Introduction

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XK469¹ (NSC 697887; (±)-2-[4-(7-chloro-2-quinoxaliny)oxy]phenoxy propionic acid) (Fig. 1), is an analog of the herbicide Assure synthesized by DuPont. In a screen for solid tumor-selective agents, it was found that XK469 possesses broad activity against murine solid tumors and human xenografts (Corbett et al., 1998; LoRusso et al., 1999). Its mechanism of antitumor activity has recently been elucidated as a selective topoisomerase II poison (Gao et al., 1999; Snapka et al., 2001). Both of the XK469 enantiomers induce reversible protein-DNA cross-links, thus stabilizing the DNA strand passing intermediates in the topoisomerase reaction. In vitro studies showed that the R(+)-enantiomer was about twice as active as the S-enantiomer (Snapka et al., 2001), with no significant interinversion found for the enantiomers in that system. The R(+)-XK469 has recently been selected for clinical evaluation by the National Cancer Institute.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Chemical structure of XK469 with the asymmetric center indicated by an asterisk (A) and chloroquinoxaline sulfonamide (the internal standard) (B).

We recently reported a validated chiral HPLC method to analyze the drug concentrations in rat urine and mouse, rat, and dog plasma samples (Zheng et al., 2002). A substantial inversion of the S()- to R(+)-enantiomer was observed in the plasma samples from all three animal species; however, the reverse inversion was not detected, at least not in the rat. Because XK469 is a new antitumor agent, pharmacokinetics have only been described in meeting abstracts using a chiral normal phase separation (Wiegand et al., 1999) and a nonchiral reverse-phase HPLC assay (Chan et al., 1999). We report here the enantiopharmacokinetics of XK469 in Fischer-344 rats following single i.v. doses of racemic XK469 and the individual enantiomers, using the chiral HPLC assay. The urinary and biliary excretion of R(+)-XK469 was also studied in the rat.

	Materials and Methods

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Chemicals and Reagents. S()-XK469 (free acid, NSC 698216), R(+)-XK469 (free acid, NSC 698215), and racemic XK469 (NSC 697887) were provided by the Drug Synthesis and Chemistry Branch of the National Cancer Institute (Rockville, MD). The optical purity of these enantiomers was found to be >99%, as assayed by a chiral HPLC method (data provided by the National Cancer Institute). The internal standard, chloroquinoxaline sulfonamide, was a gift from Dr. William Tong of Memorial Sloan-Kettering (New York, NY).

Animals. Male Fischer-344 rats were purchased from Harlan. All animals were adapted to a 12-h light/dark cycle under controlled room temperature and humidity conditions. Food and water were given ad libitum.

Pharmacokinetic Study of XK469 in Rats. Twenty-four Fischer-344 rats weighing 253-289 g were used in this study. The animals were randomly divided into four groups, with six rats in each group. The rats in the first and second groups were given S()-XK469 and R(+)-XK469, respectively, intravenously at a dose of 10 mg/kg. The rats in the third group were given a racemic mixture (50:50 of S- and R-enantiomers) of XK469 at a dose of 20 mg/kg. The right jugular vein of each Fischer-344 rat was cannulated under ketamine anesthesia (100 mg/kg) at least 17 h prior to drug administration. The animals were kept in metabolism cages 1 h after the surgery for the duration of the entire experiment. The compound was first dissolved in a small volume of sodium bicarbonate solution (1 M) then diluted in normal saline; the pH value of the dosing solution was found to be approximately 8. An appropriate volume of the dosing solution (ca. 0.5 ml) was given to each of the animals through the jugular vein cannula followed by flushing the cannula with 0.5 ml of normal saline. At the time schedule of 0 (predose), 15, 30, 60, 120, 240, 480, 720, 1440, 1800, 2160, 2880, 3300, and 4320 min after dosing, approximately 0.25 ml each of blood was withdrawn from the same cannula, and the lost fluid was replaced by flushing the cannula with an equal volume of normal saline. Plasma was separated immediately by centrifugation. The urine was collected at 24-h intervals for 72 h, and the feces were collected to 72 h. All of the samples were kept frozen at 70°C until analysis.

Biliary Excretion. The fourth group of rats was anesthetized with ketamine (100 mg/kg). The bile duct was first cannulated as previously described (Waynforth and Flecknell, 1992). R(+)-XK469 was administered via an i.v. bolus dose through the tail vein at 10 mg/kg. Bile fluid was collected at a 1-h intervals for up to 7 h. The animals were kept unconscious under ether anesthesia for the duration of the experiment.

Sample Preparations.

