Development of a V79 cell line expressing human cytochrome P450 2D6 and its application as a metabolic screening tool

ELSEVIER

Environmental

Toxicology

and Pharmacology

3 (1997) 31-39

Development of a V79 cell line expressing human cytochrome 2D6 and its application as a metabolic screening tool Reimund

Rauschenbach,

Hille Gieschen, Birgit Salomon, Michael Hildebrand” Rrseurch

Received

Laboratories,

19 April

Sdzrring

Christian

Kraus,

Gerhard

P450

Ktihne,

AG, D- 13342 Berlin, Germany

1996; revised 9 October

1996; accepted

23 October

1996

Abstract Expression of human cytochrome P4jO (CUP) in heterologous cells is a means of specifically studying the role of these enzymes was inserted into an expression vector containing the strong in drug metabolism. The complete cDNA encoding CYP2D6-VALj7, myeloproliferative sarcoma virus promotor in combination with the enhancer of the cytomegalovirus and stably expressed in V79 Chinese hamster cells. The presence of genomically integrated CYP2D6 cDNA was confirmed by polymerase chain reaction analysis. The protein expression was shown by Western blotting. Functional expression could be demonstrated by O-demethylation of dextromethorphan to dextrorphan in live cells. The enzymatic activity of 1.54 &- 16 pmol min _ ’ mg ~ ’ protein was comparable with dextromethorphan-O-demethylation activities of human liver. The metabolism of two dopaminergic ergoline derivatives was investigated in whole recombinant V79 cells. Both lisuride and terguride were monodeethylated; in case of lisuride a correlation to the in vivo situation was demonstrated comparing poor and extensive metabolizers. 0 1997 Elsevier Science B.V.

Key,$‘or&: CYP2D6; cells

Dextromethorphan;

Ergoline

derivatives;

1. Introduction Pharmacogenetics

deals

in pharmacodynamics due or

to differences functional

tablished member

expression of

enzyme

(Kalow, a

human

1992).

type

CYP2D6

of drugs The

and/

best

es-

affecting

cytochrome

debrisoquine/sparteine The

differences activity

polymorphism

of the monooxygenase

1979).

inhereted

pharmacokinetics

in individual

example

ily is the al.,

with

and

P450

(Eichelbaum

is an enzyme

a famet

which

Abhreciution.~: AUC, area under the curve; Cmax, maximum concentration; CYP2D6, cytochrome P450 2D6; K,,, Michaelis-Mentenconstant; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel; Vmax, maximum velocity. * Corresponding author. Present address: Institute of Pharmacokinetics, Schering AC, D-13342 Berlin, Germany. Tel.: + 49 30 4682731; fax: + 49 30 4681527. 1382-6689!97/$17.00 c> 1997 Elsevicr PI1 S I382-6689(96)00136-6

Science B.V. All rights

reserved

Heterologous

expression;

O-demethylation;

V79 Chinese

hamster

catalyzes both the N-oxidation of sparteine and the 4-hydroxylation of debrisoquine. Individual differences in enzyme activity led to the classification of poor and extensive metabolizers. In case of both compounds poor metabolizers might be subject to exaggerated pharmacodynamic effects. A large number of other drugs, like antiarrhythmics (e.g. flecainide, encainide), /Y-blockers and tricyclic antidepressants are, to different extents, also metabolized by this enzyme (Brosen and Gram, 1989; Eichelbaum and Gross, 1990; Spina and Caputi, 1994). This gives a twofold rationale to screen new chemical entities for their susceptibility to this enzymatic pathways. Poor metabolizers might require different treatment schemes and drug-drug interactions might be clinically relevant in case of potent compounds with narrow therapeutic windows.

