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Home , Bufetolol, CYP1A2, CYP2C8, Propranolol

Br J clin Pharmac 1995; 39: 421-431

Identification of human CYP isoforms involved in the metabolism of enantiomers- N-desisopropylation is mediated mainly by CYP1A2

K. YOSHIMOTO', H. ECHIZEN2, K. CHIBA', M. TANI3 & T. ISHIZAKI' 'Department of Clinical Pharmacology, Research Institute, International Medical Center of Japan, Tokyo, 2Department of Pharmacology, Kitasato University School of Medicine, Sagamihara and 3Division of General Surgery, International Medical Center of Japan, Tokyo, Japan

1 Studies using human microsomes and six recombinant human CYP isoforms (i.e. CYP1A2, 2A6, 2B6, 2D6, 2E1 and 3A4) were performed to identify the (CYP) isoform(s) involved in the ring 4-hydroxylation and side- chain N-desisopropylation of propranolol enantiomers in humans. 2 a-Naphthoflavone and 7-ethoxyresorufin (selective inhibitors of CYPlAl/2) inhibited the N-desisopropylation of R- and S-propranolol by human liver micro- somes by 20 and 40%, respectively, while (a selective inhibitor of CYP2D6) abolished the 4-hydroxylation of both propranolol enantiomers almost completely. In contrast, sulphaphenazole (CYP2C8/9 inhibitor), S- (CYP2C19 inhibitor), troleandomycin (CYP3A3/4 inhibitor) and diethyldithiocar- bamate (CYP2E1 inhibitor) elicited only weak inhibitory effects on propranolol metabolism via the two measured metabolic pathways. 3 Significant (P < 0.01) correlations were observed between the microsomal N-desisopropylation of both propranolol enantiomers and that for the O-deethyl- ation of among the 11 different human liver microsome samples (r = 0.98 and 0.77 for R- and S-propranolol, respectively). A marginally signifi- cant (r = 0.60, P L' 0.05) correlation was also observed between N-desisopropyl- ation of S-, but not of R-propranolol and the 4'-hydroxylation of S-mephenytoin. No significant correlations were observed between the N-desisopropylation of propranolol enantiomers and the 2-hydroxylation of , the hydroxyl- ation of or the 60-hydroxylation of testosterone. 4 Significant (P <0.01) correlations were observed between the microsomal 4-hydroxylation of R- and S-propranolol and the 2-hydroxylation of desipramine (r = 0.85 and 0.98, respectively). A weak (r = 0.66), albeit significant (P < 0.05), correlation was observed between the 4-hydroxylation of R-, but not of S-propranolol and the hydroxylation of tolbutamide. No significant correlations were observed between the 4-hydroxylation of propranolol enantiomers and the oxidation of other substrates for CYP1A2, 2C19, and 3A3/4. 5 Recombinant human CYP1A2 and CYP2D6 exhibited comparable catalytic activity with respect to the N-desisopropylation of both propranolol enantiomers; only expressed CYP2D6 exhibited a marked catalytic activity with respect to the 4-hydroxylation of both propranolol enantiomers. 6 We conclude that the side-chain N-desisopropylation of both propranolol enan- tiomers is mediated mainly by the CYPIA subfamily and to some extent by CYP2D6, whereas the ring 4-hydroxylation of the enantiomers is mediated almost

Correspondence: Dr Takashi Ishizaki, Department of Clinical Pharmacology, Research Institute, International Medical Center of Japan, 1-21-2 Toyama, Shinjuku-ku, Tokyo 162, Japan 421 422 K. Yoshimoto et al. exclusively by CYP2D6. The contribution of S-mephenytoin 4'-hydroxylase (CYP2C19) to the N-desisopropylation of propranolol enantiomers appears to be of negligible importance in human liver microsomes.

Keywords propranolol enantiomers N-desisopropylpropranolol 4-hydroxypropranolol cytochrome P450 hepatic metabolism CYP1A2 CYP2C19 CYP2D6

