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Antimicrobial Agents and Chemotherapy, July 2009, p. 2707-2713, Vol. 53, No. 7
0066-4804/09/$08.00+0     doi:10.1128/AAC.00056-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Piperaquine Pharmacodynamics and Parasite Viability in a Murine Malaria Model

Brioni R. Moore,¹ Kenneth F. Ilett,^2,3 Madhu Page-Sharp,^1,2 Jeffrey D. Jago,^4,5 and Kevin T. Batty^1,5*

School of Pharmacy, Curtin University of Technology, Bentley, Western Australia,¹ School of Medicine and Pharmacology, University of Western Australia, Crawley, Western Australia,² Clinical Pharmacology & Toxicology Laboratory, Path West Laboratory Medicine, Nedlands, Western Australia,³ School of Biomedical Sciences, Curtin University of Technology, Bentley, Western Australia,⁴ Curtin Health Innovation Research Institute, Curtin University of Technology, Bentley, Western Australia, Australia⁵

Received 14 January 2009/ Returned for modification 26 March 2009/ Accepted 9 April 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Piperaquine (PQ) is an important partner drug in antimalarial combination treatments, but the long half-life of PQ raises concerns about drug resistance. Our aim was to investigate the extended antimalarial effect of PQ in a study of drug efficacy, reinoculation outcomes, and parasite viability after the administration of a single dose of PQ in the murine malaria model. Initially, male Swiss mice were inoculated with Plasmodium berghei and at 64 h after parasite inoculation were given PQ phosphate at 90 mg/kg of body weight intraperitoneally. Parasite viability, drug efficacy, reinoculation responses, and parasite resistance were determined at 25, 40, 60, 90, and 130 days after drug administration. At each time point, six mice were reinoculated with 10⁷ P. berghei parasites and blood was harvested from another four mice for viability passage into naïve mice (n = 5 for each blood sample) and from another two mice for determination of the plasma PQ concentration. The efficacy study demonstrated that the residual PQ concentrations did not suppress the infection after 25 days. Viable parasites were present up to 90 days after PQ dosing, although only 50% and 25% of the passaged parasites remained viable at 60 and 90 days postdosing, respectively. Viable parasites passaged into the naïve hosts were generally resistant to PQ when they were exposed to the drug for a second time. PQ was found to have a substantial antimalarial effect in this model, and the effect appears to be sufficient for a host immunological response to be established, resulting in the long-term survival of P. berghei-infected mice.

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Piperaquine (PQ) is a bisquinoline antimalarial drug used in contemporary artemisinin combination treatment (ACT) strategies as a partner to dihydroartemisinin (1, 4). In order to prevent drug resistance and early parasite recrudescence associated with the short-acting artemisinin drugs, ACT partner drugs should be long-acting schizonticides with half-lives (t_1/2s) exceeding 4 days, or two asexual parasite life cycles (10, 21). Recent pharmacokinetic studies have demonstrated that PQ has biphasic elimination and a long terminal t_1/2 of 12 to 28 days in humans (10, 15, 31, 32).

Several clinical trials of the PQ-dihydroartemisinin combination have shown that it has a high degree of efficacy and good tolerability for the treatment of Plasmodium falciparum infections (1, 2, 5, 7, 11, 12, 17). While this combination is now considered the first-line antimalarial treatment in some Southeast Asian countries, the long t_1/2 of PQ raises concerns about adverse effects and drug resistance (6, 10, 20, 22). Detailed preclinical pharmacodynamic data for PQ, alone or in combination with artemisinin drugs, would complement clinical studies, especially when there is interest in the therapeutic impact of persistent, low PQ concentrations.

We have recently demonstrated that PQ has a biphasic elimination profile in mice and has a terminal elimination t_1/2 in malaria parasite-infected and healthy mice of 18 days and 16 days, respectively (18). The pharmacodynamic component of our study revealed that after the administration of a single dose of PQ phosphate (10 to 90 mg/kg of body weight) there was a rapid reduction in the level of parasitemia to a subdetectable parasite density in the groups receiving high doses and recrudescence approximately 7 days later. In the group receiving the highest dose (90 mg/kg of body weight), a subclinical infection persisted for at least 60 days, at which time the plasma PQ concentration was estimated to be 20- to 100-fold lower than earlier therapeutic levels. However, reinoculation with P. berghei parasites did not cause the standard lethal infection that was found in control mice, suggesting that the mice treated with PQ had developed a degree of immunity to the parasites (18, 19). The present study was therefore conducted to investigate drug efficacy, reinoculation outcomes, and parasite viability after the administration of a single dose of PQ in the murine malaria model.

