Br. J. clin. Pharmac. (1992), 34, 256-261

Inhibitors of imipramine metabolism by human liver microsomes ERIK SKJELBO & KIM BR0SEN Department of Clinical Pharmacology, Institute of Medical Biology, Odense University, J. B. Winsl0wsparken DK-5000 Odense C, Denmark

19,

1 The aromatic 2-hydroxylation of imipramine was studied in microsomes from three human livers. The kinetics were best described by a biphasic enzyme model. The estimated values of Vmax and Km for the high affinity site ranged from 3.2 to 5.7 nmol mg-' h-1 and from 25 to 31 pM, respectively. 2 Quinidine was a potent inhibitor of the high affinity site for the 2-hydroxylation of imipramine in microsomes from all three human livers, with apparent K1-values ranging from 9 to 92 nm. This finding strongly suggests that the high affinity enzyme is CYP2D6, the source of the sparteine/debrisoquine oxidation polymorphism. 3 The selective serotonin reuptake inhibitors (SSRI), paroxetine, fluoxetine and norfluoxetine were potent inhibitors of the high affinity site having apparent K1-values of 0.36, 0.92 and 0.33 piM, respectively. Three other SSRIs, citalopram, desmethylcitalopram and fluvoxamine, were less potent inhibitors of CYP2D6, with apparent Ks-values of 19, 1.3 and 3.9 puM, respectively. 4 Among 20 drugs screened, fluvoxamine was the only potent inhibitor of the N-demethylation of imipramine, with a K1-value of 0.14 FLM. 5 Neither mephenytoin, citalopram, diazepam, omeprazole or proguanil showed any inhibition of the N-demethylation of imipramine and the role of the S-mephenytoin hydroxylase for this oxidative pathway could not be confirmed.

Keywords imipramine CYP2D6 microsomes

sparteine

mephenytoin

genetic polymorphism

Introduction

with this, our earlier studies with human liver microsomal preparations (Br0sen et al., 1991b) have shown that CYP2D6 is a major enzyme catalyzing the 2-hydroxylation of imipramine in extensive metabolizers (EM) and that alternative low affinity P450s catalyze this oxidation

About 7% of Caucasians are poor metabolizers (PM) of sparteine/debrisoquine (Eichelbaum et al., 1979; Mahgoub et al., 1977). The sparteine/debrisoquine oxidation polymorphism is a major determinant of interindividual differences in the metabolism of tricyclic antidepressants and some neuroleptics, 13-adrenoceptor blockers, and antiarrhythmics (Br0sen & Gram, 1989; Eichelbaum & Gross, 1990). The source of the sparteine/ debrisoquine polymorphism is a particular P450 isozyme, now referred to as CYP2D6 (Nebert et al., 1991). The polymorphism is caused by the absence of the CYP2D6 from the liver of the PM (Zanger et al., 1988). Imipramine is eliminated by N-demethylation to the active metabolite desipramine and by hydroxylation to 2-hydroxyimipramine and 2-hydroxydesipramine (Gram, 1974). There is a close association between the 2-hydroxylation of imipramine and desipramine and the sparteine/ debrisoquine oxidation polymorphism (Bertilsson & Aberg-Wistedt, 1983; Br0sen et al., 1986b). In accordance

process in PM. In whites, about 3% of the population has an autosomal recessive defect in the ability to catalyze the aromatic hydroxylation of S-mephenytoin (Kupfer & Preisig, 1984). We have recently shown that the N-demethylation of imipramine is mediated in part by the enzyme responsible for mephenytoin oxidation polymorphism (Skjelbo et al., 1991). Apart from this, the pharmacokinetic importance of the mephenytoin polymorphism seems to be limited to its role in the metabolism of proguanil, diazepam, N-desmethyldiazepam, omeprazole and propranolol (Andersson et al., 1990; Bertilsson et al., 1989; Ward et al., 1989, 1991).

Thus it appears that the overall elimination of imipra-

Correspondence: Dr Kim Br0sen, Department of Clinical Pharmacology, Institute of Medical Biology, Odense University, J. B. Winsl0wsparken 19, DK-5000 Odense C, Denmark

256

In vitro metabolism of imipramine mine is determined by two distinct genetic polymorphisms in drug oxidation. The present study was undertaken to characterize further the kinetics and to identify inhibitors of the enzymes involved in the metabolism of imipramine by human liver microsomes.

