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Antimicrobial Agents and Chemotherapy, August 2007, p. 2898-2904, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00932-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Pharmacokinetics, Safety, and Hydrolysis of Oral Pyrroloquinazolinediamines Administered in Single and Multiple Doses in Rats{triangledown}

Qigui Li,* Michael P. Kozar, Todd W. Shearer, Lisa H. Xie, Ai J. Lin, Kirsten S. Smith, Yuanzheng Si, Lalaine Anova, Jing Zhang, Wilbur K. Milhous, and Donald R. Skillman

Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910

Received 27 July 2006/ Returned for modification 27 March 2007/ Accepted 31 May 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pyrroloquinazolinediamine (PQD) derivatives such as tetra-acetamide PQD (PQD-A4) and bis-ethylcarbamyl PQD (PQD-BE) were much safer (with therapeutic indices of 80 and 32, respectively) than their parent compound, PQD (therapeutic index, 10). Further evaluation of PQD-A4 and PQD-BE in single and multiple pharmacokinetic (PK) studies as well as corresponding toxicity studies was conducted with rats. PQD-A4 could be converted to two intermediate metabolites (monoacetamide PQD and bisacetamide PQD) first and then to the final metabolite, PQD, while PQD-BE was directly hydrolyzed to PQD without precursor and intermediate metabolites. Maximum tolerant doses showed that PQD-A4 and PQD-BE have only 1/12 and 1/6, respectively, of the toxicity of PQD after a single oral dose. Compared to the area under the concentration-time curve for PQD alone (2,965 ng·h/ml), values measured in animals treated with PQD-A4 and PQD-BE were one-third (1,047 ng·h/ml) and one-half (1,381 ng·h/ml) as high, respectively, after an equimolar dosage, suggesting that PQD was the only agent to induce the toxicity. Similar results were also shown in multiple treatments; PQD-A4 and PQD-BE generated two-fifths and three-fifths, respectively, of PQD concentrations, with 8.8-fold and 3.8-fold safety margins, respectively, over the parent drug. PK data indicated that the bioavailability of oral PQD-A4 was greatly limited at high dose levels, that PQD-A4 was slowly converted to PQD via a sequential three-step process of conversion, and that PQD-A4 was significantly less toxic than the one-step hydrolysis drug, PQD-BE. It was concluded that the slow and smaller release of PQD was the main reason for the reduction in toxicity and that the active intermediate metabolites can still maintain antimalarial potency. Therefore, the candidate with multiple-step hydrolysis of PQD could be developed as a safer potential agent for malaria treatment.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pyrroloquinazolinediamine {PQD; 7-(substituted)-7H-pyrrolo[3,2-f] quinazoline-1,3-diamines} has been reported to possess anticancer, antimicrobial, and antimalarial activities (15). PQD was reported to be a very active antimalarial agent both in vivo and in vitro (7, 10, 15). The compounds were potent inhibitors of fungal and human dihydrofolate reductase and were highly active against Candida albicans and an array of tumor cell lines (8, 12). The compounds were active in lung and brain tumor models and displayed in vivo activity against Pneumocystis carinii and C. albicans (8, 24). PQD acted as an inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) and could rapidly and very selectively shut down epidermal growth factor-stimulated signal transmission by binding competitively at the ATP site of the EGFR (2). Another new oral PQD derivative exerts analgesic and anti-inflammatory effects, which were related to dual inhibition of cyclo-oxygenase-2 and 5-lipoxygenase activities (13, 21).

PQD was reported to be very active as an antimalarial agent both in vivo and in vitro. However, this compound displayed "variable" toxicity in rodents, with low therapeutic indices, and was extremely toxic in the Aotus nonhuman primate model, with a very narrow therapeutic index of 1 to 2 (unpublished data). In an attempt to obviate this toxicity, two oral derivatives of PQD, tetra-acetamide PQD (PQD-A4) and bis-ethylcarbamyl PQD (PQD-BE), were designed and synthesized.

