Drug Metabolism and Disposition Fast Forward
First published on March 22, 2006; DOI: 10.1124/dmd.105.008367
0090-9556/06/3406-901-905$20.00
DMD 34:901-905, 2006
SHORT COMMUNICATION
IDENTIFICATION OF A NOVEL IN VITRO METABONATE FROM LIVER MICROSOMAL INCUBATIONS
Chun Li,
Sekhar Surapaneni,
QingPing Zeng,
Brian Marquez,
David Chow, and
Gondi Kumar
Department of Pharmacokinetics and Drug Metabolism (C.L., S.S., G.K.) and Department of Chemistry Research and Development (Q.Z., B.M., D.C.), Amgen Inc., Thousand Oaks, California
(Received November 18, 2005;
Accepted March 15, 2006)
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Abstract
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In vitro drug metabolism studies during the early drug discovery stage are becoming increasingly important. With the increasing demand for high throughput and quick turnaround time for in vitro metabolism studies, however, careful examination of the results and proper design of the experiments are still crucial. In this communication, we report the identification and mechanism of formation of a novel metabonate from incubations of a diamine-containing compound with liver microsomes. The metabonate appeared to be the major product, and its formation was NADPH- and microsomal protein-dependent. Liquid chromatography/mass spectrometry and NMR analysis of the metabonate indicated an extra carbon and unusual formation of an imidazolidine ring. Further studies revealed that this metabonate was not a true biotransformation product from the diamine compound itself in the microsomal incubation, but rather a product resulting from a condensation reaction between the compound and a metabolite of the solvent (alcohol) used in the incubation. When the microsomal incubations contained a small amount of methanol or ethanol as solvent, the alcohols were metabolized to formaldehyde or acetaldehyde, which then condensed with the diamine compound through an imine intermediate to form the metabonate. The compound itself was metabolically stable in vitro when acetonitrile or dimethyl sulfoxide was used as solvent. During the study of in vitro microsomal stability and metabolite identification of amine-containing compounds, the use of alcohol as solvent should be avoided.
In vitro metabolite identification studies during the drug discovery stage are an important part of lead optimization. Often, high metabolic "soft spots" in a molecule contribute to poor pharmacokinetic properties. Early identification of the metabolically labile sites in a particular structural series can help the medicinal chemists to better design compounds with improved metabolic stability. In addition, characterization of potentially active or toxic metabolites can potentially enable lead optimization programs to design more potent and safer drug candidates.
The advances in liquid chromatography and mass spectrometry have allowed the in vitro drug metabolism studies to be carried out in a fairly high throughput fashion. High throughput microsomal stability assays have become standard screening assays in the pharmaceutical industry for reducing metabolic liability in discovery stage. Further in vitro metabolite identification studies are carried out for lead compounds to address liabilities associated with pharmacokinetic properties or reactive metabolite formation and, therefore, have become crucial in providing synthetic directions. Typically, the in vitro incubations contain a small amount of organic solvent in which the compound of interest is dissolved. The effect of common organic solvents on in vitro cytochrome P450 enzyme activities has been reported (Chauret et al., 1998
). Dimethyl sulfoxide was found to inhibit several cytochrome P450-mediated reactions, whereas methanol and acetonitrile exhibited less inhibitory effect when present at less than 1%. As a result, methanol and acetonitrile have been used as the preferred solvents for performing in vitro metabolism studies.
In this study, we report the formation of novel metabonates of 1,2-ethylene diamine-containing compounds. Beckett has used the term "metabonate" to describe a substance which appears to be a metabolite but which, in reality, is an artifact, formed during experimental conditions, isolation, and/or storage (Beckett et al., 1971).
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Materials and Methods
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Chemicals and Reagents. Compounds A and B (R1-NH-CH2-CH(NH2)-CH2-R2) were synthesized by the Amgen Chemical Research Department (Thousand Oaks, CA). NADPH was purchased from Sigma-Aldrich (St. Louis, MO). Human and rat liver microsomes were obtained from Xenotech (Lenexa, KS).
In Vitro Incubations. The compound of interest (10 µM) was preincubated for 5 min with rat or human liver microsomes (1 mg/ml) in 0.1 M phosphate buffer (pH 7.4) at 37°C in a total volume of 0.5 ml. NADPH (1 mM) or buffer was added to the incubation and incubated at 37°C for 30 min. Reactions were terminated using an equal volume of acetonitrile. Samples were centrifuged at 14,000 rpm for 10 min and supernatants were analyzed by LC/MS.