257

processes is characterized by a distinct maximal velocity, Vmax, and a distinct Michaelis constant, Km. Mathematically, each of the partial velocities, Vpart, can be expressed according to equation 1:

Vmax X S Vpart =

(1)

Km + S

Methods

Chemicals Drugs and drug-metabolites were donated by various

pharmaceutical companies: Imipramine and metabolites, clomipramine and desmethylclomipramine: Ciba-Geigy, Basel, Switzerland; lignocaine: DAK-Laboratoriet A/S, Denmark; paroxetine and propranolol: Ferrosan, Denmark; proguanil: ICI-Pharmaceuticals, UK; pipamperone: Janssen-Pharma A/S, Denmark; fluoxetine and norfluoxetine: Eli Lilly A/S, Denmark; citalopram, desmethylcitalopram, tolbutamide and zuclopenthixol: H. Lundbeck A/S, Denmark; mephenytoin: Sandoz Pharmaceuticals, New Jersey, USA; moclobemide: Roche, Basel, Switzerland; fluvoxamine: Duphar, B. V. Weesp, Holland; mianserin: Organon, OSS, Holland; quinidine was purchased from: Sigma, Missouri, USA; diazepam: Roche A/S, Denmark; omeprazole: Astra Hassle, Sweden; perphenazine: Schering-Plough A/S, Denmark. All chemicals were of high analytic grade.

Liver microsomes Whole human livers were obtained from three kidney donor patients shortly after circulatory arrest. The sparteine and mephenytoin phenotype of the donors had not been established in vivo. The livers were immediately cut into slices, frozen in dry ice and stored at -80°C. Microsomes were prepared by a standard technique (Meier et al., 1983), and the microsomal protein concentration was measured by the method of Lowry et al. (1951). The 2-hydroxyimipramine and N-desmethylimipramine formed by human liver microsomes were assayed by an h.p.l.c. method as described by Zeugin et al. (1990). The reactions were linear with incubation time (20 min) and protein concentration (100 ,ug per tube). The limit of determination of the assay was 20 pmol per tube which corresponds to a velocity of 0.6 nmol mg-' h-1. At this level, the intraday variability for the assay of each analyte was 15%.

Analysis of kinetic data For all three livers, the velocity of 2-hydroxylation and of N-demethylation was investigated using imipramine at final concentrations from 8 to 500 FM. For 2-hydroxylation the relationship between velocity and substrate concentration was curvilinear (Figure 1). This shows that at least two distinct enzymes are responsible for the 2-hydroxylation. The total velocity, V, for 2-OHimipramine formation is therefore the sum of the partial velocities via each enzyme. Each of the enzymatic

If the imipramine concentration S is much less than Ki, the expression for the partial velocity is simplified to L x S, where L = Vmax/Km for the enzymatic process. Equation 2 describes a model which assumes that 2-OH-imipramine is formed in parallel by a high affinity enzyme (low Kin) and a low affinity enzyme (high Kin). V=

Vmax X S Km + S

+ LxS +

(2)

Vmax and Km are the apparent maximal velocity and the apparent Michaelis constant, respectively, of a high affinity enzyme, and L is a constant which relates S to the velocity via the low affinity enzyme(s). The first estimates of Km and Vmax were obtained from a graphical analysis of the lower linear part of the Eadie-Hoffstee plots representing the low substrate concentrations. Equation 2 was fitted to the data using an iterative curve fitting programme based on a procedure for non-linear regression analysis (Holford, 1990). Neither equation 1 nor equation 2 could be fitted to the data for desipramine formation. Hence, the Km and Vmax values for this reaction were estimated graphically from double reciprocal plots of 1/V against 1/S. At final concentrations of up to 100 jM a series of drugs were screened for their inhibitory effect on the 2-hydroxylation and N-demethylation of imipramine. Drugs displaying inhibition were retested at a more relevant concentration range using three substrate concentrations around the Km for 2-hydroxylation and around the Km for demethylation. For most inhibitors, a graphical analysis revealed a curvilinear relationship between reciprocal velocity and the inhibitior concentration (Dixon, 1953) (Figures 3 and 4). Hence, an equation which describes a two-enzyme model was fitted to the data: V=