This study demonstrated that the minimum dose required to clear malaria parasitemia was 2.4 µmol/kg of body weight for PQD, PQD-4A, and PQD-BE. The maximum tolerated doses of PQD-A4, PQD-BE, and PQD were found to be 190, 77, and 24 µmol/kg, yielding therapeutic indices of 80, 32, and 10, respectively, indicating that the two new derivatives, PQD-4A and PQD-BE, are 8.0-fold and 3.2-fold, respectively, safer than their parent compound when dosed for three consecutive days. Oral PQD-A4 and PQD-BE are 44 to 70 times more potent on an mg basis than intravenous artesunate (AS), which is in current use as first-line antimalarial agent. As assessed from the therapeutic index over 3 days, PQD-A4, PQD-BE, and PQD administered orally are 20, 8, and 2.5 times safer than AS given intravenously (27). In the present study, the pharmacokinetic (PK) differences between these two derivatives have been investigated and the superiority of the two derivatives to PQD in this respect has been fully evaluated. The aim of the present work was to characterize the pharmacological properties of PQD-A4 and PQD-BE in comparison to PQD in rats.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and animals. PQD (WR227825) was prepared by ChemPacific Inc. (Baltimore, MD). PQD-A4 and PQD-BE were synthesized in the Department of Medicinal Chemistry (11), Walter Reed Army Institute of Research, Silver Spring, MD. Carboxymethyl cellulose (CMC), Tween 80, and 0.9% saline solution were purchased from Abbott Labs (Chicago, IL). Sodium citrate, heparin, D-glucose, glycerol, and methanol were purchased from Sigma Chemical Co.

Male Sprague-Dawley rats obtained from Charles River Laboratories were used in this study. On arrival, the animals were acclimated for 7 days (quarantine), thereby assuring that rats were 8 weeks of age upon the initiation of dosing. The animals were housed in individual cages maintained with a temperature range of 64 to 79°F, 34 to 68% relative humidity, and 12-h light/dark cycles. Food and water were supplied ad libitum during quarantine and throughout the study. The animals were fed a standard rodent maintenance diet. The animal use protocol was approved by IACUC, WRAIR, and the research was conducted in compliance with the Animal Welfare Act and other federal statutes. Regulations relating to animals and experiments involving animals adhered to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council publication, 1996 edition.

Oral formulation and administration. Suspension formulations of PQD-A4, PQD-BE, and PQD were determined and prepared fresh daily. The suspension solution of the three PQDs was prepared in 1% (wt/vol) CMC in distilled water, using a Sonics Vibra Cell sonicator (Sonics & Materials, Inc., Danbury, CT) programmed to run with 5-second pulses and 5-second standbys for 8 min. Immediately following, 0.2% (vol/vol) Tween 80 was added to the mixture, and the mixture was resonicated for another 2 minutes. The maximum concentration for the suspension of each of the three PQDs was 50 to 100 mg/ml. The particle size of each final suspension of PQD or analog was determined by an LA-930 light-scattering particle size and distribution analyzer (HORIBA Instruments, Inc., Irvine, CA). The mean particle size for the three suspensions used in the dose range, toxicity, and PK studies ranged from 6.4 to 17.8 µm. Each drug formulation was administered via oral gavage. The vehicle (1% CMC plus 0.2% Tween 80 suspension) was administered using the same volume, duration, and route as the test compounds.

Acute toxicity of PQDs following single treatment. Single-dose 50% lethal dose (LD50) and cause of mortality were determined in rats. Three to 5 animals of each group were treated with PQD-A4, PQD-BE, and PQD at doses from 56 to 2,340 µmol/kg of body weight covering a range likely to encompass the LD50 (see Table 1). Based on the LD50 estimate from this pilot study, a dose range study was performed in order to obtain a more accurate estimate for the acute toxicity. The study was done with single dosing of' three animals of each group at five dose levels below the LD50 point estimate. Daily clinical assessments (food and water intake, behavior, activity, body weight, and death or sacrifice due to moribundity) were conducted for 14 days after dose administration.