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PQ phosphate (PQP; molecular weight, 927.5) was obtained from Yick-Vic Chemicals and Pharmaceuticals, Kowloon, Hong Kong. Sodium pentobarbitone injection (sodium pentobarbitone [30 mg/ml], propylene glycol [40%, vol/vol], ethanol [10%, vol/vol] in water; pH 9.5) was prepared in-house and diluted 50:50 with 0.9% (wt/vol) sodium chloride for injection prior to use. May-Grunwald Giemsa stain was obtained from the Department of Microbiology, Royal Perth Hospital, Western Australia, Australia. All general laboratory chemicals and solvents were of analytical grade and were obtained from Sigma-Aldrich Chemical Co. Milwaukee, WI; BDH Laboratory Supplies, Poole, England; or Merck Pty. Limited, Kilsyth, Victoria, Australia.

Mice. The study was approved by the Curtin University Animal Experimentation Ethics Committee. Male Swiss mice (age, 5 to 6 weeks; average weight, 29.8 ± 3.1 g) were obtained from the Animal Resource Centre (Murdoch, Western Australia, Australia). Male BALB/c mice (age, 7 to 8 weeks old; Animal Resource Centre) were used for the weekly passage of the malaria parasites. The animals were housed at 22°C in a 12-h light and 12-h dark cycle and had free access to sterilized commercial food pellets (Glen Forrest Stockfeeders, Perth, Western Australia, Australia) and sterilized, acidified (with HCl, pH 2.5) water to prevent bacterial infections (27, 29).

Parasites. P. berghei ANKA parasites were 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 (14) and was administered by intraperitoneal (i.p.) injection to infect the Swiss mice used in the experiment.

Enumeration of parasites in infected mice. Peripheral blood smears were prepared with blood obtained from the tail veins of the infected mice. The thin films were fixed in methanol (3 min) and then stained with May-Grunwald Giemsa by using a Hema-Tek staining machine (Ames Co., Elkhart, IN). Blood smears were examined by oil immersion light microscopy at x100 magnification under a DMLS light microscope (Leica Microsystems, Gladsville, New South Wales, Australia). The level of parasitemia was determined by counting 30 or 100 fields of view for >0.5% and <0.5% infected erythrocytes, respectively, thus ensuring a limit of detection in 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 (which was done when the level of parasitemia reached >40%, there was a >10% reduction in the mouse's body weight in less than 24 h, or termination of the experimental protocol). The mice were euthanized by sodium pentobarbitone injection (50 to 100 mg/kg i.p.).

Drug treatment. PQP was suspended in a mixture of 50% (vol/vol) glycerol-30% (vol/vol) isotonic phosphate buffer (pH 7.1)-20% (vol/vol) polysorbate 80 and administered i.p. at a dose of 2,700 µg (90 mg/kg for 30-g mice; the dose was based on that used in a previous study [18]). Infected mice (n = 50; group 1; infected treatment group) were dosed 64 h after inoculation (the anticipated level of parasitemia was 3 to 5% and was confirmed by thin film examination), while uninfected age-matched control mice received either 90 mg/kg PQP (n = 32; group 2; uninfected treatment control group) or drug vehicle (n = 20; group 3; vehicle control group).

At predetermined times of 25, 40, 60, 90, and 130 days after drug administration (day 0), a cohort of mice from each primary treatment group (groups 1, 2, and 3) was selected for the reinoculation, parasite viability, and drug efficacy investigations. Our previous study had shown that 25 days after the administration of 90 mg/kg PQP, the level of subclinical parasitemia was in the order of 0.1% infected erythrocytes and the plasma PQ concentration was approximately 14 µg/liter (18). By comparison, at 40 and 60 days after administration, the level of subclinical parasitemia was stable at approximately 0.01 to 0.1% and the plasma PQ concentrations (3 to 7 µg/liter) were considered unlikely to have an antimalarial effect. The previous study did not extend beyond 60 days, and the present study was designed to clarify the duration of antimalarial efficacy.

