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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, January 2008, p. 306-311, Vol. 52, No. 1
0066-4804/08/$08.00+0 doi:10.1128/AAC.00878-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
School of Pharmacy, Curtin University of Technology, Bentley,1 School of Biomedical Sciences, Curtin University of Technology, Bentley,2 School of Medicine and Pharmacology, University of Western Australia, Crawley,3 Clinical Pharmacology & Toxicology Laboratory, Path West Laboratory Medicine, Nedlands, Western Australia, Australia4
Received 5 July 2007/ Returned for modification 9 September 2007/ Accepted 28 October 2007
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ABSTRACT |
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INTRODUCTION |
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Contemporary clinical data for PQ-DHA suggest that this product has several advantages over established combinations such as artesunate-mefloquine and artemether-lumefantrine, including greater efficacy, improved adherence, better tolerability, and lower cost (8, 13, 20, 26, 27). Importantly, clinical studies of PQ-DHA have reported cure rates of 95 to 99% with 3-day dosage regimens in uncomplicated falciparum malaria (1, 9, 14, 24, 26, 27).
Although PQ has been used for many years in China, there are few reports with detailed preclinical and pharmacokinetic data (8, 12, 21, 23). For example, limited animal toxicity data have been published and although the therapeutic and prophylactic efficacy of PQ monotherapy was documented in early clinical trials, the therapeutic index of PQ remains poorly defined (8, 11, 14, 15, 21). Data are also lacking on the relationship between PQ serum concentrations and toxicity or efficacy (15). Nevertheless, clinical studies have demonstrated that in current antimalarial treatment strategies PQ is well tolerated, has a rapid blood schizonticidal action against P. falciparum, and shows high-level prophylactic efficacy for 3 weeks after the administration of a single dose (1, 6).
In order to address the paucity of preclinical pharmacokinetic, efficacy, and safety data that are normally required by regulatory authorities and are essential for future research, our goal was to investigate the pharmacokinetic and pharmacodynamic properties of PQ in a murine malaria model. Hence, the aim of the present study was to obtain robust pharmacokinetic and pharmacodynamic data following the administration of single doses of PQ to mice.
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MATERIALS AND METHODS |
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Mice. This study was approved by the Curtin University Animal Experimentation Ethics Committee. Male Swiss mice (5 to 6 weeks old; average weight, 29.5 ± 3.3 g) were obtained from the Animal Resource Centre (Murdoch, Western Australia, Australia). Male BALB/c mice (7 to 8 weeks old; Animal Resource Centre) were used for weekly passage of malaria parasites. Animals were housed at 22°C under a 12-h light/dark cycle with free access to sterilized commercial food pellets (Glen Forrest Stockfeeders, Perth, Western Australia, Australia) and sterilized, acidified water (HCl; pH 2.5) to prevent bacterial infections (22, 25).
Parasites. Plasmodium berghei ANKA parasites were obtained from the Australian Army Malaria Research Institute (Enoggera, Queensland, Australia) and maintained by continuous weekly blood passage in BALB/c mice. A standard inoculum of 107 parasitized erythrocytes per 100 µl was prepared by dilution of blood harvested from BALB/c mice (>30% parasitemia) in citrate-phosphate-dextrose solution (16) and administered by intraperitoneal (i.p.) injection to infect the experimental (Swiss) mice.
Parasite enumeration in infected mice. Peripheral blood smears were prepared by using blood obtained from the tail veins of infected experimental mice. The thin films were fixed in methanol (3 min) and then stained with May-Grünwald Giemsa with a Hema-Tek staining machine (Ames Co., Elkhart, IN). Blood smears were examined at a magnification of x100 by oil immersion light microscopy with a Leica DMLS light microscope (Leica Microsystems, Gladsville, New South Wales, Australia). Parasitemia was determined by counting 30 or 100 fields of view for >0.5% and <0.5% infected erythrocytes, respectively. This procedure ensured an acceptable standard error of 22% at 0.1% parasitemia (16) and a limit of detection on the order of 0.002% parasitemia. Tail vein bleeds were performed three times a day for the first 5 days after drug treatment, twice daily for the next 2 weeks, and then daily until the time of euthanasia (>40% parasitemia, >10% reduction in mouse body weight in less than 24 h or termination of the experimental protocol). Mice were euthanized by sodium pentobarbitone injection (50 to 100 mg/kg i.p.).