Vmax X S Km (1 +

+ +LxS

(3)

C) + S

According to the model, the oxidation proceeds in parallel via a high-affinity enzyme showing inhibition and a low affinity enzyme showing no inhibition. K; is the inhibitor constant for inhibition of the high affinity site and C1 is the inhibitor concentration. An initial estimate of Ki was obtained from the linear portions of Dixon plots, and the initial estimates of Km and Vmax were obtained using equation 2. The equation was fitted to the data using an iterative method (Holford, 1990). Both Dixon plots and Cornish-Bowden plots (CornishBowden, 1974) were used to indicate the type of inhibition. All incubations were carried out in duplicate.

258

E. Skjelbo & K. Br0sen Total

Results The Eadie-Hoffstee plots showed that the 2-hydroxylation of imipramine exhibited biphasic kinetics with microsomes from all three livers (Figure 1). The log likelihoods (Holford, 1990) of the estimation using a two enzyme model (equation 2) ranged from 8.8 to 12.9 as compared with 1.6 to 3.3 using a one enzyme model (equation 1). The apparent Vmax and Km values for the high affinity site and the value of L, representing the linear low affinity site, are listed in Table 1. The observed velocities of 2-hydroxyimipramine formation as well as the theoretical velocities via the high affinity and the low affinity enzyme in HL3 plotted against the substrate concentration are shown in Figure 2. The Km and Vmax values for N-demethylation determined graphically from Lineweaver-Burke plots were: HL1: 625 p1M and 100 nmol mg-' h-1, HL2: 2000 }1M

6

,................................ ~~High affinity site

,>." .5- 4 E E zC7 2

Low affinitysite

Ii 1 I i i 300 400 500 200 l00 Concentration of imipramine (>M)

- rn v vo

0

-

Figure 2 The formation of 2-hydroxyimipramine from imipramine by microsomes from human liver (HL3). Each point (0) represents the mean of duplicate determinations. ): Curve of best fit according to the model expressed by ( equation 2 (see text). (----): Theoretical velocity mediated by the high affinity isoenzyme. (- -): Theoretical velocity mediated by the low affinity isoenzyme.

Table 1 Kinetic characteristics of the 2-hydroxylation of imipramine by microsomes from three human livers (HL) (Biphasic kinetics according to equation 2 in the text)

(Vmaxm a

Kmb

LC

(nmol mg- I h-l )

(>M)

(pIlmg-' h-')

3.2 4.7 5.7

31 26 25

2.8 6.4 2.6

HL1 HL2 HL3

a Apparent maximal velocity for high affinity enzyme. b Apparent Michaelis constant for high affinity enzyme. c Ratio between the maximal velocity and the Km of 2-OH-imipramine formation by the low affinity enzyme

E w-

(cf. equation 2). EC HL-1

4

?4L4

4' 2

;3 0 1

0

2

6

HL-2

4

l'i.A. AL

1..

'

.

If

. 4l.-aW.'ti i-WO....'l-44.6.

O

cm

E

2

Figure 3 The effect of quinidine on the 2-hydroxylation of imipramine by microsomes from three human livers (HL) (Dixon plots). Imipramine concentrations: (5): 16 pLM, (0): 32 IM and (A): 64 p.M. The lines represent the best fits according to equation 3.

-5EC HL-3

6 4

2 0

1~ ~ n] u

I

I

I

0

50

100

0

150

200

250

V/S (I mg-' h-1) Figure 1 Eadie-Hoffstee plots of the appearance of 2-hydroxyimipramine after incubation of microsomes from three human livers (HL) with imipramine concentrations (S) from 8-500 F.M. V = velocity of reaction. The lines represent the best fits of a model assuming a biphasic enzyme system.