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  		TABLE 1. Anorexic indices, MTD, and mortality following intragastric administration of PQD-A4, PQD-BE, and PQD at variable daily dose for 1 and 3 days and clinical observation for 14 days for groups of three to five male rats

 
Multiple-dose subacute toxicity of PQDs. The toxicity of three PQDs was determined following daily oral administration of each compound for 3 days to groups of seven randomized, 8-week-old rats at 0, 6.0, 11.9, 23.8, 47.6, 95.0, and 190.0 µmol/kg (see Table 1). This study was designed to identify a no-observed-adverse-effect level, mild effect, and moderate effect. For this reason, the highest dose was set at one-fourth the single-dose LD50 observed in the acute-toxicity study. For these reasons, four or five dose levels with five male rats per group were required, and a vehicle control was also included. Animals were given PQD-A4, PQD-BE, and PQD by oral gavage with 2.0 ml drug solution/kg at lower dose levels (drug vehicle, 6.0, 11.9, 23.8, 47.6 µmol/kg). For high doses (95 and 190 µmol/kg), 5.0 ml/kg of the appropriate drug dilution (at concentrations of 95 µmol drug base/ml) were administered orally. General health status of the animals was monitored daily. Daily clinical assessments (food and water intake, behavior, activity, body weight, and death or sacrifice due to moribundity) were conducted for 14 days after dose administration.

Identification of metabolites of PQD-A4 and PQD-BE. Rapid hydrolysis of PQD-A4 and PQD-BE was observed in rat and human plasma following incubation at 37°C in vitro. The metabolites/degradation products were identified using high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS). Each metabolite/degradation product was isolated and compared to authentic synthetic standards for HPLC retention, MS/MS fragmentation patterns, and nuclear magnetic resonance spectrum. Three primary hydrolysis products of PQD-A4 were identified following in vitro incubation in rat and human plasma: bisacetamide PQD (PQD-A2; m/z 442.1), monoacetamide PQD (PQD-A1; m/z 400.1), and PQD (m/z 358.02). One primary hydrolysis products of PQD-BE was identified: PQD (m/z 358.02). These same hydrolysis products were also detected in rats following intravenous and intragastric administration of PQD-A4 and PQD-BE. Each hydrolysis product was quantified in rat plasma using authentic standards by liquid chromatography (LC)-MS/MS. The following selected reaction monitoring transitions were used for PQD-A4: PQD-A4, m/z 526.100->m/z 199.07; PQD-A2, m/z 442.100->m/z 358.100 m/z; PQD-A1, m/z 400.100->m/z 199.07; and PQD, m/z 358.020->m/z 199.07. For the analysis of PQD-BE the monitored transitions were as follows: PQD-BE, m/z 502.000->m/z 199.100; and PQD, m/z 358.020->m/z 199.070.

Series bioavailability of PQD-A4 and PQD. Series bioavailability profiles for rats were quite different between PQD-A4 and its parent drug, PQD. Very low gastrointestinal (GI) absorption was found for the oral PQD-A4 and PQD. In the previous efficacy studies, the drugs still showed excellent antimalarial potency in mice, rats, and monkeys at low dose levels. In order to find out dose-dependent bioavailability, the nine groups of rats treated with PQD-A4 and PQD at variable dose levels received single oral administration of 5 to 6 µmol/kg in the intravenous groups and 38 to 952 µmol/kg in the oral administration groups.

Single PK and bioavailability studies. Six groups of Sprague-Dawley rats ranging between 185 to 242 g body weight received PQD-A4, PQD-BE, and PQD at a single equimolecular dose rate of 6 µM/kg intravenously and 95 µM/kg via oral gavage. Serial plasma samples were collected during the absorption, distribution, and elimination phases for full PK analysis. Each animal was dosed, and plasma samples were obtained for up to 2 days. A total of 11 (at 0, 30, and 60 min and 2, 3, 5, 8, 12, 24, 36, and 48 h) plasma samples were collected into cooled vials by using a Culex automated blood sampler (Bioanalytical Systems Inc., West Lafayette, IN). One hundred microliters of blood per sample for intravenous groups and 200 µl for oral gavage groups were collected for all samples. During collection, blood samples were mixed with heparin-saline (50 µl) to obtain a total volume of 150 µl for intravenous samples and 250 µl for intragastric samples. All samples were analyzed by using LC-MS/MS. The Culex automatic blood sampler is a blood sampling and metabolic monitoring machine, which is controlled by its own internal computer. By using this system we were successful in evaluating PK for long-term periods. The detailed sampling process was described previously (18).

