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Doping Control Center, Korea Institute of Science and Technology, Chungryang, Seoul, Korea (H.H.Y., H.J.C., M.J.K., D.-H.K.); and Bioanalysis and Mass Spectrometry, LG Life Sciences R&D, Taejon, Korea (J.L., C.-S.L.)
(Received October 7, 2007; Accepted December 3, 2007)
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Abstract |
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; Demuth et al., 2005; Barnett, 2006; Campbell, 2007; Pratley and Salsali, 2007). As a part of the preclinical study of LC15-0133, the in vitro metabolism of LC15-0133 was investigated using rat, dog, and human liver microsomes. LC15-0133 was metabolized to 4 to 6 metabolites, via hydroxylation and carbonyl reduction, with considerable species differences noted in rat liver microsomes. C-Demethylated and carboxyl metabolites were observed only in the rat liver microsomal incubation. Because the C-demethylation reaction is not a common enzymatic reaction, the metabolic pathway for the generation of the C-demethylated metabolite requires further investigation. The purpose of the present study was to characterize the in vitro metabolism of LC15-0133 and identify the mechanism of the rat liver microsomal enzyme catalyzed C-demethylation reaction of LC15-0133.
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Materials and Methods |
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Microsomes. Human liver microsomes were purchased from BD Biosciences (Bedford, MA). Rat liver samples were taken from Sprague-Dawley rats (Daehan Animal, Daejeon, Korea) and dog liver samples from Beagle dogs (Daehan Animal). The microsomal fraction was prepared according to the method previously described elsewhere (Guengerich et al., 1986).
In Vitro Microsomal Incubation. LC15-0133 (100 µM final concentration) was incubated with 2 mg/ml liver microsomes at 37°C for 2 h, in the presence of an NADPH-generating system (10 mM glucose 6-phosphate, 0.67 mM β-NADP+ and 1 U/ml glucose 6-phosphate dehydrogenase), in a final incubation volume of 200 µl. The reaction was terminated by the addition of 400 µl of 0.1% acetic acid with 50 µl of LC15-0133-d6 (10 µg/ml) added as an internal standard solution. The samples were then passed through activated Sep-Pak C18 cartridges (96-well type OASIS HLB extraction cartridge), washed twice with 1 ml of distilled water, and eluted with 1 ml of methanol. The methanol eluate was dried under nitrogen gas. The residue was redissolved in 50 µl of acetonitrile with 10 µl injected on to an HPLC column for HPLC/MS analyses.
HPLC/MS Analysis. The HPLC/MS system consisted of an HP 1100 series binary pump HPLC system (Agilent, Palo Alto, CA) with a diode array detector (Agilent) and an ion-trap mass spectrometer equipped with an electrospray ionization source (Agilent). The chromatographic separation of LC15-0133 and its metabolites was achieved on a Capcell Pak C8 column (2.0 mm x 15 cm, 5 µm; Shiseido, Tokyo, Japan) using a linear gradient program. The mobile phases consisted of 10 mM ammonium formate at pH 6.0 (A) and 90% acetonitrile (B). The initial composition was 15% (B), programmed linearly to 65% (B) over 15 min, at a flow rate of 0.2 ml/min. Mass spectrometry and tandem mass spectrometry analyses were performed using an ion trap mass spectrometer. The entire column eluent was directly introduced into an electrospray ionization interface via a 50-cm-long section of polyetheretherketone tubing (0.13 mm i.d.). Nitrogen was used as both the nebulizing and drying gas at 35 psi and a flow rate of 8 l/min, respectively, with a temperature of 350°C. The mass spectrometer was operated in the positive ion mode. Helium was used as the collision gas for the tandem mass spectrometric experiments. Fragmentation was induced with a resonant excitation amplitude of 0.85, following isolation of the desired precursor ion over a selected mass window of 1 Da. All instrumental controls and data processing were performed using the LC/MSD Trap Software (version 4.1).
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Metabolite M3 produced a protonated molecule at m/z 387, indicating that this compound was a monohydroxylated derivative of LC15-0133. M3 generated characteristic product ions at m/z 369, 315, 258, and 143, these being 16 Da higher than those of the corresponding product ions at m/z 353, 299, 242, and 127 of LC15-0133 (Table 1). These characteristic product ions indicated hydroxylation of the tert-butyl moiety attached to the oxadiazole ring.
