0090-9556/98/2606-0528–535$02.00/0
`DRUG METABOLISM AND DISPOSITION
`Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
`
`Vol. 26, No. 6
`Printed in U.S.A.
`
`BIOTRANSFORMATION OF TOLTERODINE, A NEW MUSCARINIC RECEPTOR
`ANTAGONIST, IN MICE, RATS, AND DOGS
`
`STIG H. G. ANDERSSON, ANDERS LINDGREN, AND HANS POSTLIND
`
`Department of Drug Metabolism, Pharmacia & Upjohn AB
`
`(Received May 14, 1997; accepted February 3, 1998)
`
`This paper is available online at http://www.dmd.org
`
`ABSTRACT:
`
`Tolterodine is a new muscarinic receptor antagonist intended for
`the treatment of urinary urge incontinence and other symptoms
`associated with an overactive bladder. The in vivo metabolism of
`14C-labeled tolterodine was investigated in rats, mice, and dogs by
`analysis of blood and urine samples, whereas in vitro metabolism
`studies were performed by incubation of [14C]tolterodine with
`mouse, rat, dog, and human liver microsomes in the presence of
`NADPH. Tolterodine was extensively metabolized in vivo. Mice and
`dogs showed similar metabolite patterns, which correlated well
`with that observed in humans. In these species, tolterodine was
`metabolized along two different pathways, with the more impor-
`tant being the stepwise oxidation of the 5-methyl group to yield the
`5-hydroxymethyl metabolite of tolterodine and then, via the alde-
`hyde, the 5-carboxylic acid metabolite. The other pathway involved
`dealkylation of the nitrogen. In the subsequent phase II metabo-
`lism, tolterodine and the metabolites were conjugated with glucu-
`
`ronic acid to various degrees. Rats exhibited more extensive me-
`tabolism and a markedly different metabolite pattern, with
`metabolites also being formed by hydroxylation of the unsubsti-
`tuted benzene ring. In addition, a gender difference was observed,
`with male rats showing more extensive metabolism than females.
`Incubation of [14C]tolterodine with liver microsomes yielded a total
`of five metabolites with rat liver microsomes and three with mouse,
`dog, and human liver microsomes. The 5-hydroxymethyl metabo-
`lite of tolterodine and N-dealkylated tolterodine were major me-
`tabolites in all incubations, representing 83–99% of total metabo-
`lism. Although the extent of metabolism varied among species, the
`metabolic profiles were similar. However, rat liver microsomes
`also formed metabolites hydroxylated in the unsubstituted ben-
`zene ring. These results show that the metabolism of tolterodine in
`mice and dogs corresponds to that observed in humans, whereas
`rats exhibit a different metabolite pattern.
`
`[(R)-N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-
`Tolterodine
`phenylpropanamine] is a new muscarinic receptor antagonist that was
`specifically developed for the treatment of urinary urge incontinence
`and other symptoms associated with an overactive bladder. Treatment
`of an overactive bladder is primarily based on the use of muscarinic
`receptor antagonists, e.g. propantheline, emepronium, and oxybutynin
`(Andersson, 1988; Wein et al., 1994), and oxybutynin is currently
`considered to be the drug of choice for the treatment of such symp-
`toms (Yarker et al., 1995). Although the efficacy of oxybutynin has
`been well demonstrated, the occurrence of classic antimuscarinic
`adverse events (e.g. dry mouth) often leads to discontinuation of
`treatment (Cardozo et al., 1987). Tolterodine is characterized by
`favorable tissue selectivity for the urinary bladder over salivary glands
`(Nilvebrant et al., 1997).
`The pharmacokinetic profile of tolterodine after oral administration
`to humans is characterized by rapid absorption and a terminal half-life
`of 2–3 hr. The excretion of drug-related substances in urine and feces
`was 77 and 17% of the administered dose, respectively (Brynne et al.,
`1997). After oral administration of 14C-labeled tolterodine to mice and
`dogs, approximately equal amounts of radioactivity were recovered in
`the urine and feces, whereas rats excreted 80% of the administered
`radioactivity in the feces. The terminal half-life of tolterodine was
`approximately 2 hr in these species (Kankaanranta and Påhlman,
`
`1997). In the present study, the metabolism of [14C]tolterodine was
`investigated by characterization of the biotransformation products
`formed in vivo by mice, rats, and dogs and in vitro by liver micro-
`somes from these species and humans.