Multiple PK and bioavailability studies. Three groups of Sprague-Dawley rats ranging from 214 to 232 g body weight received three daily equimolecular oral doses (47.6 µM/kg) of PQD-A4, PQD-BE, and PQD at 25.0, 23.9, and 17.1 mg/kg, respectively. During the 3-day dose regimen, serial plasma samples were collected during the absorption, distribution, and elimination phases for a full PK analysis. Thus, each animal was dosed, and plasma samples were obtained for up to 5 days. A total of 18 samples (at 0, 30, and 60 min and 2, 3, 5, 8, 24, 36, 48, 48.5, 49, 50, 51, 53, 56, 72, and 96 h) were collected into cooled vials using an automated blood sampler (Culex; Bioanalytical Systems, Inc.). One hundred microliters of blood was collected from the oral-administration group at each time point for all samples. During collection, blood samples were mixed with heparin-saline (50 µl) to a total volume of 150 µl for the samples. Thus, a total 1.8 ml blood (11.7% of total blood volume) was withdrawn from a single rat (in the oral-administration group) during the 5-day treatment period. All plasma samples were analyzed using LC-MS/MS.

LC-MS/MS assay and validation. Rat plasma samples (50 to 100 µl each) were analyzed for PQD-A4 or PQD-BE with an LC-MS/MS procedure on a ThermoElectron Surveyor HPLC coupled to a TSQ Quantum AM triple-quadrupole mass spectrometer (ThermoElectron, San Jose, CA). The chromatographic separation was performed using an Xterra C18, 3.5-µm, 2.1-mm by 50-mm column (Waters Corp., Milford, MA). A rapid gradient was employed by using 20% A (0.5% formic acid), 75% C (H2O), and 5% D (methanol); these levels were held for 1 minute and then ramped to 20% A, 5% C, and 75% D in 5 min and held for four additional minutes, followed by a return to the original conditions and equilibration for 3 min.

The mass spectrometer was tuned and calibrated according to the manufacturer's procedure in the positive ion, electrospray ionization mode. Detection and mass scanning were performed either in the full-scan mode (m/z 150 to 650) or by selected reaction monitoring by selecting the parent ion of the drugs (M+H) in the first quadrupole, followed by collision-induced dissociation in the collision cell (99.999% argon at a pressure of 1.5 mtorr and collision energy experimentally determined) to produce the most intense, characteristic product ion.

Samples were prepared by adding 200 µl acetonitrile containing indomethacin as the internal standard (IS), vortexing for 1 min, centrifuging for 5 min, and transferring the supernatant to an autosampler injection vial prior to separation by LC-MS/MS. Standard curve and quality control (QC) samples were generated by spiking interference-free rat plasma samples (pretested via the LC-MS/MS system) with known amounts of PQD-A4 or PQD-BE and IS. Standard curve, QC, and assay samples were loaded into the autosampler, which was maintained at 4°C, with 5 µl injected into the LC-MS/MS system. The peak area ratios (PARs) for PQD-A4 and PQD-BE were determined by the peak area of drug to the IS (m/z 470.180->m/z 158.990) and were calculated for each sample. The PARs for spiked concentrations of PQD-A4 or PQD-BE to IS for the standard-curve samples were fit by 1/y weighted least-squares linear regression to the equation for the straight line (y = mx + b, where y = PAR and x = PQD-A4 or PQD-BE concentration). Drug concentrations in assay samples were calculated using the drug to IS PARs obtained by LC-MS/MS and processed by the Xcalibur Quan Browser software (Thermo Electron).

