Human liver microsome incubations

The in vitro approaches feature human liver microsome incubations that contain drug candidates at a range of concentrations that span the anticipated maximum steady state plasma levels. The microsomal incubations also contain a specific probe substrate where the concentration closely approximates that Km value for the reaction under investigation. Quantitative analysis of the specific marker metabolites and internal standards using MRM provides a simple assessment of the potential inhibitory effects drug candidates have on the metabolism of specific CYP probe substrates. 

Figure 11-13. Time-course human liver microsomal incubation profiles for a number of positive and negative controls, as well as project compounds. Metabolic stability profiles are represented both in tabular format and graphically above.

Human liver microsome incubated with ezetimibe Human jejunum microsome incubated with ezetimibe... 

Leung LY, Lhn H-K, Hicks D, et al. Metabolite characterization in rat-and human liver microsomal incubations and trough whole blood of renal transplant patients treated with sirolimus, cyclosporine, and prednisone [Abstract]. ISSXProc 1996 10 366. 

Rourick and co-workers endeavored to test the feasibility of nanoscale LC-MS on metabolite analysis with an in vitro assay based on human liver microsomal incubation [100]. Buspirone, an anxiolytic drug with a well-characterized metabolic profile [101], was chosen as a test compound. At an initial concentration of 4 pM, buspirone was incubated with microsomal protein (1 mg/mL) and 4 mM NADPH in 50 mM sodium phosphate... 

In addition to this simultaneous quantitative and qualitative function, the QTrap can be used under full-scan MS in the ion trap mode and/or CNLS, PSI as survey scans to trigger product ion scan (MS2) and MS3 experiments to obtain structural information of drug metabolites on-the-fly. Xia et al. [43] reported five metabolites of gemfibrozil were detected in a single injection. Bramwell and colleagues [44] utilized different scan functions in the QTrap to identify GSH conjugates in human liver microsome incubations. 

The glucuronide metabolite of AZT was isolated from rat and human liver microsomal incubations (177), and the formation of a toxic metabolite of AZT was demonstrated in rat hepatocytes and liver microsomes (177). The metabolism of tamoxifen was examined in human liver homogenate and human Hep G2 cell line preparations by LC/API/MS (178). Several metabolites were detected in the human liver homogenate extracts, namely, N-didesmethyltamoxifen, a-hydroxy-tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen, and tamoxifen N-oxide. All of these metabolites, except the N-didesmethyltamoxifen, were observed in the samples after incubating tamoxifen with the human Hep G2 cell line. In-vitro studies have also used MS to examine potential drug-drug interactions. For example, (179) demonstrated that the a2-agonist, dexmedetomidine, inhibited metabolism of the anesthetic alfentanil, whereas clonidine had no effect. 

FIGURE 10.2 Metabolite profiles of buspirone determined by HPLC with MSC, LSC, and RFD (Zhu et al., 2005b). Buspirone metabolites from human liver microsome incubations were separated by HPLC (1 mL/min). Radioactivity in HPLC effluent was determined by (a) TopCount 8000 DPM injected, four fractions per min and 10 min counting time (b) LSC 8000 DPM injected, two fractions per min, 10 min counting time and (c) RFD 32,000 DPM injected. 

CYP involved in human liver microsomal incubation reaction (Yao et al., 2007). 

Jonsson, G. Astrom, A. Andersson, P. Budesonide is metabolized by cytochrome P450 3A (CYP3A) enzymes in human liver. Drug Metab.Dispos., 1995, 23, 137-142 [human liver microsomal incubations extracted budesonide betamethasone is IS SPE gradient]... 

The figures that follow provide examples of some ways in which in vitro clearance data for two series can be compared and assessed to identify key questions, trends, or hypotheses. While the data presented here are for clearance in a human liver microsomal (HLM) incubation, the analysis could be applied in the same way to other data sets - including other experimental ADME or safety end points, or computationally predicted end points. 

FIGURE 6.53 Multiplexing P450 substrate analysis. Four substrates for each CYP450 were incubated in the absence (A) or presence (B) of human liver microsomes at room temperature for 20 min. The reactions were then quenched and samples were analyzed using /rPLC. The principle of this experiment is shown in the top panel. 

Cytochrome P-450 from rat or human liver microsome preparations is inactivated when incubated anaerobically with carbon tetrachloride in the presence of NADPH and an oxygen-scavenging system (Manno et al. 1988 1992). Inactivation involved destruction of the heme tetrapyrrolic structure, and followed pseudo first-order kinetics with fast and slow half lives of 4.0 and 29.8 minutes. When compared with rat liver microsomes, the human preparations were 6-7 times faster at metabolizing carbon tetrachloride, and only about one- eighth as susceptible to suicide inactivation (about 1 enzyme molecule lost for every 196 carbon tetrachloride molecules metabolized). 

Disposition Metabolic studies indicate hydrolytic release of the calicheamicin derivative from gemtuzumab ozogamicin. Many metabolites of this derivative were found after in vitro incubation of gemtuzumab ozogamicin in human liver microsomes and cytosol, and in HL-60 promyelocytic leukemia cells. Metabolic studies characterizing the possible isozymes involved in the metabolic pathway of Mylotarg have not been performed. 

The intrinsic rates of formation of monoepoxides in human, rat and mouse liver microsomes are roughly similar, when epoxide hydrolase is inhibited, whereas the amount of monoepoxides at the end of incubation is two and even 15 times higher in mouse liver microsomes than in rat and human liver microsomes, respectively (Bogaards et al., 1996). Thus, differences in epoxide hydrolase activity between different species may be of importance for toxicological outcomes. 

Figure 5.12. LC- UV (top) and LC-MS (bottom) chromatograms 30 uM human liver microsomal (HLM) incubation samples of buspirone at 30 min.

Recently, a study was conducted to compare the MDF method to PIS and NLS methods in finding oxidative metabolites in biological fluids (Zhu et al., 2004, 2006). In this study, diclofenac and clozapine (Fig. 6.8) were incubated with human liver microsomes and the metabolites generated were then spiked into pooled rat urine and bile followed by analyses using either high-resolution LC- MS -based MDF or triple-quadmpole LC-MS with NLS and PIS. 

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