32

R. Rauschenbach et al. / Environmental Toxicology and Pharmacology 3 (1997) 31-39

Studies to describe the contribution of different monooxygenase P450 enzymes to the metabolic degradation of xenobiotics can be performed by a variety of test approaches ranging from intact biosystems to isolated organs (liver), cells (hepatocytes) or sub-cellular fractions (microsomes) (Hildebrand et al., 1994). The isolation and purification of individual enzymes has been another approach. Biotechnology, however, has offered an even more promising alternative by the stable expression of these enzymes in heterologous systems, like yeast, bacteria or mammalian cells (Gonzalez and Korzekwa, 1995). Thus, donor dependent variability of enzyme contents, time consuming isolation procedures and limited practical usability of enzymes can be avoided. As mentioned before different expression systems were established for P450 enzymes, also including the mitochondrial enzymes, and all of them were characterized by different pros and cons. V79 cells, fibroblasts derived from Chinese hamsters, are an accepted toxicological model and do not exhibit endogenous P450. Doehmer introduced this system to express several P450 enzymes from rats and humans and used these cell lines for several studies on metabolic degradation and/or toxicological effects (Doehmer, 1993). Therefore V79 cells were selected to stably express a number of other P450 enzymes with special emphasis on its use for metabolic profiling (Gieschen et al., 1994). CYP2D6 seems to contribute to the biodegradation of a large number of therapeutically relevant drugs. Although this enzyme only represents approximately 2% of human liver total P450 contents (Shimada et al., 1994), it is of interest due to the inhereted difference in enzyme capacity and possible interactions with other drugs. Therefore human CYP2D6 should be expressed in V79 cell lines and applied to screen substrate properties of new and established drugs. The present paper describes the expression of the protein and the metabolic screening of two ergoline derivatives.

2. Materials

and methods

2.1. Cell line and plasmids The parental V79 Chinese hamster cell subline V79MZ was kindly supplied by Prof. J. Doehmer (Technical University, Munich) and used as recipient cell line for transfection with expression plasmids. The plasmid pCMV2 containing the human wild type fulllength CYP2D6-Val,,, cDNA (Ellis et al., 1993) was received from Prof. C.R. Wolf (Imperial Cancer Research Fund Laboratories, University of Edinburgh). The expression vector pMPSV-HE-CMV was obtained

from Dr H. Hauser (Gesellschaft fur Biotechnologische Forschung, Braunschweig) and used for expression of CYP2D6 in V79 cells. 2.2. Cell culture V79 Chinese hamster cells were cultured in Dulbecco Vogt’s modified Eagle’s medium, DMEM (Seromed, Berlin), supplemented with 5% (v/v) fetal calf serum (Gibco, Eggenstein), 1 mM sodiumpyruvat (Seromed, Berlin) and 2 mM L-glutamine (Gibco, Eggenstein) at 37°C under an athmosphere of 5% (v/v) CO, in air at 95% humidity. Selection of recombinant cell lines was maintained by addition of hygromycin B (400 fig/ml, Boehringer, Mannheim). 2.3. Genetic engineering

of V79MZ

cells

The 1.6 kb full-length CYP2D6 cDNA was removed from pCMV2 with EcoRI and inserted into the pMPSV-HE-CMV vector digested with EcoRI. In the resulting expression plasmid named pMPSV-CMVCYP2D6 the CYP2D6 was under the control of the myeloproliferative sarcoma virus long terminal repeat promotor (MPSV-LTR) and the enhancer derived from the cytomegalovirus (CMV). The CYP2D6 cDNA was flanked upstream by a SV40 splice junction and downstream by a SV40 poly A tract for stabilisation of the mRNA. The correct orientation of the CYP2D6 cDNA in the vector was determined by restriction analysis. The CYP2D6 expression plasmid was cotransfected with selection marker plasmid pSK/HMR272 by the calcium phosphate coprecipitation method described by Chen and Okayama (1988). The pSK/HMR272 plasmid was constructed by insertion of a 2.8 kb f?amHI/ Hind111 fragment containing the hygromycin B phosphotransferase gene including the thyrnidine kinase promotor and the thymidine kinase terminator of herpes simplex virus from plasmid pHMR272 (Bernard et al., 1985) into the pBluescript II vector (Stratagene, La Jolla, CA). V79 cells were seeded at a density of 2 x IO6 cells/94 mm dish and cultured for about 16 h. Plasmids pMPSV-CMV-CYP2D6 (20 pg) and pSK/HMR272 (1 pg) were diluted in 0.5 ml 50 mM N-,N-Bis[2-hydroxyethyl]-2-amino-ethanesulfonic acid (Calbiochem-Novabiochem, Bad Soden/Ts., Germany), 280 mM NaCl, 1.5 mM Na,HPO,, pH 6.95. The DNA was precipitated by addition of 0.5 ml 250 mM CaCl,. After incubation at room temperature for 20 min the mixture was added directly to the medium free cells and left at room temperature for 30 min. Medium was added and further incubated for 4 h at 37°C. Cells were subjected to 25% (v/v) DMSO shock for 1 min, washed twice with medium, and referred with 10 ml of DMEM. After further incubation at 37°C growth medium was replaced by DMEM containing hygromycin B (400 pug/