`
`Materials and Methods
`
`Chemicals. [14C]Tolterodine (labeled at the benzylic methyl group) (fig. 1),
`tolterodine (PNU-200583), 5-HM1 [(R)-N,N-diisopropyl-3-(2-hydroxy-5-hy-
`droxymethylphenyl)phenylpropanamine, PNU-200577,
`labcode DD 01],
`5-CM (PNU-200579), and N-dealkylated tolterodine (PNU-200578) were syn-
`thesized at Pharmacia & Upjohn AB (Uppsala, Sweden). b-Glucuronidase
`(Escherichia coli, product no. 127051) was obtained from Boehringer Mann-
`heim (Mannheim, Germany) and arylsulfatase (Aerobacter aerogenes, product
`no. S1629) from Sigma Chemical Co. (St. Louis, MO). All other chemicals
`were of reagent grade and were obtained from usual commercial sources.
`In Vivo Experiments. Dogs. Six beagle dogs (three male and three female)
`were each administered an oral dose of 1.5 mg/kg [14C]tolterodine (4.2
`MBq/mg) and an iv dose of 1.0 mg/kg [14C]tolterodine. Blood samples were
`collected at 1 hr after the oral dose and at 20 min after the iv dose. Serum was
`prepared by allowing the blood to coagulate for 30 min, followed by centrif-
`ugation at 1200g for 10 min. Urine was collected cumulatively at 0 – 8 hr and
`8 –24 hr after administration of the oral and iv doses.
`Mice. Male and female mice (CD-1 strain) were each administered a single
`oral dose of 4 or 40 mg/kg [14C]tolterodine (4.2 MBq/mg). Blood from the
`orbital plexus was collected into heparinized tubes 15 min after drug admin-
`
`Send reprint requests to: Dr. Stig Andersson, Department of Drug Metabo-
`lism, Pharmacia & Upjohn AB, S-751 82 Uppsala, Sweden.
`
`1 Abbreviations used are: 5-HM, 5-hydroxymethyl metabolite of tolterodine;
`5-CM, 5-carboxylic acid metabolite of tolterodine.
`528
`
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`TOLTERODINE METABOLISM IN MICE, RATS, AND DOGS
`
`529
`
`FIG. 1.Chemical structure of tolterodine.
`*, position of the radiolabel.
`
`istration. Samples from five mice in each group were pooled and centrifuged
`to prepare plasma, which was immediately stored at 220°C until analysis.
`Urine was collected in separate experiments in which mice were adminis-
`tered a single oral dose of 4 mg/kg [14C]tolterodine (0.6 MBq/mg). Animals
`were subsequently placed in individual metabolism cages and urine was
`collected at 0 – 6 hr and 6 –24 hr, in containers surrounded by solid carbon
`dioxide.
`Rats. Four Sprague-Dawley rats (two male and two female) were placed in
`individual metabolism cages and administered an oral dose of 50 mg/kg
`[14C]tolterodine (0.2 MBq/mg). Urine was collected at 0 –12 hr and 12–24 hr
`after dosing. All urine samples were stored at 220°C until analysis.
`In Vivo Sample Preparation. Urine. Dog urine was centrifuged and
`analyzed directly. Urine from mice was centrifuged, and metabolites were
`extracted by solid-phase extraction using a Supelco Visoprep SPE vacuum
`manifold and Isolute C18(EC), 1-g, 6-ml cartridges that had been sequentially
`conditioned with methanol and 20 mM ammonium acetate (pH 4.5). Urine (3
`ml) was applied, and the cartridges were washed with 2 ml of 20 mM
`ammonium acetate (pH 4.5). Metabolites were eluted with 2 ml of methanol/20
`mM ammonium acetate (pH 4.5) (80:20, v/v). The eluate was evaporated to
`dryness and then diluted in 30 ml of methanol and 270 ml of 20 mM
`ammonium acetate (pH 4.5). The extraction yield was 98 –100%.
