JAC Advance Access published online on July 23, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm280
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Antimalarial pharmacodynamics and pharmacokinetics of a third-generation antifolate—JPC2056—in cynomolgus monkeys using an in vivo–in vitro model
1 Australian Army Malaria Institute, Gallipoli Barracks, Enoggera, QLD 4051, Australia 2 University of Miami School of Medicine, Miami, Florida, USA 3 Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA 4 Jacobus Pharmaceutical Company, Princeton, New Jersey, USA
* Corresponding author. Tel: +61-7-33324930; Fax: +61-7-33324800; E-mail: mike.edstein{at}defence.gov.au
Received 28 February 2007; returned 23 April 2007; revised 23 May 2007; accepted 2 July 2007
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Abstract |
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Objectives: To assess the antimalarial pharmacodynamics and pharmacokinetics of the novel dihydrofolate reductase (DHFR) inhibitor, JPC2056 and its principal active metabolite JPC2067 in cynomolgus monkeys using an in vivo–in vitro model.
Methods: In a two-phase crossover design, five cynomolgus monkeys were administered a single dose (20 mg/kg) and multiple doses (20 mg/kg daily for 3 days) of JPC2056. Plasma samples collected from treated monkeys were assessed for in vitro antimalarial activity against Plasmodium falciparum lines having wild-type (D6), double-mutant (K1) and quadruple-mutant (TM90-C2A) DHFR–thymidylate synthase (TS) and a P. falciparum line transformed with a Plasmodium vivax dhfr–ts quadruple-mutant allele (D6-PvDHFR). Plasma JPC2056 and JPC2067 concentrations were measured by LC–mass spectrometry.
Results: The mean inhibitory dilution (ID90) of monkey plasma at 3 h after drug administration against D6, K1 and TM90-C2A was, respectively, 1253, 585 and 869 after the single-dose regimen and 1613, 1120 and 1396 following the multiple-dose regimen. Less activity was observed with the same monkey plasma samples against the D6-PvDHFR line, with a mean ID90 of 53 after multiple dosing. Geometric mean plasma concentrations of JPC2056 and JPC2067 at 3 h after drug administration were, respectively, 113 and 12 ng/mL after the single dose and 150 and 17 ng/mL after multiple dosing. The mean elimination half-life of JPC2056 was shorter than its metabolite after both regimens (single dose, 7.3 versus 11.8 h; multiple doses, 6.6 versus 11.1 h).
Conclusions: The high potency of JPC2056 against P. falciparum DHFR-TS quadruple-mutant lines provides optimism for the future development of JPC2056 for the treatment of malaria infections.
Key Words: malaria , antifolates , WR99210 , JPC2056 , DHFR inhibitors
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Introduction |
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The first-generation dihydrofolate reductase (DHFR) inhibitors, proguanil and pyrimethamine, were developed in the 1940s. These antifolates are competitive inhibitors of plasmodial DHFR, a validated drug target that is part of the bifunctional enzyme DHFR–thymidylate synthase (DHFR–TS) of both Plasmodium falciparum and Plasmodium vivax.1–6 DHFR–TS is required for interconversion of folate derivatives in the de novo biosynthesis of thymidine-5'-monophosphate. Resistance, however, appeared soon after these drugs were used for treatment of malaria infections. The efficacy of the fixed combination of sulfadoxine and pyrimethamine has also markedly declined in many malaria endemic countries throughout the world due to the emergence of DHFR–TS mutant parasite lines.1,2 Accumulation of point mutations that alter the structure of DHFR results in a reduction in the binding of antifolates to the receptor sites within the parasitic DHFR domain.5–7
In the mid-1960s, the second-generation antifolate, WR99210 [4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(2,4,5-trichlorophenoxypropyloxy)-1,3,5 triazine] was synthesized, which possessed marked activity against both pyrimethamine- and chloroquine-resistant lines of P. falciparum.8 Subsequent clinical studies showed WR99210 to cause severe gastrointestinal symptoms and have poor bioavailability in healthy subjects and, as a consequence, development of the drug was abandoned.9,10 However, interest in the dihydrotriazines remained because of exceptional potency and lack of cross-resistance to pyrimethamine and cycloguanil.8,11,12
