In vitro studies of induction and inhibition of drug oxidation in man

Pharmac. Ther. Vol. 33, pp. 101 to 108, 1987

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Specialist Subject Editor: E. E. Om~m~us

IN VITRO

STUDIES OF INDUCTION AND INHIBITION OXIDATION IN MAN

OF DRUG

A. R. Booms, S. MURRAY, C. E. SEDDON and D. S. DAVIES Department of Clinical Pharmacology, Royal Postgraduate Medical School, London W12 OHS, U.K.

INTRODUCTION The specificity of the hepatic mixed-function oxidase system, pivotal in determining the duration of action and toxicity of a large number of drugs and other chemicals, is determined by its terminal electron acceptor, cytochrome P-450. There are multiple forms of this hemoprotein, the products of separate genes, with unique but overlapping substrate specificity. However, the oxidation of a few substrates is so specific that it is competitively and completely inhibited by co-substrates for the same isozyme (1). The specific content of some of the forms of cytochrome P-450 can be selectively increased by treating animals with inducing compounds (2). Often, several related isozymes are increased coordinately by one class of inducer, of which there are at least six (3). Induction of cytochrome P-450 often leads to an increase in the oxidation of those substrates for the induced isozymes, which can be used as an indication of the specificity of the induction process. Although such studies may be performed routinely in animals, it is obviously much more difficult to conduct these investigations in man. It is not possible to perform in vitro studies on the same subjects, before and after exposure to an inducing compound. An alternative is to study different groups of subjects, one of which has been exposed to the inducing agent and another which has not. Such studies in vitro, on the induction and inhibition of monooxygenase activity, in man should help to elucidate the specificity and regulation of the different isozymes of cytochrome P-450 present. METHODS TISSUE SAMPLES

Human liver samples were obtained from patients requiring biopsy for diagnostic reasons. Tissue surplus to histological requirements, was made available for use in these studies. Local Research Ethical Committee permission was obtained to use such samples in this way (4). Prior to discharge from hospital, each patient was interviewed and details of clinical and social drug use, alcohol consumption, coffee ingestion, smoking and diet were recorded. The biopsies were assessed histologically and only those with preserved hepatic architecture were used in the studies reported here. Some subjects were phenotyped for their debrisoquine oxidation status prior to biopsy (5). Microsomal fractions were isolated by differential centrifugation and resuspended in 0.25 M potassium phosphate buffer, pH 7.25, containing 30070 (v/v) glycerol, as described in detail elsewhere (4). Samples were either assayed immediately or stored at - 80°C until required. Under such conditions of storage, monooxygenase activities showed no significant alteration over the interval from collection to use (3). ENZYME ASSAYS The Oodeethylation of phenacetin (6), the 4-hydroxylation of debrisoquine (7), the l'-hydroxylation of bufuralol (8) and the epoxidation of aldrin (3) were all determined 101

A. R, Booms et al.

102

by previously published methods. Microsomal protein content was assayed as previously described (4). RESULTS

Of eight patients who were phenotyped in vivo for their debrisoquine 4-hydroxylation status, only one was a PM subject (Table 1). When the ability of microsomal fractions from hepatic biopsy samples from these patients to 4-hydroxylate debrisoquine was determined in vitro, only the PM sample had no detectable activity. This was despite normal cytochrome P-450 content and ability to oxidize antipyrine to its three primary metabolites (data not shown, see ref. 5). The O-deethylation of phenacetin by microsomal fractions of human liver is biphasic (6). When the activity of the two components was determined in biopsy samples from EM and PM subjects, it was found that although the sample from a PM subject catalyzed the low affinity component of O-deethylase activity normally, it was deficient in the high affinity component of activity (Table 1). The effects of cigarette smoking on the two components of phenacetin O-deethylase and debrisoquine 4-hydroxylase activities were determined by comparing results obtained with biopsy samples from smokers with those from nonsmokers. Cigarette smoking was associated with a highly significant increase in the high affinity component of O-deethylase activity, which increased from 78 4- 15 pmol mg -~ to 176 ± 31 pmol mg -1 min -~ (Table 2). There was also a slight, but significant, increase in the low affinity component of this activity in smokers. However, debrisoquine 4-hydroxylase activity was not different between smokers and nonsmokers. TAnI~ 1. Debr~oquine 4-Hydroxylase and Phenacetin O-Deethylase Activities o f Extensive and Poor Metabolisers o f Debrisoquine In Vivo