Drug treatment. PQP is slightly soluble in water and was suspended in a mixture of 50% (vol/vol) glycerol, 30% (vol/vol) isotonic phosphate buffer (pH 7.1), and 20% (vol/vol) polysorbate 80 for i.p. administration at doses of 0, 300, 900, and 2,700 µg (approximately 0, 10, 30, and 90 mg/kg for 30-g mice; the PQP concentration was variable, as a standard 100-µl volume of suspension was administered to each mouse). Groups of mice were dosed 64 h after inoculation (anticipated parasitemia of 3 to 5%, confirmed by thin-film examination). For combination therapy, mice received single i.p. doses of 10 mg/kg PQP and 30 mg/kg DHA 64 h after inoculation (DHA was dissolved in a 60:40 mixture of dimethyl sulfoxide and polysorbate 80). All drug treatment groups comprised 14 mice, and control groups comprised 8 mice, unless otherwise indicated.
Parasite reinoculation. Mice that initially received a single 90-mg/kg dose of PQP were reinoculated with a second 107 P. berghei standard i.p. inoculum 60 days after drug administration. Uninfected, age-matched control mice that had received either the vehicle (n = 4) or 90 mg/kg PQP (n = 8) or remained untreated (n = 4) on day 0 were inoculated with 107 P. berghei parasites 60 days after dosing (controls for the reinoculation group). Parasitemia was monitored daily by using peripheral blood films as described above.
Pharmacokinetic study. Pharmacokinetic parameters for PQ were determined in 50 uninfected male Swiss mice (6 weeks old) given a single i.p. dose of 2,700 µg PQP (100 µl of suspension; approximately 90 mg/kg). Mice were given 50 to 100 mg/kg sodium pentobarbitone 5 to 10 min prior to blood collection. Blood was harvested from groups of mice (n = 5) by cardiac puncture at 0, 2.5, and 8 h and 1, 2, 4, 7, 10, 14, 18, and 25 days into 1-ml lithium heparin tubes (Vacutainer; Becton Dickinson, Franklin Lakes, NJ) and centrifuged at 10,000 x g for 5 min, and the plasma was separated and stored at –80°C until analyzed by high-performance liquid chromatography (11). Following this pilot study and the single-dose efficacy investigation, a larger pharmacokinetic study of PQ in infected mice was conducted. Male Swiss mice (n = 100) were inoculated with 107 P. berghei parasites and given a single i.p. dose of 2,700 µg PQP 64 h later. Blood was harvested (five mice per group) at 0, 2, 4, 6, 12, and 18 h and 1, 1.25, 2, 2.3, 3, 4, 5, 7, 9, 15, 22, 30, 40, and 50 days and processed as described above.
Statistical and pharmacokinetic analyses. Statistical analysis and data representation was performed with SigmaStat 2004 and SigmaPlot 2004 (SPSS Inc., Chicago, IL). Data are given as means ± standard deviations (SD) unless otherwise indicated. Student's t test or one-way analysis of variance (ANOVA) was used for comparison of groups, as appropriate, with a P value of <0.05 representing a significant difference.
For pharmacokinetic modeling, measured plasma concentrations were normalized to a PQP dose of 90 mg/kg (52 mg/kg PQ base), according to the weight of each mouse at the time of dosing. Consistent with the principles of destructive testing (3, 29), the mean normalized plasma concentration for each group of mice was used to estimate pharmacokinetic parameters. Pharmacokinetic analysis was performed with Kinetica version 4.2 (Thermo Fisher Scientific, Inc., Waltham, MA). Noncompartmental analysis of the plasma concentration-time data was used to estimate the area under the curve (AUC; log-linear trapezoidal method), terminal elimination half-life (t1/2), apparent clearance, and apparent volume of distribution. A two-compartment model was fitted to the data to estimate pharmacokinetic descriptors for the observed biphasic elimination (t1/2 at 
phase [t1/2] and t1/2β; weighting = 1/y2).
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RESULTS |
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and t1/2β were 0.59 and 20.7 days, respectively, in healthy mice and 0.35 and 15.4 days in malaria-infected mice.