In an effort to circumvent the poor gastrointestinal tolerability of WR99210, PS-15, the phenoxypropoxybiguanide precursor for WR99210, (also known as WR250417) N'-[3-(2,4,5-trichlorophenoxy)propyloxy]-N9-(1-methylethyl) imido-carbonimidicdiamide was synthesized. Canfield et al.10 reported WR250417 to be more active than either proguanil or WR99210 in Aotus monkey efficacy studies and was far better absorbed from the gastrointestinal tract than WR99210.13 However, further assessment of PS-15 was suspended because of regulatory restrictions in the use of the starting material, 2,4,5-trichlorophenol used to produce PS-15.14 To overcome the safety and regulatory restrictions associated with the production of PS-15, third-generation phenoxypropoxybiguanide prodrugs such as PS-26, JPC2005 and JPC2056 with their corresponding active dihydrotriazine metabolites were recently synthesized and the dihydrotriazines were shown to be as potent as WR99210, in vitro.15 Of these compounds, JPC2056 (Figure 1) which is metabolized to JPC2067,15 has been selected as the lead candidate for pre-clinical development based on equivalent efficacy to PS-15 and comparable oral tolerability in mice and monkeys to proguanil.16
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Point mutations in the dhfr region of dhfr–ts are associated with resistance of P. vivax to antifolates.17–19 Although P. vivax infections are known to respond poorly to antifolates such as pyrimethamine, either alone20 or in combination with sulfadoxine,21,22 in vitro data on the sensitivity of the parasite to DHFR inhibitors have been difficult to determine because of difficulties in culturing P. vivax continuously in vitro. Consequently, enzyme kinetic response to drugs has been studied in heterologous assay systems (e.g. yeast) to provide a better understanding of the influence that specific amino acid substitutions within the DHFR domain have on drug resistance.4,23,24 It has been shown that P. vivax DHFR quadruple mutants are more resistant to WR99210 than that of P. falciparum using an in vitro yeast expression assay system.24 As mixed infections with both P. falciparum and P. vivax are common in Asia, Oceania and South America,25,26 the development of an effective and affordable antifolate against both species would be highly advantageous.
The purpose of this study was to determine the antimalarial pharmacodynamics and pharmacokinetics of JPC2056 and its principal active metabolite, JPC2067, after oral administration of JPC2056 to cynomolgus monkeys using the in vitro–in vivo model. In vitro antimalarial pharmacodynamics of JPC2056 was determined using a monkey-in vitro model system,13 which is particularly useful for evaluating compounds that are metabolized in vivo to their active metabolite(s). The composite antimalarial activity of the monkey's plasma containing JPC2056 and its metabolites was assessed against P. falciparum lines carrying a mutant dhfr–ts and a P. falciparum line transformed with a P. vivax dhfr–ts quadruple-mutant allele.
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Compounds
JPC2056 acetate, JPC2067 hydrochloride, JPC2067-D6, WR99210 hydrochloride, chlorcycloguanil hydrochloride and pyrimethamine were supplied by Jacobus Pharmaceutical Company (Princeton, NJ, USA). JPC2056 acetate was dissolved in 0.5% hydroxyethylcellulose–0.1% Tween 80 for administration to cynomolgus monkeys. For in vitro antimalarial testing, stock solutions of drugs (JPC2056, 512 mg/L; JPC2067, 100 mg/L; WR99210, 100 mg/L; chlorcycloguanil, 200 mg/L and pyrimethamine, 400 mg/L) were prepared in methanol in glass vials coated with 0.2% (v/v) AquaSil/water solution (Pierce, Rockford, IL, USA) to minimize drug absorption to the glass surface. Subsequently, one or two dilutions of the drug stock solutions were made in drug-free human serum to obtain working solutions. The highest methanolic concentration (0.25%) used for dissolving the least active antifolates (JPC2056 and pyrimethamine) on the microtitre plates had no inhibitory effect on parasite growth.