Data analysis. The PK parameters were calculated as described previously (18). The LD50 and ID50 were calculated using the TableCurve 6.0 program (Advanced Graphics Software, Inc., Encinitas, CA). Maximum tolerant dose (MTD) was defined as the dose that caused clinical toxicity in 100% of animals but did not cause death. Means and standard deviations (SD) were calculated. Coefficients of variation were calculated as percentages of SD divided by mean values. Statistical analysis was performed with Excel (Microsoft Corporation), using Student's t test for dependent samples to compare means of paired and unpaired samples between treatment groups.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MTD, mortality, and LD50 after single oral dose. The acute-toxicity study of PQD-A4, PQD-BE, and PQD was conducted with rats following a single intragastric administration. After administration, all rats were observed for 2 weeks. The dose range and animal death rate are shown in Table 1. The LD50 data were available for PQD and PQD-BE, with 798 µmol/kg and 1,573 µmol/kg, respectively, following a single intragastric dosing. The LD50 value was not obtainable for PQD-A4 because no death was found at 2,340 µmol/kg (1,200 mg/kg), which was the highest allowable dose. Based on the acute-toxicity data, the MTDs were determined as 280 µmol/kg for PQD, 1,170 µmol/kg for PQD-BE, and 2,340 µmol/kg for PQD-A4 (Table 1).

Anorexic index and mortality after three multiple oral doses. Anorexia (reduction of food consumption and body weight) was observed in all rats with three drugs after multiple intragastric administrations at variable dose levels. Normal rats had a body weight increase of 4 to 7 g (around 2 to 3% of body weight). In anorexic drug-treated animals, the body weight either stopped increasing or decreased. An anorexic index (ratio of the body weight change to the day of the weight change) was used in this study to determine the anorexia of treated rats (5, 17), and the values, with mortalities, are exhibited in Table 1.

In all control animals from the present study, the anorexic index ranged from 2.13 to 2.66%; if the index for treated rats fell below this range significantly, it was interpreted as indicating anorexic toxicity (5, 17). For rats treated with PQD daily for 3 days, the anorexic index was in the range of 1.05 to –1.98 from doses of 11.9 to 47.6 µmol/kg, suggesting severe anorexic toxicity. In the animals treated with PQD-BE and PQD-A4, the indices were in the range of 1.88 to –0.65 and 2.06 to –0.89 from doses of 23.8 to 95.0 and 47.6 to 190.0 µmol/kg, respectively. The results show that PQD-BE and PQD-A4 have much less anorexic toxicity than their parent compound, PQD. The doses required to inhibit one-half the body weight (ID50) change were 15.96, 60.65, and 140.43 µmol/kg for PQD, PQD-BE, and PQD-A4 (Table 1), respectively, indicating that PQD-BE and PQD-A4 are 3.8-fold and 8.8-fold safer than PQD in rats during treatment by the parallel test (Fig. 1).


Figure 1
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  		FIG. 1. Mean anorexic indices following daily intragastric doses for 3 days with PQD-A4 and PQD-BE versus PQD at variable molar dosages in rats. Anorexic toxicity potencies were compared via a parallel test with measurement of anorexic indices. If the toxic potency of PQD is defined as 1, the safety margin of PQD-A4 was 8.8 and that of PQD-BE was 3.8 (n = 3 to 5).

 
Series bioavailability studies. Lower bioavailabilities were reported: 11.45% for PQD-A4 and 14.86% for PQD-BE compared to 34.56% for PQD after a single oral dose (Table 2). During the toxicity studies, increasing doses of PQD-A4 and PQD-BE showed a lower toxicity potency than those of PQD with the same dosage increases. It was suggested that the bioavailability of PQD-A4 or PQD-BE may be limited at higher dose levels. A series of bioavailability studies have been conducted, and the results showed that the bioavailabilities of PQD-A4 were dose dependent in rats (Fig. 2). The bioavailabilities of PQD-A4 were calculated as 21.04, 11.45, 8.35, and 4.30% after single intragastric doses of 38, 95, 190, and 952 µmol/kg, respectively, suggesting that the bioavailability was significantly reduced as the oral dose was increased. A similar result was also found for PQD administered alone: after giving doses of 56, 95, and 140 µmol/kg intragastrically, the absolute bioavailabilities were 39.63, 34.56, and 23.70%, respectively.