Subject 1 2 3 4 5 6 7 8

In vitro

Metabolic ratio

Phenotype

0.6 1.3 1,6 0.8 1,1 4.8 4.5 48

EM EM EM EM EM EM EM PM

Cytochrome P-450 Debrisoquine content 4-hyroxylase P O D 1" (nmol m g -1) activity activity 0.50 0.36 0.67 0.38 0.27 --0.41

80.7 91.9 34.7 49.0 35.0 78.5 74.0 0

POD 2 activity

l" 93 -38 ---0

-1250 -510 ---580

* P O D 1, P O D 2: The high affinity and low affinity components, respectively, of phenacetin O-deethylase activity. t - - : value was not determined. Monooxygenase activities in vitro have been expressed in pmol m g - 1 m i n - l .

TABLE 2. Effect o f Cigarette Smoking on Hepatic Microsomal Monooxygenase Activities Monooxygenase activity Debrisoquine 4-hydroxylase Phenacetin Odeethylase 1~' Phenacetin Odeethylase 2

N

Activity (pmol m g - ~ m i n - l) Smokers N Nonsmokers

7

72.5

q- 12.9

9

176

±

31

9

1190 ±

110

9

2p*

74.2 ±

14.4

NS

16

78

±

15

<0.005

16

840

±

110

<0.05

*Values for smokers were compared with those for nonsmokers by Student's t test. 1"1,2, The high affinity and low affinity components, respectively, o f phenacetin O-deethylase activity. Values are m e a n ± SD.

Induction and inhibition of drug oxidation in man

103

TmsL~ 3, Effect of Anticonvulstant Treatment on Hepatic Phenacetin O-Deethylase Activity

Sample "Controls"~t ACI AC2

Cytochrom¢ P-450 content (nmol rag- 1)

Asyl hydrocarbon hydroxylase activity*

POD 1"}" activity

POD 2 activity

0.52 ± 0.12 0.61 0.74

29.9 4- 11.0 68.0 64.8

120 4-90 335 145

1050 4- 350 2280 1400

*Monooxygenase activities have been expressed in pmol rag-~ min-~. ~POD 1, POD 2: The high affinity and low affinity components, respectively, of phenacetin O-deethylase activity. $Controls were hepatic biopsy samples from patients not receiving any known inducer of monooxygenase activity. AC1, AC2: Two patients who were being treated with anticonvulsant therapy (AC1 = primidone and phenobarbitone; AC2 = phenytoin). Values for "controls" are mean 4- SD (n > = 17). Further details of this study can be found in (3).

TABLF. 4. ICzo Values for Inhibition of Debrisoquine 4-Hydroxylase Activity of Human Liver Inhibitor Nortriptyline Bufuralol Guanoxan Sparteine Phenformin Acetanilide Phenytoin Antipyrine Phenacetin Tolbutamide

Impaired Oxidation in PM phenotype?

IC50" ( ~ )

Yes Yes Yes Yes Yes No No No Yest No

15 70 100 250 550 I100 1200 1800 > 2500 > 8000

*Concentration of inhibitor causing 50% inhibition of debrisoquine 4-hydroxylasc activity. tBut see (12) and elsewhere in this article.

TAn~ 5. Ki Values for Inhibitors of Debrisoquine 4.Hydroxylase Activity of Human Liver Inhibitor Bufuralol Phenformin Guanoxan Sparteine Acetanilide Antipyrine

Nature of inhibition

K i (igM)*

Competitive Competitive Competitive Competitive Noncompetitive Noncompetitive

19 205 30 85 1230 19300

*Ki values were determined as described in (1).

The effects of treatment with anti-convulsant drugs on O-deethylase activity were investigated in biopsy samples from two patients (Table 3). Despite obvious elevations of cytochrome P-450 content and in the activity of aryl hydrocarbon hydroxylase, there was no consistent effect of anticonvulsant treatment on either component of phenacetin Odeethylase activity. The forms of cytochrome P-450 involved in the 4-hydroxylation of debrisoquine and the high affinity component of phenacetin O-deethylation were further characterized by studying the effects of potential inhibitors. Most compounds with impaired oxidation in the PM phenotype, such as sparteine, guanoxan and phenformin, were potent inhibitors of debrisoquine 4-hydroxylase activity (Table 4). Compounds which do not show impaired oxidation in the ~M phenotype, such as acetanilide and antipyrine, were only very weak inhibitors of this activity. The nature of the inhibition was investigated and in the case of sparteine, guanoxan, phenformin and the racemate of bufuralol, it was competitive, whereas with acetanilide and antipyrine it was noncompetitive (Table 5).

