Materials and Methods

Source of Chemicals.

Human liver microsomes were obtained from the International Institute for the Advancement of Medicine (Exton, PA). The microsomes were thawed, pooled, aliquoted, and refrozen at −80°C before use. Insect cell microsomes containing cDNA-expressed CYP2D6-Val (Supersomes) were obtained from Gentest Corporation (Woburn, MA). Quinidine, dextromethorphan, and β-NADPH were purchased from Sigma Chemical Company (St. Louis, MO). Imipramine, trifluperidol, and propranolol were purchased from ICN Biomedicals (Aurora, OH). The substrate for the MTP assay, 3-cyano-7-ethoxycoumarin and its metabolite, 3-cyano-7-hydroxycoumarin, were obtained from Molecular Probes, Inc. (Eugene, OR). Pluronic F-68 was purchased from Life Technologies (Gaithersburg, MD). NCEs for the enzyme inhibition studies were synthesized at Schering-Plough Research Institute as part of a drug discovery program.

Enzyme Assays.

The dextromethorphan O-demethylase assay for CYP2D6 in HLM was performed according to a modification of a previously published method (Chen et al., 1990; Palamanda et al., 1998). Initial studies in our laboratory have established a K_m of approximately 18 μM. The substrate concentration used in the inhibition studies is 16 μM, which approximates theK_m. Also, it is estimated that the reaction mixture contains 1 pmol of CYP2D6. Metabolite production was quantified by comparison to a standard curve of known concentrations of dextrorphan.

The microtiter plate assay for CYP2D6 was performed with a CytoFluor Series 4000 Multi-well Plate Reader (PerSeptive Biosystems, Framingham, MA) according to Crespi et al. (1997) with modifications. All compounds for analysis were initially dissolved in methanol. Stock solutions were made in 20% aqueous methanol and the final methanol concentration in the assay was 0.5%. A 20 mM 3-cyano-7-ethoxycoumarin solution was prepared in acetonitrile. After the addition of 5 μl of the test compound, 95 μl of a 100 mM potassium phosphate buffer, pH 7.4, containing 3 mM β-NADPH, 0.02% Pluronic F-68 (a nonionic surfactant of polyoxyethylene-polyoxypropylene copolymer used to help solubilize the substrate), and 100 μM 3-cyano-7-ethoxycoumarin, was added to each well. The plate was preincubated at 37°C for 5 min, then 100 μl of a 100 mM potassium phosphate buffer, pH 7.4, containing 50 pmol/ml of insect cell-expressed CYP2D6-Val (Supersomes) was added. Therefore, the reaction mixture contained 5 pmol CYP2D6 as reported byCrespi et al. (1997). Initial studies in our laboratory have shown that the K_m for 3-cyano-7-ethoxycoumarin was 45 μM, similar to a K_m of 67 μM reported by Crespi et al. (1997). The final substrate concentration used was 50 μM, which approximates the K_m. The plate was incubated for 45 min at 37°C followed by the addition of 100 μl of a reaction-stop solution containing 60% acetonitrile and 40% 0.1 M Tris, pH 9. In some experiments, after the addition of the recombinant enzyme to start the reaction, fluorescence data were acquired every 10 min for 50 min by scanning while the plate remained in the instrument.

Data Analysis.

Relative CYP2D6 activity was calculated from the fluorescence data for each time point using CytoCalc Data Analysis Software (PerSeptive Biosystems, Framingham, MA). The IC₅₀ values were estimated by comparison to control values, i.e., enzyme in the reaction mixture and 0.5% methanol.

Results and Discussion

We previously reported the IC₅₀ values for 62 NCEs using Supersomes and human liver microsomes for CYP2D6 inhibition (Palamanda et al., 1998). Of these structurally related compounds, the IC₅₀ values for the two assays closely matched for 53 of the 62 (85%) compounds (difference equal to or less than 5-fold). The remaining nine compounds had IC₅₀ values in the MTP assay that differed from the IC₅₀ values in HLM assay by more that 5-fold (compounds 54 to 62, Table 1). The IC₅₀ values for six compounds were 9- to 28-fold higher in the MTP assay, whereas the IC₅₀ values for the other three compounds were 6- to 93-fold higher in the HLM assay (Palamanda et al., 1998). A difference of 5-fold or less in the IC₅₀ was considered acceptable because we were comparing data from a single cytochrome P-450 isoform (Supersomes) to data from a mixture of cytochrome P-450 enzymes (HLM) and the substrates were different. Also, literature IC₅₀values for quinidine inhibition of CYP2D6 using dextromethorphan (Rodrigues and Roberts, 1997) and bufuralol (Halliday et al., 1995) as substrates were 0.22 μM and 0.04 μM, respectively, a difference of slightly over 5-fold. However, it should be noted that the [S]/K_m ratios for the two reactions may be different, which could have contributed to the difference in the IC₅₀ values. In our studies, the [S]/K_m ratio was approximately 1 for both the MTP and HLM assays.

To determine if the IC₅₀ values observed at the 45-min endpoint in the MTP assay remained constant throughout the incubation, fluorescence data were acquired at 10, 30, and 50 min intervals. The IC₅₀ values for the six compounds that showed higher IC₅₀ in the MTP assay shifted markedly to higher values over time (compounds 54–59, Table 1), whereas the IC₅₀ values for the three compounds that showed lower IC₅₀ values in the MTP assay remained relatively stable (compounds 60–62, Table 1). Also, three additional compounds (compounds 18, 21, and 26; Table 1) whose IC₅₀ values were within 5-fold of the IC₅₀ values determined in HLM showed relatively stable IC₅₀ (2- to 3-fold differences in the IC₅₀ values; Table 1). Upon using the IC₅₀ values observed at 10 min in the MTP assay, all but the three compounds that showed lower IC₅₀ in the MTP assay were found to have IC₅₀ values within a 5-fold difference from the HLM assay, i.e., 95% match (Table 1).