and 143 nmol mg-' h-' and HL3: 300 01m and 80 nmol mg-' h-1. The Dixon plots of the quinidine inhibition kinetics for 2-hydroxyimipramine formation confirmed the biphasic model (Figure 3) and the apparent Kis for quidinine inhibition of the high affinity enzyme ranged from 9-92 nM. Replicate experiments with HL3 showed good reproducibility of the estimated parameters (Table 2). The inhibition characteristics of about 20 drugs for 2-hydroxylation and N-demethylation are listed in

In vitro metabolism of imipramine

259

Table 2 Inhibition of the formation of 2-hydroxyimipramine by quinidinea in microsomes from three human livers (HL)

HL1 HL2 HL3 mean (s.d.)c

Vmax

Km (>M)

Ki

Lb

(nmolmg- h)

(nM)

(plmg-'h-')

3.0 (2.9-3.2) 4.8 (4.6-5.0) 3.9 (0.94)

30 (22-35) 24 (23-26) 35 (10.0)

92 (42-181) 9 (7-11) 78 (16.5)

24 (13-34) 10 (9-10) 8.9 (3.5)

aVmax, Km, Ki refer to the high affinity enzyme site (equation 3). Mean values and ranges determined at three imipramine concentrations are given. b Ratio between maximal velocity and the Km of 2-OH-imipramine formation

by

the low affinity enzyme (equation 3). c Mean and (s.d.) for four independent experiments each using three imipramine concentrations and HL3 microsomes. Table 3 The effect of various drugs on the 2-hydroxylation and N-demethylation of imipramine by microsomes from human liver HL3

2-hydroxylation Kia (>LM) Citalopram Desmethylcitalopram Fluoxetine Norfluoxetine Fluvoxamine Paroxetine Mianserin Clomipramine Desmethylclomipramine Moclobemide Perphenazine Pipamperone Zuclopenthixol Lignocaine Mephenytoin Diazepam Propranolol Tolbutamide Omeprazole Proguanil Quinidine

19 1.3 0.92 0.33 3.9 0.36 6.7 16 7.9 140 0.16

N-demethylation Kia (,UM)

0.1i

E E0.05

0.14 b

*

c

-* 40 * * * * * d

E

10

a

According to equation 3 where the Kis refer to the high affinity enzyme site. b At a paroxetine concentration of 50 ,UM there was a 45% reduction in desipramine formation (imipramine concentration: 256 ,UM). c At a pipamperone concentration of 100 p,M, there was a 27% reduction in 2-OH-imipramine formation (imipramine concentration: 16 ,UM). d See Table 2. -No inhibition. * Not tested owing to interfering chromatographic peaks.

Table 3. Cornish-Bowden plots and Dixon plots of the inhibition kinetics of all inhibitors were consistent with competitive inhibition. The only potent inhibitor of N-demethylation was fluvoxamine (Table 3) having an apparent Ki of about 0.14 5LM (Figure 4); paroxetine and propranolol were relatively weak inhibitors of N-demethylation (Table 3). Discussion

Using an enzyme kinetic approach (Figures 1, 2, 3) we have shown that the formation of 2-OH-imipramine is

0

0

500

1000

Concentration of fluvoxamine (nM) Figure 4 Dixon plots of fluvoxamine inhibition of desipramine formation from imipramine by human liver microsomes (HL3). The imipramine concentrations were 128 ,M (0), 256 ,UM (0), and 500 ,UM (A). Each point represents the mean of duplicate determinations, and the curves represent the best fits according to equation 3. The mean apparent Km and Vmax values for the high-affinity enzyme catalyzing the N-demethylation (equation 3) were 565 ,UM and 53 nmol mg-' h-1, respectively. The average value of the apparent inhibition constant, Ki, was 0.14 ,UM for the high affinity site (range: 0.05-0.25 ,UM).

best described by a two-enzyme-model. The apparent Kis for quinidine inhibition of the high affinity enzyme ranged from 9 to 92 nm (Table 2), and this confirms that the high affinity enzyme is CYP2D6 (Otton et al., 1984). The results of a recent in vitro study (Br0sen et al., 1991b) are in agreement with these assumptions. The clinical implications of the two-enzyme kinetics of 2-hydroxylation have been discussed by Sindrup et al.