Drug administration and blood collection in cynomolgus monkeys
At the University of Miami (FL, USA), five male cynomolgus monkeys (mean age 2.1 ± 0.2 years and weight 2.9 ± 0.5 kg) were sequentially administered a single dose (20 mg/kg) and multiple doses (20 mg/kg daily for 3 days) of JPC2056 via oral gavage, with a 3 week washout period between drug administrations. Blood samples (1.5 mL) were collected into heparinized Vacutainer tubes before drug administration of the single dose and immediately before the last dose of the 3 day regimen. Subsequent blood samples were then obtained at 1, 3, 6, 24, 48 and 72 h after the administration of both regimens. Blood samples were centrifuged (1200 g for 5 min) and the separated plasma stored at –80°C. Plasma samples were then shipped on dry-ice to the Australian Army Malaria Institute (Brisbane, Australia) and the Walter Reed Army Institute of Research (Washington, DC, USA) and stored at –80°C until in vitro and LC-MS/MS analysis, respectively. Approval for the monkey study was obtained from the University of Miami Animal Care and Use Committee (Miami, FL, USA), which is in full compliance with all USDA and NIH regulations governing the use of vertebrate animals in teaching and research.
Parasites and drug preparation
Four P. falciparum lines were used in this study: (i) K1, double-mutant dhfr–ts allele (59R/108N) is chloroquine and pyrimethamine resistant but sensitive to mefloquine;27 (ii) TM90-C2A dhfr–ts quadruple-mutant allele (51I/59R/108N/164L) is resistant to chloroquine, pyrimethamine, mefloquine and atovaquone;28 (iii) D6 wild-type dhfr–ts allele (N51/C59/S108/I164) is sensitive to chloroquine and pyrimethamine;29 (iv) the D6-PvDHFR mutant line is sensitive to chloroquine and resistant to pyrimethamine. The D6-PvDHFR line was developed by transforming D6 parasites so that they express the dhfr–ts quadruple-mutant (57L/58R/61M/117T) allele of the AMRU1 strain of P. vivax,30,31 which is resistant to chloroquine and pyrimethamine.31,32 The D6-PvDHFR line expresses PvDHFR from an episomal plasmid; therefore, D6-PvDHFR remained under pyrimethamine pressure to ensure the plasmid was not lost during culturing. Pyrimethamine drug pressure was removed 96 h before assaying with DHFR inhibitors. Routinely, plasmid DNA was recovered from transfected parasites and the plasmid-bound dhfr was sequenced (data not shown) to ensure new mutations were not introduced. Parasites were cultured continuously in LPLF 1640 RPMI (GIBCO, Grand Island, NY, USA), as described previously.12,13 Cultures were synchronized with 5% sorbitol, so that the parasites were 6–12 h into the schizogony cycle at the start of the in vitro assay.
