throbber
0090-9556/98/2604-0289–293$02.00/0
`DRUG METABOLISM AND DISPOSITION
`Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
`
`Vol. 26, No. 4
`Printed in U.S.A.
`
`TOLTERODINE, A NEW MUSCARINIC RECEPTOR ANTAGONIST, IS METABOLIZED BY
`CYTOCHROMES P450 2D6 AND 3A IN HUMAN LIVER MICROSOMES
`
`HANS POSTLIND, ÅSA DANIELSON, ANDERS LINDGREN, AND STIG H. G. ANDERSSON
`
`Department of Drug Metabolism, Pharmacia & Upjohn AB
`
`(Received February 11, 1997; accepted January 9, 1998)
`
`This paper is available online at http://www.dmd.org
`
`ABSTRACT:
`
`Tolterodine, a new muscarinic receptor antagonist, is metabolized
`via two pathways: oxidation of the 5-methyl group and dealkylation
`of the nitrogen. In an attempt to identify the specific cytochrome
`P450 enzymes involved in the metabolic pathway, tolterodine was
`incubated with microsomes from 10 different human liver samples
`where various cytochrome P450 activities had been rank ordered.
`Strong correlation was found between the formation of the 5-hy-
`droxymethyl metabolite of tolterodine (5-HM) and CYP2D6 activity
`(r2, 0.87), as well as between the formation of N-dealkylated
`tolterodine and CYP3A activity (r2, 0.97). When tolterodine was
`incubated with human liver microsomes in the presence of com-
`pounds known to interact with different P450 isoforms, quinidine
`was found to be the strongest inhibitor of the formation of 5-HM.
`
`Ketoconazole and troleandomycin were found to be the strongest
`inhibitors of the formation of N-dealkylated tolterodine. A weak
`inhibitory effect on the formation of N-dealkylated tolterodine was
`found with sulfaphenazole, whereas tranylcypromine did not inhibit
`the formation of this metabolite. Microsomes from cells overex-
`pressing CYP2D6 formed 5-HM, whereas N-dealkylated tolterod-
`ine was formed by microsomes expressing CYP2C9, -2C19, and
`-3A4. The Km for formation of N-dealkylated tolterodine by CYP3A4
`was similar to that obtained in human liver microsomes and higher
`for CYP2C9 and -2C19. We conclude from these studies that the
`formation of 5-HM is catalyzed by CYP2D6 and that the formation
`of N-dealkylated tolterodine is predominantly catalyzed by CYP3A
`isoenzymes in human liver microsomes.
`
`[(R)-N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-
`Tolterodine
`phenylpropanamine] is a new muscarinic receptor antagonist specif-
`ically developed for the treatment of urinary urge incontinence and
`other symptoms associated with overactive bladder (Nilvebrant et al.,
`1997). Following oral administration, tolterodine is rapidly absorbed
`from the gastrointestinal tract and exhibits extensive first-pass metab-
`olism. Metabolites are formed via two pathways: oxidation of the
`5-methyl group to a 5-hydroxymethyl derivative (5-HM1) (PNU-
`200577; labcode DD 01) and dealkylation of the nitrogen (fig. 1). In
`humans, about 80% of an administered oral dose of tolterodine is
`excreted in the urine, the main metabolites being the 5-carboxylic
`acids of tolterodine, N-dealkylated tolterodine, and their glucuronide
`conjugates. Less than 1% of the parent compound is excreted un-
`changed (Brynne et al., 1997).
`In a previous study in healthy volunteers, one subject showed
`notably lower systemic clearance than the overall average. This dis-
`similarity was probably due to differences in metabolic capacity
`(Brynne et al., 1997). However, the specific P450 enzymes involved
`in the metabolism of tolterodine have not been identified. Such
`knowledge is of great importance to predict potential drug interactions
`and genetic variations in drug metabolism. In this study, we per-
`formed experiments in which the formation of metabolites of toltero-
`dine was correlated with marker P450 activities in human liver mi-
`
`FIG. 1.Main metabolic pathways of tolterodine in human liver microsomes.