`Hydrolysis of conjugated metabolites in rat urine was performed by incu-
`bation of urine samples (450 ml) with 450 ml of 0.1 M ammonium acetate (pH
`5.5) and either 20 ml of b-glucuronidase or 30 ml of arylsulfatase, for 16 hr at
`37°C. The hydrolyzed metabolites were extracted with Sep-Pak PLUS C8,
`125-Å, solid-phase cartridges, which were conditioned before use by the
`sequential passing of 5 ml of methanol and 5 ml of 20 mM ammonium acetate
`(pH 4.5) through the cartridges. Urine was applied, the cartridges were washed
`with 4 ml of 0.1 M ammonium acetate (pH 5.5)/methanol (95:5, v/v), and
`metabolites were eluted with 3 ml of methanol/0.1 M ammonium acetate (pH
`5.5) (50:50, v/v). The extract was evaporated to dryness and dissolved in 400
`ml of 20 mM ammonium acetate (pH 4.5) before analysis.
`Plasma and Serum. The plasma and serum samples were treated with
`acetone (2 times the sample volume) and centrifuged to precipitate proteins.
`The supernatants were transferred to new vials, and the pellets were washed
`twice with aliquots of acetone/20 mM ammonium acetate (pH 4.5) (1:1, v/v).
`The combined supernatant and pellet extract was evaporated to dryness and
`dissolved in 20 mM ammonium acetate (pH 4.5) containing 10% methanol.
`The extraction yield was 101 6 11% (mean 6 SD).
`In Vitro Experiments. Preparation of Microsomes from Mouse, Rat, and
`Dog Liver. Livers from untreated male mice (CD-1 strain), male Sprague-
`Dawley rats, and male Beagle dogs were used. The microsomal fraction was
`prepared from a 20% (w/v) liver homogenate in 0.25 M sucrose containing 1
`mM EDTA and 10 mM Tris-HCl buffer (pH 7.4). The homogenate was
`centrifuged at 20,000g for 20 min, and the resulting supernatant was then
`centrifuged at 100,000g for 60 min. The microsomal pellet was suspended in
`one half the original volume of 0.1 M potassium pyrophosphate buffer (pH 7.4)
`with 1 mM EDTA, homogenized, and centrifuged at 100,000g for 60 min. The
`resulting microsomal pellet was subsequently suspended and homogenized in
`100 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol and 0.1
`mM EDTA, to a total volume of 0.5–1 ml/g of liver. Microsomes were
`immediately stored at 270°C. During the preparation procedure, the temper-
`ature was maintained as close to 14°C as possible.
`Protein Determination. The protein content of microsomal fractions was
`determined as described by Lowry et al. (1951), using bovine serum albumin
`as the standard. The measured protein concentrations in the liver microsomes
`were as follows: mouse, 3.2 mg/ml; rat, 7.3 mg/ml; dog, 39.9 mg/ml.
`
`Human Liver Microsomes. Human liver microsomes were obtained from
`Human Biologics, Inc. (Phoenix, AZ). Microsomes were prepared from five
`frozen liver samples. Approximately equal amounts of microsomal protein
`from the samples were pooled, and the protein concentration was determined
`to be 20 mg/ml, as described by Lowry et al. (1951). Microsomes were stored
`at 270°C until use in the incubation experiments.
`Incubations. For each species, incubations were performed in triplicate. The
`incubation mixtures contained 100 mM potassium phosphate buffer (pH 7.4),
`1 mM NADPH, 50 mg of [14C]tolterodine, and 1 mg of liver microsomal
`protein, in a final volume of 1 ml. The reaction was started by the addition of
`25 ml of an aqueous solution of [14C]tolterodine, to yield a final concentration
`of 154 mM. The incubations were performed at 37°C for 60 min and were
`terminated by the addition of 1 ml of acetone. Control incubations in which
`NADPH was omitted were performed as described above. The samples were
`stored at 220°C for at least 1 hr before analysis. Before analysis, microsomal
`protein was precipitated by centrifugation at room temperature, and the acetone
`was evaporated with a stream of nitrogen at 37°C. A 200-ml aliquot of the
`remaining supernatant from each incubation was analyzed.
`Analysis. HPLC Analysis. Quantification of the in vitro incubation products
`was performed by HPLC using two LKB 2150 pumps, an LKB 2152 LC
`controller, a Beckman 171 radioisotope detector, a Beckman 110B solvent-
`diluting module, 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 (pH 4.5) in methanol. The solvent flow rate was 1 ml/min, and a
`gradient of decreasing polarity (0 min, 10% methanol; 5 min, 20% methanol;
`35 min, 45% methanol; 40 min, 100% methanol; 50 min, 100% methanol) was
`used.