Introduction Methods Propranolol is metabolised extensively in human liver Chemicals and reagents via three major metabolic pathways (aromatic ring hydroxylation at the 4-position, side-chain N-desiso- R- and S-propranolol HCI, diethyldithiocarbamate, propylation and direct ) [1]. In vitro testosterone, desipramine HCI, phenacetin, pheno- [2] and in vivo studies [3] have suggested that aro- barbitone, troleandomycin, and tol- matic ring 4-hydroxylation of the drug cosegregates butamide were purchased from Sigma Chemical Co. with debrisoquine 4-hydroxylase (CYP2D6) activity, (St Louis, MO, USA). 60-Hydroxytestosterone was which exhibits a major genetic polymorphism in vari- obtained from Steraloids Inc. (Wilton, NH, USA). ous ethnic groups [4,5]. The side-chain oxidation of Acetonitrile, methanol and other reagents of analyti- propranolol has been suggested to cosegregate with cal grade were purchased from Wako Pure Chemical S-mephenytoin 4'-hydroxylase (CYP2C19) activity Industries, Ltd (Osaka, Japan). NADP, glucose-6- [6] which also exhibits a marked genetic polymor- phosphate, and glucose-6-phosphate dehydrogenase phism [7]. Thus, the mean in vivo partial metabolic were obtained from Oriental Yeast (Tokyo, Japan). clearance of propranolol to a-naphthoxylactic acid Microsomal preparations from six recombinant (NLA) in the poor metaboliser phenotype (PMs) for human CYP (i.e. CYP1A2, CYP2A6, S-mephenytoin 4'-hydroxylation was about half that CYP2B6, CYP2D6, CYP2E1 and CYP3A4) in extensive metabolisers (EMs) [6]. NLA is a major expressed in human B lymphoblastoid cell line AHH- urinary product of the side-chain oxidation pathway 1 were purchased from Gentest Corp. (Woburn, MA, in man [1]. A possible cosegregation of S-mepheny- USA). The following compounds were generous gifts toin 4'-hydroxylase with one of the major metabolic from the respective pharmaceutical companies: rac-4- pathways of propranolol would be especially relevant hydroxypropranolol HCl from ICI Pharm (Maccles- in Oriental populations, because they have a greater field, UK), rac- HCl from Yoshitomi frequency of PMs (12.6-22.5%) [8,9] than European Pharmaceutical Co. (Osaka, Japan), 2-hydroxyde- and North American Caucasian populations sipramine oxalate from Ciba-Geigy (Basel, Switzer- (2.7-4.2%) [4,7]. However, Walle et al. [10] reported land), propericiazine and nitrazepam from Shionogi that the mean partial metabolic clearance of propra- Pharmaceutical Co. (Osaka, Japan), hydroxy- nolol to NLA in smokers was about twice that in non- tolbutamide from Hoechst (Frankfurt, Germany) and smokers, suggesting that one of the cigarette from Ono Pharmaceutical Co. smoke-inducible hepatic (s) is also involved (Osaka). Rac-desisopropylpropranolol HCI was in the side-chain oxidation of propranolol. kindly donated by Dr Narimatu (University of Chiba, Until recently, the identification of CYP isoform(s) Chiba, Japan), and rac-mephenytoin, 4'-hydroxy- involved in particular metabolic pathways has been mephenytoin, and N-desmethylmephenytoin (i.e. done using human liver microsomes and relatively nirvanol) by Dr Kupfer (University of Bern, Bern, specific inhibitor and probes for the differ- Switzerland). S- and R-mephenytoin were separated ent CYP forms [11, 12]. Using recombinant DNA on a Chiralcel OJ column (10 gm, 4.6 x 250 mm, technology it is now possible to utilise cloned human Daicel Chemical Co. Ltd, Tokyo, Japan) according to CYP isoforms expressed in heterologous or ortholo- the method of Yasumori et al. [18]. gous cells to confirm the role of specific CYPs [13]. To our knowledge, except for an in vitro study of Preparation of human liver microsomes Rowland et al. [14] using yeast microsomes, this strategy has not been used for identifying human Human liver specimens were obtained from 11 CYP isoform(s) involved in the metabolism of pro- patients (aged 45 to 75 years, seven males and four pranolol. Because propranolol is used as a racemate females) who underwent partial hepatectomy for and the individual enantiomers differ in their pharma- metastatic liver tumour(s) in the Division of General cokinetics [6, 15] and P-adrenoceptor antagonist Surgery, International Medical Center of Japan. There activity [16, 17], we intended to identify the human was no evidence of chronic liver injury. Serological CYP isoform(s) involved in the side-chain oxidation tests for hepatitis B and C virus antibodies were neg- and ring 4-hydroxylation of both enantiomers using ative for all patients. Three patients were smokers: human liver microsomes and six different recombin- donors of HL-6, HL-26 and HL-29 smoked about 5, ant human CYP isoforms. 15 and 30 cigarettes per day, respectively. Liver Human CYP isoforms and propranolol metabolism 423 samples obtained from patients with ard genase. After incubation at 370 C in a shaking water those who were taking drugs known to induce or bath for 30 to 60 min, the reaction was terminated by inhibit hepatic activity were not adding 25 gl 2 N HC104 (for propranolol) or 100 gl included in the study. All prescribed drugs were dis- acetonitrile (for other substrates). Incubation times continued at least 48 h before surgery. In all patients used were: 20 min for phenacetin and testosterone, anaesthesia was achieved by a combination of nitrous 30 min for propranolol enantiomers, desipramine oxide, and pancuronium bromide. The liver and tolbutamide, and 45 min for S-mephenytoin. sample was fixed in formalin for histological Experiments were performed in duplicate on the same examination and surplus tissue was taken for the day using the same microsomal preparation for each present study under the supervision of a clinical substrate. pathologist. The tissues were frozen in liquid Incubation conditions used for microsomes from nitrogen within 5 min after excision and stored at B lymphoblastoid cells expressing the different -80°C until used. Subsequent examination con- recombinant human CYP isozymes were essentially firmed that all liver specimens used were histopatho- similar to those used for human liver microsomes logically normal. The use of human tissue samples except for the incubation time (120 min) and the for the study was approved by the Institutional Ethics quantity of microsomes used (equivalent to 0.125 mg Committee, International Medical Center of Japan. protein). Human liver microsomes were prepared by differ- ential centrifugation as described in detail elsewhere Inhibition study [12, 19]. The protein content of each microsomal preparation was determined by the method of Lowry The following selective inhibitors were used: a- et al. [20]. The microsomal samples were aliquoted, naphthoflavone and 7-ethoxyresorufin for CYPlA1/2 frozen and stored at -80° C until used. [22], sulphaphenazole for CYP2C8/9 [30], S-mephenytoin for CYP2C19 [25-27], quinidine for Incubation conditions CYP2D6 [11, 31], diethyldithiocarbamate for CYP2E1 [32], and troleandomycin for CYP3A [33]. The microsomal incubation conditions employed Each propranolol enantiomer (5 JM) was incubated were similar to those reported previously [12, 19, 21]. with and without one of the inhibitors at concentra- However, the quantity of microsomes used for the tions ranging from 0.001 to 1000 JM under the incu- incubation of each substrate differed by 8-fold bation conditions described above. The effects of (equivalent to 0.0125 to 0.1 mg protein) because of each compound on the formation of the 4-hydroxy differences in the catalytic activities of microsomes and N-desisopropyl metabolites from each propra- with respect to substrates. For instance, while incuba- nolol enantiomer (5 JM) at the respective inhibitor tion with propranolol enantiomers was performed concentrations were compared with the control values using microsomes equivalent to 0.0125 mg protein, determined with the incubation of propranolol alone those with desipramine, tolbutamide, phenacetin, and and expressed as the percentage of the respective testosterone used 0.025 mg protein and that with control values. The inhibitory potency