Mass Spectrometric Analysis. LC/MS analysis was carried out using a YMC ODS-AQ column (3 µm, 2.0 x 100 mm; Waters, Milford, MA) at a flow rate of 0.25 ml/min. Mobile phase contained A: H2O, 0.1% formic acid and B: CH3CN, 0.1% formic acid. A linear gradient elution (0-5 min, 5% B, 5-25 min, linear to 65% B) was used for separation. The entire flow after UV detector was directed to the electrospray source of a Q-TOF mass spectrometer (Waters), operated in the positive ion mode and V configuration of time of flight. The electrospray voltage was set to 3.5 kV, whereas the cone voltage was tuned to 35 V. The Z-spray source block and desolvation temperatures were at 100°C and 350°C, respectively. Neubulizing gas flow was set to maximum, and desolvation gas flow was set to 750 l/h. A solution containing 2 µM leucine enkephalin in 50:50 acetonitrile and water was infused to the lock spray source. MH+ of leucine enkephalin at m/z 556.2771 was used as lock mass for exact mass measurement correction. Mass spectra were acquired from 100 to 1000 amu with a 1-s scan cycle time. For tandem mass spectrometry experiments, collision energy was set to 25 eV with argon as collision gas.
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FIG. 1. HPLC/UV trace of in vitro human liver microsomal incubation of compound A with (a) or without (b) NADPH.
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NMR Analysis. Isolated metabolite was dissolved in MeOH-d4 and manually loaded into a Protasis MRM (Marlboro, MA) microcoil NMR probe with a total volume of 5 µl. Spectra were acquired on a Bruker (Newark, DE) AV600 NMR spectrometer.
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Results and Discussion
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Compound A (R1-NH-CH2-CH(NH2)-CH2-R2) and several structural analogs showed moderate in vitro clearance in rat and human microsomal stability assays, and moderate to high in vivo clearance in rats. In vitro metabolism studies were then carried out using rat and human liver microsomes to identify the metabolic liabilities. Compound A appeared to be metabolized to one major metabolite (Ma) in both human and rat liver microsomes, as shown in Fig. 1. The formation of Ma was microsomal protein-, time-, and NADPH-dependent. Metabolite Ma appeared to be less hydrophilic than the parent compound A, because it eluted after compound A on the reversed phase HPLC. Full scan and tandem mass spectra of compound A and metabolite Ma are shown in Fig. 2. Metabolite Ma displayed a molecular ion 12 amu higher than that of compound A. Accurate mass measurement indicated an additional carbon and one extra double bond equivalence over the parent compound A. Tandem mass spectrum of Ma was identical to that of parent drug A, with major fragment ions at m/z 232, 244, and 348 which corresponded to R1-NH3+, R1-NH-CH2+, and R1-NH-CH2-CH+-CH2-R2. The addition of one extra carbon of Ma was probably on the primary amine of compound A based on the tandem mass spectra. Metabolite Ma was isolated from the human liver microsomal incubation of compound A in the presence of NADPH. NMR analysis of isolated Ma showed all proton signals as in compound A, plus two additional doublets 10' and 10'', as shown in Fig. 3. The two doublets had chemical shifts at 4.34 and 4.58 ppm, and a coupling constant of 7.9 Hz, indicative of the methylene protons adjacent to two heteroatoms (imidazolidine protons at C-10 position). The NMR data are consistent with information obtained from mass spectral data. The formation of Ma was quite unusual and cannot be explained from any common biotransformation pathways.
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FIG. 2. Accurate mass LC/MS spectra of compound A (a) and metabolite Ma (b), and tandem mass spectra of compound A (c) and metabolite Ma (d).
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The in vitro microsomal incubations contained 1% methanol in which compound A was dissolved. We hypothesized that the source of one additional carbon might be the solvent methanol. The incubations were then repeated using different solvents, as illustrated in Fig. 4 for a structural analog, compound B. The formation of the metabolite was clearly solvent-dependent, as shown in Fig. 5. When the incubation contained 1% methanol, metabolite Mb1, the molecular ion of which was 12 amu higher than that of parent compound B, was detected. When the incubation contained 1% d4-methanol, a similar metabolite (Mb2), which had the same retention time as Mb1, but had 2 amu mass increments over Mb1, was observed. When the incubation contained 1% ethanol, a metabolite (Mb2) with longer retention time than Mb1 and a mass increment of 26 amu over the parent compound was detected, but was formed to a lesser extent.