Plasma To a set of 13 mm × 100 mm glass tubes, an appropriate amount of the internal standard in 50 µl of methanol was added, followed by an addition of 0.2 ml of plasma sample or plasma spiked with XK469 standards. These samples were acidified with 0.2 ml of acetic acid (2 N). One milliliter of acetonitrile was then used to precipitate the plasma proteins. Following centrifugation at 1000g for 10 min at 4°C, the supernatant was transferred to a clean tube, and the content was evaporated to dryness under a stream of nitrogen. The residue was reconstituted with 100 µl of the mobile phase A, consisting of 30% (v/v) methanol in 20 mM ammonium nitrate buffer, pH 4.0, and a 50-µl aliquot was injected into the HPLC.

Urine. To 0.5 ml of rat urine sample, a fixed amount of the internal standard was added, followed by the addition of 0.5 ml of acetic acid (2 N). The acidified urine sample was extracted with 4 ml of ethyl acetate. After centrifugation at 1000g for 10 min at 4°C, the ethyl acetate extract was separated, and the organic solvent was evaporated to dryness by a stream of N₂. The residue was dissolved in 100 µl of the mobile phase B before analysis by HPLC.

Feces. The wet feces was weighed, and a 2-g aliquot was used for drug analysis. To each sample, an appropriate amount of the internal standard was added, followed by the addition of 8 ml of distilled water and 100 µl of acetic acid. The mixture was homogenized with an SDT 1610 model homogenizer (Tekmar-Dohrmann, Mason, OH) three times at 20 s each. Another 2 ml of distilled water was used to rinse the homogenizer and the inner wall of the tube. The homogenate and washings were combined, and the mixture was centrifuged at 1000g for 30 min at 4°C (Bartels and Smith, 1989). The supernatant of the homogenate was transferred to a clean tube and processed by the same method used for the urine samples.

Bile. The total volume of the bile samples was measured, and a 20-µl aliquot of each was used for drug analysis. An appropriate amount of the internal standard was added to each sample, which was then diluted with 80 µl of distilled water, followed by the addition of 100 µl of 2 N acetic acid. The acidified bile samples were extracted with ethyl acetate as described above for urine sample.

Protein Binding. Plasma protein binding of R(+)- and S()-XK469 was determined using the ultrafiltration technique. Appropriate amounts of R(+)- and S()-XK469 were separately added to the rat plasma samples to achieve 50 and 100 µg/ml, respectively. The plasma samples were incubated at 37°C for 1 h. One milliliter of each sample was then loaded onto a preconditioned Centrifree tube (Amicon, Beverly, MA). The tube assembly was centrifuged at 1000g for 40 min at 25°C. The protein-free ultrafiltrate was collected for drug analysis. Before centrifugation, another 0.1 ml of the plasma sample was saved for analysis of the total drug concentration. Protein binding was calculated by the following equation:

Chromatographic Conditions. A Schimadzu (Columbia, MD) HPLC system, consisting of an SCL-10Avp system controller, two LC-10ATvp pumps, a SIL-10ADvp autoinjector, a C-R5A Chromatopac recorder, and a Spectroflow 757 UV detector (ABI Analytical Kratos Division, Chestnut Ridge, NY), was used for the analysis.

Data Analysis. Plasma concentration-time profiles were analyzed by WinNonlin software version 3.0 (Pharsight, Mountain View, CA).

	Results

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Pharmacokinetics of XK469 in Fischer-344 Rats. Representative plasma concentration-time profiles of XK469 in rats are shown in Fig. 2. As shown in Fig. 2A, following i.v. dosing of S()-XK469 at 10 mg/kg, high concentrations of R(+)-XK469 ranging from 2 to 72 µg/ml were detected in the circulation for up to 72 h. The concentration of the S-enantiomer declined rapidly and was below the detection limit after only 5 to 8 h. As shown in Fig. 2C, similar results were obtained following an i.v. bolus dose of racemic XK469 at 20 mg/kg. In contrast, no detectable S()-XK469 was observed following i.v. administration of R(+)-XK469 at 10 mg/kg (Fig. 2B). The plasma R(+)-XK469 levels ranged from 3 to 93 µg/ml and were detectable for up to 72 h.

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Representative plasma concentration-time profiles of R(+)-XK469 () and S()-XK469 () in Fischer-344 rats given i.v. bolus S()-XK469 at 10 mg/kg (A), i.v. bolus R(+)-XK469 at 10 mg/kg (B), and i.v. bolus racemic XK469 at 20 mg/kg (C).