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A . R . BooBIs et al.

T.~LE 6. Comparison of K i Values for Inhibition of Debrisoquine 4-Hydroxylase and Bufuralol l'-Hydroxylase Activities of Human Liver g i value Q,tM) Bufuralol Debrisoquine l'-hydroxylase 4-hydroxylase

Inhibitor Bufuralol Debrisoquine Phenformin Guanoxan Sparteine Acetanilide Amylobarbitone

(12.87)* 60.0 59.0 29.6 60.2 8. lmMl" Stimulation

15.6 (140)* 205 30.0 85.0 1.23 mMt" Stimulation

*Values are Km for the respective reactions. tlnhibition was noncompetitive. In all other cases where inhibition occurred it was competitive.

The l'-hydroxylation of bufuralol is impaired in PM subjects (9) and in studies of the purified forms of cytochrome P-450 catalyzing the 4-hydroxylation of debrisoquine it was found that this isozyme also catalyzes the l'-hydroxylation of bufuralol (10,11). The effects of inhibitors of debrisoquine 4-hydroxylase activity on bufuralol l'-hydroxylase activity were therefore determined (Table 6). There was very close agreement between Ki for inhibition of debrisoquine 4-hydroxylase activity and Ki for inhibition of bufuralol l'-hydroxylase activity. In addition, the Ki for inhibition of each reaction by the other compound was in very good agreement with the Km for oxidation of that compound (8). In all cases, where inhibition was competitive for one activity, it was competitive for the other. Acetanilide was a noncompetitive inhibitor of both reactions, and amylobarbitone stimulated both activities. Although debrisoquine inhibits the high affinity component of phenacetin O-deethylase activitiy (12), this compound, even at very high concentrations, had no appreciable effect on the 4-hydroxylation of debrisoquine (Table 4). During the course of such studies, it was found that many solvents are potent inhibitors of the 4-hydroxylation of debrisoquine, whereas activity is stimulated in the presence of even modest concentrations of salt. The effects of these two factors on other monooxygenase activities of human liver were determined. Both bufuralol l'-hydroxylase and debrisoquine 4-hydroxylase activities were substantially reduced by DMSO, by 80-95°70 (Fig. 1). Aldrin epoxidase and the high affinity component of phenacetin O-deethylase activities were inhibited only very slightly, by 20 to 30070. The inclusion of 10mM sodium chloride in the incubation medium stimulated both bufuralol l'-hydroxylase and debrisoquine 4-hydroxylase activities, by approximately 50070, whilst having no effect whatsoever on the other two activities studied. 180

160 140 120 I00 "5 80 o 60 40 20 0

lOmM Noel

25/~t DMSO

Addition

FIo, 1. Effect of dimethylsulphoxide (DMSO)" and sodium chloride (NaCI) on monooxygenase activities of human liver. The activities studied were debrisoquine 4-hydroxylase ( [ ]), the high affinity component of phenacetin O-deethylase (~r-fff~), bufuralol l'-hydroxylase ( ~ ) and aldrin epoxidase ( [~[~J~l ). Activities have been expressed as a percentage of the appropriate control.

Induction and inhibition of drug oxidation in man

105

120 I00

80

60 t)

40

20 0

2

5

20

200

50

500

2000

Concentration (~M)

FIa. 2. Effect of quinidine and quinine on debrisoquine 4-hydroxylase activity. Quinidine ( ~ ; ~ ; ~ ) was tested at concentrations from 2 to 2000/z~ and quinine ([7"7"~) at concentrations from 5 to 2000/tu. Activities have been expressed as a percentage of the corresponding vehicle control.

120

IOC

8C

o 40 oc

60

(.3

40' 2C

2

5

20

I [[Lo 50

200

500

2000

5000

Concentration (FM)

FIG. 3. Effect of quinidine and quinine on the high affinity component of phenacetin O-deethylase activity. Both quinidine ( 0 7 7 ~ ) and quinine ( ~ ) were tested at concentrations from 2 to 5000 p~. Activities have been expressed as a percentage of the corresponding vehicle control.