`MS. Metabolic profiles and mass spectra of the metabolites in the in vivo
`samples from mice and dogs and from in vitro incubations were obtained by
`analysis by HPLC coupled to electrospray-ionization MS. The system con-
`sisted of an autosampler and a quaternary pump (HP1050; Hewlett-Packard)
`connected to a Supelco PKB 100 (2-cm) precolumn and a Supelco PKB 100
`(150- 3 4.5-mm) column. A stream splitter diverted approximately 80% of the
`flow to a UV detector (Pharmacia UV-M monitor, set at 280 nm) coupled to
`a radioactivity detector (Packard Radiomatic A525) and 20% to a triple-stage
`quadrupole mass spectrometer equipped with an electrospray-ionization inter-
`face (TSQ 700; Finnigan MAT). The solvent flow rate was set to 1 ml/min, and
`the mobile phase and gradient described above were used. Aliquots of 100 ml
`were analyzed. The sheath gas was set to 80 psi, and the electrospray voltage
`and capillary temperature were 4.5 kV and 200°C, respectively.
`Rat urine, hydrolyzed by b-glucuronidase, was fractionated by the HPLC
`method used for the in vitro samples. Collected fractions were evaporated and
`fractionated using a second HPLC system with a Zorbax SB-CN guard column
`(12.5 3 4 mm) and a Zorbax SB-CN column (150 3 4.6 mm). The gradient
`was as follows: 0 min, 10% methanol; 20 min, 50% methanol; 25 min, 100%
`methanol.
`Metabolites in the HPLC fractions of rat urine were analyzed by GC/MS, as
`trimethylsilyl derivatives, after treatment with 50 ml of N,O-bis(trimethylsi-
`lyl)trifluoroacetamide overnight. Mass spectra of the trimethylsilyl-derivatized
`metabolites were obtained using an HP5890A gas chromatograph connected to
`a TSQ 70 mass spectrometer with electron impact ionization at 70 eV. The gas
`chromatograph was equipped with a on-column injector and a DB-1 capillary
`column (15 m 3 0.32 mm; film thickness, 0.25 mm). One-microliter aliquots
`of the derivatization mixture were injected, and the column temperature was
`maintained at 110°C for 1 min after injection and then increased to 290°C in
`7 min. The mass spectrometer was scanned between 35 and 700 amu, with a
`cycle time of 0.7 sec.
`
`Results
`Dog Urine. The general appearance of the radiochromatograms,
`with respect to the relative concentrations of the metabolites, did not
`reveal any obvious intraindividual or gender differences. However,
`the total amount of radioactivity recovered in the urine samples did
`vary. Fig. 2a shows a typical radiochromatogram of urine collected at
`0 – 8 hr after a 1.5 mg/kg oral dose. The chromatogram contained
`major metabolite peaks at retention times of 15–16 min and 18 –20
`min, and minor peaks were evident at 11–13 min and 28 –30 min.
`
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`530
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`ANDERSSON ET AL.
`
`FIG. 2.Radiochromatograms of urine collected at 0 – 8 hr ( a) and serum
`collected at 1 hr (b) after oral administration of [14C]tolterodine (1.5 mg/kg) to
`a female dog.
`
`Electrospray-ionization MS of metabolites showed prominent peaks
`corresponding to the protonated molecular ion ([M1H]1) for each
`metabolite and was useful in distinguishing the metabolites in the
`unresolved peaks at 15–16 min and 28 –30 min. 5-HM, 5-CM, and
`N-dealkylated tolterodine metabolites were identified by comparing
`the retention times and product-ion mass spectra (obtained by colli-
`sion-induced dissociation of the [M1H]1 ions) with those of the
`corresponding reference standards (figs. 3–5). The 14C-labeled frag-
`ment ions of the metabolites appeared 2 amu larger than the nonla-
`beled fragment ions of the reference standards. The identities of most
`of the other metabolites were deduced from the fragmentation patterns
`of the product-ion mass spectra. Thus, the product-ion mass spectra of
`glucuronide conjugates contained peaks showing the loss of the gluc-
`uronide moiety (2176 amu) and intense peaks identical to those of the
`product-ion mass spectra of the corresponding aglycon. Several key
`fragments in the mass spectra of the N-dealkylated metabolites were
`identical to those of metabolites that were not dealkylated (table 1).