The "metabolites" identified were not true biotransformation products from the parent compound itself, but, rather, from chemical reaction between the parent compound and metabolites of the solvents used. We propose a mechanism of formation of the novel metabonates as condensation reaction between formaldehyde (or acetaldehyde) and the amine containing xenobiotic through an imine intermediate, as shown in Fig. 6. Formaldehyde (or acetaldehyde) was formed as a consequence of alcohol dehydrogenase-catalyzed metabolism of methanol or ethanol (Dawidek-Pietryka et al., 1998), which was present as solvent for the compound in the microsomal incubations. A similar finding also has been reported by Yin et al. (2001) for compounds containing 1,2-diamino and 1,2-amino hydroxyl groups in microsomal incubations containing methanol.
To test the hypothesis that the reactive species is aldehyde, a small amount of formaldehyde was added to the solution containing the diamine compound A (or B), which resulted in the instantaneous formation of product Ma (or Mb1). This reaction is a typical Mannich reaction (Thompson, 1968). When acetonitrile or dimethyl sulfoxide was used as solvent in the microsomal incubation, compounds A and B had little turnover, and were metabolically stable. A very minor Mb1 was detected for compound B (Fig. 4), but it was also present in similar amount in all control incubations without NADPH, probably because of a trace amount of formaldehyde in the incubation solutions or in HPLC mobile phases.
There have been a few examples of metabonate formation in the literature. A new imidazolidone metabolite was discovered during the course of studying the biotransformation of lidocaine in humans (Breck and Trager, 1971; Nelson et al., 1973). This metabolite was a product of the condensation reaction between acetaldehyde and a major N-dealkylated metabolite of lidocaine. A diarylimine was detected as a metabonate of N-benzyl-4-chloroaniline, which was a chemical artifact from two debenzylation metabolites (Low et al., 1994).
Our results indicated that if careful examination of the metabolite structure and its mechanism of formation were not done, wrong conclusions might be drawn. The moderate to high in vitro micromal clearance (in methanol) of the diamine compounds was initially thought to be the cause of moderate to high in vivo clearance of these compounds in rats. The clearance mechanism was explored further using bile duct-cannulated rats, and a significant amount of unchanged parent drug was detected in the bile; metabolism played a minor role in the elimination of the diamine compounds. In the Caco-2 assay, these compounds also showed significant efflux ratios. Whether or not the imidazolidine metabonate is formed in vivo or has any implication in the in vivo clearance of the diamine compounds is not clear. The presence of alcohol can strongly affect the liability and interpretation of in vitro metabolism of these diamine-containing compounds. During the study of in vitro microsomal stability and metabolite identification of amine-containing compounds, the use of alcohol as solvent should be avoided.
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Footnotes
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Current affiliation: Brian Marquez, Pharmaceutical Sciences NMR Laboratory, Pfizer Inc., Groton, CT.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.105.008367.
ABBREVIATIONS: LC/MS, liquid chromatography/mass spectrometry; amu, atomic mass unit(s); HPLC, high pressure liquid chromatography.
Address correspondence to: Chun Li, Mail Stop: 30E-2-C, Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Dr., Thousand Oaks, CA, 91320-1799. E-mail: chunl{at}amgen.com
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References
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Breck G and Trager WA (1971) Mannich intermediate in the formation of a new metabolite of Lidocaine in man. Science (Wash DC) 173: 544-546.[Abstract/Free Full Text]
Chauret N, Gauthier A, and Nicoll-Griffith DA (1998) Effect of common organic solvents on in vitro cytochrome P450-mediated metabolic activities in human liver microsomes. Drug Metab Dispos 26: 1-4.[Abstract/Free Full Text]
Dawidek-Pietryka K, Szczepaniak S, Dudka J, and Mazur M (1998) In vitro studies of human alcohol dehydrogenase inhibition in the process of methanol and ethylene glycol oxidation. Arch Toxicol 72: 604-607.[CrossRef][Medline]
Low CM, Ulgen M, and Gorrod JW (1994) The recognition of a diarylimine as a metabonate produced during incubation of N-benzyl-4-chloroaniline with hepatic microsomal preparations. J Pharm Pharmacol 46: 585-590.[Medline]
Nelson S, Breck G, and Trager W (1973) In vivo metabolite condensations. Formation of N1-ethyl-2-methyl-N3-(2,6-dimethylphenyl)-4-imidazolidinone from the reaction of a metabolite of alcohol with a metabolite of Lidocaine. J Med Chem 16: 1106-1112.[Medline]
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