In vitro antimalarial pharmacodynamics
The in vitro antimalarial pharmacodynamics of JPC2067 in cynomolgus plasma samples was assessed by bioassay.33 Samples with high antimalarial activity (determined by preliminary screening) were pre-diluted (range, 4–60 times) before they were bioassayed. Drug-treated monkey plasma (25 µL) was serially diluted 2-fold on microtitre plates (range, 8–8192) with drug-free human serum. All wells were then inoculated with 75 µL of infected erythrocyte suspension in culture medium. In control experiments, we observed that drug-free cynomolgus plasma diluted eight times with human serum and culture medium had no effect on parasite growth. The sensitivity of the four P. falciparum lines to JPC2056, JPC2067, WR99210, chlorcycloguanil and pyrimethamine was determined in parallel with the bioassay analysis. Working drug solutions (25 µL) were serially diluted 2-fold on microtitre plates using drug-free human serum, followed by the addition of 75 µL of infected red blood cells suspended in the culture medium. The final cell suspension (100 µL) for bioassay and sensitivity tests had a haematocrit of 1.5%, of which 0.3% were infected erythrocytes (>95% rings). Tritiated hypoxanthine incorporation was used to determine the extent to which parasite growth was inhibited by different drug concentrations or different dilutions of monkey plasma during 72 h of incubation. These data were used to generate concentration (or dilution)–response curves (TableCurve 2D, Jandal Scientific Software, CA, USA). IC50 and IC90 (or ID50 or ID90) values were defined as the drug concentrations (or dilutions) producing 50% or 90% inhibition of uptake of tritiated hypoxanthine by intra-erythrocytic malaria parasites when compared with drug-free serum samples (controls). The JPC2067 equivalent concentrations obtained by bioassay were estimated by multiplying the ID50 by IC50 of the K1 line.
The LC-MS/MS [selected reaction monitoring (SRM)] method for the measurement of JPC2056 and JPC2067 in plasma using JPC2067-D6 as the internal standard will be published elsewhere. The deuterated internal standard is JPC2056 with the gem dimethyl group fully substituted with deuterium atoms. Briefly, monkey plasma samples (200 µL) spiked with internal standard were passed through cyano SPE cartridges conditioned with methanol and ammonium hydroxide. After washing with a methanol–water solution, the samples were eluted with acidic methanol and the recovered eluant dried. Following reconstitution with an acetonitrile–water solution, samples were separated on a Restek Allure C18 column using a formic acid:methanol:water gradient driven by a Surveyor pump coupled to a LEAP autosampler. Mass spectrometric detection was accomplished on a ThermoElectron TSQ AM triple quadrupole mass spectrometer equipped with an electrospray ionization source in the positive ion mode. The peak area ratios of JPC2056 (product at m/z 328.1 from parent ion at m/z 412.0) and JPC2067 (product at m/z 126.2 from parent ion at m/z 410.1) to JPC2067-D6 (product at m/z 225.0 from parent ion at m/z 416.2) were calculated for each sample from the measured peak areas obtained by SRM. The retention time for JPC2056 was 1.56 ± 0.13 min, for JPC2067 was 1.31 ± 0.01 min and for JPC2067-D6 was 1.2 ± 0.05 min. Linear regression of the concentration data (range, 2.5–250 ng/mL) yielded a correlation coefficient of >0.994 for both JPC2056 and JPC2067. The lower limit of quantification (LLOQ) for each compound was 2.5 ng/mL. The overall precision for analysis of JPC2056 and JPC2067, as defined by the percent coefficient of variation (%CV) of quality control samples, was 
10% for all concentrations (n = 5). The accuracy of the method was within the accepted tolerance of ±20% relative standard deviation (RSD) at the LLOQ and ±15% RSD at the remaining concentrations through the linear range.
Pharmacokinetic and statistical analysis
JPC2056 and JPC2067 pharmacokinetics were determined by non-compartmental analysis (PK Solutions 2.0; Summit Research Services, Montrose, CO, USA). The elimination rate constant (kel) was determined by least-squares regression analysis of the plasma drug concentration–time curve. The area under the concentration–time curve (AUC0
t) was computed to the last sample point (t) using linear trapezoidal rule. The AUC0
was calculated as the sum of AUC0t and the quotient of the last measurable concentration (Ct) and kel. The half-life (t
) was calculated by dividing the constant 0.693 by kel. The apparent oral clearance (CL/F), expressed as a function of bioavailability (F), was calculated as the dose divided by AUC0. The apparent volume of distribution (V) was calculated as the ratio of CL/F to kel. Accumulation ratio was calculated by dividing the AUC0 after the last dose of the 3 day regimen by the AUC0 following the single dose. The metabolic ratio was calculated by dividing the AUC0 values of JPC2067 by those of JPC2056.
