Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Cytochromes P450 (CYPs¹) comprise a superfamily of hemoproteins that play an important role in the metabolism of a wide variety of xenobiotics and endogenous compounds. The human CYP genes have been characterized and classified into various families and subfamilies based on their structures. The gene families CYP1, CYP2, and CYP3 are currently thought to be the major CYP enzymes responsible for the oxidative metabolism of drugs or xenobiotics (Spatzenegger and Jaeger, 1995).

Inhibition of CYP-mediated metabolism, often the mechanism for drug-drug interactions, can limit the use of a drug because of adverse clinical effects. In past years, substantial progress has been made in the development of in vitro tools that can be used by the pharmaceutical industry to predict drug-drug interactions. The potential for CYP enzyme inhibition is addressed routinely by measuring the rates of metabolism of a probe biotransformation (a specific substrate to a specific metabolite) in human liver microsomes (HLM) or heterologously expressed enzymes (rCYP) in the presence and absence of new chemical entities (NCEs) (as summarized in Parkinson, 1996). Usually, multiple inhibitor and/or probe substrate concentrations are tested to generate quantitative inhibition parameters [apparent inhibition constant (K_i) or inhibitor concentration that produces 50% inhibition (IC₅₀ value)].

A limitation with most of the existing probes is that they require HPLC analysis for their metabolite quantification, which severely limits sample throughput. Also, it is not uncommon to observe interference of the inhibitor with the assay endpoint (e.g., coelution with the metabolite) when UV detection is employed. The use of LC with mass spectrometry (MS) detection obviously circumvents the selectivity issue,but the sample preparation, including the selection of appropriate internal standard, can still be problematic. In general, analytical methodology is the rate-limiting step in the acquisition of in vitro data.

Recently, efforts have been made to develop microtiter plate assays with nonfluorescent CYP probes that produce fluorescent metabolites (Crespi et al., 1997; Chauret et al., 1999). Most published assays refer to the use of these fluorogenic probes in microsomes expressing individual CYPs (rCYP) because the selectivity of the probes is poor or unknown (Crespi et al., 1997). In this regard, a novel fluorogenic probe 3-[2-(N,N-diethyl-N-methyl-ammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC; see Fig. 1) was reported to be a useful substrate to address CYP2D6 inhibition in a microtiter plate assay using rCYP2D6 microsomes (Crespi et al., 1999).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Chemical structures of AMMC (substrate), AMHC (metabolite), AHMC (external standard).

This article reports the studies that were conducted in HLM to address the selectivity of AMMC toward CYP2D6. Results generated with AMMC are compared with those obtained with bufuralol, the classical probe that is known to be hydroxylated at the 1'-position by CYP2D6 in HLM (Kronbach et al., 1987).

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. Quinidine, dextromethorphan, perhexeline, fluoxetine, imipramine, sparteine, norfluoxetine, and quinine were purchased from Sigma/RBI (St. Louis, MO). The compounds AMMC, bufuralol, 3-[2-(N,N-diethylamino)ethyl]-7-hydroxy-4-methylcoumarin (AHMC), and 1'-OH-bufuralol were from GENTEST Corp. (Woburn, MA) and BD Biosciences (San Jose, CA). Inhibitory monoclonal antibodies against CYP2D6 prepared in mouse ascites were obtained from Merck (Whitehouse Station, NJ) (Shou et al., 2000). All other reagents were of highest purity commercially available or HPLC grade.

Tissue and Microsomes. Human tissues were obtained from various sources (F.P. Guengerich, Vanderbilt University School of Medicine, Nashville, TN; IIAM, Exton, PA; Quebec Transplant, Montreal, QC, Canada). Human liver microsomes were either from GENTEST Corp., BD Biosciences, or prepared from frozen (80°C) tissue as described in the literature (Lu and Levin, 1972). Protein concentrations of the microsomal fractions were determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Microsomes prepared from lymphoblast or baculovirus/insect cells cDNA expressing individual cytochrome (Supersomes) were obtained from GENTEST Corp and BD Biosciences.