of probe sub- S-mephenytoin used 0.1 mg protein. Substrate con- strates was defined by IC50 values. Diethyldithio- centrations were: 10 gM for phenacetin, desipramine and troleandomycin were not preincubated and tolbutamide, 100 gM for S-mephenytoin and with the NADPH-generation system before initiating 30 gM for testosterone. It has been shown that the propranolol metabolism. Experiments were performed catalytic activities of human liver microsomes with with microsomal preparations obtained from three to respect to phenacetin O-deethylase, tolbutamide four different human . Microsomal preparations hydroxylase, S-mephenytoin 4'-hydroxylase, desipra- used for the inhibition study were selected randomly. mine 2-hydroxylase, and testosterone 6p-hydroxylase at the substrate concentrations employed herein are Assay attributable to CYPlA1/2 [22], CYP2C8/9 [23,24], CYP2C19 [25-27], CYP2D6 [28] and CYP3A [29], Propranolol metabolites were determined by h.p.l.c. respectively. Because a previous report [2] and a with fluorescence detection as described by Otton et pilot study showed that the affinity constants for 4- al. [2] with minor modifications. To each reaction hydroxylation and N-desisopropylation of both pro- mixture, 50 gl internal standard solution (equiva- pranolol enantiomers ranged from 2 to 5 JM, we used lent to 50 Jg rac-bufetolol HCI) was added and a substrate concentration of 5 JM. In addition, we the mixture was centrifuged at 10,000 g for 5 min. determined the R/S ratio for mephenytoin 4'-hydr- The supernatant was passed through a 0.45 gm (pore oxylase activity at 1 mm for all of the microsomal size) filter membrane (Gelman Science, Tokyo, samples in order to assess their putative phenotypes Japan), and 100 gl of the filtrate was injected into the according to the criteria of Yasumori et al. [18] (i.e. h.p.l.c. system, consisting of a model L-6000 pump an R/S ratio < 0.3 designated as an EM and > 0.7 as a (Hitachi Ltd, Tokyo, Japan), a model 655A-20 PM). A previous study [21] has supported the validity autosample injector (Hitachi), a reversed-phase col- of the criteria. umn (Eicompak MA-ODS, 250 x 4.6 mm internal The enzyme reactions were initiated by adding 50 diameter, 5 gm particle size, Eicom, Kyoto, Japan) gl of an NADPH-generating system consisting of 20 and a model F-1050 fluorescence detector (Hitachi) mM glucose-6-phosphate, 5 mM NADP, 40 mM MgCl2 set at excitation and emission wavelengths of 230 and and 10 units ml-' of glucose-6-phosphate dehydro- 380 nm, respectively. Column temperature was main- 424 K. Yoshimoto et al. tained at 350 C by a model CTC-100 water circulator Metabolite formation (Eicom). Metabolites of other substrates were determined by The appearance rates of both propranolol metabolites h.p.l.c. and u.v. absorbance methods reported else- were linear (r > 0.95, P < 0.01) over 60 min. Dupli- where [21, 24, 34] or developed in our laboratory cate results did not differ by more than 10%. The (unpublished data). The internal standards used were: mean R/S ratio for the 4-hydroxylation pathway was phenobarbitone for O-deethylphenacetin, properi- 1.24 ± 0.41 and the corresponding value for the ciazine for 2-hydroxydesipramine, chlorpropamide N-desisopropylation pathway was 1.88 ± 0.32. In the for hydroxytolbutamide, phenobarbitone for 4'- absence of the NADPH-generation system no appre- hydroxymephenytoin and nitrazepam for 6i-hydr- ciable formation of 4-hydroxy or N-desisopropyl oxytestosterone. The h.p.l.c. column used was a products was observed. In addition, the incubation of CAPCELL PAK C18 AG 120 (250 x 4.6 mm internal rac-4-hydroxypropranolol (0.2 nM) with human liver diameter, Shiseido Co. Ltd, Tokyo). microsomes in the presence or absence of the The mobile phases used to assay the metabolites of NADPH-generation system for 60 min resulted in no propranolol, desipramine and S-mephenytoin were appreciable loss of the metabolite. However, when 20/80, 24/76 and 26/74 (v/v) mixtures of acetonitrile rac-N-desisopropylpropranolol (0.05 nM) was incu- and 0.05 M K2PO3 buffer (pH-4.0), respectively. An bated with human liver microsomes, an appreciable amine modifier, triethylamine (1% (v/v)), was added loss with a mean first order rate constant of 0.047 to the mobile phase for assay of propranolol metabo- min-' (n = 3) was observed regardless of the presence lites. A 60/40 mixture of methanol and 0.05 M K2P03 or absence of the NADPH-generation system. In con- buffer (pH 3.4) was used for the 6f-hydroxytestos- trast, no appreciable loss was observed, when rac-N- terone assay. The mobile phase was delivered at 0.7 desisopropylpropranolol (0.05 nM) was incubated to 1.0 ml min-'. All chromatograms were recorded with recombinant human CYP1A2 in the presence or with a model D-2500 Chromato-Integrator (Hitachi). absence of the NADPH-generation system. The mean R/S ratio for the N-desisopropylation of propranolol by recombinant human CYP1A2 was 1.18. Data analysis The metabolite formation rates of other CYP substrates employed in the present study were Data are expressed as mean ± s.d. unless stated other- also linear (r > 0.95, P < 0.01) over the respective wise. Correlations between metabolite formation rates incubation periods. The mean recoveries of para- of the respective CYP isoform-selective substrates cetamol, 2-hydroxydesipramine, hydroxytolbutamide, and of propranolol enantiomers were determined by 4'-hydroxymephenytoin and 6,3-hydroxytestosterone least-squares linear regression. Comparisons between and their respective internal standards from micro- the 4-hydroxylation and N-desisopropylation of somal incubation mixtures were >95%, with co- propranolol enantiomers by microsomes from the efficients of variation of < 5%. putative EMs and PMs of the S-mephenytoin 4'- hydroxylation were made using Student's t-test for Inhibition study unpaired data. A P value of < 0.05 was considered statistically significant. The effects of co-incubating CYP-selective inhibi- tors or substrates on the N-desisopropylation of propranolol enantiomers are shown in Figure 1. a-Naphthoflavone and 7-ethoxyresorufin were potent inhibitors of the N-desisopropylation of both pro- pranolol enantiomers. The mean IC50 values for Results a-naphthoflavone were 0.04 and 0.06 gM for R- and S-propranolol, and those for 7-ethyoxyresorufin were Assays 0.63 and 4.5 gM, respectively. The mean maximum inhibition produced by a-naphthoflavone on the side- No chromatographic interference with the determina- chain oxidation of R- and S-propranolol was 81 and tion of the two propranolol metabolites was observed 59%, respectively. Corresponding values for 7- in the presence or absence of the putative inhibitors. ethoxyresorufin were 60 and 65%, respectively. Otton et al. [2] and others [35, 36] reported In contrast, other inhibitors or substrates had only a chromatographic separation of 4- and 5-hydroxypro- weak inhibitory effect on the N-desisopropylation of pranolol using similar conditions to those employed both propranolol enantiomers. None produced in the present study. The minimum determinable con- inhibitory effects in excess of 50%. centrations of the 4-hydroxy and N-desisopropyl Figure 2 shows the results of inhibition studies metabolites of propranolol were 80 and 40 pmol ml-, with respect to 4-hydroxylation. Quinidine inhibited respectively, with a signal to noise ratio of 3. The the 4-hydroxylation of both propranolol enantiomers coefficients of variation for the assay of these almost completely. Mean IC50 and maximum metabolites were < 5% across the concentration inhibitory effects for R- and S-propranolol were 0.11 ranges studied. The mean (± s.d.) recoveries of both and 0.09 gM and 93 and 99%, respectively. Other propranolol metabolites and the internal standard compounds produced only weak inhibition. The mean from incubation mixtures were >95% with coeffi- maximum inhibition elicited by 7-ethoxyresorufin cients of variation of < 6%. (10 gM) for R- and S-propranolol was 61 and 66%, Human CYP isoforms and propranolol metabolism 425