`The proposed molecular structures of identified metabolites are sum-
`marized in fig. 6. The most abundant peak (19.7 min) in the urinary
`metabolic profile of dog urine represented 5-CM, with its correspond-
`ing glucuronide conjugate (18.7 min). The other major peaks, at
`15–16 min, were not completely resolved and contained two metab-
`olites, 5-HM and N-dealkylated 5-CM. These major metabolites con-
`stituted approximately 70 – 80% of the total radioactivity in the urine
`samples. The peak at 12.4 min was N-dealkylated 5-HM. The unre-
`solved peaks at approximately 29 min contained intact tolterodine and
`its corresponding glucuronide conjugate. Trace amounts of 5-HM
`glucuronide and N-dealkylated tolterodine were also observed in some
`of the samples.
`The metabolite pattern in urine from dogs that had received an iv
`dose of 1 mg/kg was similar to that observed after the 1.5 mg/kg oral
`dose. However, the concentration of intact tolterodine in urine was
`much higher after iv administration.
`Dog Serum. The metabolite pattern observed in serum collected 1
`hr after oral administration of 1.5 mg/kg contained three major peaks,
`at retention times of approximately 16, 20, and 29 min (fig. 2b). The
`peak at 16 min represented 5-HM and N-dealkylated 5-CM, whereas
`the peak at 20 min represented 5-CM. These metabolites constituted
`
`FIG. 3.Product-ion mass spectra of 5-HM (a) and the corresponding synthesized
`reference standard (b).
`
`30 – 60% of the total radioactivity in serum samples. The most abun-
`dant peak (24 – 65% of total radioactivity), at a retention time of 29
`min, corresponded to intact tolterodine. The 1 mg/kg iv dose yielded
`a metabolite pattern that exhibited a higher relative concentration of
`intact tolterodine but was otherwise similar to that observed after oral
`administration.
`Mouse Urine. The major metabolites found in dog urine were also
`observed in mouse urine (fig. 7a), although the relative concentrations
`of N-dealkylated and conjugated metabolites were higher in mouse
`urine. The radiochromatogram of mouse urine collected at 0 – 6 hr
`after oral administration of 4 mg/kg [14C]tolterodine contained major
`peaks at retention times of 15–20 min, i.e. 5-CM and the correspond-
`ing glucuronide at 20 and 18 min, respectively, N-dealkylated 5-CM
`and 5-HM at 16 min, and N-dealkylated 5-HM glucuronide and a
`small amount of N-dealkylated 5-CM glucuronide at 15 min. The
`abundance of the [M1H]1 ion for N-dealkylated 5-CM glucuronide at
`490 amu was too low to allow a product-ion mass spectrum to be
`obtained (table 1).
`Mouse Plasma. The radiochromatograms for plasma samples col-
`lected from mice given the 4 mg/kg dose contained two major peaks
`(fig. 7b). The peaks with a retention time of 15–17 min contained
`three different metabolites, i.e. 5-HM, N-dealkylated 5-HM glucuro-
`
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`TOLTERODINE METABOLISM IN MICE, RATS, AND DOGS
`
`531
`
`FIG. 4.Product-ion mass spectra of 5-CM (a) and the corresponding synthesized
`reference standard (b).
`
`nide, and N-dealkylated 5-CM. The other major peak corresponded to
`5-CM glucuronide. Minor peaks identified represented N-dealkylated
`tolterodine and the corresponding glucuronide and intact tolterodine,
`at retention times of 27, 29.5, and 30.5 min, respectively. After an oral
`dose of 40 mg/kg, the relative concentrations of the metabolites in the
`radiochromatograms were clearly different, in comparison with the
`metabolic profile after the lower dose. The concentrations of intact
`tolterodine and N-dealkylated tolterodine, together with its glucuro-
`nide conjugate, were increased more than proportional to dose, to-
`gether representing 11% of the radioactivity in plasma at the lower
`dose and 45% at the higher dose.