Conditions for Individual Incubations. Microsomal incubations were prepared on ice in Eppendorf polypropylene tubes containing an appropriate amount of human liver microsomal protein, the substrate, and a typical NADPH-regenerating system (100 mM phosphate buffer at pH 7.4 containing 20 mM glucose 6-phosphate, 2.0 mM NADP⁺, 2.0 mM magnesium chloride, and 4 units/ml glucose-6-phosphate dehydrogenase) in a total volume of 500 µl. The substrate, AMMC, was dissolved in acetonitrile in such concentrations that the total organic solvent content did not exceed 1% when added to microsomal incubations, because it is known that organic solvents can affect the activity of the enzyme (Chauret et al., 1998; Hickman et al., 1998; Busby et al., 1999). Incubations in phosphate buffer were used as controls, and incubations with no AMMC were used as blanks. The tubes were transferred then to a water bath set at 37°C. After a specific period of time, the reactions were quenched by adding an equivalent volume of acetonitrile to precipitate the proteins. The incubation mixtures were then centrifuged for 10 min at 13,000 rpm in an Eppendorf centrifuge 5415C. An aliquot of the supernatant was analyzed by HPLC/UV, HPLC/MS, or HPLC/fluorescence. To evaluate the linearity of the reaction rate for AMMC, the amount of microsomal protein and incubation time varied from 0 to 2 mg/ml and 0 to 90 min, respectively. To determine the kinetic parameters (apparent K_m and V_max using Lineweaver-Burk calculations), incubations containing 0.5 mg/ml microsomal protein (obtained from pooled donors) were conducted for 45 min with various concentrations AMMC (0-30 µM).

HPLC Analysis of Incubations. To investigate the overall metabolism profile of AMMC, analysis of the microsomal incubations was carried out using LC/MS on a Waters Alliance 2690 HPLC interfaced to a Micromass Quattro LC triple quadrupole mass spectrometer (Waters Corp., Milford, MA). The sample (5 µl) was injected onto a YMC-AQ C₁₈ column (4.6 × 50 mm, Waters Corp.). A gradient mobile phase consisting initially of 90:10, solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) was brought to a composition of 60:40 A/B in 10 min at a flow rate of 1 ml/min. Positive ion electrospray over the range m/z 200 to 400 was used for detection. Selected incubations were analyzed also by UV (Waters Photodiode Array, model 994) or fluorescence (Shimadzu RF-551, Kyoto, Japan). UV detection was monitored at 325 nm and fluorescence was read at an excitation wavelength of 395 nm and an emission wavelength of 460 nm.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   HPLC analysis of a human liver microsomal incubation of AMMC using electrospray in total ion mode and positive ion mass spectrum of the metabolite (AMHC).

Conditions for Inhibition Studies.

A) Incubations using the fluoregenic probe AMMC Incubations using HLM were conducted in 96-well microtiter plates using experimental conditions reported in Table 1 and according to published methodology (Chauret et al., 1999). A Biomek 1000 automated laboratory workstation controlled with Nemesis version 1.0.5 software (Beckman Coulter, Inc., Fullerton, CA), was used for serial (1:3, v/v) dilutions of the test compounds and for the addition of all of the reagents in the various steps of the incubations (microsomes in buffer, substrate, inhibitor, and NADPH, 0.1-ml total volume). An enzymatic reaction, as described in Chauret et al. (1999), was performed at the end of the incubation to remove excess NADPH because the fluorescence of this reagent interferes with the detection of AMHC. Reactions were terminated by the addition 0.1 ml of 4:1 (v/v), acetonitrile/Tris base solution (0.5 M). Fluorescence of samples was monitored using a cytofluorimeter (Cytofluor II, Applied Biosystems, version 4.1 or 4.2; Foster City, CA) with excitation and emission filters set at 395/40 and 460/40 nm, respectively. Typically, ten concentrations of inhibitors were tested (0.03-100 µM) for all inhibitors except quinidine (0.0015-5 µM). The inhibitors were selected on the basis that they covered a wide range of IC₅₀ values for CYP2D6 inhibition (Crespi et al., 1999). The analysis and calculations of the microtiter plate data to generate IC₅₀ values were achieved as described previously (Chauret et al., 1999).