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In the 90-mg/kg group, the mean parasitemia declined rapidly and was undetectable by 36 h after dosing. Recrudescence occurred after 7 to 8 days in all mice, with a mean peak parasitemia of 1.8% ± 1.6% observed 16 days after dosing. The parasitemia declined and generally remained below 0.1% until the mice were reinoculated on day 60. All mice were active and alert and had stable body weights throughout the course of the study.
Linking the parasitemia-time profiles for the 90-mg/kg PQ dose group (Fig. 2 and 3) to the corresponding pharmacokinetic data (Fig. 1) indicates that plasma PQ concentrations fell from a mean of 250 µg/liter at 2 h after administration of the dose to 45 µg/liter approximately 36 h after administration of the dose, at which time the parasitemia was below the limit of detection (0.002%). From 2 to 7 days after administration of the dose, when parasites were undetectable, the plasma PQ concentration was approximately 20 to 50 µg/liter. In most mice, concentrations of >10 µg/liter persisted for at least 30 days and the extrapolated mean plasma concentration 60 days after dosing (the time of the reinoculation experiment) was 3 µg/liter in malaria-infected mice and 5.5 µg/liter in control mice.
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PQ-DHA combination study. The effects of a single dose of PQP alone (10 mg/kg), DHA alone (30 mg/kg), or combination therapy (PQP at 10 mg/kg plus DHA at 30 mg/kg) are summarized in Fig. 4 (data have been normalized for clarity, expressing parasitemia as a proportion of the initial parasitemia). The starting parasitemia was 4.5% ± 1.1% for control mice (n = 8), 4.6% ± 1.1% for DHA-treated mice (n = 14), 1.5% ± 0.6% for PQP-treated mice (n = 14; P < 0.001 compared to other groups; ANOVA), and 3.7% ± 1.5% for PQP-DHA combination-treated mice (n = 14). Nadirs for the DHA, PQP, and PQP-DHA treatment groups were 11.9-fold ± 4.8-fold, 12.8-fold ± 3.1-fold, and 22.4-fold ± 11.8-fold lower than the initial parasitemia (P = 0.007; PQP-DHA compared to both DHA and PQP alone; ANOVA).
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DISCUSSION |
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Interpretation of the pharmacodynamic effects of PQ was improved by characterization of the pharmacokinetic properties of PQ in mice. Recent human studies have shown that PQ has a t1/2β of 23 days in adults and 14 days in children (12, 23), and a previous study that used 14C-labeled PQ to investigate drug disposition reported a t1/2 of 9 days in mice. By contrast, by using a specific high-performance liquid chromatography method of analysis (11), we have shown that PQ has a biphasic elimination profile with similar terminal t1/2 values in both malaria-infected (17.8 days) and healthy (16.1 days) mice.
The pharmacodynamic profile following the administration of a dose of 90 mg/kg PQP (Fig. 2) showed relatively rapid initial parasite elimination (with a plasma PQ concentration of >50 µg/liter during this time), followed by a low, subclinical parasitemia over the period of about 30 to 60 days (plasma PQ concentration of 3 to 10 µg/liter). The subclinical parasitemia following the administration of 90 mg/kg PQP was further investigated by rechallenging the mice with a fresh inoculum of 107 P. berghei parasites 60 days after the first treatment. Since the parasitemia did not redevelop, as in the parallel control animals, we suggest that the mice previously treated with PQ had developed a degree of immunity to the parasites. Our data show that the residual PQ concentration at 60 days was not effective in clearing the established infection or suppressing a new infection, thus excluding a pharmacokinetic explanation for the outcome of the reinfection protocol.
Using the smaller, less effective 30-mg/kg PQP dose, we deduce from the survival data (70% at 30 days) that the apparent dose of PQ producing a 50% response is probably 20 to 30 mg/kg in this treatment model (survival at 10 mg/kg PQP was zero). By comparison, murine malaria suppression studies that used the Peters 4-day test with chloroquine-sensitive strains of P. berghei showed that the 50% effective doses of PQ ranged from 4.5 to 6.4 mg/kg (5, 19). Regardless of the efficacy model used, these doses are substantially lower than the reported 50% lethal dose of 1,098 mg/kg PQ for mice (8) and indicate that effective doses of PQ are considerably less than those likely to cause toxicity.