For statistical analysis, the paired Student's t-test was used and a value of P < 0.05 was taken as significant. Data are presented in the text as geometric means and means ± SD and graphically as mean ± SEM.
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In vitro antimalarial pharmacodynamics
Table 1 shows IC50s and IC90s of JPC2056, JPC2067, WR99210, chlorcycloguanil and pyrimethamine against the four P. falciparum lines, including D6 parasites transformed with the P. vivax dhfr–ts AMRU1 quadruple-mutant allele (D6-PvDHFR). JPC2056 possessed low intrinsic antimalarial activity, with mean IC90s ranging from 858 to 1895 ng/mL. In contrast, mean IC90s of 0.010–0.029 ng/mL were obtained when JPC2067 was incubated with the D6, K1 and TM90-C2A lines and 0.51 ng/mL when incubated with the D6-PvDHFR line. Slightly higher values were observed with WR99210. The in vitro activity of the active dihydrotriazines was similar to that reported in previous studies.12,15 The IC90s for chlorcycloguanil and pyrimethamine correlated with the number of mutations present in DHFR in the P. falciparum lines. For instance, compared with the D6 (wild-type), K1 (double-mutant), TM90-C2A (quadruple-mutant) and D6-PvDHFR (quadruple-mutant) were about 6080-, 16 060- and 18 620-fold less susceptible to pyrimethamine, respectively.
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Figure 2 shows that plasma antimalarial activity in monkeys given JPC2056 tended to be higher after the multiple-dose regimen than the single dose against the four P. falciparum lines. The mean ID90s at 3 h after multiple dosing were 1120 ± 255 for K1, 1396 ± 480 for TM90-C2A, 1613 ± 449 for D6 and 53 ± 13 for D6-PvDHFR. These values were approximately 1.9-, 1.6-, 1.3- and 1.2-fold higher following multiple dosing when compared with the single dose. K1, TM90-C2A and D6 were similarly inhibited by monkey plasma samples collected up to 72 h after JPC2056 administration. In contrast, inhibition of D6-PvDHFR was markedly less pronounced, with no activity at 72 h after drug administration.
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Pharmacokinetics of JPC2056 and JPC2067 in cynomolgus monkeys
Figure 3 shows mean plasma concentrations of JPC2056 and JPC2067 at various times after administration of the third dose of JPC2056. It shows that mean equivalent concentrations of JPC2067 measured by bioassay were slightly higher than those estimated by LC–MS/MS and also shows the concordance between the two analytical methodologies.
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Table 2 shows that the mean elimination half-life of JPC2056 was comparable for both regimens (single dose, 7.3 h versus multiple doses, 6.6 h). The geometric mean Cmax of JPC2056 at 3 h after drug administration was 1.3-fold higher following the multiple-dose regimen than the single-dose regimen (149.6 versus 113.4 ng/mL). The AUC following the single dose of JPC2056 was 2.14 versus 2.38 µg·h/mL after multiple dosing, suggesting negligible accumulation after once-daily dosing for a drug with a
7 h half-life in the cynomolgus monkey. JPC2056 is widely distributed to tissues, with a mean V/F of 56 L/kg.
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JPC2056 was biotransformed to JPC2067 in vivo, with geometric mean Cmax of 11.5 and 16.8 ng/mL after the single- and multiple-dose regimens, respectively (Table 2). The AUC of JPC2067 increased from 0.26 µg·h/mL after the single dose to 0.31 µg·h/mL following multiple dosing, with an accumulation ratio of 1.19. The elimination half-life of JPC2067 was longer than that of the prodrug, with mean values of 11.8 and 11.1 h after the single- and multiple-dose regimens, respectively. Similar to the LC–MS/MS data, the mean elimination half-life of JPC2067 was 11.9 h by bioassay.