Recent reports (13) that quinidine is a selective inhibitor of the form of cytochrome P-450 catalyzing the oxidation of sparteine were tested with debrisoquine as a substrate. Quinidine was, indeed, an extremely potent inhibitor of this reaction, whereas quinine was aproximately 100-fold less potent (Fig. 2). When the effects of the two compounds on the high affinity component of phenacetin O-deethylase activity were determined, their potency was reversed, in that quinidine produced less inhibition than quinine (Fig. 3). The more dramatic comparison, however, was between the effect of quinidine on the two monooxygenase activities. The ICs0 value for quinidine was almost 700 times greater for phenacetin O-deethylase activity than for debrisoquine 4-hydroxylase activity, whereas the ICs0 values for quinine were almost the same for the two reactions (Table 7). TABLE 7. ICso Valuesfor Quinine and Quinidine Inhibition of Phenacetin O-Deethylaseand Debrisoquine 4-Hydroxylase Activities Monooxygenase Debrisoquine 4-hydroxylase Phenacetin O-deethylase

ICso Oa~)

Quinidine

Quinine

0.7 500

65 120

Details of this study can be found in (22). JPT

33:1-H

106

A . R. BooBIs et al.

DISCUSSION Although studies of induction and inhibition of monooxygenase activity in vitro in man are more difficult to perform than in animals, the foregoing illustrates how a combination of careful documentation of the samples, together with appropriate methodology permits both the specificity and regulation of the isozymes of cytochrome P-450 in man to be investigated. The 4-hydroxylation of debrisoquine is polymorphic in man, as reflected by considerably greater values for the metabolic ratio of the metabolite to the parent compound in urine (14). This impairment can be detected in vitro with liver microsomal samples, there being no detectable 4-hydroxylase activity in those samples from subjects phenotyped PM. All samples from EM subjects have detectable activity (5). Although the samples from PM subjects have normal cytochrome P-450 content and oxidizing ability towards several other substrates such as antipyrine, the high affinity component of phenacetin O-deethylase activity is also impaired, there being no detectable activity in such samples (12). At the time that these studies were performed, some four or five years ago, these data strongly supported observations in vivo by others (15) and ourselves (16), that the Odeethylation of phenacetin was catalyzed by the same enzyme as that responsible for the polymorphism in the 4-hydroxylation of debrisoquine. However, amongst several discrepancies which subsequently came to light was the observation that although cigarette smoking has long been known to induce the O-deethylation of phenacetin in vivo (17) and was shown to cause a significant increase in the high affinity component of this reaction in vitro, the 4-hydroxylation of debrisoquine is not altered in cigarette smokers as compared to nonsmokers. Interestingly, although O-deethylation appears to be readily inducible by constituents of cigarette smoke, presumably polycyclic aromatic hydrocarbons, anticonvulsant drugs have no consistent effect on either component of O-deethylase activity. Similar observations have been made in vivo on the effects of rifampicin, which is a much less potent inducer of the oxidation of phenacetin than cigarette smoke whereas the opposite is true for the oxidation of antipyrine (3). Thus, it appears that the O-deethylation of phenacetin, particularly the high affinity component, is selectively inducible by cigarette smoking and not by other categories of inducing compound. This suggests that the O-deethylation reaction in vivo might well provide a useful means of determining induction by such compounds. A further implication of this is that at least the induced form of the O-deethylase enzyme is not responsible for the 4-hydroxylation of debrisoquine. The question then arose as to whether the constitutive activities were catalyzed by the same isozyme. Guengerich and his colleagues (10) have recently purified two different isozymes of cytochrome P-450 from human liver, representing the major forms catalyzing these two reactions. This separation of activities is supported by our own data on the effects of inhibitors of monooxygenase activity. For the majority of compounds with impaired metabolism in PM subjects, there is an excellent correlation with their abilities to competitively inhibit the 4-hydroxylation of debrisoquine in vitro. With the exception of phenacetin, all such compounds fall into this category. Conversely, those compounds, the oxidation of which is not impaired in the PM phenotype, are either weak or non-inhibitors of the 4-hydroxylase reaction in vitro. Amongst reactions that have been shown to be impaired in PM subjects is the l'-hydroxylation of bufuralol (9). Although the magnitude of impairment of this activity in biopsy samples from PM subjects is not as great as that for the 4-hydroxylation of debrisoquine (18), nevertheless each compound is a potent competitive inhibitor of the oxidation of the other. In addition, there is an excellent correlation between their inhibitory constants, Ki, and the Km for the oxidation of that compound. This would suggest that the same isozyme catalyzes the hydroxylation of both substrates. This has been substantiated by studies on the purified isozymes (10,11). In contrast, although debrisoquine could inhibit the high affinity component of the O-deethylation of phenacetin, the latter compound was virtually without effect on the 4-hydroxylation reaction (12). This supports the data from the purification studies (10), that these two reactions are catalyzed primarily by different