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Figure 1 Effects of CYP-selective inhibitors on the side-chain oxidation of propranolol enantiomers (5 gM) by human liver microsomes. Data are the mean values of experiments performed with microsomes from three to four different livers. * R-propranolol, S-propranolol. and that elicited by troleandomycin (1000 g1M) was P c 0.05) with the 4'-hydroxylation of S-mephenytoin 43 and 42%, respectively. (Figure 3b). The y intercept of the regression line was significantly (P < 0.01) different from zero Correlation study (95% confidence interval was 0.006 to 0.009) and accounted for 76% of the mean S-propranolol There were significant (P < 0.01) correlations N-desisopropylase activity. between phenacetin O-deethylation and the N-desiso- Three human livers provided R/S ratios of > 0.7 propylation of both propranolol enantiomers (r = 0.98 with respect to microsomal S-mephenytoin 4'- and 0.77 for R- and S-propranolol, respectively) hydroxylation and eight an R/S ratio of < 0.3. The (Figure 3a and Table 1). While the y intercepts for former were considered to be from PMs. Although the regression lines for R- and S-propranolol were the mean S-mephenytoin 4'-hydroxylase activity in significantly different from zero (95% confidence microsomes from the putative EMs (0.055 ± 0.032 intervals were 0.002 to 0.063 and 0.005 to 0.017, nmol min-' mg-' protein) was about 10 times respectively), they accounted for only 20 and 44% of (P < 0.01) greater than that obtained from the overall mean N-desisopropylase activity for R- and putative PMs (0.006 ± 0.001 nmol min- mg- S-propranolol, respectively. protein) (Figure 4b), there were no significant The N-desisopropylation of S- but not that of interphenotypic differences in N-desisopropylase R-propranolol was correlated marginally (r = 0.60, activities for both propranolol enantiomers between 426 K. Yoshimoto et al.