R. Rauschenbach et al. i Environmental Toxicology and Pharmacology 3 (1997) 31-39

ml) on the third day colonies appearing every cotton buds and grown studies. 2.4. DNA

isolation

after transfection. Resistant 12- 14 days were picked with in mass culture for further

and PCR

analysis

The presence of genomically integrated CYP2D6 cDNA in recombinant cells was confirmed by PCR analysis. Preparation of chromosomal DNA was performed according to a modified method described by Sambrook et al. (1989). Cells from a well of a six-wellplate were incubated with proteinase K in 400 ~1 digestion buffer (100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 25 mM EDTA, 0.5% (w/v) SDS, 100 pg/ml proteinase K) at 50” for 16 h. The viscous lysate was drawn 5 times through a hypodermic needle to shear the chromosomal DNA into smaller fragments. Samples were extracted twice with one volume of phenol/ chloroform/isoamyl alcohol 25/24/l (v/v/v). DNA was recovered by ethanol precipitation and resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, at room temperature overnight. Amplification was performed with 35 cycles using 1 pg chromosomal DNA as template. The 5’ primer corresponded to residues 886 to 909 within the CYP2D6 cDNA and the 3’ primer to a sequence within the proximal vector region resulting in a 0.7 kb fragment after amplification. 2.5. Western

33

HRP to catalyze oxidation of luminol, which then emitted light. The light emission was visualized on film (exposure for 2 min). Additionally 8 ,ug and 12 pug of male human liver microsomes, prepared from surgical waste, were applied for comparison. 2.6. Enzyme

assay and analysis

of dextromethorphan

Enzymatic activity of CYP2D6 in whole cells was determined by O-demethylation of dextromethorphan to dextrorphan. Cells were seeded at a density of 1 x lo6 cells per 94 mm petri dish in 10 ml of DMEM medium and grown for 24 h at 37°C. Medium was replaced by 10 ml of fresh medium containing 50 pM dextromethorphan and further grown at 37°C for 24 h. Cell culture supernatant was removed and 2 ml were extracted with 4 ml acetone/methanol 75/25 (v/v). Precipitated proteins were pelleted by centrifugation and the supernatant was dried under a gentle stream of nitrogen. Metabolic products were analysed by reverse phase HPLC (Spherisorb Phenyl, 250 x 4.6 mm, 5 pm, Muder und Wochele, Berlin) with an isocratic solvent system (10 mM KH,PO,/acetonitrile, pH 4.0, 55/45, v/v) at ambient temperature, applying a flow rate of 1.2 ml/min and UV-detection at 280 nm. Protein concentration of cytosol free pellets from cells used in enzyme assays was determined according to Lowry et al. (1951).

blotting 2.7. Enzyme

Microsomal fractions were prepared from the parental and the transfected V79 cell clone with the highest O-demethylation activity of dextromethorphan (s. 2.6). The fractions were resuspended to about 10 mg protein/ml sample buffer (125 mM Tris-HCl pH 6.8, 20% glycerine, 10% 2-mercaptoethanol and 4% SDS), diluted to 2 mg/ml and heated to 95°C for 5 min. Ten micrograms, 5 pg and 2.5 pg microsomal protein of each cell line were separated by SDS-PAGE (12.5%), then transferred to nitrocellulose (Towbin et al., 1979) in Laemmli buffer 40 mM Tris, 380 mM Glycin, pH 8.3, and 0.2% SDS) containing 20% methanol and 30% water for 1 h at 300 mA. For immunodetection the reagents of Rat cytochrome P450 ECL (enhanced chemoluminescent) Western blotting kit (Amersham Life Science, Braunschweig) were used except the included antibodies. The applied primary monoclonal anti-2D6-antibody BI-11412 (Zanger et al., 1988), derived in mouse, was kindly provided by Dr U.M. Zanger (Dr Margarete Fischer-Bosch-Institute, Stuttgart). In a second step the membrane was incubated with a biotinylated secondary anti-mouse-antibody (Boehringer Mannheim), which, in turn, was detected by a streptavidin-horseradish peroxidase (HRP), kit content conjugate. The ECL-reagent utilized the bound