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Resistance to the antifolate Fansidar® (sulfadoxine–pyrimethamine) is now widespread because of the emergence and spread of parasites containing amino acid substitutions in the DHFR region of DHFR–TS. These mutations accumulate in a stepwise fashion, usually first with a point mutation at codon 108 (S108N), followed by N51I or C59R and then I164L. The additional mutations create parasites that are increasingly resistant to pyrimethamine. Patients who are infected with P. falciparum carrying dhfr–ts double-mutant alleles (59R/108N) or 51I/108N usually have an adequate clinical response to sulfadoxine–pyrimethamine.34,35 Currently, the most common pyrimethamine-resistant dhfr–ts allele in Africa is the triple-mutant (51I/59R/108N), which has a borderline sensitivity to sulfadoxine–pyrimethamine.36–38 Unlike Southeast Asia,39 the highly pyrimethamine-resistant dhfr–ts quadruple-mutant allele (51I/59R/108N/164L) is still rare in Africa.40–43 These parasites are insensitive to chlorproguanil–dapsone, highlighting the need for new antifolate drugs.44
In our study, we showed that chlorcycloguanil (the active metabolite of chlorproguanil) is considerably more active than pyrimethamine against both the DHFR–TS double-mutant (59R/108N) and quadruple-mutant (51I/59R/108N/164L) lines of P. falciparum in vitro, and it is more active against the DHFR–TS double mutant than the quadruple mutant. In contrast, JPC2067 and WR99210 were >100-fold more potent than chlorcycloguanil and had comparable activity against both mutants. The increased potency of JPC2067 and WR99210 against the P. falciparum mutant lines is probably due to the dihydrotriazines having a more flexible side chain than that of chlorcycloguanil and so can adopt a conformation that fits well in the active site of the mutant DHFR enzyme.6 Our findings indicate that JPC2067 is far more active than JPC2056 in vitro and that JPC2067 is likely responsible for most of the antimalarial activity in vivo.
In accord with the intrinsic antimalarial activities of JPC2067, comparable potency was observed in monkey plasma samples tested in vitro against the two antifolate-resistant lines. At 3 h following JPC2056 administration, the ID90s of the monkeys' plasma against the DHFR–TS double- and quadruple-mutant lines were, respectively, 585 and 869 after the single dose and 1120 and 1396 following multiple dosing (Figure 2). The fact that 50-fold dilutions of monkey plasma can still inhibit parasite growth at 72 h (or 5 days after commencement of the 3 day regimen of JPC2056) suggests that this drug may play a very useful role in controlling highly antifolate-resistant falciparum malaria. Our in vitro pharmacodynamic data are in good agreement with earlier findings that assessed the activity of WR99210 and/or JPC2067 against novel dhfr quadruple-mutant alleles of P. falciparum.24,41,45,46
On the basis of the LC-MS/MS data, the prodrug JPC2056 is converted to its active metabolite, JPC2067, in monkeys. The geometric mean plasma Cmax of JPC2056 was 150 ng/mL after multiple dosing, but owing to the limited number of samples collected following the last dose, this is probably an underestimation. The corresponding Cmax of JPC2067 was 17 ng/mL, with a metabolic ratio using AUC values of 13%. The elimination half-life of JPC2056 is less than that of JPC2067 (7 versus 12 h). Similarly, following PS-15 administration to Macaca mulatta (rhesus) and Saimiri sciureus (squirrel) monkeys, the prodrug is eliminated faster than its metabolite, WR99210.13,47 The slower elimination of JPC2067 parallels the prolonged plasma antimalarial activity observed in this study. The 3 day regimen of JPC2056 is advantageous with accumulation of the active metabolite (accumulation ratio of 1.19) after daily administration of the prodrug.