`
`crosomes. We also used inhibitors and isoenzymes expressed using
`recombinant
`technology to determine the individual enzymes in-
`volved in the metabolism of tolterodine.
`
`1 Abbreviations used are: P450, cytochrome P450; 5-HM, 5-hydroxymethyl
`metabolite of tolterodine; HPLC, high pressure liquid chromatography; Cli, intrin-
`sic clearance (Vmax/Km); V, rate of metabolite formation.
`
`Send reprint requests to: Dr. Hans Postlind, Department of Drug Metabolism,
`Pharmacia & Upjohn AB, S-751 82 Uppsala, Sweden.
`
`Materials and Methods
`Chemicals. [14C]Tolterodine 4.2 and 0.85 MBq/mg, 5-HM [(R)-N,N-diiso-
`propyl-3-(2-hydroxy-5-hydroxymethylphenyl)-phenylpropanamine], N-deal-
`kylated tolterodine, and N-dealkylated 5-HM were synthesized at Pharmacia &
`Upjohn AB (Uppsala, Sweden). b-NADPH was obtained from Merck KGaA
`(Darmstadt, Germany), whereas a-naphthoflavone, quinidine, sulfaphenazole,
`289
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`290
`
`POSTLIND ET AL.
`
`tranylcypromine, and troleandomycin were obtained from Sigma. Ketocon-
`azole was kindly provided by the Janssen Research Foundation (Beerse,
`Belgium). All other chemicals were of high purity and were obtained from
`usual commercial sources.
`Human Liver Microsomes. A HepatoScreen test kit with 10 different
`human liver microsomal samples was obtained from Human Biologics, Inc.
`(Phoenix, AZ) and used in correlation experiments. Four of the samples were
`from males. The microsomes had been characterized with respect to the
`following enzyme activities: 7-ethoxyresorufin O-dealkylation (CYP1A), caf-
`feine 3-demethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), tol-
`butamide methyl-hydroxylation (CYP2C9), S-mephenytoin 4-hydroxylation
`(CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone
`6-hydroxylation (CYP2E1), testosterone 6b-hydroxylation (CYP3A), lauric
`acid 11-hydroxylation (CYP2E1) and lauric acid 12-hydroxylation (CYP4A),
`benzphetamine N-demethylation (unknown), and total P450 content. Pooled
`human liver microsomes were obtained from XenoTech LLC (Kansas City,
`KS) and used in enzyme kinetic and inhibition experiments.
`Overexpressed P450 Isoenzymes. Microsomes containing expressed P450
`isoforms from human lymphoblast cells (CYP1A1 and -1A2) or insect cells
`(CYP2C8, -2C9-Arg, -2C19, -2D6, and -3A4) and nontransfected cells were
`purchased from Gentest (Woburn, MA).
`Incubation Conditions. All incubations with liver microsomes were car-
`ried out at a protein concentration corresponding to 1 mg/ml in 100 mM
`potassium phosphate buffer (pH 7.4) and 1 mM b-NADPH at 37°C. In the
`experiments measuring the enzyme kinetics, 5–200 mM tolterodine was incu-
`bated in a final volume of 250 ml for 15 min. In correlation experiments, the
`HepatoScreen test kit was incubated at 150 mM concentration in a final volume
`of 1 ml for 30 min. Inhibition experiments were carried out at tolterodine
`concentrations equal to the apparent Km for formation of 5-HM and N-
`dealkylated tolterodine (7 and 50 mM, respectively) in a final volume of 1 ml
`for 15 min. The different inhibitors were dissolved in methanol and then added
`in 10 ml (1% v/v final concentration) to the microsomes (10 ml of methanol
`was used in control experiments). a-Naphthoflavone, sulfaphenazole, tranyl-
`cypromine, and troleandomycin were added at a final concentration of 1, 10,
`and 50 mM, quinidine at 0.1, 1, and 10 mM (5-HM) or at 1, 10, and 50 mM
`(N-dealkylated tolterodine), and ketoconazole at 0.01, 0.1, and 1 mM. Incuba-
`tions containing troleandomycin were preincubated with microsomes and 1
`mM b-NADPH for 15 min before addition of tolterodine. All other substances
`tested for inhibition were added just before tolterodine.