(1990). Using a two-enzyme model, we confirmed that paroxetine and fluoxetine had Kis of less than 1 pLM (Table 3) (Br0sen et al., 1991a; Br0sen & Skjelbo, 1991; Crewe et al., 1992). Clinical studies have shown that the two antidepressants, which are selective serotonin reuptake inhibitors (SSRI), also are very potent inhibitors of CYP2D6 catalyzed drug oxidations in vivo and hence may cause serious drug-drug interactions when coadministered with a substrate of the enzyme, unless very low doses of the other drug is given (Bell & Cole, 1988; Br0sen et al., 1991a; Sindrup et al., 1992; Vaughan, 1988). Citalopram, another SSRI, was a relatively weak inhibitor of CYP2D6 (Table 3) and in accordance with this, citalopram is also a weak inhibitor in vivo (Gram, personal communication). Perphenazine is a potent inhibitor of 2-hydroxylation, but not of the N-demethylation of imipramine in vivo

260

E. Skjelbo & K. Br0sen

(Br0sen et al., 1986a; Gram, 1975). This finding was confirmed here in vitro (Table 3). Perphenazine is itself metabolized by CYP2D6 in vivo (Dahl-Puustinen et al., 1989). Zuclopenthixol is also metabolized in part by sparteine/debrisoquine oxidase (Dahl et al., 1991), and hence is a substrate for CYP2D6. Nevertheless, we were unable to show any inhibition of 2-hydroxylation (Table 3). The reason for this in vivolin vitro discrepancy is not clear. The S-mephenytoin hydroxylase is assumed to be one of a group of closely related isoenzymes in the 2C subfamily (Ged et al., 1988), but it is still not known which one of these is defective in mephenytoin poor metabolizers. In vivo co-segregation with the mephenytoin oxidation polymorphism may be considered indicative that a drug is metabolized by the S-mephenytoin hydroxylase. On the basis of an earlier panel study, we hypothesized that the N-demethylation of imipramine is partially catalyzed by the S-mephenytoin hydroxylase (Skjelbo et al., 1991), and we therefore expected that other putative substrates of this isoenzyme would inhibit the formation of desipramine by human liver microsomes. Apart from propranolol this was not the case (Table 3), and the role of CYP2C isoenzymes for the formation of desipramine was not confirmed by the present study. It is possible that the N-demethylation of imipramine is catalyzed by a P450 which is distinct from but functionally coupled to the S-mephenytoin hydroxylase. Among the 20 drugs screened, fluvoxamine was the only potent inhibitor of the N-demethylation of imipra-

mine as well as being a relatively weak inhibitor of the 2-hydroxylation. The best description of the inhibition kinetics of fluvoxamine on the N-demethylation was obtained using equation 3 (Figure 4). These data suggest that fluvoxamine is a potent, selective inhibitor of one of the P450-isoenzymes catalyzing the N-demethylation of imipramine. This P450 remains to be identified. It has previously been shown that the plasma concentrations of two other tertiary amine antidepressants, amitriptyline and clomipramine increase during fluvoxamine coadministration (Bertschy et al., 1991). Fluvoxamine is also a potent inhibitor of propranolol metabolism in vivo (Benfield & Ward, 1986), and in combination with the findings of the present study it may be hypothesized that propranolol metabolism and N-demethylation of imipramine are in part catalyzed by the same isoenzyme. In conclusion, the present study has confirmed the importance of CYP2D6 for the 2-hydroxylation of imipramine. Studying the formation of 2-OH-imipramine in human liver microsomes is therefore useful for the identification of inhibitors and possible substrates of this enzyme. The study has not clarified which P450s catalyze the N-demethylation of imipramine and further studies on this issue are needed. This study was supported by the Danish Medical Research Council, grant no. 12-9206 and 12-0282-1 and represents a contribution to the aims of the COST-B 1 action. The technical assistance of Miss Lone Lindal Hansen and Mrs Annelise Casa as well as the excellent secretarial assistance of Mrs Pia Poulsen and Mrs Bente Kuhlmann is appreciated.