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Table 1 Correlations between the catalytic activity of human liver microsomes with respect to the ring 4-hydroxylation and side-chain N-desisopropylation of propranolol enan- tiomers and metabolic reactions of five selective substrates of distinct human CYP isoforms Propranolol Metabolic reaction CYP 4-Hydroxylation N-Desisopropylation ofsubstrate isoform R S R S Phenacetin O-deethylation CYPlAl/2 0.46 0.10 0.98** 0.77** Desipramine 2-hydroxylation CYP2D6 0.85** 0.98** 0.45 0.44 Tolbutamide hydroxylation CYP2C8/9 0.66* 0.46 0.51 0.45 S-Mephenytoin 4'-hydroxylation CYP2Cl9 0.27 0.29 0.43 0.60* Testosterone 6,B-hydroxylation CYP3A 0.54 0.36 0.48 0.30 *P <0.05, **P

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U. I 0.2 04 0.6 08 1 0 0.02 0.04 0.06 0.08 01 0.12 0.14 Phenacetin O-deethylase activity S-Mephenytoin 4'-hydroxylase activity (nmol min-1 mg-1 protein) (nmol min-1 mg-1 protein) Figure 3 Relationship between phenacetin O-deethylase activity (10 gM) and propranolol N-desisopropylase activity (5 tM) (a), and that between S-mephenytoin 4'-hydroxylase activity (100 gM) and propranolol N-desisopropylase activity (5 gM) (b) in microsomes from 11 human livers. * R-propranolol, S-propranolol.