Parasite reinoculation. The parasite reinoculation arm of the study was designed to evaluate the effects of parasite reinoculations in previously infected, treated mice (i.e., potentially immune to reinfection; group 1) and parasite inoculations in uninfected, treated mice (i.e., to assess drug efficacy; group 2). At each predetermined time, mice from the original infected treatment group (group 1; n = 6), were reinoculated with 10⁷ P. berghei parasites. Simultaneously, a group of vehicle-treated (n = 4; group 3) and drug-treated (n = 6; group 2) uninfected, age-matched mice were inoculated with 10⁷ P. berghei parasites (as controls for the reinoculation group). The level of parasitemia was monitored by evaluation of peripheral blood films twice daily for 30 days after each predetermined time of 25, 40, 60, 90, and 130 days, as described above.

Parasite viability. The parasite viability arm of the study was designed to evaluate the viability of parasites from previously infected, treated mice with subclinical parasitemia (group 1). Included in this component was an evaluation of P. berghei resistance to PQ. At each designated time point (25, 40, 60, 90, and 130 days), blood was harvested from a group of treated mice (group 1; n = 4) by cardiac puncture. The blood was centrifuged at 3,000 x g for 10 min (Biofuge Primo; Heraeus Instruments/Kendro Instruments Australia Pty. Ltd., Lane Cove, New South Wales, Australia); and the plasma was separated, measured, and stored at –80°C for later analysis of the PQ concentration. The red blood cells were washed three times in 0.9% (wt/vol) sodium chloride to deplete antibodies and to ensure that no residual PQ was present in the matrix (after each wash, the cells were centrifuged at 3,000 x g for 5 min), and care was taken to remove all of the buffy coat. The packed red blood cells were then resuspended in 0.9% (wt/vol) sodium chloride by using the same volume as that of the plasma that was removed. A blood smear was prepared by using the red blood cell suspension to estimate the level of parasitemia in the donor sample. As the level of parasitemia in the donor sample was normally <0.1%, recipient mice were inoculated with a 1:5 dilution of the original suspension (200 µl i.p. injection; inoculum, <10⁵ P. berghei parasites).

An inoculum of blood from each donor mouse was passaged into a group of naïve male mice (age, 5 weeks; n = 5), resulting in a total group size of 20 for each time point. Five days after inoculation, three of the mice from each donor cohort were treated with a single 90-mg/kg i.p. dose of PQP and the remaining two mice per group remained untreated. Hence, at each time point both parasite viability and the effect of PQ were evaluated. The level of parasitemia was monitored by evaluation of peripheral blood films twice daily, as described above.

Plasma PQ concentrations. The plasma PQ concentration was determined in both malaria parasite-infected mice (n = 4) and uninfected age-matched control mice (n = 2) at each time point (25, 40, 60, 90, and 130 days). Blood was harvested by cardiac puncture and centrifuged at 3,000 x g for 5 min, and the plasma was separated and stored at –80°C until it was analyzed by a validated high-pressure liquid chromatography assay with limits of quantification and detection of 1.6 µg/liter and 0.7 µg/liter, respectively, and a coefficient of variation of <12% (9, 13). The mean plasma concentration was compared with data from a previous pharmacokinetic study (18).

Data analysis. Data analysis and representation were performed with the SigmaStat (version 3.1) and the SigmaPlot (version 9) programs (Systat Software Inc., San Jose, CA). Data are given as the means ± standard deviations (SDs), unless otherwise indicated.

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PQ pharmacodynamics. Administration of a single dose of 90 mg/kg PQP resulted in a rapid decline in the level of parasitemia to below the limit of detection (0.002%) by 36 h after dosing (Fig. 1). In the group 1 mice, recrudescence occurred 7 to 8 days after drug administration, with a mean peak level of parasitemia of 1.32% ± 0.56% at 13 days. The level of parasitemia remained relatively stable for approximately 8 days and declined to a mean of 0.03% by day 25 (Fig. 1). Thereafter, the mean level of parasitemia remained below 0.02% until 80 days after dosing, beyond which there were no detectable parasites in any mice up to and including the experimental endpoint of 130 days after the initial dosing.

