Introduction

Top Abstract Introduction Experimental Procedures Results and Discussion References

Metabolism plays an important role in drug disposition, which can have pharmacological and toxicological implications in the use of therapeutics (Lin and Lu, 1997). Of the many classes of drug-metabolizing enzymes, the cytochromes P-450 (CYPs) superfamily is the major enzyme system often involved in the rate-limiting step of drug biotransformation processes. Over 35 human CYPs have been identified and are classified into families, subfamilies, and specific isoforms by amino acid sequence homology (Nelson et al., 1993). Although the catalytic mechanism of all CYPs is similar due to a conserved haem-thiolate functionality, amino acid variations in the substrate binding sites confer compound-, regio-, and stereoselectivity of metabolism (Guengerich and Macdonald, 1990). From experimental observations, it has become apparent that most biotransformation of xenobiotics is done by enzymes in the first three families (CYP1, 2, and 3) with CYPs in other families being involved in "housekeeping" metabolism of endogenous molecules (Gonzalez, 1989).

The relative abundance of the hepatic CYPs has been determined as: CYP1A2 (13%), 2A6 (4%), 2B6 (<1%), 2C (20%), 2D6 (2%), 2E1 (7%), and 3A4 (30%) (Shimada et al., 1994; Rendic and Carlo, 1997). The relative abundance/activities of each isoform are subject to variation due to the various modes of CYP regulation: 1) induction (CYP1A1/1A2, 2A6, 2E1, 2C, and 3A4); 2) inhibition (all CYPs); and 3) genetic polymorphism (CYP2A6, 2C9, 2C19, and 2D6) (Rendic and Carlo, 1997). Variability in CYP content and activities can have profound influence on the in vivo response of humans to drugs. Over 90% of human drug oxidation can be attributed to the following CYPs: 1A2 (4%), 2A6 (2%), 2C9 (10%), 2C19 (2%), 2E1 (2%), 2D6 (30%), and 3A4 (50%) (Rendic and Carlo, 1997; Bertz and Granneman, 1997).

It has been observed that up to 40% of new chemical entities investigated in humans were withdrawn due to serious pharmacokinetic problems (Prentis et al., 1988). These observations prompted pharmaceutical companies to address important pharmacokinetic parameters such as absorption and metabolism in the early stages of drug discovery and development (Lin and Lu, 1997). In vitro metabolism systems are now in routine use to establish the metabolic stability of drug candidates (DCs), identify specific enzymes involved in the metabolism of DCs, and assess the inhibitory and inductory effects of DCs on drug-metabolizing enzymes (Parkinson, 1996).

Models for the quantitative extrapolation of in vitro data to the human in vivo situation are in continual development to improve their predictive power (Ito et al., 1998). Use of experimental animal (rat, dog, and rabbit) tissue (liver slices, S-9 fractions, microsomes, and primary hepatocyte cultures) is limited by the existence of profound interspecies differences between the animals and humans in CYP isoforms, regulation, and activity profiles (Boobis et al., 1990). The use of human tissues is now recommended although availability and variability are limiting factors in meeting the need for metabolism studies of an increasing number of DCs generated through combinatorial chemistry. Advances in molecular biology have led to the possibility of cloning and heterologously expressing genes of the human CYPs. Enzymes with catalytic properties comparable to those of human liver microsomes have now been expressed in Escherichia coli, insect cells, lymphoblastoid cells, Hep G₂ cells, and yeast (Gonzalez and Korzekwa, 1995; Friedberg and Wolf, 1996). These enzymes can be produced in large amounts to meet the demand of the automated high-throughput screening systems for drug metabolism research.

The aim of this study was to set-up an "in-house" capacity to produce recombinant CYPs for metabolism studies in the drug discovery and development processes. The cDNAs of nine human CYPs were cloned and expressed in yeast. Enzymes were produced from 20-liter yeast fermentations and their catalytic properties were assessed.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results and Discussion References

Materials.