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L o S-MP Figure 4 Mean (± s.d.) N-desisopropylase activity with respect to propranolol (PL) enantiomers (a) and S-mephenytoin (S-MP) 4'-hydroxylase activity (b) in liver microsomes from putative extensive metaboliser (EM, open bars, n = 8) and poor metaboliser (PM, hatched bars, n = 3) phenotypes for S-mephenytoin 4'-hydroxylation (designated according to the in vitro criteria of Yasumori et al. [18]). NS = no significant difference between the corresponding columns; * = significant (P < 0.05) difference between the corresponding columns. the two groups. The mean activities for R-propranolol hydroxylation, and testosterone 6f-hydroxylation obtained from PMs and EMs were 0.033 ± 0.005 and between the two groups (data not shown). No signifi- 0.039 ± 0.015 nmol min-1 mg-1 protein, and those for cant correlation was observed between N-desiso- S-propranolol were 0.016 ± 0.002 and 0.021 ± 0.007 propylase activity for each propranolol enantiomer nmol min-1 mg-' protein, respectively. No significant and the activities of CYP2D6 (desipramine), differences were observed in phenacetin O-deethyl- CYP2C8/9 (tolbutamide) or CYP3A (testosterone) ation, desipramine 2-hydroxylation, tolbutamide (Table 1). 428 K. Yoshimoto et al.

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ND o"d. ..: *__. t srtI DNi I r."LIW-- lI- -r `_ .:-- A.1- rn-- 2 2 6- 2E -34 1A2 2A 296 6i ^2EI 3A4 Figure 5 The mean (± s.d.) N-desisopropylase (a) and ring 4-hydroxylase (b) activities with respect to propranolol enantiomers in microsomes from recombinant human CYPs expressed in human B lymphoblastoid cells. * R-propranolol, Fl S-propranolol. ND = not detected.