`Microsomes containing expressed P450 isoforms from human lymphoblast
`cells or insect cells (20 pmol, respectively) were incubated with 10 mM
`tolterodine, 100 mM potassium phosphate buffer (pH 7.4) and 1 mM
`b-NADPH in a final volume of 250 ml at 37°C for 20 min. Microsomes from
`nontransfected cells were used as controls. In enzyme kinetic experiments,
`incubations were performed as described above with 10 –200 mM tolterodine.
`Under the conditions used, the formation of tolterodine metabolites was
`linear with respect to incubation time and protein concentrations.
`The reactions were terminated by addition of acetone (1:1 v/v) and stored at
`220°C. Before analysis, the microsomal protein was precipitated by centrif-
`ugation at 3200 rpm, and the acetone in the collected supernatant evaporated
`with a stream of nitrogen. A 100 –200-ml aliquot of the remaining supernatant
`was used for HPLC analysis.
`HPLC Analysis. The incubations were analyzed for the parent drug and its
`metabolites by HPLC using two LKB 2150 pumps, an LKB 2152 LC control-
`ler, Pharmacia UV-M monitor set at 280 nm, Beckman 171 radioisotope
`detector, a Supelco PKB 100 (2 cm) precolumn, and a Supelco PKB 100
`(150 3 4.5 mm) column. The mobile phase was 20 mM ammonium acetate in
`methanol (pH 4.5). The solvent flow rate was 1 ml/min, and a gradient of
`decreasing polarity [time (min)/% methanol: 0/10, 5/20, 35/45, 40/100, 50/
`100) was used.
`Calculations. The amount of each metabolite was calculated from the
`radiochromatogram as the (% area of the metabolite)/(% area of all metabolites
`1 parent compound), and the results are expressed as in relation to milligram
`of microsomal protein or picomole of P450 per minute. The retention times of
`formed metabolites were compared with the retention times of synthesized
`reference standards, and their identity was further confirmed by electrospray
`ionization mass spectrometry. The enzymatic constants were calculated using
`
`FIG. 2.A representative radiochromatogram from incubation of tolterodine with
`human liver microsomes.
`
`nonlinear regression and correlation coefficients using linear regression and
`GraphPad Prism software.
`
`Results
`Metabolite Identification. Human liver microsomes converted
`[14C]tolterodine into several products in the presence of b-NADPH.
`Fig. 2 shows a representative radiochromatogram containing four
`major peaks at retention times of 11.5, 14, 23, and 26 min. The
`identity of the metabolites was confirmed by comparison with the
`retention times and product-ion mass spectra, obtained by collision-
`induced dissociation of protonated molecular ions ([M1H]1), of
`synthesized reference standards (data not shown). N-Dealkylated
`5-HM (retention time, 11.5 min) had a [M1H]1 ion at m/z 300, 5-HM
`(retention time, 14 min) at m/z 342, and N-dealkylated tolterodine
`(retention time, 23 min) at m/z 284. The peak at 26 min corresponded
`to intact [14C]tolterodine.
`Enzyme Kinetics. Pooled human liver microsomes were used to
`calculate the apparent Km, Vmax, and Cli values, which are shown in
`table 1. Fig. 3 shows Eadie-Hofstee plots for the formation of 5-HM
`and N-dealkylated tolterodine. Plots for both metabolites were linear,
`indicating the involvement of single P450 isoforms in the reactions.
`Correlation Experiment. Tolterodine was incubated with ten dif-
`ferent human liver microsomal samples, where different P450 activ-
`ities had been rank ordered (HepatoScreen test kit). The rate of
`formation of 5-HM and N-dealkylated tolterodine (fig. 1) varied
`approximately 6- and 7-fold, respectively, among the samples (fig. 4).
`Correlations between the rate of formation of tolterodine metabolites
`and different P450 marker activities are shown in table 2. The for-
`mation of 5-HM correlated strongly with dextromethorphan O-dem-
`ethylation (r2, 0.87), a marker for CYP2D6 activity (Schmid et al.,
`1985), whereas no correlation was obtained for other P450 activities.