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  		TABLE 2. PK parameters for PQD following single (equimolar) dose and daily dosing for 3 days of intragastric administrations of PQD-A4, PQD-BE, and PQD to male ratsa

 

Figure 2
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  		FIG. 2. Mean AUC values (top) and mean bioavailability (bottom) of PQD-A4 following single intragastric administration at 20, 50, 100, and 500 mg/kg in rats (markers), and computer simulation (solid lines) predicting the AUC and bioavailability through whole-dose levels (n = 3 to 6).

 
Metabolic pathways of PQD-A4 and PQD-BE. In the present study, the hydrolysis metabolites of PQD-A4 and PQD-BE were observed in in vitro and in vivo studies. After a short incubation (30 min) in plasma at room temperature or oral administration in animals with PQD-A4, the drug was almost undetectable in all samples. For the analysis of PQD-A4, only three metabolites (PQD-A2, PQD-A1, and PQD) were separated and identified from these samples. The primary observed metabolite of PQD-A4 was PQD, suggesting that PQD-A4 undergoes three-step degradation to PQD (Fig. 3), and the rate of conversion of PQD-A4 to PQD was greater for in vivo (84 to 95%) than in vitro (49 to 76%) samples (our unpublished data). In addition, PQD was also the only metabolite of PQD-BE detectable in all samples, indicating that PQD-BE is likewise degraded to PQD (Fig. 3). Detailed in vivo biotransformation data were obtained from single and multiple PK rat studies and bioavailability studies as described below.


Figure 3
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  		FIG. 3. Schematic demonstration of a three-step metabolic pathway of PQD-A4 sequentially to PQD-A2, PQD-A1, and then to its parent compound (PQD), as well as a one-step metabolic pathway of PQD-BE directly to PQD, as detected by LC-MS/MS. The structures were identified in rat blood following intragastric or intravenous administration of PQD-A4 and PQD-BE.

 
Single PK analysis. The conversions of PQD-BE and PQD-A4 to PQD were determined at 30 min (first collected sample) after oral administration in rats. PQD was shown as the primary product (over 84 to 95%) present in this study compared to other metabolites (4.8 to 15.3%). Therefore, PQD-A4 and PQD-BE were active (11) and prodrugs of PQD. The PK analysis of PQD was performed for the three drugs following the oral administrations. The results revealed that there was a slow decline in the plasma drug concentration and that the elimination half-lives were 12.64 to 17.13 h for the three drugs after a single administration. The mean area under the curve (AUC) of PQD ranged from 1,047 to 1,381 ng·h/ml for both PQD-A4 and PQD-BE, which was less than one-half of the AUC for PQD administered alone (2,965 ng·h/ml) at single equimolar dosage. The bioavailabilities of PQD-A4 (11.45%) and PQD-BE (14.86%) were much less than that of PQD alone (34.56%). PQD-A4 also generated two additional products, PQD-A2 and PQD-A1, in rat blood. Although the two products had low concentrations in plasma, both compounds exhibited long half-lives: 13.2 h and 12.6 h, respectively (Table 2). The two degradation products (PQD-A1 and PQD-A2) of PQD-A4 were reported to possess potent antimalarial effects, similar to those of PQD, PQD-BE, and PQD-A4, in vitro (11). PQD-BE seems to have only one active product, PQD.

