Human liver microsomes assays

Comparative study of fluorescence CYPs assays of niclosamide with that obtained by conventional HPLC assay using human liver microsomes and recombinant CYPs was developed [72]. 

Di, L. et al. 2006. Comparison of cytochrome P450 inhibition assays for drug discovery using human liver microsomes with LC-MS, rhCYP450 isozymes with fluorescence, and double cocktail with LC/ S. Int. J. Pharmaceut. http //dx.doi.org/10.1016/j.ijpharm.2006.10.039. 

Draper, A., Madan, A., Smith, K. and Parkinson, A. (1998). Development of a non-high pressure liquid chromatography assay to determine testosterone hydroxylase (CYP3A) activity in human liver microsomes. Drug Metab. Dispos. 26 299-304. 

In addition, compound 15 also had good metabolic stability in human liver microsome in vitro assay (hLM ti/2 = 39min) and in rat in vivo pharmacokinetic studies (ty2 = 3.3 h, po), with a rat oral bioavailability of 15%, showing a significant improvement in these PK parameters over the lead compound 1. The observed improvement in PK during the optimization was another validation of the strategy discussed above. This part of the optimization process is summarized in Scheme 19.2. 

Human liver microsome stability testing Broad-spectrum selectivity screen then, rat receptor affinity assay... 

C. Nomeir, A. A. Validation of higher-throughput high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s CYP2D6 and CYP3A4 enzyme inhibition studies in human liver microsomes. Rapid Commun Mass Spectrom 2000, 14, 207-214. 

A critical parameter for the development and application of this approach is the use of enzyme-specific assays for the assessment of the catalytic activities of individual enzymes in human liver microsomes. Cytochrome P450 form-specific assays have been developed for in vitro measurement of most human cytochrome P450 forms. Many of these specific assays will be discussed in Section 6. 

Vinylidene chloride has been shown to be activated by human liver S9 supernatant in the Ames assay, suggesting the presence of a cytochrome P450 enzyme. Also, the formation of dichloroacetaldehyde was demonstrated in two human liver microsomal preparations the rate of formation was approximately the same as in rat liver microsomes (WHO, 1990). 

Chauret N, Tremblay N, Lackman R, et al. Description of a 96-well plate assay to measure cytochrome P4503A inhibition in human liver microsomes using a selective fluorescent probe. Anal Biochem 1999 276 215-226. 

Ogilvie BW, Carrott PW, Yang C, et al. A convenient non-HPLC marker assay for measuring CYP2C8 activity in human liver microsomes 5- and 6-chloromethyl-fluorescein diethyl ether (CMFDEE) O dealkylation. Drug Metab Rev 2001 33 (suppl 1) 230. 

In some cases the rate of enzyme inactivation can be quantified without an assay for enzyme activity. For example, inactivation of CYPs due to MIC formation can be directly quantified spectrophotometrically, which avoids the potential artifacts introduced by the measurement of catalytic activity. Microsomes, or purified enzymes, are incubated with a substrate and NADPH and monitored for MIC formation over time in a spectrophotometer. An example of MIC formation by diltiazem in human liver microsomes (HLMs) is shown in Figure 3 (36). The MIC exhibits an absorbance maximum between 448 nm and 456 nm when the heme iron is in the reduced state (17). Extinction coefficients of MIC are approximately 64mM/cm (79). Thus, MIC formation by diltiazem in the example is 59% of the total CYP, which would be consistent with inactivation of most of the CYP3A in the microsomes. 

Lee SH, Slattery JT (1997) Cytochrome P450 isozymes involved in lisofylline metabolism to pentoxifylline in human liver microsomes. Drug Metab Dispos 25 1354-1358 Li AP (1999) Cryopreserved human hepatocytes characterization of DME activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability and drug-drug interaction potential. Chem Biol Interact 121 17-35... 

Busby WF, Ackermann JM, Crespi CL (1999) Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450. Drug Metab Dispos 27 246-249 Clohs L, Wong J (2002) Validation of a capillary electrophoresis assay for assessing the metabolic stability of verapamil in human liver microsomes. J Cap Elec Microchip Tech 7 113 Coughtrie MWH, Fisher MB (2003) The role of sulfotransferase and UDP-glucuronosyltransferases. In Lee JS, Obach RS, Fisher MB (eds) Drug Metabolizing Enzymes. Marcel Dekker, New York pp 541-575... 

Ethell BT, Anderson GD, Beaumont K et al. (1998) A universal radiochemical HPLC assay for the determination of UDP-glucuronosyltransferase activity. Biochem 255 142-147 Fisher MB, Campanale K, Ackermann BL et al. (2000), In vitro glucuronidation using human liver microsomes and the pore-forming peptide alamethicin. Drug Metab Dispos 28 560-566... 

In an in vitro study of midazolam biotransformation using human liver microsomes, midazolam metabolism was competitively inhibited by the antifungal azoles keto-conazole, itraconazole, and fluconazole, and the antidepressant fluoxetine and its metabolite norfluoxetine (55). The degree of inhibition was consistent with the inhibition reported in pharmacokinetic studies, and suggests that in vitro assay is useful for predicting significant interactions. 

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... 

The three-in-one assay is conducted using pools of human liver microsomes from 10 to 20 donors. The microsomes are frozen in small aliquots and stored at -80°C until use. In this assay, hydroxytolbutamide, dextrorphan, and 6-p-hydroxytcstos-terone that are formed from tolbutamide hydroxylase (CYP 2C9 reaction) [53], dextromethorphan 0-demethylase (CYP 2D6 reaction) [53,54], and testosterone 6-P-hydroxylase (CYP 3A4 reaction) [55], respectively, are quantified by LC-MS/ MS in an approximately 1 min run time. 

Yao, M. et al., Development and full validation of six inhibition assays for five major cytochrome P450 enzymes in human liver microsomes using an automated 96-well microplate incubation format and LC-MS/MS analysis, J. Pharm. Biomed. Anal., 44, 211, 2007. 

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