`Testosterone 6b-hydroxylation, a marker for CYP3A activity (Wax-
`man et al., 1988), showed a strong correlation with the formation of
`N-dealkylated tolterodine (r2, 0.97). A relatively strong correlation
`was also obtained toward total P450 content and the formation of this
`metabolite (r2, 0.76).
`Chemical Inhibition Experiments. The effect of various sub-
`stances, at different concentrations, on the formation of metabolites in
`pooled human liver samples is shown in fig. 5. a-Naphthoflavone, an
`inhibitor of CYP1A isoenzymes (Guengerich, 1992; Tassaneeyakul et
`
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`TOLTERODINE METABOLISM BY HUMAN LIVER MICROSOMES
`
`291
`
`Enzyme kinetics for the formation of the 5-hydroxymethyl and N-dealkylated
`metabolites of tolterodine in pooled human liver microsomes
`
`Correlation between the rate of formation of tolterodine metabolites and total
`P450 content and different P450 marker activities in human liver samples
`
`TABLE 1
`
`TABLE 2
`
`Parameter
`
`Metabolite
`
`5-HM
`
`N-Dealkylated Tolterodine
`
`Vmax (pmol/mg protein 3 min)
`Km (mM)
`CIi (ml/min 3 mg protein)
`
`317
`7
`47
`
`687
`52
`13
`
`Metabolism
`
`P450 Isoenzyme
`
`Total P450 content
`7-Ethoxyresorufin O-dealkylation
`Caffeine 3-demethylation
`Coumarin 7-hydroxylation
`Tolbutamide methyl-hydroxylation
`S-Mephenytoin 4-hydroxylation
`Dextromethorphan O-demethylation
`Chlorzoxazone 6-hydroxylation
`Testosterone 6b-hydroxylation
`Lauric acid 11-hydroxylation
`Lauric acid 12-hydroxylation
`Benzphetamine N-demethylation
`
`1A
`1A2
`2A6
`2C9
`2C19
`2D6
`2E1
`3A
`2E1
`4A
`Unknown
`
`Correlation (r2)
`
`5-HM
`
`N-Dealkylated
`Tolterodine
`
`—a
`—a
`—a
`—a
`—a
`—a
`0.87b
`—a
`—a
`—a
`—a
`—a
`
`0.76c
`—a
`—a
`—a
`0.64d
`—a
`—a
`0.22
`0.97b
`0.29
`0.43
`0.27
`
`a p . 0.2.
`b p , 0.0001.
`c p , 0.001.
`d p , 0.01.
`
`FIG. 3.Eadie-Hofstee plots for the formation of the 5-HM and N-dealkylated
`tolterodine in human liver microsomes ([S], substrate concentration).
`
`FIG. 4.Rate of formation of the 5-HM and N-dealkylated tolterodine in human
`liver microsomes from different individuals.
`
`al., 1993), did not significantly inhibit the metabolism of tolterodine
`at concentrations used in this study. Quinidine, which is regarded as
`a specific inhibitor of CYP2D6 (Inaba et al., 1985), almost completely
`inhibited the formation of 5-HM at 10 mM. High concentrations of
`quinidine also produced slight inhibition of the formation of N-
`dealkylated tolterodine. The strongest inhibition of the formation of
`N-dealkylated tolterodine was observed with ketoconazole, which
`inhibited formation of this metabolite by .70% at a concentration of
`1 mM but did not affect the formation of 5-HM. A strong inhibition
`was also observed for the formation of N-dealkylated tolterodine with
`troleandomycin, which is known to specifically interact with CYP3A
`isoenzymes (Guengerich and Shimada, 1991). Troleandomycin had
`
`FIG. 5.Effect of various cytochrome P450 inhibitors on the rate of formation of
`the 5-HM (A) and N-dealkylated tolterodine (B) at a 7 and 50 mM concentration
`of tolterodine, respectively, in human liver microsomes.