Multiple PK analysis. Three PQDs were administered intragastrically as daily multiple equimolar doses for 3 days in rats. The PK parameter estimates obtained on day 1 and day 3 after multiple equimolar doses of 47.6 µmol/kg intragastrically for the three PQDs are summarized in Table 2. The results revealed that the PK parameters for day 1 and day 3 were not similar and that a significant accumulation and slow elimination of PQD were found for the plasma concentration during the 3-day dosing. Plasma concentration on day 1 indicated that the maximum concentrations (Cmax) of the three PQDs (31.23 to 84.36 ng/ml) were similar to the plasma concentrations on day 3 (30.84 to 87.23 ng/ml). However, the AUCs of PQD-A4, PQD-BE, and PQD (706, 876, and 1,570 ng·h/ml, respectively) on day 1 were about one-half those on day 3 (1,250, 1,530, and 2,436 ng·h/ml), suggesting greater drug accumulation in plasma with 1.7 to 1.9 ratios of AUC on day 3 (AUCDay3)/AUCDay1. The elimination half-lives on day 1 for the three PQDs ranged from 12.9 to 14.3 h, much shorter than the 16.9 to 23.2 h observed on day 3 (Table 2). The accumulations of the three PQD drugs were derived from their long half-lives and were perhaps augmented by the increasing half-lives on day 3, which may be due to the anorexic toxicity in the present study.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PQD and its derivatives induced dose-dependent GI toxicity including reductions of body weight gain in rats. To evaluate the anorexic toxicity, an anorexic index has been previously used with reductions of body weight. Body weight was more sensitive, more objective, and more accurate in reflecting anorexic toxicity than food and water consumption (17). Rats have about 2% body weight (5 to 7 g) gain every day, which is dependent on the intake of 14 to 21 g food and 25 to 32 ml water daily (17). Therefore, the body weight could indicate the consumption of food and water to evaluate the anorexic toxicity alone (3, 6, 16). In this study, the decrease of body weight was calculated as a sole index of anorexic toxicity to evaluate the GI toxicity of the PQD drugs in rats (Table 1). The index results showed that (i) anorexic toxicity was dose dependent for the three PQDs; (ii) GI toxicity was significantly reduced by the derivatives, PQD-A4 and PQD-BE, compared to PQD at any dose level; and (iii) the GI toxicity of PQD was about 3.8- and 8.8-fold higher than those of PQD-BE and PQD-A4, respectively, which had similar safety margins (3.2- and 8.0-fold), as shown in our therapeutic-index study, with the same multiple doses (27).

The two derivatives demonstrated improved toxicity profiles in comparison to their parent, PQD. PQD seems to be the only agent to induce toxicity because toxicity was found to be dependent on PQD concentration in plasma in the present study. For example, at an equimolar dose level PQD-A4 produced less PQD (AUC, 1,047 ng·h/ml) and resulted in mild toxicity; PQD-BE generated a little more PQD (1,381 ng·h/ml) and caused moderate toxicity, while PQD alone with high plasma concentration (2,965 ng·h/ml) induced severe toxicity in rats. At a single oral dose, PQD-A4 and PQD-BE were about 12-fold and 6-fold safer than PQD. As expected, PQD-A4 was even slower than PQD-BE since PQD-A4 generated three metabolites and each metabolite had an elimination half-life of more than 12 h. Although the PQD concentration in plasma was significantly less from PQD-A4 and PQD-BE administration, the efficacies of derivatives were not affected because the derivatives (PQD-A4 and PQD-BE) and their intermediate metabolites (PQD-A1 and PQD-A2) all have antimalarial potencies similar to that of PQD (11). An increased therapeutic index was the aspiration for the approaches in the design as well as the evaluation criteria. The PQD derivatives showed excellent oral activity, with low toxicity and a higher therapeutic index for the treatment of rodent malaria in the present study.

Theoretically, PQD-A4 could be hydrolyzed to PQD by a sequential metabolic pathway to trisacetamide PQD, to PQD-A2, to PQD-A1, and then to PQD. Surprisingly, the trisacetamide PQD was not detected in any of the samples from our in vitro and in vivo studies, suggesting that PQD-A4 is the only three-step derivative resulting from a triple-hydrolysis pathway, and not from a four-step process (4, 26). PK parameters showed that although the three metabolites have different concentrations (AUCs) in rat blood, they have very close half-lives, in the range of 12.6 to 13.2 h (Table 2). Since PQD-A4 and trisacetamide PQD were not measurable in these samples at any time point, the rapid hydrolysis of PQD-A4 to PQD-A2 should be considered because no concentration of PQD-A4 was detected from any sample.