assay and analysis

of ergoline derivatives

The metabolism studies were performed in live cell cultures. Cells were seeded at a density of 5 x 105cells/ 75 cm2 flask and incubated for 24 h in 10 ml medium. After medium exchange 0.3 PM of r4C-labeled substrate, lisuride or terguride (Fig. I), was added and incubated for 48 h. Supernatant was removed and 2 ml were extracted with 4 ml ice-cold acetone/methanol (3:l; v/v). After centrifugation, decanting and removal of the liquid phase by a gentle stream of nitrogen the residues were dissolved in start-eluate and analyzed by HPLC. Metabolites were separated on a Spherisorb ODS II-column (125 x 4.6 mm, 5 pm, M & W-Chro-

Fig. 1. Investigated and terguride (B).

“C-labeled

(,) ergoline

derivatives

lisuride

(A)

34

R. Rauschenhach

et al.

i Environmental

Toxicology

matographietechnik, Berlin) as stationary phase at ambient temperature. The mobile phase consisted of acetonitrile (Eluate A) and aqueous ammoniumcarbonate 0.01 M (Eluate B). A linear gradient was applied for 30 min from 20% A to 100% A at a flow rate of 1.0 ml/min. The HPLC-chromatograms were recorded both by fluorescence (extinction 282 nm, emission 370 nm) and by on-line radioactivity detection (RAMONA LS4, Raytest, Germany).

and Pharmacology

3 (1997) 31-39

pMPSV-CMV-CYPPDG

2.8. Clinical study in volunteers During a Phase I study the influence of concomitant food-intake on the pharmacokinetics of a peroral standard dose of lisuride (given as Doperginm tablet with 0.2 mg lisuride hydrogen maleate) was studied in 30 healthy male volunteers (age: 19928 years, bw, 55590 kg). Plasma samples were taken to generate a lisuride day-profile under fasted conditions and after food-intake. Bioanalysis were performed by RIA (Huempel et al., 1981). Retrospectively, the participating test subjects were phenotyped for CYP2D6 using dextromethorphan as a probe drug (Hildebrand et al., 1989). Each poor and intermediate metabolizer was characterized according to the metabolic ratio (MR) of dextromethorphan/dextrorphan of 6.9 and 0.15 respectively. All other subjects were extensive metabolizers with MR-values of 0.0003-0.015.

3. Results 3.1. Construction of a CYP2D6 expressing V79 cell line The 1.6 kb full length cDNA, encoding human CYP2D6-Val,,,, was cut out of the plasmid pCMV2 with EcoRI and inserted into expression vector pMPSV-HE-CMV and placed under the control of the strong MPSV-LTR promotor and the CMV enhancer contained in the vector (Fig. 2). The resulting expression plasmid pMPSV-CMV-CYP2D6 was cotransfected with the pSK/HMR272 selection plasmid containing the gene of the hygromycin B phosphotransferase into V79 cells by the calcium phosphate procedure (Chen and Okayama, 1988). Twenty one hygromycin B resistant colonies were picked 20 days after transfection and further grown for analysis. The presence of the genomically integrated CYP2D6 cDNA in twelve hygromycin B resistant cell clones was demonstrated by PCR analysis. Chromosomal DNA was isolated and subjected to PCR using a 5’ primer hybridizing internal of the CYP2D6 cDNA and a 3’ primer homologous to the proximal vector sequence, respectively. Genomic DNA from eleven recombinant V79 cell clones showed the expected 0.7 kb amplifica-

Fig. 2. Structure of plasmid pMPSV-CMV-CYPZD6 human cytochrome P450 2D6.