There were significant (P < 0.05 or 0.01) correla- family in humans (Figures 1, 3 and 5, Table 1). Thus, tions between desipramine 2-hydroxylation and the the two selective inhibitors of CYPlA activity, a- 4-hydroxylation of R- and S-propranolol (r = 0.85 or naphthoflavone and 7-ethoxyresorufin [22], were 0.98, respectively). The y intercepts of the regression potent inhibitors of the N-desisopropylation of both lines for R- and S-propranolol (0.029 and 0.001, propranolol enantiomers (Figure 1). While CYP1A2 respectively) did not differ significantly from zero; is constitutionally expressed in the liver, only 50% of the respective 95% confidence intervals were -0.026 human livers appear to have the mRNA for CYPlAl to 0.086 and -0.010 to 0.012. There was a weak (r = and the level of expression of CYPlAl is substan- 0.66), albeit significant (P <0.05), correlation tially lower than that of CYP1A2 [37]. This indicates between the 4-hydroxylation of R- but not that of that CYP1A2 rather than CYPlAl is mainly respon- S-propranolol and tolbutamide hydroxylation. No sible for the hepatic N-desisopropylation of propra- significant correlations were observed between the nolol enantiomers. Tassaneeyakul et al. [22] showed 4-hydroxylation of either propranolol enantiomer and that a-naphthoflavone was a 10 times more potent activities for each of the remaining three probe sub- inhibitor than 7-ethoxyresorufin of recombinant strates for distinct CYP isoforms (Table 1). human CYP1A2, whereas both inhibitors had comparable potency with respect to recombinant Recombinant CYP isoform study CYPlAl. Our finding that a-naphthoflavone was an order of magnitude more potent inhibitor than 7- Figure 5 shows the catalytic activities of the recombi- ethoxyresorufin of the side-chain oxidation of propra- nant human CYP isoforms with respect to the 4- nolol enantiomers (Figure 1) also supports the hydroxylation and N-desisopropylation of propranolol contention that metabolism by CYP1A2 rather than enantiomers (5 ,UM). Only CYP1A2 and CYP2D6 CYPlAl is the more important pathway. In addition, showed substantial N-desisopropylation of both pro- the strong correlation was observed between pranolol enantiomers. Although recombinant phenacetin O-deethylase activity (CYP1A2) and CYP1A2 showed some 4-hydroxylase activity for R- and S-propranolol N-desisopropylase activity both propranolol enantiomers, it accounted for only (Table 1 and Figure 3). However, the y intercepts of 8.1 and 3.5% of the corresponding CYP2D6 activi- the regression lines indicated that about 20 and 44% ties, respectively. The other CYP isoforms and con- of the total N-desisopropylase activity resides in CYP trol microsomes exhibited no appreciable propranolol isoform(s), other than the CYPlA subfamily. Poor 4-hydroxylase activity. correlations between microsomal N-desisopropylase activities and those for desipramine 2-hydroxylase, tolbutamide hydroxylase and testosterone 6p-hyroxy- lase (Table 1) exclude the possibilities that this Discussion enzyme might be CYP2D6, 2C8/9 or 3A, respec- tively. The results provide strong in vitro evidence that the Our finding that microsomal S-propranolol N- side-chain N-desisopropylation of both propranolol desisopropylase activity showed a weak correlation enantiomers is catalysed mainly by the CYPIA sub- with S-mephenytoin 4'-hydroxylase activity (Figure Human CYP isoforms and propranolol metabolism 429