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FIG. 1. Parasitemia-time profile in Swiss mice (group 1) following the administration of a single dose of 90 mg/kg PQP administered 64 h after inoculation with 107 P. berghei-parasitized erythrocytes (day 0). Data are shown as the total level of parasitemia (mean percentage of erythrocytes infected ± SD), commencing from the time of PQP administration. Symbols: •, control (n = 4); , 90 mg/kg (n = 50 initially and then less 10 mice following each of the predetermined study points of 25, 40, 60, and 90 days, indicated by ).

Parasite reinoculation study. Control mice that were initially uninfected and untreated (group 3) and then inoculated at each predetermined time (25, 40, 60, 90, or 130 days) showed parasite density-time profiles similar to those found in previous studies (3, 18, 19), with rapidly rising levels of parasitemia being detected until the time of euthanasia (6 to 8 days after inoculation; peripheral level of parasitemia, >40%; Fig. 2A). Among the uninfected control mice that received 90 mg/kg PQP on day 0 (group 2) and that were inoculated for the first time at the predetermined times, a modest suppressive effect against the malaria parasites was demonstrated only in the cohort inoculated at 25 days (Fig. 2B). This cohort of mice had a mean level of parasitemia approximately 10-fold lower than that for the controls and a median survival time of 15 days (Fig. 2B). In contrast, no apparent parasite suppression was seen at any other time point; and the median survival times were 8, 8, 8, and 7 days for the cohorts inoculated at 40, 60, 90, and 130 days, respectively. In the last four groups, the parasite density-time profiles and survival times were comparable to those for control mice that received only the drug vehicle.

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FIG. 2. Parasitemia-time profiles from the parasite reinoculation arm of the study. Data are shown as the total level of parasitemia (mean percentage of erythrocytes infected ± SD), commencing from the time of inoculation. (A) Control group (group 3) consisting of untreated, uninfected age-matched mice (n = 4 at each time point) that were inoculated at predetermined times; (B) drug efficacy group (group 2), consisting of age-matched uninfected mice given 90 mg/kg PQP at day 0 (n = 6 mice at each predetermined time point); (C) reinoculation group (group 1), consisting of infected mice treated with 90 mg/kg PQP at day 0 and then reinoculated with 107 P. berghei parasites at each study point (n = 6 mice at each predetermined time point). The predetermined inoculation times were 25 days (•), 40 days (), 60 days (), 90 days (), and 130 days ().

Data for mice that were previously inoculated and that received 90 mg/kg PQP on day 0 (group 1) are shown in Fig. 2C. The cohort that was reinoculated on day 25 maintained a low, subclinical level of parasitemia (<0.015%), with all mice remaining asymptomatic for 4 weeks (the predetermined endpoint of the reinoculation study; Fig. 2C). A similar outcome was seen at 40, 60, and 90 days, with all mice remaining asymptomatic and with the peak levels of parasitemia being 0.1%, 0.3%, and 0.9%, respectively. In contrast, mice reinoculated 130 days after initial drug administration (and which had been aparasitemic for >50 days) showed rapidly increasing levels of parasitemia and a median survival time of 10 days (Fig. 2C).

Parasite viability and drug resistance study. Blood harvested from the group 1 mice inoculated at 25 days (mean level of parasitemia, 0.03% ± 0.04%) was shown to contain viable parasites when naïve mice were inoculated with red blood cell suspensions. All naïve mice developed infections, and the mice that received drug vehicle 5 days after inoculation progressed to the experimental endpoints (high levels of parasitemia) and had a median survival time of 19 days (Fig. 3A). Following the administration of 90 mg/kg PQP 5 days after inoculation, the level of parasitemia in the treated mice was not markedly different from that in the group treated with the vehicle, and the median survival time was also 19 days. A similar outcome was observed for the mice inoculated at 40 days (Fig. 3B), and the data from the two groups inoculated at 25 days and 40 days indicated that the parasites were resistant to PQ.