The PQ doses used in the murine model were up to 10-fold higher than single doses used clinically (PQ at 7 to 12 mg/kg/day, equivalent to PQP at 12 to 20 mg/kg/day) and up to 3-fold higher than the maximum total PQ dose of 25 to 35 mg/kg used in most clinical studies (8, 17). Currently, PQ is only used in a combination formulation with either artemisinin or DHA. However, as the artemisinins are short-acting drugs, combination with a second antimalarial drug is recommended for clinical use (8). Further, the partner antimalarial should be a long-acting drug with a t1/2 that covers at least two asexual erythrocytic life cycles (>4 days for P. falciparum), as well as having good patient tolerability, low cost, and limited preexisting drug resistance (2, 8, 12, 28). PQ has therefore been regarded as a good partner drug for the artemisinins because it has a very long t1/2 and rates well on tolerability and cost (12, 24). Notably, in combination with artemisinin compounds, resistance to PQ has not become a clinical problem (8, 27).
The present study demonstrates that a murine malaria treatment model can be used for detailed preclinical investigation of antimalarial combinations. As shown in Fig. 4, the combination of subtherapeutic doses of PQP (10 mg/kg) and DHA (30 mg/kg) produced a greater decline in parasitemia and a longer survival time than either drug alone. Although higher doses of PQ alone were effective as monotherapy (Fig. 2), the rate of parasite decline was enhanced when the PQ dose was given in combination with DHA. Hence, an additive or synergistic in vivo effect is plausible.
The results from our study support the contention that in animal models DHA and PQ show additive efficacy (1), despite recent in vitro evidence that DHA either showed no interaction or was mildly antagonistic when combined with PQ (7). One limitation of the present investigation might be that only single doses of DHA and PQ were used. However, a single-dose study design facilitates the use of subtherapeutic doses and enables both additive and antagonistic effects to be detected. A multiple-dose study would most likely yield a similar therapeutic result, albeit more rapidly.
A general limitation of all murine studies is that direct extrapolation to human malaria is normally not possible. Nevertheless, as demonstrated in the present study, murine models have the advantage of investigating outcomes of drug therapy alone and in combination in the whole animal. These models also offer an opportunity for detailed investigations of the mechanisms of disease and/or therapeutic response. For example, our results suggest that persistently low plasma PQ concentrations may not impede the development of a malaria infection. Furthermore, it is now well accepted that the long t1/2 of PQ predisposes to the emergence of drug resistance, as was evidenced with PQ monotherapy in China in the 1970s (8, 10, 21, 27). However, when used in combination with artemisinin derivatives, which accelerate therapeutic responses and reduce parasite biomass, PQ is thought to prevent recrudescence by killing residual parasites and reducing the number of surviving mutant parasites, which in turn prevents resistance to either drug (4, 10, 14, 26). Indeed, clinical studies have indicated that posttreatment prophylaxis from PQ may reduce both relapse and reinfection for a period of 4 to 6 weeks after treatment (1, 10, 20). As a result, patients remain asymptomatic for a longer period, increasing the time for hematological recovery, halving the risk of anemia, and reducing the gametocyte carriage rate (10, 20). This suggestion that posttreatment prophylaxis provided by PQ when used in combination therapy may delay but not prevent a subsequent relapses (20) could be investigated in detail with a murine model.
In conclusion, our study has shown that the P. berghei murine malaria model provides a valuable conceptual model for the comparison of single-dose and combination therapies, which is generally not feasible in the clinical setting. Importantly, where immune and other regulatory mechanisms may be integral features, the murine model is a useful extension of more-rapid, comparatively high-throughput in vitro studies. We have shown that in the murine malaria model, PQ has a pharmacokinetic profile comparable to that in humans and a potent antimalarial effect after single-dose treatment. Finally, as in human studies, PQ efficacy was enhanced when it was combined with DHA, suggesting an additive antimalarial effect.
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ACKNOWLEDGMENTS |
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This work was supported by the NHMRC New Investigator grant (141103) and the NHMRC Biomedical (Dora Lush) Postgraduate Research Scholarship (323251) from the National Health and Medical Research Council of Australia.
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FOOTNOTES |
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Published ahead of print on 5 November 2007.
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) and malaria-infected (
) mice. The lines represent the best fit of a two-compartment model to the respective data sets (extrapolated beyond the last data point for healthy mice [25 days] to facilitate comparisons).
, 30 mg/kg (n = 13); 
, 90 mg/kg (n = 14). Panel B shows an expanded view for the first 5 days after drug administration.