3b) is compatible with the in vivo data of Ward et al. addition, recent studies [43-45] have shown that [6] suggesting a role for the latter enzyme in the side- recombinant human CYP2D6 expressed in different chain oxidation of propranolol [6,38]. However, the vectors (lymphoblastoid cells and yeast) has N- high y intercept of the regression line indicated that dealkylating activity with respect to deprenyl and contribution of S-mephenytoin 4'-hydroxylase to this . Thus, further studies are required to pathway is small. Moreover, as shown in Figure 4a, resolve the role of CYP2D6 in the side-chain data obtained with microsomal sanples from the oxidation of propranolol. Our in vitro data indicated putative EMs and PMs of S-mephenytoin disclosed that other recombinant human CYP isoforms (2A6, no significant interphenotypic differences in mean 2B6, 2E1 and 3A4) had no measurable N-desiso- microsomal N-desisopropylase activities. We also propylation activity for propranolol enantiomers observed that S-mephenytoin did not inhibit the side- (Figure 5). chain oxidation of either propranolol enantiomer The mean R/S ratio of N-desisopropylpropranolol (Figure 2), in agreement with Otton et al. [2]. Thus, appearance observed in the present study (i.e. 1.9) we suggest that although CYP2C19 may contribute to was almost identical to that reported by Otton et al. some extent to the N-desisopropylation of S-propra- [2] and von Bahr et al. [46] (i.e. 1.8 and 1.9, respec- nolol, this contribution is minor, at least in human tively), whereas the corresponding value reported by liver microsomes. However, two further metabolic Nelson & Shetty [47] was 0.91. Otton et al. [2] sug- reactions, oxidative deamination of N-desisopropyl- gested that the preferential appearance of the N- propranolol to an aldehyde intermediate and its sub- desisopropyl metabolite of R-propranolol may be sequent oxidation to NLA are also involved in the associated not only with its enantioselective forma- side-chain metabolism of propranolol [1, 2]. While tion but also with its further metabolism. Indeed, it the former reaction was suggested to be mediated by has been suggested that the metabolism of N-desiso- monoamine oxidase (MAO) [39], the enzyme(s) propylpropranolol to its aldehyde intermediate may involved in the latter reaction remain unknown. Thus, occur enantioselectively in rat, dog and human liver there is a possibility that CYP2C19 activity might be microsomes [47,48]. This pathway may be mediated involved in this reaction, thereby explaining the by MAO in humans, because the reaction was observation that PMs for S-mephenytoin 4'-hydroxyl- inhibited by the MAO inhibitor, (1 gM), in ation excreted less NLA than EMs [6]. human liver microsomes [2]. Our finding that the R/S In accordance with the data obtained with human ratio for N-desisopropylation of propranolol by liver microsomes, that obtained with recombinant recombinant human CYP1A2 (i.e. 1.18) was much human CYP1A2 indicated substantial N-desisopro- less than that observed with human liver microsomes pylase activity with respect to both propranolol enan- would also support the above explanation. We also tiomers (Figure 5). However, the observation that observed that the recombinant CYP1A2 used had no recombinant CYP2D6 also exhibited significant measurable catalytic activity with respect to the N-desisopropylase activity for both propranolol further metabolism of N-desisopropylpropranolol (see enantiomers was at variance with those made in Results). Thus, we assume that the apparent enantio- studies using human liver microsomes. Thus, quini- selectivity in the N-desisopropylation of propranolol dine, a selective CYP2D6 inhibitor [31], caused no with human liver microsomes may be exaggerated to substantial inhibition of propranolol N-desisopro- some extent by further enantioselective metabolism pylase activity at concentrations associated with of as yet uncharacterized N-desisopropylated metabo- marked inhibition of 4'-hydroxypropranolol produc- lite(s) by MAO. tion (Figure 2). In addition, no significant correlation The ring 4-hydroxylation of both propranolol enan- was observed between microsomal N-desisopropyla- tiomers by human liver microsomes was inhibited tion of propranolol enantiomers and CYP2D6-medi- almost completely by the CYP2D6-selective ated desipramine 2-hydroxylation. Our findings with inhibitor, quinidine, with an IC50 value of about human liver microsomes support those of Otton et al. 0.1 gM (Figure 2). In addition, there were highly [2] who reported that the side-chain oxidation of pro- significant (P <0.01) correlations between desi- pranolol by human liver microsomes did not proceed pramine 2-hydroxylation, known to be mediated by when cumene hydroperoxide was used as the oxidant CYP2D6 [28], and the 4-hydroxylation of R- and S- instead of NADPH and molecular oxygen. This oxi- propranolol (Table 1). These findings are consistent dant has been proposed to sustain CYP2D6 activity with those of previous in vitro [2] and in vivo [3] preferentially [40]. Furthermore, Raghuram et al. [41] studies, indicating that the ring 4-hydroxylation of showed that the mean plasma AUC of propranolol propranolol is mediated by CYP2D6. However, we glycol, a product of side-chain oxidation [1, 2], was cannot offer any explanation for the observation that similar in PMs and EMs of debrisoquine. On the tolbutamide hydroxylase activity correlated weakly other hand, Anthony et al. [42] reported that debriso- with R-propranolol 4-hydroxylase activity (Table 1). quine decreased the metabolic clearance of propra- Nonetheless, the observation that sulphaphenazole, a nolol by 35% not only via inhibition of ring selective inhibitor of tolbutamide hydroxylase 4-hydroxylation but also via the side-chain oxidation, (CYP8/9) [30], elicited only a weak inhibitory effect as measured by the urinary of NLA. As on propranolol 4-hydroxylase activity (Figure 2) suggested by Otton et al. [2], the decreased portion of suggests that the correlation between the two the metabolic clearance of propranolol mediated via catalytic activities could be a chance finding. the side-chain oxidation by debrisoquine could reflect Although recombinant human CYP1A2 exhibited the MAO inhibition activity of debrisoquine. In some activity for this pathway, it was an order of 430 K. Yoshimoto et al. magnitude less than the corresponding activity medi- pranolol. The use of recombinant human CYP ated by CYP2D6 (Figure 5b). isoforms confirmed the results obtained from in vitro In conclusion, our findings indicate that the side- [2] and in vivo studies [1,3,41,42], indicating that chain oxidation of both propranolol enantiomers is the ring 4-hydroxylation of both propranolol mediated mainly by CYP1A2, whereas their ring enantiomers is mediated mainly by CYP2D6. hydroxylation is mediated almost exclusively by CYP2D6. Assuming that the results of these in vitro This study was supported by a grant-in-aid from the Japan studies can be extrapolated to in vivo drug disposi- Health Science Foundation (1-7-1-C), Drug Innovation Sci- tion, the findings cast doubt on any significant or ence Project (1-2-10) and the Ministry of Human Health dominant contribution of S-mephenytoin 4'-hydroxyl- and Welfare, Tokyo, Japan. ase (CYP2C19) to the side-chain oxidation of pro-

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