`Rat Urine. The metabolic profile in urine collected at 0 –12 hr from
`rats that had received an oral dose of 50 mg/kg differed from that for
`dogs and mice. In addition to small amounts of metabolites formed by
`dealkylation and oxidation of the 5-methyl group, rat urine contained
`several more-polar metabolites with short retention times (fig. 8).
`Urine was treated with either b-glucuronidase or arylsulfatase; only
`treatment with the former affected the appearance of the radiochro-
`matogram. After hydrolysis of glucuronic acid conjugates, the metab-
`olites were tentatively identified from retention times and mass spec-
`tra obtained by using GC/MS with trimethylsilyl derivatization.
`Trimethylsilyl derivatives were formed with the hydroxy and deal-
`kylated amino groups. The mass spectra contained diagnostic frag-
`
`FIG. 5.Product-ion mass spectra of N-dealkylated tolterodine (a) and the
`corresponding synthesized reference standard (b).
`
`ments of the nitrogen moiety and of the diphenyl cation from cleavage
`of the aliphatic side chain, which facilitated the determination of
`N-dealkylation and hydroxylation in the diphenyl moiety. These rat-
`specific metabolites were most likely formed by mono- and dihy-
`droxylation of the unsubstituted benzene ring. Three of the metabo-
`lites also contained a methoxy group. However, it was not possible to
`determine the exact sites of hydroxylation from these data. The key
`MS fragments and tentatively assigned metabolite structures are sum-
`marized in table 2. A gender difference was also observed, with urine
`from male rats containing relatively higher concentrations of these
`metabolites.
`In Vitro Studies. Liver microsomes from mice, rats, dogs, and
`humans converted [14C]tolterodine into several products in the pres-
`ence of NADPH. Five metabolites were detected in the radiochro-
`matograms for incubations with rat liver microsomes and three for
`those with mouse, dog, and human liver microsomes. The major
`metabolites of [14C]tolterodine were identified by comparison of their
`chromatographic retention times with those of reference standards
`and/or by MS.
`Table 3 shows the chromatographic retention times and the proton-
`ated molecular ions from the MS analyses, as well as the rates of
`formation of metabolites. N-Dealkylated tolterodine and 5-HM were
`
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`ANDERSSON ET AL.
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`TABLE 1
`
`Summary of key MS fragmentation for tolterodine and its metabolites in dog and
`mouse urine after electrospray ionization and collision-induced dissociation
`
`Metabolite
`
`[M1H]1
`
`CIDa Fragments
`
`Tolterodine
`5-HM
`
`5-CM
`
`N-Dealkylated tolterodine
`N-Dealkylated 5-HM
`
`N-Dealkylated 5-CM
`
`Tolterodine glucuronide
`5-HM glucuronide
`5-CM glucuronide
`
`N-Dealkylated tolterodine
`glucuronide
`N-Dealkylated 5-HM glucuronide
`N-Dealkylated 5-CM glucuronide
`
`a CID, collision-induced dissociation.
`
`328
`344
`
`358
`
`286
`300
`
`316
`
`504
`518
`534
`
`460
`
`476
`490
`
`286, 199, 182, 149, 123, 91
`302, 225, 215, 197, 154, 133,
`105, 91
`316, 239, 229, 211, 194, 181,
`165, 153, 133, 105, 91
`199, 181, 149, 123, 91
`223, 213, 195, 177, 165, 167,
`152, 133, 105, 91
`239, 229, 211, 181, 165, 153,
`133, 105, 91
`328, 286, 199, 182, 149
`342, 233, 213, 195
`358, 316, 239, 229, 211, 179,
`153
`284, 196, 147, 119
`
`213, 167
`
`FIG. 7.Radiochromatograms of urine collected at 0 – 6 hr ( a) and plasma
`collected at 15 min (b) after oral administration of [14C]tolterodine (4 mg/kg)
`to mice.
`
`FIG. 6.Metabolic pathway of tolterodine in mice (given 4 mg/kg orally) and
`dogs (given 1.5 mg/kg orally or 1 mg/kg iv).