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FIG. 3. Parasitemia-time profiles in naïve Swiss mice (n = 5) after inoculation with red blood cell suspensions harvested from previously inoculated and PQP-treated mice (n = 4; group 1) at 25, 40, 60, and 90 days after initial drug administration (A to D, respectively). Data are shown as the total level of parasitemia (mean percentage of parasitized erythrocytes infected ± SD), commencing from the time of red blood cell passage. Five days after inoculation, three of five mice from each group of naïve recipient mice received a single i.p. dose of 90 mg/kg PQP (indicated by ). Inocula consisting of erythrocytes harvested at day 25 from the 4 PQP-treated mice in group 1 produced a viable infection in all 20 recipient mice (A). The untreated mice (•; n = 8) and the PQP-treated naïve recipient mice (; n = 12) had similar parasite density-time profiles, apart from those on days 7 and 8 (2 to 3 days after PQ administration), and the median survival time was 19 days in both groups. (B) Inocula consisting of erythrocytes harvested at day 40 from the mice in group 1 also produced viable infections in all recipient mice (, untreated, n = 8; , treated, n = 12) and a profile similar to that for the mice inoculated at day 25. (C) In the group inoculated with erythrocytes at day 60, viable infections were produced with erythrocytes from only two of the four group 1 donor mice. Untreated recipient mice (; n = 4) had a median survival time of 10 days. Treated recipient mice (; n = 12) showed different profiles, with the cohort receiving erythrocytes from one donor mouse having a progressive infection (dashed line) and with the other cohort having a strong response to PQP treatment (dotted line). (D) In the group inoculated with erythrocytes at day 90, a viable infection was found in the recipients of erythrocytes from only one group 1 donor mouse, and although the development of the infection was delayed, the parasite density-time profile for the untreated mice (; n = 2) was similar to that for the treated mice (; n = 3).

Blood harvested from the group 1 mice inoculated at 60 days (mean level of parasitemia, 0.004% ± 0.006%) showed that viable parasites were present in only two of the donor mice, with the blood from two of the donor mice not producing detectable parasitemia in any of the naïve mice inoculated. In the 10 naïve recipient mice that developed an infection from the red blood cell suspension, mice that received drug vehicle 5 days after inoculation had a median survival time of 10 days (n = 4; Fig. 3C). The administration of PQP to the remaining mice (5 days after inoculation) resulted in a prompt decline in the level of parasitemia and recrudescence in all recipients (n = 6; Fig. 3C). The recipients of blood from one donor (n = 3) had a progressive infection that reached 10% parasitemia within 25 days. However, the three recipients of blood from the other donor showed a response to PQ that was consistent with successful treatment.

Among the group 1 mice inoculated with red blood cell suspensions at 90 days, the recipients of blood from only one donor mouse developed a detectable infection (Fig. 3D), while all recipients of blood from the other three donors remained free of infection throughout the 30-day monitoring period. The lag period for the infection to develop suggested a low parasite count in the red blood cell suspension (inoculum), but following PQ administration, the drug had little effect and the median survival time of 20 days was similar to that for the mice given the vehicle. Among the final set of group 1 mice, which were inoculated at 130 days, none of the recipient mice developed an infection during the 30-day monitoring period, thus indicating that no viable parasites were present 4 months after the mice were inoculated and treated with PQP.

PQ concentrations. The mean plasma PQ concentrations in the malaria parasite-infected mice (n = 4) at each time point were 15 ± 8, 11 ± 1, and 2 ± 2 µg/liter for mice inoculated with blood from infected mice 25, 40, and 60 days after PQP administration, respectively, while the plasma PQ concentrations at 90 and 130 days were undetectable (Fig. 4). The plasma PQ concentrations in the present study were found to be comparable to the mean plasma concentration-time profile obtained in our previous study (18). The plasma PQ concentrations in uninfected control mice were 13.3 µg/liter at 25 days (n = 1), 4.9 µg/liter at 40 days (n = 1), 1.4 and 1.6 µg/liter at 60 days (n = 2), 1.9 µg/liter and undetectable at 90 days (n = 2), and undetectable at 130 days (n = 2) after PQP administration.