`
`no effect on the formation of 5-HM. Sulfaphenazole, a competitive
`inhibitor of CYP2C9 (Baldwin et al., 1995), was found to have a weak
`inhibitory effect on the formation of N-dealkylated tolterodine at a
`concentration of 50 mM (,30%), whereas tranylcypromine, an inhib-
`
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`292
`
`POSTLIND ET AL.
`
`TABLE 3
`
`TABLE 4
`
`Metabolism of tolterodine by microsomes from cells overexpressing P450
`isoenzymes
`
`Enzyme kinetics for the formation of N-dealkylated tolterodine in microsomes
`from cells overexpressing P450 2C9-Arg, -2C19, and -3A4
`
`P450 Isoenzyme
`
`1A1
`1A2
`2C8
`2C9-Arg
`2C19
`2D6
`3A4
`
`Rate of Formation (pmol/pmol P450 3 min)
`
`5-HM
`
`ND
`ND
`ND
`ND
`ND
`5.0 6 0.35
`ND
`
`N-Dealkylated Tolterodine
`
`ND
`ND
`ND
`0.25 6 0.05
`1.51 6 0.06
`ND
`0.23 6 0.03
`
`Data are mean 6 SD of two experiments. ND, not detectable.
`
`itor of CYP2C19 (Inaba et al., 1985; Wienkers et al., 1996), did not
`affect the formation of this metabolite at concentrations used in this
`study.
`Metabolism by Overexpressed P450 Isoenzymes. Microsomes
`from cells overexpressing CYP1A1, -1A2, -2C8, -2C9-Arg, -2C19,
`-2D6, or -3A4, together with microsomes from nontransfected cells,
`were incubated with 10 mM tolterodine in the presence of b-NADPH.
`Microsomes from cells overexpressing CYP2D6 catalyzed the forma-
`tion of 5-HM, whereas N-dealkylated tolterodine was formed in
`microsomes overexpressing CYP2C9-Arg, -2C19, and -3A4 (table 3).
`The enzyme kinetic constants for the formation of N-dealkylated
`tolterodine by microsomes are shown in table 4. No metabolites were
`formed in incubations with microsomes from nontransfected cells
`(data not shown).
`
`Discussion
`The results presented in this study provide evidence for the involve-
`ment of CYP2D6 in the formation of 5-HM and CYP3A as the major
`enzyme involved in the formation of N-dealkylated tolterodine. The
`formation of 5-HM is dependent on CYP2D6, based on a high
`correlation with dextromethorphan O-demethylation, inhibition by
`quinidine, and formation of the metabolite only in cell microsomes
`overexpressing CYP2D6. The formation of N-dealkylated tolterodine
`correlated strongly with testosterone 6b-hydroxylation. A relatively
`strong correlation was also obtained toward total P450 content. This
`is in accordance with immunochemical and inhibition studies indicat-
`ing that as much as 60% of the total P450 content in human liver may
`be CYP3A isoenzymes (Guengerich, 1990).
`According to the results from the HepatoScreen test kit, the in-
`volvement of CYP2C9 in the formation of N-dealkylated tolterodine
`could be substantial. This was, however, not confirmed by experi-
`ments with sulfaphenazole, an inhibitor described as specific for this
`P450 isoenzyme (Baldwin et al., 1995). Only high concentrations (50
`mM) showed a weak inhibitory effect (,30%) on the formation of
`N-dealkylated tolterodine. Furthermore, the activities of CYP2C9 and
`-3A in the test kit were found to correlate with each other (r2, 0.60).