for expression of

tion fragment which was not detected in DNA from non-transfected cells (Fig. 3). An amplification product with the identical length was obtained from pMPSVCMV-CYP3A4 DNA used as a positive control. 3.2. The expressed protein Protein expression of CYP2D6 was confirmed by Western blots (Fig. 4) where the anti-2D6-antibody recognized proteins in human liver microsomes and the transfected cell line, but no protein in non-transfected V79 cells. In fact, the observed protein bands reflect the different applied protein concentrations. Functional expression of CYP2D6 in recombinant V79 cell clones was demonstrated by 0-demethylation of 50 ,uM dextromethorphan to dextrorphan (Fig. 5). Furthermore, to a minor extent, other unidentified degradation products were present. Within 67 h eight CYP2D6 expressing V79 cell clones 0-demethylated 14-33 ,uM dextromethorphan to dextrorphan. The specific enzymatic activity determined from 24 h incubation of substrate and recombinant V79h2D6 was about 154 +_ 16 pmol/min per milligrams protein of cytosol free pellet. Fig. 6 shows the time dependence of Odemethylation of dextromethorphan in live V79h2D6 cells. Dextrorphan appears after 10 h of incubation. After 42 h of incubation 50% of dextromethorphan were converted. 0-demethylation of dextromethorphan was not derived from endogenous activity in recipient V79MZ cells. Human CYP2D6 expressing V79 cells were further investigated by determining their metabolic activity towards two ergoline derivatives, lisuride and terguride, as unknown substrates. Terguride was monodeethylated by human CYP2D6 (Fig. 7). Other minor peaks

R. Rauschenbach

et al. /Environmental

Toxicology and Pharmacology 3 (1997) 31-39

35

Fig. 3. PCR analysis of the genomic DNA of twelve V79h2D6 cell clones. Total genomic DNA was isolated from the cells and subjected to PCR using two synthetic primers amplifying a 0.7 kb fragment. Lanes 1 and 16, /i DNA PstI fragments as size markers; lane 2, pMPSV-CMV-CYP2D6 plasmid (positive control); lane 3, non-transfected V79 cells (negative control); lanes 4-15, V79h2D6 cell clones,

observed in the chromatograms were also detectable with non-transfected V79 cells. This reflected that additional, probably non-P450 metabolic activity was present. Due to its total amount and the interest in CYP2D6 mediated reactions, which could unequivocally be attributed to monodeethylation, the minor products were not identified. In case of lisuride the same reaction was observed. The N-monodeethylated metabolites of both substrates were identified by cochromatography of the synthetic reference compound. Metabolic pattern obtained by fluorescence and radioactivity detection were similar.

tremely different plasma level time profiles with high peak plasma levels and correspondingly higher AUCvalues for lisuride (Fig. 8). These two subjects could be classified as poor and intermediate metabolizers of dextromethorphan and thus, exhibit a lower enzymatic capacity of 2D6 mediated biotransformation. The relevance of the in-vitro finding that 2D6 is involved in the biodegradation of lisuride was demonstrated in this volunteers experiment, which also showed that decreased metabolic capacity of 2D6 resulted in an increase of systemic body burden which accounted for a factor of up to 15 in case of AUC (Table 1).

3.3. Clinical relevance of 206 lisuride pharmacokinetics

4. Discussion

polymorphism

for

Retrospectively, volunteers of a food-effect study with lisuride were phenotyped for the 2D6-polymorphism. Two of twenty-three volunteers exhibited ex-

The present paper describes the establishment of a V79 cell line stably expressing cytochrome P450 2D6 and its application in studying the metabolism of the two ergoline derivatives terguride and lisuride as examA 280

DC )P

1

HO \ ‘/ : N,CH I

%

Fig. 4. Western blot analysis of microsomal fractions of 10, 5.0 and 2.5 pg protein of parental (lane 224) and CYP2D6 (lane 5-7) expressing V79 cells, respectively. Lane 8 and 9 show 8 pg and 12 pg protein of human liver microsomes, respectively. Lane 1, ECL molecular weight markers.