References Andersson, T., Reg'ardh, C.-G., Dahl-Puustinen, M.-L. & Bertilsson, L. (1990). Slow omeprazole metabolizers are also poor S-mephenytoin hydroxylators. Ther. Drug. Monit., 12, 415-416. Bell, I. R. & Cole, J. 0. (1988). Fluoxetine induces elevation of desipramine level and exacerbation of geriatric nonpsychotic depression. J. clin. Psychopharmac., 8, 447-448. Benfield, P. & Ward, A. (1986). Fluvoxamine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in depressive illness. Drugs, 32, 313-334. Bertilsson, L. & Aberg-Wistedt, A. (1983). The debrisoquine hydroxylation test predicts steady-state plasma levels of desipramine. Br. J. clin. Pharmac., 15, 388-390. Bertilsson, L., Henthorn, T. K., Sanz, E., Tybring, G., Sawe, J. & Villen, T. (1989). Importance of genetic factors in the regulation of diazepam metabolism: Relationship to S-mephenytoin, but not debrisoquin, hydroxylation phenotype. Clin. Pharmac. Ther., 45, 348-355. Bertschy, G., Vandel, S., Vandel, B., Allers, G. & Volmat, R. (1991). Fluvoxamine-tricyclic antidepressant interaction. An accidental finding. Eur. J. clin. Pharmac., 40, 119-120. Br0sen, K., Gram, L. F., Klysner, R. & Bech, P. (1986a). Steady-state levels of imipramine and its metabolites: Significance of dose-dependent kinetics. Eur. J. clin. Pharmac., 30, 43-49. Br0sen, K., Otton, V. & Gram, L. F. (1986b). Imipramine demethylation and hydroxylation: Impact of the sparteine oxidation phenotype. Clin. Pharmac. Ther., 40, 543-549. Br0sen, K. & Gram, L. F. (1989). Clinical significance of the sparteine/debrisoquine oxidation polymorphism. Eur. J. clin. Pharmac., 36, 537-547.

Br0sen, K., Gram, L. F. & Kragh-S0rensen, P. (1991a). Extremely slow metabolism of amitriptyline but normal metabolism of imipramine and desipramine in an extensive metabolizer of sparteine, debrisoquine and mephenytoin. Ther. Drug. Monit., 13, 177-182. Br0sen, K. & Skjelbo, E. (1991). Fluoxetine and norfluoxetine are potent inhibitors of P450IID6-the source of the sparteine/debrisoquine oxidation polymorphism. Br. J. clin. Pharmac., 32, 136-137. Br0sen, K., Zeugin, T. & Meyer, U. A. (1991b). Role of P450IID6, the target of the sparteine/debrisoquine oxidation polymorphism, in the metabolism of imipramine. Clin. Pharmac. Ther., 49, 609-617. Cornish-Bowden, A. (1974). A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem. J., 137, 143-144. Crewe, H. K., Lennard, M. S., Tucker, G. T., Woods, F. R. & Haddock, R. E. (1992). The effect of selective serotonin re-uptake inhibitors on cytochrome P4502D6 (CYP2D6) activity in human liver microsomes. Br. J. clin. Pharmac., 34, 262-265. Dahl-Puustinen, M.-L., Liden, A., Alm, C., Nordin, C. & Bertilsson, L. (1989). Disposition of perphenazine is related to polymorphic debrisoquin hydroxylation in human beings. Clin. Pharmac. Ther., 46, 78-81. Dahl, M.-L., Ekquist, B., Widen, I. & Bertilsson, L. (1991). Disposition of the neurolephic zuclopenthixol cosegregates with the polymorphic debrisoquine hydroxylation in humans. Acta psychiatrica Scand., 84, 99-100. Dixon, M. (1953). The determination of enzyme inhibitor constants. Biochem. J., 55, 170-171. Eichelbaum, M., Spannbrucker, N., Steincke, B. & Dengler,