`
`major metabolites in all species. Although considerable interspecies
`differences in the extent of metabolism were apparent, the metabolic
`profiles were similar. In mice, 5-HM and N-dealkylated tolterodine
`represented approximately 20 and 64% of total metabolism, respec-
`tively. One minor product, N-dealkylated 5-HM, was also detected. In
`rats, three major metabolites were formed, i.e. 5-HM, didealkylated
`tolterodine, and N-dealkylated tolterodine, representing approxi-
`mately 5, 11, and 78% of total metabolism, respectively. Minor
`metabolites included a dihydroxylated product, N-dealkylated 5-HM,
`and a metabolite hydroxylated in the unsubstituted benzene ring,
`
`FIG. 8.Radiochromatogram of urine collected at 0 –12 hr after oral
`administration of [14C]tolterodine (50 mg/kg) to a rat.
`
`which together represented about 6% of total metabolism. The dihy-
`droxylated product and the product hydroxylated in the unsubstituted
`benzene ring were detected only in incubations with rat liver micro-
`somes. In dogs and humans, 5-HM represented approximately 39 and
`21% and N-dealkylated tolterodine represented approximately 47 and
`71% of total metabolism, respectively. One minor product, N-
`dealkylated 5-HM, was detected in both species.
`
`Discussion
`Tolterodine is extensively metabolized and similar metabolic pro-
`files were obtained for urine and plasma after oral and iv administra-
`tion to mice and dogs, indicating that biotransformation takes place
`predominantly in the liver. Biotransformation products were formed
`via two major pathways, i.e. oxidation of the 5-methyl group in the
`benzene ring and dealkylation of the nitrogen. Furthermore, conju-
`gates of both tolterodine and its metabolites were formed by glucu-
`
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`TOLTERODINE METABOLISM IN MICE, RATS, AND DOGS
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`533
`
`Summary of key MS fragmentation for trimethylsilyl derivatives of tolterodine and its metabolites in rat urine
`
`TABLE 2
`
`Metabolite
`
`M1
`
`[M215]1
`
`a
`
`b
`
`Metabolite
`
`M1
`
`[M215]1
`
`a
`
`b
`
`397
`
`382
`
`269
`
`114
`
`603
`
`588
`
`445
`
`144
`
`573
`
`558
`
`445
`
`114
`
`427
`
`412
`
`269
`
`144
`
`385
`
`370
`
`269
`
`102
`
`603
`
`588
`
`475
`
`114
`
`515
`
`500
`
`357
`
`144
`
`473
`
`458
`
`357
`
`102
`
`515
`
`500
`
`357
`
`144
`
`473
`
`458
`
`357
`
`102
`
`633
`
`618
`
`475
`
`144
`
`545
`
`530
`
`387
`
`144
`
`561
`
`546
`
`445
`
`102
`
`Petitioner Torrent Pharmaceuticals Limited - Exhibit 1014 - Page 6
`
`

`
`534
`
`ANDERSSON ET AL.
`
`TABLE 3
`
`Metabolism of tolterodine by mouse, rat, dog, and human liver microsomes
`
`Metabolite
`
`Retention Time
`
`[M1H]1
`
`Rate of Formation
`
`Mouse
`
`Rat
`
`Dog
`
`Human
`
`Dihydroxylated tolterodine
`N-Dealkylated 5-HM
`5-HM
`Hydroxylated tolterodine
`Didealkylated tolterodine
`N-Dealkylated tolterodine
`
`Data are mean 6 SD of three incubations.
`
`min
`9.0
`13.2–13.7
`16.7–17.3
`20.0–21.0
`23.5–23.8
`26.3–27.4
`
`358
`300
`342
`342
`242
`284
`
`0
`54 6 2
`203 6 19
`0
`0
`652 6 2
`
`pmol/mg protein/min
`,20
`41 6 6
`75 6 1
`48 6 5
`174 6 6
`1206 6 14
`
`0
`19 6 2
`249 6 13
`0
`0
`298 6 16
`
`0
`19 6 6
`130 6 10
`0
`0
`442 6 26
`
`ronidation. In contrast, rats also formed metabolites by oxidation of
`the unsubstituted benzene ring to form mono- and dihydroxylated
`metabolites, and the urinary metabolite pattern showed gender differ-
`ences. The major metabolites in dogs and mice, 5-CM and N-deal-
`kylated 5-CM, were not detected in rat urine. A possible cause of the
`different metabolite pattern in rats might be a metabolic switch
`resulting from the relatively high administered dose (50 mg/kg).