Induction and inhibition of drug oxidation in man

107

isozymes. By implication, therefore, the O-deethylase isozyme appears to be inducible by cigarette smoking whereas that catalyzing the 4-hydroxylation of debrisoquine is not. A further distinction between the two enzyme activities can be made on the basis of their reactions to in vitro modifiers, such as solvents and high concentrations of salt. The debrisoquine 4-hydroxylase form of cytochrome P-450 is extremely sensitive to most organic solvents, and particularly to DMSO. This contrasts with the effects on other activities, such as the epoxidation of aldrin and the O-deethylation of phenacetin. There is good evidence that these two activities are catalyzed by different isozymes, and this would therefore suggest the involvement of at least three different forms of cytochrome P-450 in these reactions. Debrisoquine 4-hydroxylase activity is also inhibited by moderate concentrations of salt (sodium chloride). Again, other activities such as the epoxidation of aldrin and the O-deethylation of phenacetin are unaffected by this compound. However, an activity that is believed to be catalyzed by the same isozyme as that involved in the metabolism of debrisoquine, the l'-hydroxylation of bufuralol, is similarly affected by the presence of sodium chloride. The mechanisms involved in these effects have yet to be determined. A final means of distinguishing the two activities, the O-deethylation of phenacetin and the 4-hydroxylation of debrisoquine, is the potency and selectivity of inhibition by quinidine. This compound inhibits all activities studied to date catalyzed by the debrisoquine 4-hydroxylase form of cytochrome P-450, at submicromolar concentrations (13,19-22). In contrast, it requires some several hundred times this concentration to inhibit activities catalyzed by other forms of cytochrome P-450, such as the O-deethylation of phenacetin. The diastereoisomer, quinine, is much less potent and does not show the same degree of selectivity of effect, with similar inhibitory potencies for both the O-deethylation of phenacetin and the 4-hydroxylation of debrisoquine. The potency of inhibition of the 4-hydroxylation of debrisoquine by quinidine is such that it should impair activitiy in vivo, even at very modest doses. Recently it has been shown that this, indeed, is the case and that doses of as little as 50mg can cause substantial impairment of this activity in vivo (22). It is probable that the isozyme of cytochrome P-450 catalyzing the 4-hydroxylation of debrisoquine is somewhat unusual in the degree of specificity for its substrates. This has enabled detailed studies to be performed by using selective inhibitors of the reaction and comparing their effects with those on other activities. This has proven a very powerful means of distinguishing activities and in characterizing the specificity of the isozyme. Other groups have recently published results of a similar approach (23,24). An alternative to the use of competitive substrates, where specificity overlaps to such an extent that such an approach would not provide definitive information, is the use of monoclonal antibodies. These can be tailored to very precise specificity and will undoubtedly prove valuable in defining the contribution of a particular isozyme to the metabolism of any given substrate (25). It is possible to determine induction of monooxygenase activity in vitro, but this is only possible with detailed knowledge of the history of the patients from whom the samples are obtained. The development of liver banks comprising large numbers of well characterized samples should prove invaluable in pursuing such studies further.