`Tranylcypromine, which has been described as an inhibitor of S-
`mephenytoin 4-hydroxylation (CYP2C19) (Inaba et al., 1985; Wien-
`kers et al., 1996), did not inhibit the formation of N-dealkylated
`tolterodine at concentrations used in this study. Ketoconazole and
`troleandomycin, both known to interact with CYP3A (Guengerich and
`Shimada, 1991; Maurice et al., 1992), were found to be the strongest
`inhibitors of the formation of N-dealkylated tolterodine. A marginal
`inhibitory effect on the formation of this metabolite was also observed
`at high concentrations of quinidine. This is probably not an effect on
`CYP2D6 but rather a result of nonspecific inhibition of CYP3A, as
`quinidine is a known substrate for this P450 isoenzyme (Guengerich
`et al., 1986). The formation of N-dealkylated tolterodine was detected
`
`Parameter
`
`Vmax (pmol/pmol P450 3 min)
`Km (mM)
`CIi (ml/min 3 pmol P450)
`
`P450 Isoenzyme
`
`2C9-Arg
`
`1.86
`74
`0.025
`
`2C19
`
`12.3
`70
`0.176
`
`3A4
`
`1.48
`47
`0.031
`
`in microsomes overexpressing not only CYP3A4 but also CYP2C9
`and -2C19. The Km for formation of N-dealkylated tolterodine with
`CYP3A4 was 47 mM, similar to that obtained in human liver micro-
`somes (52 mM), whereas Km values for CYP2C9 and -2C19 was about
`70 mM. Estimates of the relative Cli values per picomole of P450
`using these overexpressed enzymes indicated that formation of N-
`dealkylated tolterodine was about 6- and 7-fold higher for CYP2C19,
`respectively, compared with CYP3A4 and -2C9. However, human
`liver does not contain equimolar concentrations of the different P450
`isoforms, and the levels of b-NADPH-P450 reductase may vary
`considerably among different cDNA-expressed preparations and also
`differ from that found in human liver. Consequently, enzyme turnover
`numbers may vary substantially not only between the different prep-
`arations but also in comparison to human liver. Thus, there is no firm
`basis for extrapolation of relative Cli values obtained with cDNA-
`expressed enzymes to human liver. CYP3A4 had the lowest Km
`compared with CYP2C9 and -2C19. As CYP3A4 is also the major
`P450 isoenzyme expressed in human liver, it is reasonable to assume
`that the Vmax in human liver microsomes is higher for CYP3A4 than
`for CYP2C9 and -2C19. If this is the case, then the contribution of
`lower Vmax, higher Km enzymes, i.e. CYP2C9 and 2C19, will be
`completely obscured. Taken together, the results of the present study
`indicate that the formation of N-dealkylated tolterodine is predomi-
`nantly catalyzed by CYP3A4 in human liver microsomes. Estimates
`of the Cli values from data obtained in pooled human liver micro-
`somes also showed good agreement with the results of a study in
`healthy volunteers that reported that about 80% of tolterodine is
`predominantly metabolized via formation of 5-HM (Brynne et al.,
`1997).
`Clinical studies have demonstrated that individuals with reduced
`CYP2D6-mediated metabolism represent a high-risk group in the
`population with a propensity to develop adverse drug effects (Smith,
`1986). The number of drugs identified as being affected by CYP2D6
`polymorphism has increased steadily over the years and includes
`diverse classes such as b-adrenoreceptor antagonists, tricyclic antide-
`pressants, neuroleptics, and other miscellaneous drugs like dextro-
`methorphan and codeine (Daly et al., 1993; Murray, 1992). CYP3A is
`the major P450 subfamily in human liver and is involved in the
`metabolism of .50% of pharmaceutical drugs on the market. In
`addition, CYP3A enzymes have been reported to be involved in
`interactions with several drugs such as macrolides, ketoconazole,
`cyclosporin, and others (Honig et al., 1993; Periti et al., 1992; Pichard
`et al., 1990; Wrighton and Stevens, 1992). The possibility of clinical
`drug interaction at the enzyme level thus exists, especially if toltero-
`dine is administered at the same time as a compound that is prefer-
`entially metabolized by CYP2D6 or to individuals associated with the
`CYP2D6 poor metabolizer phenotype. However, the large amount of
`CYP3A in the liver and the fact that tolterodine is predominantly
`eliminated via oxidation by CYP2D6 makes it less likely that clini-
`cally significant drug-drug interactions would occur with CYP3A
`substrates in individuals with the CYP2D6 extensive metabolizer
`phenotype.
`
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`
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`
`Petitioner Torrent Pharmaceuticals Limited - Exhibit 1010 - Page 5
`
`

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