Chemicals The Saccharomyces cerevisiae strain INVSc1-HR bearing the human reductase gene was a gift from the LINK Project (a program of the University of Dundee/Biotechnology and Biology Research Council/Department of Trade and Industry/Pharmaceutical Industry). The galactose-inducible expression vector pYeD60 was a gift from Dr. Magnus Ingelman-Sundberg (Karolinska Institute, Stockholm, Sweden). cDNAs for CYP1A1, CYP1A2, CYP2C19, CYP2E1, and CYP2D6 were also gifts from Dr Magnus Ingelman-Sundberg. cDNAs for CYP2C9, CYP3A4, and CYP3A5 were received from the LINK Project and the cDNA of CYP2C8 was cloned from a human liver cDNA library. Isoform-specific primers were synthesized in our laboratory. Polymerase chain reaction and sequencing chemicals were of the highest grade commercially available.

Equipment. Primers were synthesized on a Beckman Oligo 100 M DNA synthesizer and polymerase chain reaction done on a PTC-200 Engine (MS Research, Inc., Watertown, NY) and automated sequencing on an ABI Prism 377 DNA Sequencer (Perkin-Elmer Cetus Instruments, Eden Prairie, MN). Fermentations were done in a 20-liter Biostat C Fermentor (B. Braun Biotech International GmbH, Melzungen, Germany). Centrifugations were done on an Avanti J-20 centrifuge (Beckman Instruments, Berkeley, CA). A Rannie high pressure laboratory homogenizer 8.30H (ADV Homogeniser Group, Copenhagen, Denmark) and Ultraturrax homogenizer (Janke and Kunkel IKA Labortechnik, Germany) were used in the preparation of microsomes.

Methods.

Construction of expression plasmids The INVSc1-Hr yeast strain modified by the integration of the human NADPH-P-450 reductase gene into its genome (LINK project) was used for the expression of human CYPs. Figure 1 shows the structure of the expression plasmid pYeDP60 (Urban et al., 1990) into which cDNAs of individual CYPs were cloned. The CYP cDNAs' restriction sites at the 5-prime end were modified to be BamHI and the 3-prime modified to be EcoRI or KpnI sites. The cDNAs were cloned into BamHI-EcoRI or BamHI-KpnI sites of the pYePD60 vector (Fig. 1). The yeast bearing the human reductase was transformed with the pYeDP60-CYP constructs to give a panel of yeast strains for the production of the following CYPs: 1A1, 1A2, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Structure of the pYeDP60 vector with cloning sites.

Enzyme expression and microsomal preparation. Selective medium (yeast nitrogen base, glucose, 20 µg/ml His, 20 µg/ml Leu) was inoculated with yeast cells, INVSc1-HR, transformed with plasmids carrying specific CYPs, and incubated at 30°C for 24 h. A rich fermentation medium (200 g yeast extract/10 liters, 200 g peptone/10 liters, 2% glucose) at pH 6.7 was kept at 30°C in a 20-liter fermentor. The fermentation was started by inoculation with a preculture grown in selective medium at 1 liter innoculum/10 liter rich medium and kept at pH 6.7. After 12 h of fermentation, 600 ml of 50% glucose and 300 ml of ethanol were added. After another 18 h of fermentation, induction of CYP expression was initiated by addition of 1 liter of 20% galactose. After an induction period of 8 h, the cells were harvested by centrifugation (10,000g, 10 min).

Enzyme Kinetics.

General incubation conditions All incubations were done in a total volume of 200 µl. Final concentrations of 1 mM NADPH and 0.1 M potassium phosphate buffer (pH 7.4) were used. The substrates were dissolved in ethanol, water, or methanol and 2 to 5 µl was added to the incubation mixture. The stock enzyme concentrations were diluted to 0.5 to 1 pmol CYP/µl with 0.1 M potassium phosphate buffer (pH 7.4), and the appropriate volume was added to get the required enzyme concentration. Each reaction was optimized for CYP concentration and incubation time linearity (Table 4). The stop solutions used were: 1) 50 µl of a 94% acetonitrile, 6% glacial acetic acid solution for CYP2C9; 2) 100 µl of 6% perchloric acid for CYP2C19; 3) 10 µl of 60% perchlorate for CYP2D6; and 4) 100 µl methanol for all other CYPs. The protein was then sedimented by centrifugation at 10,000g for 5 min. Analysis of the metabolites formed was done as described in Table 1. Quantitation of metabolite formed was done from external standard curves made using authentic metabolites.