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FIG. 4. Plasma PQ concentrations in mice given PQP at approximately 90 mg/kg i.p. (normalized for pharmacokinetic analysis). Data are given as the mean plasma PQ concentration ± SD for malaria parasite-infected mice at 25, 40, and 60 days after treatment with PQP (; n = 4). The lines show the mean concentration-time profile (two-compartment model) for healthy (dashed line) and malaria parasite-infected mice (solid line) from a previous study (18).

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Our pharmacodynamic study of PQ in a P. berghei murine malaria model has demonstrated that PQ had an extended antimalarial effect and eventually resolved the infection, although this outcome cannot be attributed to PQ alone. After the administration of a single dose of 90 mg/kg PQP (Fig. 1), a rapid initial decline in the parasite level was observed, followed by parasite recrudescence and an extended (10-week) period of low levels of parasitemia. Parasites were undetectable for the remaining 7 weeks of the study, which concluded 4 months after PQP administration.

Uninfected, untreated control mice developed lethal infections when they were inoculated with blood from infected and treated mice at each experimental time point (25, 40, 60, 90, and 130 days), suggesting that mouse age does not affect the course of infection in this model. The uninfected, treated mice (90 mg/kg PQP at day 0) also developed lethal infections at each time point, with any degree of antimalarial effect being shown only in the group inoculated at 25 days (Fig. 2B). This finding demonstrates that low plasma PQ concentrations are unlikely to have therapeutic benefits.

The long t_1/2 of PQ demonstrated in our previous study (18) was reflected in the present study. The contribution of PQ concentrations was evaluated by inoculation of PQ-treated, uninfected control mice and relation of the outcomes to the plasma PQ concentrations at predetermined time points of 25, 40, 60, 90, and 130 days. When PQ-treated uninfected mice were inoculated, parasite suppression was observed only in the group inoculated at 25 days, and this was associated with plasma PQ concentrations in the order of 10 to 20 µg/liter (18) (Fig. 4). The plasma PQ concentrations at 40 and 60 days (means, 10.5 µg/liter and 2.1 µg/liter, respectively) failed to suppress the development of infection after inoculation, and by day 90, PQ was no longer detectable in the mice. Therefore, we conclude that in this murine malaria model the contribution of residual PQ concentrations of approximately 10 to 20 µg/liter to the suppression of parasites was low, while PQ concentrations of <10 µg/liter were ineffective. Although a direct comparison of the findings of the present study to those of studies with humans is not plausible, our findings are consistent with those in a recent report suggesting that plasma PQ concentrations of >30 µg/liter are required for the successful treatment of clinical malaria (23). Hence, the malaria model could provide evidence for the concentration efficacy thresholds of other antimalarial drugs.

The persistence of residual drug is also of concern in the context of the selection of parasites resistant to an ACT partner drug with a long t_1/2. On the basis of the findings of our study, it is feasible that the reemergence of PQ-resistant strains of P. falciparum could occur as a result of the presence of residual PQ following ACT, because antimalarials with long t_1/2s are considered to be more likely to select for drug resistance (4, 22, 33). However, to our knowledge, there are no clinical reports of resistance to PQ as a result of PQ-based ACT, which may reflect the high success rates associated with this regimen (1, 2, 5, 11, 12, 20, 24, 28, 30).

A further issue related to the residual PQ concentrations and the subclinical parasitemia in the present study was the viability of the P. berghei parasites. At 25 and 40 days, when the mean level of parasitemia was in the range of 0.01 to 0.04%, a viable infection ensued in the naïve mice and there was an indication that the parasites were resistant to PQ (Fig. 3). Furthermore, at both the 25- and 40-day time points, the reinoculation of P. berghei parasites into the previously infected, treated mice did not lead to lethal infections and the mean levels of parasitemia remained below 0.01%. Given that the plasma PQ concentrations were shown to be ineffective at both of these time points, it is most likely that the mice mounted an immunological response to the infection. This acquired immunity presumably suppressed (and eventually cleared) the parasitemia and prevented the establishment of infection upon reinoculation. Antibody-mediated mechanisms have been described to be responsible for parasite clearance during infection and for the prevention of parasite recrudescence and/or reinfection with the same parasite strain (16, 25), and the present study demonstrates that this murine malaria model has a potential role in investigations of immunity to malaria parasites.

