Microsomal incubation

E. M. Benson, A. J. Tomlinson and S. Nayloi, Time course analysis of a microsomal incubation of a therapeutic dmg using preconcenti ation capillary electrophoresis (Pc-CE) , 7. High Resolut. Chromatogr. 17 671-673 (1994). 

Figure 5.49 (a) Total-ion-current trace, and (b) the reconstructed ion chromatogram of mjz 510.2 0.5 (monooxygenated metabolites) from LC-MS analysis of human microsomal incubation of Glyburide. Reprinted with permission from Zhang, H., Henion, J., Yang, Y. and Spooner, N., Anal. Chem., 72, 3342-3348 (2000). Copyright (2000) American Chemical Society. 

Analysis of reaction mixtures for 1-propanol and 2-propanol following incubation of NDPA with various rat liver fractions in the presence of an NADPH-generating system is shown in Table I ( ). Presence of microsomes leads to production of both alcohols, but there was no propanol formed with either the soluble enzyme fraction or with microsomes incubated with SKF-525A (an inhibitor of cytochrome P450-dependent oxidations). The combined yield of propanols from 280 ymoles of NDPA was 6.1 ymoles and 28.5 ymoles for the microsomal pellet and the 9000 g supernatant respectively. The difference in the ratio of 1- to 2-propanol in the two rat liver fractions may be due to differences in the chemical composition of the reaction mixtures (2) Subsequent experiments have shown that these ratios are quite reproducible. For comparison, Table I also shows formation of propanols following base catalyzed decomposition of N-propyl-N-nitrosourea. As expected (10,11), both propanol isomers were formed, the total yield in this case being almost quantitative. 

The metabolism of NPYR is summarized in Figure 1. a-Hy-droxylation (2 or 5.position) leads to the unstable intermediates and decomposition of gives 4-hydroxybutyraldehyde [ ]. The latter, which exists predominantly as the cyclic hemiacetal 1, has been detected as a hepatic microsomal metabolite in rats, hamsters, and humans and from lung microsomes in rats (9-13). The role of 1 and as intermediates in the formation of 6 and 7 is supported by studies of the hydrolysis of 2-acetoxyNPYR and 4-(N-carbethoxy-N-nitrosamino)butanal, which both gave high yields of 7 (9,14). In microsomal incubations, can be readily quantified as its 2,4-dinitrophenylhydrazone derivative (15). The latter has also been detected in the urine of rats treated with NPYR ( ). 

The effects of changing Jt-conjugation at the 4-position on both the rate of isomerization of the initially formed o-quinones to QMs and the reactivity of the quinoids formed from 4-propylcatechol, 2,3-dihydroxy-5,6,7,8-tetrahydronaphtha-lene (2-THNC), hydroxychavicol, and 4-cinnamylcatechol were studied (Fig. 10.6).9 These catechols were selectively oxidized to the corresponding o-quinones or QMs and trapped with GSH. Microsomal incubations with the parent catechols produced only o-quinone-GSH conjugates. However, if GSH was added after an initial incubation period both o-quinone- and QM-GSH conjugates were observed. The results indicate that the extended Jt-conjugation at the para position enhances the rate... 

Reactive Metabolites of PAHs. A wide variety of products have been identified as metabolites of PAHs. These include phenols, quinones, trans-dihydrodiols, epoxides and a variety of conjugates of these compounds. Simple epoxides, especially those of the K-region, were initially favored as being the active metabolites responsible for the covalent binding of PAH to DNA. Little direct experimental support exists for this idea (62.63,64) except in microsomal incubations using preparation in which oxidations at the K-region are favored (65,66). Evidence has been presented that a 9-hydroxyB[a]P 4,5-oxide may account for some of the adducts observed in vivo (67.68) although these products have never been fully characterized. 

Fluoranthenes. With the exception of 3-methylcholanthrene, much less work has been undertaken on nonalternant PAHs. Several recent studies have reported on the major metabolites and mutagenicity of various fluoranthenes (181-185), but little is known about the DNA adduct which they form. Some studies on dibenzo[a,e]fluoranthene showed that several adducts are formed by microsomal incubations (185) and additional studies will be required to provide complete structural elucidation of the products formed. 

Korfmacher, W. A. et al. 1999. Development of an automated mass spectrometry system for the quantitative analysis of liver microsomal incubation samples A tool for rapid screening of new compounds for metabolic stability. Rapid Commun. Mass Spectrom. 13 901. 

Recent work in our laboratories has confirmed the existence of a similar pathway in the oxidation of vindoline in mammals (777). The availability of compounds such as 59 as analytical standards, along with published mass spectral and NMR spectral properties of this compound, served to facilitate identification of metabolites formed in mammalian liver microsome incubations. Two compounds are produced during incubations with mouse liver microsome preparations 17-deacetylvindoline, and the dihydrovindoline ether dimer 59. Both compounds were isolated and completely characterized by spectral comparison to authentic standards. This work emphasizes the prospective value of microbial and enzymatic transformation studies in predicting pathways of metabolism in mammalian systems. This work would also suggest the involvement of cytochrome P-450 enzyme system(s) in the oxidation process. Whether the first steps involve direct introduction of molecular oxygen at position 3 of vindoline or an initial abstraction of electrons, as in Scheme 15, remains unknown. The establishment of a metabolic pathway in mammals, identical to those found in Strep-tomycetes, with copper oxidases and peroxidases again confirms the prospective value of the microbial models of mammalian metabolism concept. 