Fig. 5. HPLC-chromatograms of a lipophilic extract following bations of a V79h2D6 cell clone with 50 ,uM dextromethorphan 67 h. DMP, dextromethorphan; DOP, dextrorphan.

incufor

R. Rauschenbach et al. 1 Environmental Toxicology and Pharmacology 3 (1997) 31-39

36

60-

40-

FM alo-

O-

Fig. 6. Time dependent dextromethorphan O-demethylation of a recombinant V79 cell line expressing human CYP2D6. Reactions were performed in whole cells with initial substrate concentrations of 50 PM. DMP, dextromethorphan; DOP, dextrorphan.

pies for substrates of this enzyme. V79 Chinese hamster cells are a suitable host for heterologous expression of any cytochrome P450 and their use in metabolic and mutagenicity studies, because they lack P450-mediated metabolic activity of xenobiotics (Glatt et al., 1987; Kiefer and Wiebel, 1989). Therefore, cytochrome P450 activity of recombinant V79 cells is only derived from P450 cDNA mediated gene transfer. Stably expression of rat CYP2Bl (Doehmer et al., 1988) rat CYPlAl (Dogra et al., 1990), human CYPIAI (Schmalix et al., 1993), rat CYPlA2 (Woelfel et al., 1991), human CYPlA2 (Woelfel et al., 1992), human CYP2E1, human CYP2A6 (Doehmer et al., 1994), human CYP2D6 called RT2D6 (Fischer et al., 1992) and human CYP3A4 (Rauschenbach et al., 1995) in V79 cells were reported. Polymorphic CYP2D6 is one of the most relevant drug metabolizing enzymes and catalyses a broad range of clinically important drugs (Cholerton et al., 1992). Besides human liver, where the majority of metabolites was produced, CYP2D6 has been also identified in Table 1 Area under the curve values (AUC) and peak plasma levels (Cmax) of lisuride in plasma of 2D6-phenotyped human volunteers after peroral administration of one tablet Dopergina [0.2 mg lisuride hydrogen maleate] in fasted state Phenotype

AUC (pg/h

PM IM EM

3103 (n = 1) 1064 (n = 1) 194 f 105 (n = 15)

PM, poor metabolizer; metabolizer.

per ml)

IM, intermediate

Cmax

(pg/ml)

869 (n = 1) 681 (n= 1) 94 + 95 (n = 23) metabolizer;

EM, extensive

human kidney, small intestine and brain (Kaminsky and Fasco, 1991; Britto and Wedlund, 1992). Dextromethorphan is a standard substrate for this enzyme and used for investigation of the debrisoquine hydroxylation polymorphism (Kuepfer et al., 1984; Schmid et al., 1985). Therefore, the specific enzymatic activity in the V79-derived cell line expressing the human CYP2D6 was demonstrated by O-demethylation of dextromethorphan to dextrorphan. Different systems for heterologous expression had been established (Gonzalez and Korzekwa, 1995). Functional expression of CYP2D6 had been shown in COS cells (Gonzalez et al., 1988) yeast (Ellis et al., 1992), Hep G2 cells (Aoyama et al., 1990), SF9 insect cells (Gonzalez et al., 1994) and human B-lymphoblastoid cells (Crespi et al., 1991). However, comparisons are difficult because enzymatic activities were either determined with different substrates or referred to different cell fractions and different specific values. The activity of the V79h2D6 cell line of about 154 + 16 pmol/min per milligram protein of cytosol free pellet was comparable with dextromethorphan-O-demethylation activities of human liver (Rodrigues et al., 1994; Kronbach et al., 1987), CYP2D6 expressing human B-lymphoblastoid cells (Dayer et al., 1987; Dayer et al., 1989) and RT2D6 cells (Fischer et al., 1992). The observed rates of O-demethylation in human liver microsomes were within the range of 9.1-16.7 nmol/h per milligram. Microsomes prepared from CYP2D6 expressing human B-lymphoblastoid cells exhibit activities of about 12 nmol/h per milligram (Rodrigues et al., 1994), whereas microsomes of RT2D6 cells reached a Vmax of 10.3 nmol/hour per milligram. These results showed that the MPSV promoter in combination with the CMV enhancer in V79h2D6 cells as well as the CMV promoter/enhancer unit used for RT2D6 cells was capable of sufficiently promoting the expression of CYP2D6 cDNA in V79 cells. Fischer et al. (1992) cotransfected the pSV2-Neo gene for selection of successful transfection. As this neomycin-resistance-encoding gene was shown to mediate a negative effect on promoters in stable transfectants of mammalian cell lines (Artelt et al., 1991), we used the hygromycin B-resistance-encoding gene instead. The current cDNA sequence encoded the wild type protein containing valine in position 374 (Ellis et al., 1993) whereas the cDNA used in RT2D6 cells encoded the amino acid sequence published by Gonzalez et al. (1988) which contained a methionine at that position. As O-demethylation activities of dextromethorphan were similar in both cell lines, this amino acid difference seems to have no influence on this particular catalytic activity. Crespi et al. (1995) showed a higher turnover rate and/or lower Km-values for CYP2D6-Val towards different substrates. They postulated CYP2D6-