In vitro metabolism of imipramine H. J. (1979). Defective N-oxidation of sparteine in man: a new pharmacogenetic defect. Eur. J. clin. Pharmac., 16, 183-187. Eichelbaum, M. & Gross, A. S. (1990). The genetic polymorphism of debrisoquine/sparteine metabolism-clinical aspects. Pharmac. Ther., 46, 377-394. Ged, C., Umbenhauer, D. R., Bellew, T. M., Bork, R. W., Srivastava, P. K., Shrinriki, N., Lloyd, R. S. & Guengerich, F. P. (1988). Characterization of cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4'-hydroxylase. Biochemistry, 27, 6929-6940. Gram, L. F. (1974). Metabolism of tricyclic antidepressants. A review. Dan. Med. Bull., 21, 218-231. Gram, L. F. (1975). Effects of perphenazine on imipramine metabolism in man. Psychopharmac. Commun., 1, 165-175. Holford, N. (1990). MK Model, version 4 Biosoft. Cambridge, UK. Kupfer, A. & Preisig, R. (1984). Pharmacogenetics of mephenytoin: a new drug hydroxylation polymorphism in man. Eur. J. clin. Pharmac., 26, 753-759. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. biol. Chem., 193, 265-275. Mahgoub, A., Idle, J. R., Dring, L. G., Lancaster, R. & Smith, R. L. (1977). Polymorphic hydroxylation of debrisoquine in man. Lancet, ii, 584-586. Meier, P. J., Mueller, H. K., Dick, B. & Meyer, U. A. (1983). Hepatic monooxygenase activities in subjects with a genetic defect in drug oxidation. Gastroenterology, 85, 682-692. Nebert, D. W., Nelson, D. R., Coon, M. J., Estabrook, R. W., Feyereisen, R., Fujii-Kyriyama, Y., Gonzalez, F. J., Guengerich, F. P., Gunsalus, I. C., Johnson, E. F., Loper, J. C., Sato, R., Waterman, M. R. & Waxman, D. J. (1991). The P450 superfamily: Update on new sequences, gene mapping, and recommended nomenclature. DNA and Cell Biology, 10, 1-14.

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Otton, S. V., Inaba, T. & Kalow, W. (1984). Competitive inhibition of sparteine oxidation in human liver by P-adrenoceptor antagonists and other cardiovascular drugs. Life Sci., 34, 73-80. Sindrup, S. H., Br0sen, K. & Gram, L. F. (1990). Nonlinear kinetics of imipramine in low and medium plasma level ranges. Ther. Drug Monit., 12, 445-449. Sindrup, S. H., Br0sen, K., Gram, L. F., Hallas, J., Skjelbo, E., Allen, A., Allen, G. D., Cooper, S. M., Mellows, G., Tasker, T. C. G. & Zussmann, B. D. (1992). The relationship between paroxetine and the sparteine oxidation polymorphism. Clin. Pharmac. Ther., 51, 278-287. Skjelbo, E., Br0sen, K., Hallas, J. & Gram, L. F. (1991). The mephenytoin oxidation polymorphism is partially responsible for the N-demethylation of imipramine. Clin. Pharmac. Ther., 49, 18-23. Vaughan, D. A. (1988). Interaction of fluoxetine with tricyclic antidepressants. Am. J. Psychiat., 145, 1478. Ward, S. A., Walle, T., Walle, U. K., Wilkinson, G. R. & Branch, R. A. (1989). Propranolol's metabolism is determined by both mephenytoin and debrisoquin hydroxylase activities. Clin. Pharmac. Ther., 45, 72-79. Ward, S. A., Helsby, N. A., Skjelbo, E., Br0sen, K., Gram, L. F. & Breckenridge, A. M. (1991). The activation of the biguanide antimalarial proguanil co-segregates with the mephenytoin oxidation polymorphism-a panel study. Br. J. clin. Pharmac., 31, 689-692. Zanger, U. M., Vilbois, F., Hardwick, J. P. & Meyer, U. A. (1988). Absence of hepatic cytochrome P450bufl causes genetically deficient debrisoquine oxidation in man. Biochemistry, 27, 5447-5454. Zeugin, T. B., Br0sen, K. & Meyer, U. A. (1990). Determination of imipramine and seven of its metabolites in human liver microsomes by a high-performance liquid chromatographic method. Anal. Biochem., 189, 99-102.

(Received 25 November 1991, accepted 16 March 1992)

Inhibitors of imipramine metabolism by human liver microsomes.

1. The aromatic 2-hydroxylation of imipramine was studied in microsomes from three human livers. The kinetics were best described by a biphasic enzyme...
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