Segall and coworkers described the in vitro mouse hepatic microsomal metabolism of the alkaloid senecionine (159) (Scheme 34). Several pyrrolizidine alkaloid metabolites were isolated from mouse liver microsomal incubation mixtures and identified (222, 223). Preparative-scale incubations with mouse liver microsomes enabled the isolation of metabolites for mass spectral and H-NMR analysis. Senecic acid (161) was identified by GC-MS comparison with authentic 161. A new metabolite, 19-hydroxysenecionine (160), gave a molecular ion consistent with the addition of one oxygen atom to the senecionine structure. The position to which the new oxygen atom had been added was made evident by the H-NMR spectrum. The three-proton doublet for the methyl group at position 19 of senecionine was absent in the NMR spectrum of the metabolite and was replaced by two signals (one proton each) at 3.99 and 3.61 ppm for a new carbinol methylene functional group. All other H-NMR spectral data were consistent for the structure of 160 as the new metabolite (222). 

Many factors may confound the assessment of the D DI potential of early discovery compounds [93], Limited or no solubility data exist to understand the likelihood that the compound will precipitate out of an in vitro incubation. The compounds have generally not been analyzed from a spectroscopic perspective their characteristics may interfere with a fluorogenic DDI assay. Metabolism data are typically not available. The binding of a compound to plasma proteins or microsomal incubation constituents is not well understood, which may lead to underprediction of its inhibitory potential. The compounds are typically delivered in DMSO, which may cause solvent-related inhibition of the enzymatic assay. Also, since little is known about in vivo concentrations or projected dose, framing the consequences of an early DDI in vitro experiment may be difficult. With these factors in mind, general experimental paradigms have been developed to help minimize their potential impact. 

When 4C-benzo(a)pyrene (100 nmol) was incubated with the reconstituted MFO system, the reaction components were increased 10-fold (maintaining the original incubation volume, and substrate and NADPH concentrations). Metabolites were extracted and analyzed by HPLC as described for the microsomal incubations. 

Several other minor metabolites of phenol have been identified in vitro. The formation of 1,4-dihydroxybenzene and 1,2-dihydroxybenzene in a 20 1 molar ratio was observed in isolated rat liver microsomes incubated with phenol (Sawahata and Neal 1983). Further catalysis to / -benzoquinone, 4,4 -biphenol, and biphenoquinone has been demonstrated in microsomes and in in vitro peroxidase preparations. The benzoquinone products react nonenzymatically with nucleophiles, including cysteine and reduced glutathione, to yield. V-conjugates of 1,4-dihydroxybenzene and 4,4-biphenol (Eastmond et al. 1986 Lunte and Kissinger 1983 Subrahmanyam and O Brien 1985). 

Lunte SM, Kissinger PT. 1983. Detection and identification of sulfhydryl conjugates of p-benzoquinone in microsomal incubations of benzene and phenol. Chem-Biol Interact 47 195-212. 

Note Control 2 was used for testing substrate stability during microsomal incubation it was very helpful in prediction of outcomes for synthesis, especially in the case of HOTYR, which is easily oxidized under neutral and basic aqueous conditions. 

S. F. Comparison of SPF and fast LC to eliminate mass spectrometric matrix effects from microsomal incubation products. J Pharm Biomed Anal 2002, 28, 279-285. 

Figure 6.20 (a) LC-MS total ion current of neutral loss 175 and (b) HPLC-radioactivity profile of a rat liver microsomal incubation of radioactive compound A. Reproduced from [37J with permission from Elsevier. 

The classic hard nucleophile used in trapping hard metabolite electrophiles is cyanide. Indeed, with modern detection sensitivities it is now often possible to detect cyanide adducts from microsomal incubations that were quenched with acetonitrile, with the residual cyanide in the acetonitrile reacting with the electrophilic species. In an experiment designed specifically to generate and detect cyanide adducts, millimolar concentrations of cyanide may be included in a microsomal incubation with no detrimental effect on the metabolic turnover. 

Reiter R, Burk RF. 1988. Formation of glutathione adducts of carbon tetrachloride metabolites in a rat liver microsomal incubation system. Biochem Pharmacol 37 327-331. 

Butenediol can be oxidized to 3,4-epoxybutanediol (epoxybutanediol), as has been shown in rat liver microsomes. Incubation for 30 min with butadiene gave concentrations of butenediol and epoxybutanediol which were nearly three-fold and 10-fold, respectively, higher than the corresponding concentration of epoxybutene (Cheng Ruth, 1993). Epoxybutanediol can, however, also be a product of diepoxybutane hydrolysis. 

Figure 5.1. Full-scan MS and data-dependent MS/MS spectra of verapamil generated from a microsomal incubation sample.

Figure 5.9. LC-MS (TIC) and UV (220 nm) chromatograms for microsomal incubation of verapamil containing parent molecule and detected metabolites. Data-dependent accurate mass measurements for verapamil, metabolite parents and fragment ions are shown in the inset.

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