The extended period of our study ensured that the full scope of the drug efficacy, parasite viability, and host response components could be evaluated. We found that the mean level of parasitemia beyond 60 days was very low (<0.01%), and possibly due to the low parasite densities, the inoculation success rates were 50% and 25% at 60 and 90 days, respectively. Treatment with PQ also showed a variable response, with some infected recipients in the group inoculated at 60 days showing evidence of drug resistance and others showing susceptibility to the drug (although the immediate drug effects were less than the standard drug effects in control mice). The reinoculation of experimental mice with P. berghei parasites also failed to establish lethal infections at these time points, suggesting the persistence of immunity in the presence of low levels of parasitemia and in mice with recently cleared infections.

The peripheral parasitemia remained undetectable from 80 days after the original treatment with PQP, suggesting that the infections had resolved or that the residual parasite biomass was very low. At the final time point of our study, the passaging of blood into naïve mice failed to establish an infection, and in contrast to the earlier predetermined reinoculation time points, the second P. berghei inoculum at the 130-day time point led to a typical, lethal infection. These results suggest that any immunity or immunological memory was absent, a finding that is also consistent with the view that the maintenance of immunological memory requires continuous exposure to the parasites (8, 25).

The present study may have been limited by the relatively small number of donor mice used to demonstrate parasite viability at each time point studied. At the time points of 25 and 40 days, when the responses to the parasites were consistent, the small sample size was not a conflicting issue. However, as time progressed the variability of parasite viability increased, and by 60 days, only 50% of the parasites proved to be viable upon reinoculation. It is possible that if the number of donor mice had been increased at each time point, we may have been able to reduce the variability, particularly as the number of viable parasites decreased, thus providing more defined outcomes and conclusions.

A general limitation of all studies of malaria in mice is that direct extrapolation to malaria in humans is not usually possible. Nevertheless, murine malaria models have a role in the investigation of the mechanisms of disease, the outcomes of drug therapy, and/or in vivo therapeutic responses. In the present study, it was shown that although PQ remained detectable in plasma for more than 50 days, the drug concentrations were not sufficient to suppress parasites more than 25 days after drug administration. Despite the presence of subtherapeutic drug concentrations, we observed parasite suppression after 25 days, indicating that the immune system plays an important role in the maintenance and the eventual resolution of the parasitemia. Furthermore, we have shown that in our P. berghei model the parasites had the capacity to rapidly become resistant to PQ, as indicated by inadequate parasite suppression upon retreatment. These findings are similar to clinical observations that showed that the long t_1/2 of PQ predisposes parasites to the emergence of drug resistance, as was evident with PQ monotherapy in China in the 1970s (4, 6, 7, 26).

In conclusion, the present study has demonstrated that the murine P. berghei malaria treatment model can be a valuable conceptual tool for the preclinical investigation of the pharmacodynamic effects of antimalarial drugs such as PQ. We have found that PQ has a substantial antimalarial effect in this model and that this effect appears to be sufficient for establishment of a host immunological response. The findings of our study also suggest that the presence of residual PQ could lead to the development of PQ-resistant parasites if the initial ACT is not curative and/or a new infection arises during the early posttreatment period.

 
This work was supported by a New Investigator Grant (grant 141103) and a Biomedical (Dora Lush) Postgraduate Research Scholarship (323251) from the National Health and Medical Research Council of Australia.

 
* Corresponding author. Mailing address: School of Pharmacy, Curtin University of Technology, G.P.O. Box U1987, Perth, Western Australia 6845, Australia. Phone: (61-8) 9266 7369. Fax: (61-8) 9266 2769. E-mail: Kevin.Batty{at}curtin.edu.au

Published ahead of print on 20 April 2009.

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Antimicrobial Agents and Chemotherapy, July 2009, p. 2707-2713, Vol. 53, No. 7
0066-4804/09/$08.00+0     doi:10.1128/AAC.00056-09
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