`However, mice given a similar dose (40 mg/kg) did not form metab-
`olites by oxidation of the unsubstituted benzene ring. The metabolites
`containing vicinal diols were also methylated. Methylation is not
`unexpected and occurs mainly with phenols containing vicinal diols,
`e.g. catechols (Mulder, 1982) and metabolites of terodiline (Nore´n et
`al., 1985). Terodiline has a molecular structure similar to that of
`tolterodine. Extensive metabolism and a gender difference are well
`documented in rats (Shapiro et al., 1995). In connection with toxico-
`logical studies, rats are a well-researched species and are often used as
`a primary species in preclinical safety evaluations during drug devel-
`opment. However, rats can be a poor choice in some situations. One
`such case is when rats metabolize and excrete the drug so extensively
`that relevant systemic levels of intact drug are difficult to maintain
`during toxicological studies. Another case is when the metabolite
`pattern in blood does not reflect that of humans as a result of more
`extensive metabolism; consequently, rats are not properly exposed to
`the drug and/or active metabolites, as are human subjects. General
`pharmacokinetic studies in rats have indeed shown that the systemic
`levels of tolterodine are very low, considering the administered dose
`(Kankaanranta and Påhlman, 1997). Furthermore, as a result of the
`different metabolic profile in rats, systemic levels of the pharmaco-
`logically active metabolite 5-HM are also very low. Rats were there-
`fore excluded as a main species in the preclinical safety evaluation of
`tolterodine.
`The metabolic profiles of mice and dogs showed similarities to
`those of human subjects (Brynne et al., 1997), and the major metab-
`olites in the urine from these species were 5-CM and N-dealkylated
`5-CM. The only phase II metabolites that were identified were gluc-
`uronide conjugates, and the concentrations of these were highest in
`mice. At higher doses in mice, the relative concentrations of intact
`tolterodine and N-dealkylated tolterodine were increased, whereas the
`concentrations of 5-HM and the acid metabolites were decreased,
`indicating dose-dependent biotransformation.
`Recently, we reported that the formation of 5-HM and N-dealkyl-
`ated tolterodine in humans is catalyzed by cytochrome P450 2D6 and
`3A4, respectively (Postlind et al., 1998). Further biotransformation to
`the acid metabolites via the aldehydes is most likely catalyzed by
`alcohol and aldehyde dehydrogenases. However, the metabolic capac-
`ity of cytochrome P450 2D6 in humans is much lower, in comparison
`with the 3A4 isoenzyme (Shimada et al., 1994). A possible explana-
`tion for the altered metabolism observed with the higher oral dose in
`mice is therefore that the isoenzymes involved in the formation of
`
`5-HM and the acid metabolites were saturated. Consequently, the
`relative concentrations of tolterodine and N-dealkylated tolterodine, as
`well as their glucuronide conjugates, were increased.
`In vitro studies have become an increasingly important tool in
`pharmaceutical research (Chiu, 1993; Rodrigues, 1994). Subcellular
`fractions, slices, recombinant enzymes, and cell cultures are routinely
`used in the screening for candidate drugs, the selection of species for
`toxicological studies, and the prediction of the situation in humans.
`Although results obtained from the use of microsomes are limited, the
`data often yield a good representation of the in vivo situation, in terms
`of the major metabolic pathways in which cytochromes P450 are
`involved. Incubation of tolterodine with liver microsomes in vitro
`yielded a good qualitative prediction of in vivo metabolism, although
`the carboxylated metabolites were missing because of the lack of
`alcohol and aldehyde dehydrogenases in the microsomal system.
`Furthermore, the metabolites formed in incubations with liver micro-
`somes from mice and dogs were very similar to those observed with
`human microsomes, which is in accordance with the results from in
`vivo studies.
`In conclusion, the metabolism of tolterodine was extensive. Mice
`and dogs showed similar metabolite patterns, which correlated with
`that observed for humans. Tolterodine was metabolized along two
`different pathways in these species, with the more important being the
`stepwise oxidation of the 5-methyl group attached to the benzene ring
`to yield 5-HM and then, via the aldehyde, 5-CM. The other pathway
`involved dealkylation of the nitrogen. In the subsequent phase II
`metabolism, tolterodine and the metabolites were conjugated with
`glucuronic acid to various degrees. Rats exhibited a different meta-
`bolic profile, with metabolites also being formed by hydroxylation in
`the unsubstituted benzene ring of the tolterodine molecule.
`
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