JPC2056 relies on metabolic oxidation and cyclization to produce the active dihydrotriazine, JPC2067. Human liver microsomal studies have shown that JPC2056 is primarily (60% to 70%) metabolized by cytochrome P450 3A4. On the basis of the liver microsomal studies of phenoxypropoxybiguanides, metabolism of JPC2056 is similar in cynomolgus monkeys and humans, with 70% to 75% of the total metabolites as active dihydrotriazine, 24% to 30% dealkylated and <2% hydroxylated (T. W. Shearer, personal communication). In the present study, JPC2067 equivalent concentrations obtained by bioassay were slightly higher than the LC-MS/MS values of JPC2067, indicating that JPC2067 was responsible for most of the plasma antimalarial activity. Additional activity (additive and/or synergistic) may have come from dealkylated and/or hydroxylated metabolites.
In Latin America, the Indian subcontinent, the Southwest Pacific and Southeast Asia, P. vivax malaria is highly prevalent and is a major public health problem causing morbidity and occasional death.48 As observed with P. falciparum, amino acid changes in the DHFR region of DHFR–TS cause P. vivax resistance to pyrimethamine. In Indonesia and Papua New Guinea, the DHFR–TS double mutant (58R/117N) and quadruple mutants (57L/58R/61M/117T) and (57L/111L/117T/173F) are common.19,49 All highly pyrimethamine-resistant alleles carry both the 57L and 117T mutations.19
As it is difficult to culture P. vivax long-term, we assessed the in vitro sensitivity of antifolate drugs against a P. vivax dhfr–ts quadruple-mutant (57L/58R/61M/117T) allele transfected into the D6 line of P. falciparum (D6-PvDHFR). Similar to the P. falciparum DHFR–TS quadruple-mutant, D6-PvDHFR is highly resistant to chlorcycloguanil and pyrimethamine, but is far more susceptible to WR99210 and JPC2067. Nevertheless, D6-PvDHFR is about 68- and 24-fold less susceptible than TM90-C2A to WR99210 and JPC2067, respectively. Using an in vitro yeast expression system, WR99210 and JPC2067 were also less active against a 57L/111L/117T/173F allele from P. vivax than a 51I/59R/108N/164L allele from P. falciparum.24 In our monkey-in vitro model system, we observed that after treating monkeys with JPC2056, the monkey plasma samples were less active in inhibiting the D6-PvDHFR line when compared with the TM90-C2A line. Despite this reduced activity against D6-PvDHFR, with an ID90 of about 53 at 3 h after the multiple-dose regimen, our data suggest that sufficient drug should still be present to kill parasites containing P. vivax dhfr–ts quadruple-mutant alleles. This observation is in accord with Hastings et al.'s49 prediction that drugs of the WR99210 class are likely to demonstrate good clinical efficacy against P. vivax parasites carrying the dhfr–ts quadruple-mutant allele.
The present study shows that JPC2067 is an extremely effective inhibitor of the P. falciparum DHFR, particularly against quadruple mutants, which lead to high-level resistance to the antifolate combinations, sulfadoxine–pyrimethamine and chlorproguanil–dapsone. Although not as potent against a D6 line expressing a highly pyrimethamine-resistant P. vivax DHFR–TS, the JPC2067 concentrations observed in the monkeys were still effective in inhibiting parasite growth. As in vitro antimalarial activity of JPC2056 persisted for up to 72 h after completing drug administration, JPC2056 may prove to be a very useful combination partner for treating highly resistant DHFR mutant P. falciparum and P. vivax infections.
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The University of Miami and the Australian Army Malaria Institute received financial support from Jacobus Pharmaceutical Company for the animal studies and in vitro pharmacodynamic investigations, respectively.
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With the exception of D. P. J. and G. A. S., no other authors have financial interests in Jacobus Pharmaceutical Company.
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We are most grateful to Mr Anthony Kotecki and Miss Kerryn Rowcliffe for in vitro and bioassay analyses. We would also like to thank the Australian Red Cross Blood Service (Brisbane) for providing human erythrocytes and serum for the in vitro cultivation of P. falciparum lines. The material has been reviewed by the Walter Reed Army Institute of Research, and there is no objection to its publication. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army (USA), the Department of Defense (USA), the Australian Defence Health Service or any extant Australian Defence Force policy.
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References |
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