B) Incubations with the standard probe, bufuralol. Inhibition studies using bufuralol as a probe were performed similar to those described previously except that the specific experimental conditions summarized in Table 1 were used. Typically, five concentrations of inhibitors [0.1-100 µM for all inhibitors, except quinidine (0.01-10 µM)] were used to generate an IC₅₀ value. Because both bufuralol and its 1'-hydroxy metabolite are fluorescent, HPLC analysis, as described previously, was used for the quantification of the metabolite.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Metabolism Profile of AMMC. Human liver microsome incubations containing AMMC resulted in the formation of one major metabolite as detected by UV, fluorescence, or MS analysis (Fig. 2). Based on MS analysis, the metabolite M1 (m/z = 290.2) corresponded to the loss of 14 atomic mass units from the parent compound (m/z = 304.2) consistent with O- or N-demethylation (-CH₃ + H). Based on the fragmentation pattern, the amino alkyl side chain of the metabolite was not modified by metabolism, indicating that the demethylation had occured on the coumarin moiety. From this MS evidence, the metabolite M1 was identified as AMHC. The rate of metabolism of AMMC was linear with time and protein concentration up to 45 min and at least 1 mg of protein. Using a microsomal protein concentration of 0.5 mg/ml (obtained from pooled human liver donors), the apparent K_m and V_max were determined to be ~3 µM and 20 pmol/(min · mg of protein), respectively.

Involvement of CYP in the Metabolism of AMMC. The involvement of CYP in the O-demethylation of AMMC was determined using several approaches including metabolism studies (using microsomes containing recombinant enzymes and human liver microsomes from various donors) and inhibition studies.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Involvement of rCYP in the metabolism of AMMC. , 1.5 µM AMMC; , 25 µM AMMC.

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Effect of inhibitory CYP2D6 antibody on the metabolism of AMMC in rCYP2D6 and in various human liver microsomal preparations. , 1.5 µM AMMC; , 25 µM AMMC.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Comparison of AMMC (0.5 mg/ml microsomal protein, low NADPH-regenerating system, 45 min) and bufuralol (25 µM bufuralol, 0.8 mg/ml microsomal protein, typical NADPH-regenerating system, 20 min) turnover in human liver microsomes prepared from various donors. , 1.5 µM AMMC; , 25 µM AMMC.

Inhibition of CYP2D6. The 96-well plate assay, as described above, was used to evaluate the inhibitory potential of various chemical inhibitors using AMMC as a probe. With an HLM preparation high in CYP2D6 (as determined in previous studies; (Chauret et al., 1997), the signal corresponding to 100% activity (incubations containing HLM, AMMC, and NADPH) was approximately 3-fold above the signal corresponding to 0% activity (incubations containing HLM, AMMC without NADPH). In Table 2, the IC₅₀ values obtained for various inhibitors in HLM using the fluorescent probe AMMC are compared with those determined with the classical probe bufuralol. There was a good correlation between the values obtained with both probes (r² = 0.98, n = 8). The IC₅₀ values obtained with AMMC and bufuralol using rCYP2D6 as the enzyme source were also determined, and the results are presented in Table 2. Once again, there was a good correlation between the values obtained with both probes (r² = 0.96, n = 7).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

This paper describes the metabolism studies that were performed in HLM to determine the selectivity of AMMC, a novel fluorescent probe (Crespi et al., 1999), toward CYP2D6. When AMMC was incubated in human liver microsomes, one major metabolite was detected using UV, fluorescence, or MS detection. It was identified as the O-desmethyl metabolite, AMHC, by LC-MS. The AMMC-O-demethylation in HLM occurred at an apparent K_m of 3 µM and a V_max of 20 pmol/(mg of protein · min).