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Urinary and Fecal Elimination. Urine was collected at 24-h intervals for 72 h. No detectable S()-XK469 was observed in the pooled 24-h urine or in the urine collected afterwards in any of the rats (based on the limit of detection of 0.2 µg/ml in 0.5 ml of rat urine). About 7 to 10% of the dose administered was excreted as the R-enantiomer in urine over 72 h.

Biliary Excretion. About 1.1 to 5.2 ml of bile fluid was obtained from each rat receiving i.v. dosing of R(+)-XK469. It was found that about 3% of the administered dose was recovered cumulatively in the bile fluid collected up to 7 h (Fig. 3). No S()-XK469 was detected in the bile fluid. However, excretion of R(+)-XK469 in bile was previously observed, following an i.v. bolus dose of S()-XK469 in the rat (data not shown).

View larger version (46K): [in this window] [in a new window]   Fig. 3.   Cumulative excretion of R(+)-XK469 (µg/ml) in bile fluid after i.v. administration of R(+)-XK469 at 10 mg/kg; all n = 6 except , n = 5 and &, n = 4.

Protein Binding. XK469 was found to be highly bound to rat plasma proteins. The mean values of the percentage of bound drugs were found to be 97.0 and 95.4% for S()-XK469 and 98.5 and 96.1% for R(+)-XK469, at 50 and 100 µg/ml, respectively. Thus, no significant difference in protein binding between the R(+)- and S()-enantiomer was found.

	Discussion

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We previously studied the pharmacokinetics of XK469 in Fischer-344 rats and CD2F1 mice using a nonchiral HPLC method (Chan et al., 1999). Following i.v. administration of S()-XK469 at 10, 20, 50, and 75 mg/kg in rats and 10, 20, 50, and 100 mg/kg in mice, respectively, the pharmacokinetics was essentially found to be linear within the dose range. The achievable circulating level of XK469 reached 1.4 mM at 100 mg/kg in the mouse (Chan et al., 1999), which is within the biologically active level (Gao et al., 1999). The present study examined the detailed pharmacokinetics of XK469 enantiomers in Fischer-344 rats using a chiral HPLC method. The most important phenomenon we observed was the rapid and predominant inversion of the S-enantiomer to the more active R(+)-XK469 upon administration to rats. However, inversion from R(+)-XK469 to the S-enantiomer was not detected. The extent of inversion can be estimated by calculation of the fractional inversion, f_R, by employing the drug metabolite pharmacokinetic principles (Pang and Kwan, 1983; Kerr et al., 1991), since the R(+)-XK469 can be considered as a metabolite-transformed product of the S()-enantiomer. This was accomplished by using the mean AUC_{R from S}, the mean dose, and the mean value of CL_R, as described previously. The value of f_R was estimated to be 0.93. The data indicated that S()-XK469 was cleared rapidly and the major route was through inversion to the R-enantiomer. In contrast, the clearance of the R(+)-XK469 was slow, and elimination was probably through metabolism, urinary excretion, and biliary excretion. The stereoselective inversion of S()- to R(+)-XK469 has also been observed in mice, dogs, and monkeys (Wiegand et al., 1999; Zheng et al., 2002). The unidirectional inversion substantiates the importance for the current clinical development of the R(+)-enantiomer.

Being a phenoxy analog of the nonsteroidal anti-inflammatory drugs, this inversion is probably mediated by an enzymatic reaction similar to the well studied metabolic inversion of the nonsteroidal anti-inflammatory drugs, such as ibuprofen, ketoprofen, and fenoprofen (Nakamura et al., 1981; Lee et al., 1985; Sanins et al., 1991; Cheng et al., 1994; Davies, 1998), although we do not have the direct data to substantiate this conclusion. For ibuprofen, it was found that the R()-enantiomer was preferentially converted to the S(+)-enantiomer, which possesses most of the anti-inflammatory activity, via a number of enzymatic processes (Nakamura et al., 1981; Sanins et al., 1991). In the present case, S()-XK469 was found to convert to the R-enantiomer. This was a result of the nomenclature of the stereochemical designation due to the Cahn-Ingold-Prelog priority. The absolute configurations of S()- and R(+)-XK469 are identical to those of the R()- and S(+)-ibuprofen, respectively.

No evidence of an interaction between XK469 enantiomers was found in the current study, on the basis of the mean terminal decay rate constants , the harmonic mean of terminal t_1/2, or the clearance values. When the mean terminal decay rate constant of the R(+)-enantiomers given as such was compared with that derived from the racemic mixture, no difference was found. Similarly, no difference was found between the mean clearance value of S()-XK469 when administered alone and that derived from the racemic mixture.