Introduction

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Tolterodine [(R)-N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)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 symptoms (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 microsomes from these species and humans.

	Materials and Methods

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Chemicals. [¹⁴C]Tolterodine (labeled at the benzylic methyl group) (fig. 1), tolterodine (PNU-200583), 5-HM¹ [(R)-N,N-diisopropyl-3-(2-hydroxy-5-hydroxymethylphenyl)phenylpropanamine, PNU-200577, labcode DD 01], 5-CM (PNU-200579), and N-dealkylated tolterodine (PNU-200578) were synthesized at Pharmacia & Upjohn AB (Uppsala, Sweden). -Glucuronidase (Escherichia coli, product no. 127051) was obtained from Boehringer Mannheim (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.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Chemical structure of tolterodine. *, position of the radiolabel.

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 centrifugation 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 administration. Samples from five mice in each group were pooled and centrifuged to prepare plasma, which was immediately stored at 20°C until analysis.

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 20°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 µl of methanol and 270 µl of 20 mM ammonium acetate (pH 4.5). The extraction yield was 98-100%.

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 ± 11% (mean ± 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 70°C. During the preparation procedure, the temperature was maintained as close to +4°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 70°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 µg 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 µl of an aqueous solution of [¹⁴C]tolterodine, to yield a final concentration of 154 µM. 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 20°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-µl 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- × 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 consisted 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- × 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 interface (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 µl 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.

	Results

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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. Electrospray-ionization MS of metabolites showed prominent peaks corresponding to the protonated molecular ion ([M+H]⁺) 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 collision-induced dissociation of the [M+H]⁺ ions) with those of the corresponding reference standards (figs. 3-5). The ¹⁴C-labeled fragment ions of the metabolites appeared 2 amu larger than the nonlabeled 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 glucuronide moiety (176 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 summarized in fig. 6. The most abundant peak (19.7 min) in the urinary metabolic profile of dog urine represented 5-CM, with its corresponding glucuronide conjugate (18.7 min). The other major peaks, at 15-16 min, were not completely resolved and contained two metabolites, 5-HM and N-dealkylated 5-CM. These major metabolites constituted approximately 70-80% of the total radioactivity in the urine samples. The peak at 12.4 min was N-dealkylated 5-HM. The unresolved 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.

View larger version (23K): [in this window] [in a new window]   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.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Product-ion mass spectra of 5-HM (a) and the corresponding synthesized reference standard (b).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Product-ion mass spectra of 5-CM (a) and the corresponding synthesized reference standard (b).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Product-ion mass spectra of N-dealkylated tolterodine (a) and the corresponding synthesized reference standard (b).

View larger version (19K): [in this window] [in a new window]   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).

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 30-60% of the total radioactivity in serum samples. The most abundant 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 corresponding 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 [M+H]⁺ 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).

View larger version (23K): [in this window] [in a new window]   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.

Mouse Plasma. The radiochromatograms for plasma samples collected 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 glucuronide, 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 glucuronide conjugate, were increased more than proportional to dose, together 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 -glucuronidase or arylsulfatase; only treatment with the former affected the appearance of the radiochromatogram. After hydrolysis of glucuronic acid conjugates, the metabolites were tentatively identified from retention times and mass spectra obtained by using GC/MS with trimethylsilyl derivatization. Trimethylsilyl derivatives were formed with the hydroxy and dealkylated amino groups. The mass spectra contained diagnostic fragments 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 dihydroxylation of the unsubstituted benzene ring. Three of the metabolites 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 summarized in table 2. A gender difference was also observed, with urine from male rats containing relatively higher concentrations of these metabolites.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Radiochromatogram of urine collected at 0-12 hr after oral administration of [14C]tolterodine (50 mg/kg) to a rat.

In Vitro Studies. Liver microsomes from mice, rats, dogs, and humans converted [¹⁴C]tolterodine into several products in the presence of NADPH. Five metabolites were detected in the radiochromatograms 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.

	Discussion

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Tolterodine is extensively metabolized and similar metabolic profiles were obtained for urine and plasma after oral and iv administration 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, conjugates of both tolterodine and its metabolites were formed by glucuronidation. 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 differences. The major metabolites in dogs and mice, 5-CM and N-dealkylated 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 metabolites 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 (Norén 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 toxicological studies, rats are a well-researched species and are often used as a primary species in preclinical safety evaluations during drug development. 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 pharmacologically active metabolite 5-HM are also very low. Rats were therefore 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 metabolites in the urine from these species were 5-CM and N-dealkylated 5-CM. The only phase II metabolites that were identified were glucuronide 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-dealkylated 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 capacity of cytochrome P450 2D6 in humans is much lower, in comparison with the 3A4 isoenzyme (Shimada et al., 1994). A possible explanation 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 microsomes 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 metabolic profile, with metabolites also being formed by hydroxylation in the unsubstituted benzene ring of the tolterodine molecule.