protection was related to the prechallenge antibody titres induced. The data are indicative that MSP-3 and GLURP can induce protective immunity against an experimental P. falciparum infection using adjuvants that are acceptable for human use and this should trigger further studies with those new antigens.     

Cytophilic immunoglobulin responses to Plasmodium falciparum glutamate-rich protein are correlated with protection against clinical malaria in Dielmo, Senegal

The goal of this study was to analyze antibody responses to Plasmodium falciparum glutamate-rich protein (GLURP) using clinical data and plasma samples obtained from villagers of Dielmo, Senegal. This molecule was chosen because it is targeted by human antibodies which induce parasite growth inhibition in antibody-dependent cellular inhibition (ADCI) assays. The results showed a strong correlation between protection against malaria attacks and levels of immunoglobulin G2 (IgG2) and IgG3 against GLURP(94-489) (R0) and IgG3 against GLURP(705-1178) (R2) when corrected for the confounding effect of age-related exposure to malaria. Thus, GLURP may play a role in the induction of protective immunity against P. falciparum malaria.     

Merozoite surface protein-3: a malaria protein inducing antibodies that promote Plasmodium falciparum killing by cooperation with blood monocytes

We have previously found that the acquired protection against malaria implicates a mechanism of defense that relies on the cooperation between cytophilic antibodies and monocytes. Accordingly, an assay of antibody-dependent cellular inhibition (ADCI) of parasite growth was used as a means of selecting for molecules capable of inducing protective immunity to malaria. This allowed us to identify in the sera of clinically protected subjects an antibody specificity that promotes parasite killing mediated by monocytes. This antibody is directed to a novel merozoite surface protein (MSP-3) of a molecular mass of 48 kD. Purified IgG from protected subjects are effective in ADCI and those directed against MSP-3 are predominantly cytophilic. In contrast, in nonprotected individuals, whose antibodies are not effective in ADCI, anti-MSP-3 antibodies are mostly noncytophilic. A region in MSP-3 targetted by antibodies effective in the ADCI assay was identified and its sequence was determined; it contains an epitope not defined by a repetitive structure and does not appear to be polymorphic. Antibodies raised in mice against a peptide containing this epitope, as well as human antibodies immunopurified on this peptide, elicit a strong inhibition of Plasmodium falciparum growth in ADCI assay, whereas control antibodies, directed to peptides from other molecules, do not. The correlation between isotypes of antibodies produced against the 48- kD epitopes, clinical protection, and the ability of specific anti-MSP- 3 antibodies to block the parasite schizogony in the ADCI assay suggests that this molecule is involved in eliciting protective mechanisms.     

Absence of Association between Piperaquine In Vitro Responses and Polymorphisms in the pfcrt,pfmdr1, pfmrp,and pfnhe Genes in Plasmodium falciparum

We have analyzed the profiles of 23 of Plasmodium falciparum strains for their in vitro chemosusceptibilities to piperaquine (PPQ), dihydroartemisinin (DHA), chloroquine, monodesethylamodiaquine, quinine, mefloquine, lumefantrine, atovaquone, pyrimethamine, and doxycycline (DOX) in association with polymorphisms in genes involved in quinoline resistance (Plasmodium falciparum crt [pfcrt], pfmdr1, pfmrp, and pfnhe). The 50% inhibitory concentrations (IC₅₀s) for PPQ ranged from 29 to 98 nM (geometric mean = 57.8 nM, 95% confidence interval [CI] = 51 to 65) and from 0.4 to 5.8 nM for DHA (geometric mean = 1.8 nM, 95% CI = 1.4 to 2.3). We found a significant positive correlation between the responses to PPQ and DHA (r² = 0.17; P = 0.0495) and between the responses to PPQ and DOX (r² = 0.41; P = 0.001). We did not find a significant association between the PPQ IC₅₀ (0.0525 < P < 0.9247) or the DHA IC₅₀ (0.0138 < P < 0.9018) and polymorphisms in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 genes. There was an absence of cross-resistance with quinolines, and the IC₅₀s for PPQ and DHA were found to be unrelated to mutations in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 transport protein genes, which are involved in quinoline antimalarial drug resistance. These results confirm the interest in and the efficacy of the combination of PPQ and DHA for areas in which parasites are resistant to chloroquine or other quinolines.Over the past 20 years, many strains of Plasmodium falciparum have become resistant to chloroquine (CQ) and other antimalarial drugs (32). This development has prompted the search for an effective alternative antimalarial drug with minimal side effects. One strategy for reducing the prevalence of malaria is the combinatorial use of drugs, which is thought to protect against the development of resistance to each drug and to reduce the overall rate of transmission of malaria (52). Since 2001, more than 60 countries have officially adopted artemisinin-based combination therapies (ACTs) for the treatment of falciparum malaria (40), and the official first-line antimalarial treatment in Africa is now ACT (18). The artemisinin derivatives cause rapid and effective reductions in the parasite biomass and the level of gametocyte carriage, while the partner drug, which has a longer duration of action, achieves effective clinical and parasitological cure. Several different ACTs have been evaluated, including artesunate-sulfadoxine-pyrimethamine (PY) (50), artesunate-amodiaquine (7), artemether-lumefantrine (LMF) (53), artesunate-mefloquine (MQ) (2), artesunate-chlorproguanil-dapsone (42), artesunate-atovaquone (ATV)-proguanil, artesunate-pyronaridine (44), and dihydroartemisinin (DHA)-piperaquine (PPQ) (1, 22, 47).However, individual P. falciparum isolates resistant to artemisinin in vitro and the first clinical failures have been described in Cambodia (10, 16, 39, 45). In addition, prior therapy with an amodiaquine-containing ACT has been found to select for a reduced response to monodesethylamodiaquine (MDAQ), suggesting that amodiaquine-containing regimens may rapidly lose efficacy in Africa (38). This emergence of parasites resistant to ACTs underlines the fact that novel compounds and combinations must be discovered and developed.DHA-PPQ is an inexpensive, safe, and highly effective treatment for uncomplicated falciparum and vivax malaria (36, 43). DHA-PPQ has been shown to offer a longer posttreatment prophylactic effect following therapy than artemether-lumefantrine (27, 53, 54) or artesunate-amodiaquine (23). The significantly lower risk of recurrent parasitemia after treatment with DHA-PPQ is likely explained by differences in the pharmacokinetics of the nonartemisinin partner drugs. PPQ, a bisquinoline, is estimated to have an elimination half-life of 17 to 33 days (28, 47, 48), while the elimination half-life of lumefantrine is 4 to 10 days (19) and that of amodiaquine is 1 to 6 h (the half-life of its active metabolite, monodesethylamodiaquine, is 1 to 10 days). Bisquinolines are compounds with two quinoline nuclei bound by a covalent aliphatic or aromatic link.The first aim of the present work was to assess the in vitro cross-resistance of PPQ with other quinoline drugs, whether they are artemisinin partners or not. The following drugs were tested: CQ, quinine (QN), MQ, MDAQ, LMF, DHA, ATV, PY, and doxycycline (DOX). The second aim was to identify genetic polymorphisms in the Plasmodium falciparum crt (pfcrt) pfcrt, pfmrp, pfmdr1, and pfnhe-1 genes, which are known to be associated with reduced quinoline susceptibility, that could be associated with decreased susceptibility to PPQ with the goal of identifying molecular markers of PPQ resistance for use in resistance surveillance.