Evaluation of enzyme-substrate specificity and Michaelis-Menten kinetics. The Michaelis-Menten kinetics of each CYP with respect to its marker substrate (Table 4) were done using modifications of analytical methods reported in the literature (Table 1): 7-ethoxyresorufin O-dealkylation (Burke and Mayer, 1985), paclitaxel 6-hydroxylation (Rahman et al., 1994), diclofenac 4-hydroxylation (Leemann et al., 1993), bufuralol 1'-hydroxylation (Kronbach et al., 1987), S-mephenytoin 4-hydroxylation, chlorzoxazone 6-hydroxylation, and testosterone 6-hydroxylation (Pearce et al., 1996). The catalytic activities of all the CYPs were assessed on each reaction (Table 3).

Evaluation of inhibitor-enzyme selectivity. After a cursory review of the literature on inhibitor concentrations that would show inhibitor-CYP selectivity, an average of 10 µM was considered suitable for a one-concentration screening study. The inhibitors evaluated, -naphthoflavone, quercetin dihydrate, quinidine, sulfaphenazole, and ketoconazole, were dissolved in methanol. Two microliters of the 1 mM stock solutions were added to the 200-µl incubation mixtures to give a final inhibitor concentration of 10 µM and an added methanol content of 1%. The effect of the methanol was assessed for each set of inhibition studies as a baseline activity level. The inhibitory effect of each compound was assessed on the catalytic activity of each CYP toward its maker reaction at conditions similar to those used in the enzyme-substrate specificity evaluation.

Data Analysis. K_m and V_max were determined by nonlinear least-squares fitting using Graphfit Version 3.0 (Erithacus Software Limited, Middlesex, UK). Sigmaplot 4.0 (SPSS, Inc., Chicago, IL) was used for graphical representations.

	Results and Discussion

Top Abstract Introduction Experimental Procedures Results and Discussion References

We have successfully cloned and expressed human CYPs in S. cerevisae. The CYPs have been characterized with respect to their Michaelis-Menten kinetics, substrate specificity, and inhibitor-enzyme selectivity properties and have been found to be comparable to those of human liver microsomes and other recombinant CYPs. This means that we can use our enzymes in metabolism research and obtain valid in vitro data for extrapolation to in vivo metabolism.

The choice of yeast (S. cerevisiae) for the heterologous expression of human CYPs was based on a number of factors: 1) simplicity of setting up, 2) high yield production of stable enzymes without having to modify the cDNA sequence for most of the CYPs, and 3) the presence of an endoplasmic reticulum in yeast. The alternative systems where E. coli, which, although easy to grow and extract enzyme, requires modification of the CYP protein amino terminus for efficient enzyme production and also lacks an endoplasmic reticulum (Iwata et al., 1998). The lymphoblastoid and Hep G₂ cell systems with the apparent advantage of being mammalian are, however, difficult to optimize for maximal CYP expression (Gonzalez and Korzekwa, 1995). The baculovirus-insect cell system produces large amounts of enzyme per milligram of protein but is technically more difficult to set up and maintain because CYP expression is transient (Friedberg and Wolf, 1996). Many university laboratories, pharmaceutical companies, and biotech companies selling or using recombinant CYPs are using one or a combination of the above mentioned heterologous systems with success after appropriate modifications unique to each system. To avoid having to add exogenous NADPH-P-450-reductase to reaction mixtures, the systems have been engineered to coexpress the NADPH-P-450 reductase and cytochrome b₅ with the human CYP of interest (Gonzalez and Korzekwa, 1995).

In our system, the CYP yield ranged from 22 to 132 pmols/mg microsomal protein (Table 2). No CYP was detectable in the microsomes from yeast not transformed with recombinant CYP, implying that the yeast has very little constitutive CYP. The NADPH-P-450 reductase activity averaged 75 nmols reduced cytochrome c/min/mg (Table 2). In comparison, human liver microsomes have an average total CYP content of 300 pmols/mg microsomal protein and average reductase activity of 150 nmols reduced cytochrome c/min/mg (Pearce et al., 1996). By comparing the CYP content-reductase activity ratio in yeast and human liver microsomes, it can be concluded that the reductase will probably not be a limiting factor in supporting individual CYP reactions in our system. Expression system-related factors and incubation conditions can, however, have an effect on the coupling efficiency of the reductase and CYPs.