The [M+H]+ ion of M1 was observed at m/z 401, which gained 30 Da from that of protonated LC15-0133, and the MS2 spectrum yielded product ions at m/z 357, 247, and 228. This metabolite was postulated to be a carboxylic acid derivative generated due to further oxidation of M3. The product ion at m/z 357 was postulated to have been generated due to dissociation of a carboxyl moiety from the protonated molecular ion. The MS3 spectrum of the ion at m/z 357 was consistent with the MS2 spectrum of M4, the C-decarboxylated metabolite (Fig. 2, B and C), which strongly indicated that M1 was a carboxylic acid metabolite.
M5 and M6 produced [M+H]+ ions at m/z 373, and both generated characteristic product ions at m/z 355, 301, 283, 244, and 175, suggesting that M6 was a stereoisomer of M5. The protonated molecular and the product ions of these metabolites were 2 Da higher than those of the corresponding ions of the parent compounds, suggesting that these metabolites were carbonyl-reduced metabolites. M2 produced a [M+H]+ ion at m/z 403, which was 32 Da higher relative to that of LC15-0133, with major product ions at m/z 385 and 357. This metabolite was postulated to be a dihydroxylated metabolite. M4 generated an [M+H]+ ion at m/z 357, which was 14 Da less than that of LC15-0133, suggesting that this metabolite was a demethylated derivative. All the product ions were also 14 Da less than the corresponding ions of the parent compounds, suggesting that demethylation had occurred at the tert-butyl moiety. The structure of M4 was confirmed by comparison with a synthetic standard.
The structure of M4 was somewhat unexpected, as C-demethylation is not common during metabolism, whereas O- and N-demethylations are frequently observed in the biotransformation of xenobiotics (Burke et al., 1994; Coutts et al., 1994; Ertl et al., 1999). In the case of terfenadine possessing a tert-butyl moiety on the backbone of the compound, similarly to LC15-0133, both alcohol and acid metabolites have been identified, but the C-demethylated metabolite has never been reported (Jurima-Romet et al., 1994; Ling et al., 1995; Rodrigues et al., 1995; Terhechte and Blaschke, 1995). Initially, it was postulated that M4 was able to be generated via the sulfate conjugate of M1, from which the carboxyl sulfate moiety might easily be dissociated. However, this possibility was ruled out, as not even a trace amount of a sulfate conjugate was detected in the incubation along with phosphoadenosyl phosphosulfate (data not shown).
It was subsequently hypothesized that the C-demethylated metabolite (M4) could be spontaneously generated from the acid metabolite (M1) via decarboxylation, where the removal of the carboxyl group was attributed to the oxadiazole ring attached to the tert-butyl moiety. To test this hypothesis, the acid metabolite (M1) was collected using preparative HPLC, treated, and then reinjected under different conditions. The treatment of M1 under high pH or high temperature conditions accelerated the formation of M4, supporting the notion that the C-demethylated metabolite was generated by the nonenzymatic decarboxylation of M1 (Fig. 3).
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A considerable amount of the C-demethylated metabolite M4 (
10% of the parent drug found) was observed in the urine and plasma samples from rats administered LC15-0133 (data not shown). The apparent kinetic parameters for M4 formation from LC15-0133 in rat liver microsomes were Km = 72.9 µM and Vmax = 5.03 pmol/min/mg protein (Supplemental Data Fig. 2). However, neither the acid or demethylated metabolite (M1 and M4, respectively) was found in the incubations with dog and human liver microsomes (Supplemental Data Fig. 3), which suggests this biotransformation reaction, alcohol to carboxylic acid, is species-specific. The research on the enzyme(s) involved in carboxyl formation is progressing to identify which enzyme reaction is species-specific.
In conclusion, LC15-0133 was metabolized to a carboxylic acid metabolite, via an alcohol metabolite, by a rat microsomal enzyme, which subsequently decarboxylated to yield a demethylated metabolite at the tert-butyl moiety. This unusual C-demethylation reaction was thought to occur due to the resonance stabilization due to the oxadiazole ring attached to the tert-butyl moiety.
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Acknowledgments |
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Footnotes |
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: LC15-0133, 1-[2-(5-tert-butyl-[1,3,4] oxadiazole-2-carbonyl)-4-fluoro-pyrrolidin-1-yl]-2-(2-hydroxy-1,1-dimethyl-ethylamino)-ethanone; HPLC, high-performance liquid chromatography; MS, mass spectrometry.
The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dong-Hyun Kim, Doping Control Center, Korea Institute of Science and Technology, PO Box 131, Chungryang, Seoul 136-791, Korea. E-mail: dhkim{at}kist.re.kr
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357 ions.