The higher IC₅₀ values in the MTP assay at 45 to 50 min compared to 10 min for the six compounds suggest that the higher concentration of CYP2D6 and the longer incubation period in the MTP assay may be more rapidly inactivating the test compounds than in HLM. This is evident because the amount of CYP2D6 used in each assay and the duration of the incubation are different. In HLM, it is estimated that the concentration of CYP2D6 is 5 pmol/mg protein (Shimada et al., 1996). Under our assay conditions for HLM, i.e., 1 mg/ml microsomal protein and a 200 μl volume, there is 1 pmol enzyme present. In the MTP assay, 5 pmol enzyme per well (in 200 μl) are required due to the low turnover rate of the enzyme for the substrate (Crespi et al., 1997). The incubation times are 15 and 45 min for the HLM assay and the MTP assay, respectively. Therefore, in the MTP assay, there is 5 times more enzyme present and the length of the incubation is approximately 3 times longer than in the HLM assay; this may result in a more rapid metabolism in the MTP assay. This is consistent with our finding that for those six compounds that showed higher IC₅₀values in the MTP assay at 45 to 50 min, the IC₅₀at 10 min matched more closely with the IC₅₀ in HLM. To confirm that the shift to higher IC₅₀values over time is an indicator of metabolism, known substrates of CYP2D6 were tested (Spatzenegger and Jaeger, 1995). When dextromethorphan, imipramine, propranolol, and trifluperidol were evaluated in the MTP assay, the IC₅₀ curves shifted to the right for all compounds, i.e., higher IC₅₀ values over time (Fig.1). In contrast, when quinidine was analyzed, the IC₅₀ value did not change over time. This is expected because quinidine is a competitive inhibitor of CYP2D6 but not a substrate (Nielson et al., 1995). Nevertheless, this hypothesis needs to be further confirmed by monitoring the reaction mixture by liquid chromatography-mass spectrometry analysis. Using this generalization, a shift of inhibition curves to theleft over time (i.e., lower IC₅₀) would suggest metabolism- or mechanism-based inhibition. Because there are no known specific metabolism-based inhibitors of CYP2D6 (Parkinson, 1996), preliminary experiments were carried out with the MTP assay using recombinant CYP3A4 from baculovirus-infected insect cells as described by Crespi et al. (1997; data not shown). The inhibition curves for erythromycin and troleandomycin, both of which are mechanism-based inhibitors of CYP3A4, shifted to the left, i.e., lower IC₅₀ values over time, whereas the curve for ketoconazole, a nonmechanism-based inhibitor of CYP3A4, remained relatively stable over time. Therefore, these observations may have general application in rapidly screening NCEs for inhibition of cytochrome P-450 enzymes.

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Figure 1

Inhibition curves for dextromethorphan (A), propranolol (B), imipramine (C), trifluperidol (D), and quinidine (E) at selected time points in the MTP assay.

In comparing the results from the MTP assay to those obtained from CYP2D6 inhibition in HLM, it is important to point out that insect cell microsomes containing the expressed enzyme do not contain any other cytochrome P-450 isoforms. Thus, the potential for metabolism by any of the other cytochrome P-450 isoforms in liver microsomes does not exist in the Supersomes. If a compound is metabolized in HLM by one isoform to a less potent inhibitor for a second isoform, it is expected that its IC₅₀ obtained with HLM would be higher than that obtained in the MTP assay. Therefore, it is reasonable to speculate that the three compounds with higher IC₅₀ values in HLM might have been subjected to metabolism to less potent inhibitors by other cytochrome P-450 isoforms. However, it should be mentioned that using the recombinant CYP2D6 preparation results in a more intrinsically accurate estimate of the IC₅₀ of a particular compound for this isoform, but may not reflect the apparent IC₅₀determined in HLM, which is probably more relevant in predicting drug-drug interactions. Furthermore, in the prediction of the in vivo inhibition potential for of a specific cytochrome P-450 and the consequent clinically relevant drug-drug interaction, plasma levels (better yet concentrations in the liver) of the compound and the potency of the inhibitor (better expressed asK_i) all must be taken into consideration.

Results of the current study indicate that the risk of overestimating the IC₅₀ (false positive) for a test compound is minimized by using data acquired after shorter incubation time (10 min instead of 45 min). This results in increased predictability of the MTP from 85 to 95%. However, there is still a concern for the compounds that showed lower IC₅₀ in the MTP assay (3 of 62, approximately 5%) because of the potential of discarding these three NCEs (false negatives). Nevertheless, in the final analysis, utilization of Supersomes coupled with the MTP technology has greatly helped us select appropriate NCEs quickly by enhancing throughput. This is a “balancing act” between discarding a small number of false negatives versus enhanced throughput, but the overall benefit derived by accelerating our discovery program outweighs the loss of approximately 5% of NCEs during the first supersomal screen.

In conclusion, the results of the present study demonstrate that the MTP assay using recombinant enzyme may be useful for screening large numbers of compounds for potential inhibition of CYP2D6. Furthermore, the IC₅₀ values obtained at early time points in the MTP assay more closely predict the IC₅₀values obtained in HLM assay. However, the IC₅₀values of selected candidates should still be confirmed in HLM.