It was clear from the study using rCYP (Fig. 3) that, at a concentration of AMMC close to its K_m, CYP2D6 was the major enzyme involved in the formation of AMHC. This was further confirmed in experiments where the AMMC-O-demethylation was completely abolished (96%) in the presence of a CYP2D6 antibody. At a concentration of AMMC well above its K_m (25 µM), other enzymes, especially the CYP1 family, also contributed to the metabolism of AMMC as shown by the study with rCYPs. An experiment with inhibitory antibody (Fig. 4), where only partial inhibition was obtained with donor 2, provided further evidence of the role of P4501A in the metabolism of AMMC because it had been shown, in a previous study, that this particular donor was high in CYP1A2 (data not shown).

The AMMC-O-demethylase activity in HLM obtained from various donors was highly correlated with the 1'-hydroxylation of bufuralol, a highly selective CYP2D6 probe (Fig. 5). It did not correlate with any other selective CYP substrates. At a concentration of 1.5 µM AMMC, the linear regression line passed through the origin and no activity was observed in two specimens of hepatic microsomes that were deficient in CYP2D6 [as determined by Western blot and bufuralol-1'-hydroxylase activity (results not shown)]. These observations imply that there was a minimal contribution of other enzymes to AMMC demethylation under these conditions. In contrast, at a concentration of 25 µM AMMC, the linear regression line did not pass through the origin and significant activities were observed in the two specimens of hepatic microsomes that were deficient in CYP2D6. This is in accordance with the fact that there is a contribution from other enzymes (putatively CYP1A2) to AMMC demethylation at this higher concentration.

Knowing the experimental conditions under which AMMC was selectively metabolized by CYP2D6 in HLM, a CYP2D6 inhibition assay was developed in a 96-well plate format using the fluorescence of AMHC as the endpoint. Two approaches were used to eliminate the interference originating from the fluorescence of NADPH. In HLM, an enzymatic NADPH removal step using glutathione reductase and oxidized glutathione was used (Chauret et al., 1999), whereas for the rCYP, an NADPH-regenerating system was refined to minimize the concentration of NADPH. In both cases, the interference of NADPH was minimal. The assay was performed in an HLM rich in CYP2D6 and at a concentration of AMMC equal to approximately double the K_m to achieve a good signal-to-noise ratio (at this concentration, AMMC-O-demethylation is selective for CYP2D6). Similar experimental conditions were adopted for inhibition using bufuralol as a probe. The IC₅₀ values obtained for eight inhibitors were all within 4-fold using the two probe substrates, which is good considering that the inhibitors chosen covered a range of greater than 5 log units of IC₅₀ values. Similar results were obtained in experiments conducted using rCYP2D6 as the enzyme source. In general, the values obtained with AMMC were slightly lower than those obtained with bufuralol. The fact that the amount of protein and the incubation time in the inhibition protocols with AMMC in HLM were higher may account for the difference, especially if the test compounds are extensively metabolized by microsomal proteins.

In conclusion, it has been clearly demonstrated that, under proper experimental conditions, the novel fluorogenic substrate, AMMC, can be used as a selective CYP2D6 probe in HLM. It has been recommended that new chemical entities be evaluated in different in vitro systems for a better understanding of the CYP2D6 inhibition phenomenon (Palamanda et al., 1998). This present study demonstrates that AMMC can be added to the arsenal of currently accepted CYP2D6 probes such as bufuralol for studying CYP2D6 inhibition in human liver microsomes. The great advantage of AMMC over standard probes is that HPLC analysis is not required for metabolite quantification, increasing sample throughput.