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6. Booms, A. R., KARN, G. C., WHYT~, C., BKODn~,M. J. and DAVIES,D. S. (1981) Biphasic O-deethylation of phenacetin and 7-ethoxycoumarin by human and rat liver microsomal preparations. Biochem. Pharmac. 30: 2451-2456. 7. KAHN,(3. C., Booms, A, R., MURRAY,S., BKODm,M. J. and DAV~S, D. S. (1982) Assay and characterisation of debrisoquine 4-hydroxylase activity of microsomal fractions of human liver. Br. J. clin. Pharmac. 13: 637-645. 8. Booms, A. R., MURRAY, S., HAMPDEN, C. E. and DArt, s, D. S. (1985) Genetic polymorphism in drug oxidation: In vitro studies of human debrisoquine 4-hydroxylase and bufuralol l'-hydroxylase activities. Biochem. Pharmac. 34: 65-71. 9. DAYER,P., BALANT,L., COURVOISmR,F., KtrP~R, A., KuBu, A., GOKGIA,A. and FABR~, J. (1982) The genetic control of bufuralol metabolism in man. Eur. J. Drug Metab. Pharmacokin. 7: 73-77. 10. DISTLERATH,L. M., REniY, P. E. B., MARTIN,M. V., DAWS, G. G., WILKn~SON,(3. R. and GU~NO~R~CH, F. P. (1985) Purification and characterization of the human liver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phenacetin O-deethylation, two prototypes for genetic polymorphism in oxidative drug metabolism, o'. Biol. Chem. 260: 9057-9067. II. GUT, J., CATIN,T., DA~.R, P., I(RONBACH,T., Z-~NGER,U. and MEYER,U. A. (1986) Debrisoqnine/sparteinetype polymorphism of drug oxidation. Purification and characterization of two functionally different human liver cytochrome P-450 isozymes involved in impaired hydroxylation of the prototype substrate bufuralol. £. biol. Chem. 261: 11734-11743. 12. KAHN,(3. C., Booms, A. R., BRODI~,M. J., TOVEKUD,E.-L., MURRAY,S. and DAVIES,D. S. (1985) Phenaeetin O-deethylase: an activity of a cytochrome P-450 showing genetic linkage with that catalysing the 4-hydroxy]ation of debrisoquine7 Br. J. clin. Pharmac. 20: 67-76. 13. OTTON, S. V., INABA,T. and KALOW, W. (1984) Competitive inhibition of sparteine oxidation in human liver by fl-adrenoceptor antagonists and other cardiovascular drugs. Life Sci. 34: 73-80. 14. MAHC,Otm, A., IDLE, J. R., DRING, L. (3., LANCASTER,R. and S~'rH, R. L. (1977) Polymorphic hydroxylation of debrisoquine in man. Lancet U: 584-586. 15. SLOAN,T. P., MAHOOUB,A., LANCASTER,R., IDLE, J. R. and S~TH, R. L. (t978) Polymorphism of carbon oxidation of drugs and clinical implications. Br. Med. J'. 2: 655-657. 16. Toe,RUb, E. L. (1981) PhD thesis, University of London. 17. PANTUCK,E. J., KUNTZ~taN,R. and CONNEY,A. H. (1972) Decreased concentration of phenacetin in plasma of cigarette smokers. Science 175: 1248-1250. 18. MINDEK,E. I., MIrroR, P. J., MULL]~R,H. K., MINDER, C. and M~YER, U. A. (1984) Bufuralol metabolism in human liver: a sensitive probe for the debrisoqnine-type polymorphism of drug oxidation. Eur. J. clin. Invest. 14: 184-189. 19. HoRI, R., OKU~trRA, K., INUI, K.-I., YASUHARA,M., YA~DA, K. and SAKURAI,T. (1984)Quinidine-induced rise in ajmaline plasma concentration. J'. Pharm. Pharmac. 36: 202-204. 20. L E ~ N N , T., DAY,R, P. and M~Y~R, U. A. (1986) Single-dose quinidine treatment inhibits metoprolol oxidation in extensive metabolizers. Eur. £. clin. Pharmac. 29: 739-741. 21. INASA, T., TYNDALl, R. E. and MAHON, W. A. (1986) Quinidine: potent inhibition of sparteine and debrisoquine oxidation in rive. Br. J. eli,. Pharmac. 22: 199-200. 22. SPEnds, C. J., MURRAY, S., BoOmS, A. R. and SEDDON, C. E. (1986) Quinidine and the identification of drugs whose elimination is impaired in subjects classified as poor metabolizers of debrisoquine. Br. J. din. Pharmac. 22: 739-743. 23. SPn~A, E., BmGERSSON,C., YON BARR, C., ERICSSON,O., MEI.LSTROM,B., STEINER, E. and S~OQWST,F. (1984) Phenotypic consistency in hydroxylation of desmethylimipramine and debrisoquine in healthy subjects and in human liver microsomes. Clin. Pharmac. Ther. 36: 677-682. 24. INABA,T., JURnO~, M., MAHON,W. A. and KA~OW, W. (1985) In vitro inhibition studies of two isozymes of human liver cytochrome P-450. Mephenytoin p-hydroxylase and sparteine monooxygenase. Drug Metab. Dispos. 13, 443-448. 25. THews, P. E., R~n~, L. M., RYAN, D. E. and L~vis, W. (1984) Characterization of nine monoclonai antibodies against rat hepatic cytochrome P-450. Delineation of at least five spatially distinct epitopes. J. biol. Chem. 259: 3890-3899.