The CYPs showed expected substrate specificity (Table 3) with respect to the established marker reactions (Ono et al., 1996). We have also shown for the first time that chlorzoxazone 6-hydroxylation is catalyzed by CYP1A1 (V_max = 1.88) with a high turnover besides the documented role of CYP2E1 and 1A2 (Ono et al., 1995). Chlorzoxazone 6-hydroxylation is used as an in vivo probe for hepatic CYP2E1 activity (Frye et al., 1997). The use of chlorzoxazone as an in vivo probe for CYP2E1 activity might not be affected because CYP1A1 is not constitutively expressed in the liver, but results in subjects pre-exposed to CYP1A1 and 1A2 inducers should be interpreted with caution. Reaction conditions for each CYP-marker reaction were optimized with respect to time and enzyme concentration for Michaelis-Menten kinetic studies. Table 4 summarizes the apparent Michaelis-Menten constants (K_m) and the maximum turnover of the reactions (V_max) for the different CYPs with respect to specific marker reactions. The K_m values obtained for the same reactions using human liver microsomes are also shown for comparison. The values we obtained are within the ranges of those reported in the literature. It can also be noted that recombinant CYP2E1 in our study and others (Ono et al., 1995; Kudo et al., 1997) has a higher K_m (177 µM) than that obtained using human liver microsomes (40 µM; Peter et al., 1990). Additional investigations on this observation are in progress.

Chemical inhibitors showed significant selectivity toward specific CYPs as reported in literature (Ono et al., 1996); -naphthoflavone (CYP1A1 and 1A2), quercetin (CYP2C8), sulfaphenazole (CYP2C9), quinidine (CYP2D6), and ketoconazole (CYP3A4 and 3A5) (Fig. 2). Methanol (Fig. 2) and acetonitrile (not shown) are good solvents for substrates and inhibitors because they have minimal inhibitory effects on CYP at final concentration of less than 2%.

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Selective inhibition of CYPs by several chemicals.

Ticlopidine has been previously shown to inhibit phenytoin metabolism (Donahue et al., 1997) and to inhibit the CYP2C19-catalyzed omeprazole metabolism more than the CYP3A4 activity in vivo (Tateishi et al., 1999). Using human liver microsomes, Donahue et al. (1997) showed that ticlopidine was a potent competitive inhibitor of S-mephenytoin hydroxylation and a weak inhibitor of tolbutamide hydroxylation, and concluded that the drug inhibited CYP2C19 more than CYP2C9. Screening for the inhibitory effect of ticlopidine on all the CYPs in this study showed the drug to be a potent inhibitor of CYP2C19 (IC₅₀ = 4.5 µM) and CYP2D6 (IC₅₀ = 3.5 µM) activities (Fig. 3). Coadministration of ticlopidine with drugs that are substrates of CYP2C19 (Donahue et al., 1997) or CYP2D6 can result in clinically important drug-drug interactions.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Inhibition of CYP2C19 and CYP2D6 activity by ticlopidine.

The Michaelis-Menten parameters from different published studies show a tremendous variation in the apparent K_m and V_max values. Variation in inhibition constants (K_i and IC₅₀) can also be observed in published literature. The causes of these differences could be: 1) interindividual variation from using liver microsomes from donors with different genetic/disease/drug backgrounds, 2) differences causedby the specific expression system used owing to physiological factors unique to each system affecting the CYP quantity/affinity/activity profiles, and 3) interlaboratory variations due to differing experimental conditions used. These variations and their causes have important implications on the validity of metabolic in vitro-in vivo quantitative extrapolations. It has been shown that incubation conditions such as type of buffer (Tris or phosphate) and ionic strength, amounts of added NADPH-P-450 reductase, cytochrome b₅, protein content, glutathione, phospholipids, and detergents can have a profound effect on enzyme kinetics (Shet et al., 1995; Crespi, 1998; Mäenpää et al., 1998). Although there are great variations in the enzyme kinetic values, the ranking with respect to substrate specificity and inhibitor selectivity for the panel of drug-metabolizing CYPs is generally the same irrespective of source of enzyme or incubation conditions used. This means that the different laboratories can still reach the same qualitative conclusions with respect to metabolism studies but very different quantitative deductions.

In conclusion, our recombinant CYPs have passed a three level validation criterion test with respect to enzyme kinetic properties. This unlimited source of enzyme will satisfy the demands from our automated drug-drug interaction high-throughput screening procedures, metabolic pathway identification studies, and elucidation of structure/functional properties of each CYP for better prediction of the roles of CYPs in drug metabolism.