Comparable results were also found for PQD-BE, which theoretically should sequentially metabolize to monoethylcarbamyl PQD and then to PQD. However, PQD was the only metabolite measurable in all samples at any time point; the monoethylcarbamyl PQD was not detected. PQD-BE appears to be a one-step hydrolysis derivative (1, 9). Unlike PQD-A4, which was undetectable in blood, PQD-BE was demonstrable in most of the blood samples, with a mean elimination half-life of 9.3 ± 5.1 h. This suggests that PQD-BE was directly hydrolyzed to PQD without any steps in between. The one-step hydrolysis of PQD-BE to PQD should be considered; the one-step rate of conversion to PQD could be faster than the three-step hydrolysis of PQD-A4 in rats.

During three multiple oral doses (47.6 µmol/kg) of PQD-A4, PQD-BE, and PQD, a significant drug accumulation was observed in the blood. The twofold drug accumulation in the blood could affect the PK parameters in the rats if it was not due to the everyday normal trough accumulation. The change in PK parameters may have resulted from daily dose accumulation, prolonged half-lives, and systemic toxicity, which could be a pathophysiological factor to change the slopes of the distribution and elimination due to biotransformation, excretion, and protein or tissue binding (19, 25). In the present study, the half-lives of three PQDs have been changed and extended 3 to 6 h longer on day 3 than on day 1, suggesting that the systemic toxicity encountered in these animals was due to mild to severe anorexic toxicity and body weight loss presented in this study.

On day 3 after the daily treatments for 3 days, the drug exposure levels (AUCs) increased about twofold and the drug exposure time (half-life) was extended 25 to 37% in all cases compared to that on day 1. It is usually considered that both the factors (high drug exposure level and long drug exposure time) cause an increase of toxicity. An investigation of drug exposure indicated that continued exposure over time (drug exposure time) rather than a high concentration (drug exposure level) over a short time (interval time) contributed to toxicity (14, 20, 22, 23). This result demonstrated that systemic toxicity with multiple doses could be greater than that with a single administration. Therefore, longer-term (more than three daily doses) administrations of PQD and its derivatives need further and detailed investigations of toxicity.

The two derivatives showed exceptionally less toxicity and higher therapeutic indices than their parent drug, which successfully reduced side effects associated with PQD in our previous study (27) and in the present studies. However, the two derivatives of PQD also reduced the bioavailability by one-half compared to PQD in rats. The series bioavailability studies showed that the limitation of bioavailability was more affected at higher dose levels (>20 mg/kg) and was dose dependent. This could be a reason why the efficacy was not affected very much at therapeutic dose levels (low doses of <10 mg/kg) in malaria-infected rats (27).

In conclusion, the PK studies demonstrated that PQD-A4 and PQD-BE are active derivatives of PQD with three- and one-step hydrolysis pathways, respectively. At a single oral dose, PQD-A4 and PQD-BE were about 12-fold and 6-fold safer than PQD on the basis of their MTD data. The slow and smaller release of PQD could be the reason for reduced toxicity, and the active intermediate metabolites can still maintain antimalarial potency. Therefore, the sequential three-step derivative, PQD-A4, is considered a safer candidate than the parent compound (PQD).


   ACKNOWLEDGMENTS
 
This study was supported by the United States Army Research and Materiel Command.

The opinions or assertions contained herein are the private views of the authors and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pharmacology, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20307-5100. Phone: (301) 319-9351. Fax: (301) 319-7360. E-mail: qigui.li{at}na.amedd.army.mil Back

{triangledown} Published ahead of print on 11 June 2007. Back


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Antimicrobial Agents and Chemotherapy, August 2007, p. 2898-2904, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00932-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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