R. Rauschmbacll et al. / Enoironmental Toxicology and Pharmacology 3 (1997) 31-39

3,

Fig. 7: Rauschenbach

et al.:

Fig. 7. Metabolic pattern of terguride after incubation Monodeethylterguride.

Development

of a V79 cell line.....

with nontransformed

Val to be the more common allele in human populations, because the CYP2D6-Met allele was not found among 83 individuals tested. Human CYP2D6 is involved in N-dealkylation processes as N-demethylation of amiflavine, desmethylcitalopram, imipramine and amitriptyline and in the O-demethylation of codeine (Coutts et al., 1994; Mikus et al., 1994). As the investigated ergoline derivatives lisuride and terguride were known to be N-dealkylated in vivo and in other in vitro systems, human CYP2D6 was screened for being responsible for this reaction (Toda and Oshino, 1981; Huempel et al., 1984, 1989; Fbrster, 1987). V79 cells expressing functional human CYP2D6applied for these studies. Val,,, could be successfully

V79 cells (A) and V79h2D6 cells (B). TER, terguride; MDT,

With CYP2D6 metabolic activity was limited to single N-dealkylation, although a second identical reaction to a dideethylated metabolite is known to occur in vivo (Huempel et al., 1984, 1989; Forster, 1987). RT2D6 cells were shown to be metabolically active towards clozapine, fluperlapine, tropisetron and ondansetron (Fischer et al., 1992, 1994). All characterized metabolic reactions mediated by CYP2D6 were identified as hydroxylations. Although these reactions were found partially CYP2D6 dependent in human liver microsomes as well, there is only evidence that CYP2D6 is important for overall metabolism of clozapine and fluperlapine in vivo. In the case of tropisetron, CYP2D6 is involved in metabolism, but clearly an enzyme metabolism is also catalyzed by the CYP3A and the CYPlA subfamily (Dixon et al., 1995).

38

R. Rauschenhach

Lisuride

0

et al.

I Environmental

and Pharmacology

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References

Lug/ml]

2

Toxicology

4

6

8

10

12

twne [h]

Fig. 8. Plasma levels of lisuride in 2D6-phenotyped healthy volunteers after peroral administration of one tablet Doperginm (EM, extensive metabolizer; IM, intermediate metabolizer; PM, poor metabolizer).

Similarly, lisuride and terguride metabolism as well was dependent of CYP3A4 (Rauschenbach et al., 1995) and additionally of CYPl Al, whereas neither CYPlA2 (Gieschen et al., 1994) nor CYP2A6 and CYP2El expressing V79 cell lines exhibited any activity towards these compounds (Gieschen and Hildebrand, submitted). Thus in-vitro a number of CYP enzymes could be shown to be involved in ergoline biodegradation. However, the aforementioned study in 2D6 phenotyped volunteers revealed that this polymorphic enzyme might play an important role in in vivo metabolism. Extensive and non-extensive metabolizers exhibited clearly different pharmacokinetics of lisuride which is considered as a hint that the contribution of other CYP enzymes may be of less importance. Relating to these observations in vivo, in-vitro studies with single enzymes may be helpful for the planning of human trials like interaction studies with phenotyped volunteers. In the case of an early development compound without knowledge about its behaviour in man, additional in-vitro studies have to be performed. For example incubation with human liver microsomes and CYP enzyme specific inhibitors will verify the actual role of a single enzyme in biotransformation of that compound. In conclusion, V79 cells expressing P450 enzymes can contribute to a profound insight and understanding of complex biodegradation processes.

Acknowledgements We would like to thank H. Fischer and A. Neumann for their excellent technical assistence, D. Henschel for synthesis of oligonucleotides and Dr Biere for synthesis and preparation of various formulations of the 14C-labeled ergoline derivatives. The work was part of a research project sponsored by the Schering AG, Berlin, Germany.

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