Introduction

Abstract Introduction Results Discussion References

Tacrine [THA²; Cognex] is a potent centrally active cholinesterase inhibitor approved for treatment of mild to moderate Alzheimer's disease (1-3). Commonly observed side effects associated with tacrine treatment are cholinergic in nature and involve gastrointestinal discomfort (1). Also observed is a relatively high incidence of elevations in serum ALT, a marker enzyme for hepatotoxicity, which is reversible upon cessation of drug administration (3). The mechanism(s) involved in the elevation of serum ALT after THA treatment are unknown; however, formation of hepatotoxic metabolites or a drug-induced hypersensitivity reaction may play a role (4-6). THA bioavailability and plasma concentrations are low and variable in Alzheimer's patients, with peak plasma concentrations reached in 1 to 2 hr postdose (7-9). THA studies in rat and dog show species-dependent pharmacokinetic parameters with rapid absorption and metabolism (10). No evidence of ALT elevations has been observed in rats or dogs after THA administration, suggesting a species difference in hepatotoxic potential (4). In vitro metabolism studies conducted using animal and human liver microsomal preparations have shown THA to be metabolized to stable, protein-reactive, and cytotoxic metabolites with 1-OH-THA identified as the major stable metabolite (6, 11, 12). Also observed in these studies were species differences in the formation of NADPH-dependent protein reactive and cytotoxic metabolites. Enzymology investigations have implicated a major role of CYP1A2 in the metabolism and bioactivation of THA (6,11-13). Species differences in hepatotoxic potential of THA led us to conduct extensive in vivo animal and human studies to assess the potential of species differences in metabolism. The relevance of these results with respect to hepatotoxicity and CYP1A2 activity is discussed.

Materials and Methods

Chemicals. [9-¹⁴C]THA was prepared in the Parke-Davis Pharmaceutical Research Laboratories (Ann Arbor, MI) (14) and shown to have radiochemical and chemical purities of >99%. Unlabeled THA and 1-, 2-, and 4-OH-THA (all of chemical purity >99%) were prepared by methods previously described (15). 7-OH-THA and the N-hydroxylamine derivative of THA (N-hydroxyl-THA) were synthesized using the method of Woolf et al. (12). All other chemicals were reagent grade or better.

Animals. Male and female Wistar rats weighing 228 ± 10 g and 190 ± 15 g, respectively, were purchased from Charles River Laboratories (Wilmington, MA). Male and female beagle dogs weighing 10.1 to 11.0 kg were obtained from Marshall Research Animals (North Rose, NY). All animals were allowed free access to standard diet and water.

Dosing and Sample Collection. THA doses were prepared in distilled water.

Rats. Six male and six female fasted rats were administered orally a 2 mg/kg (13 µCi, specific activity; 5.66 mCi/mMol free base equivalents) solution dose of [¹⁴C]THA. Animals were housed in individual stainless-steel metabolism cages for collection of urine and feces up to 168 hr after dosing. A satellite group of three animals were administered the aforementioned dose and killed at 4 hr for plasma metabolic profiling.

Dogs. Two male and two female fasted dogs were administered orally 2 mg/kg [¹⁴C]THA (116 µCi, specific activity; 0.99 mCi/mMol free base equivalents) as a solution dose in gelatin capsules. Dogs were housed in stainless-steel metabolism cages equipped with urine collection pans. Urine and feces were collected for 96 hr. Blood samples were taken at 4 hr for plasma metabolic profiling. For metabolite isolation, two male and two female dogs were administered unlabeled THA (2 mg/kg) in gelatin capsules on study days 1, 2, 4 and 5. [¹⁴C]THA (2 mg/kg; 109 µCi, specific activity; 1.02 mCi/mMol free base equivalents) was administered via capsule on study day 3. Dogs were housed as described with urine and feces collected for 96 hr after the [¹⁴C]THA dose.

Humans. The study was conducted in accordance with recommendations for clinical studies after approval by an Institutional Review Board. Six healthy male volunteers, with an average age of 57 years and an average weight of 77.8 kg participated in the study. Each subject received a single 10 mg (100 µCi, specific activity; 2.16 mCi/mMol free base equivalents) oral solution dose of [¹⁴C]THA, followed by a single 40 mg (100 µCi, specific activity; 0.54 mCi/mMol free base equivalents) oral solution dose to the same subjects 28 days later. Medications were administered in water. Subjects were fasted for 8 hr before dosing and an additional 4 hr postdose. Urine and feces were collected for 96 hr postdose. Blood samples were collected at 1 and 4 hr for plasma metabolic profiling.

Analytical Methods. Total radioactivity was measured in a Packard model 3375 or 4530 liquid scintillation counter (Downers Grove, IL). Amounts of radioactivity in urine and cage wash were determined directly in Research Products International 3a70b (Elk Grove, IL) or Beckman Ready Safe (Fullerton, CA) scintillation cocktails. Feces were homogenized with water and samples mixed with Soluene 350 or combusted in a Packard model 306 sample oxidizer. The resulting ¹⁴CO₂ was trapped with Carbosorb and mixed with Permafluor before counting. Urine samples were injected directly onto the HPLC system or were lyophilized and reconstituted in mobile phase before analysis. Gradient HPLC radioactivity profiling and TSP/MS analyses were accomplished by using methods described previously (12).

Plasma Extraction. Rat plasma (0.5 ml) was treated with ice-cold absolute ethanol (2.0 ml), vortexed, and stored at 20°C for a minimum of 2 hr to assist in precipitation. The mixtures were then centrifuged at 700 g for 10 min, supernatants transferred, and the pellet washed with ice-cold ethanol (1.0 ml). The combined supernatents were evaporated to dryness with a gentle stream of nitrogen at 40°C and reconstituted in mobile phase for HPLC analysis. Dog plasma (5-10 ml) was treated with ice-cold ethanol (4 volumes) dropwise with stirring and extracted as described. Aliquots of pooled human plasma (3 ml) were precipitated by dropwise addition of ice-cold ethanol (60 ml) while stirring and treated as described. Plasma extraction efficiencies ranged from 70 to 90%.

Glucuronidase Treatment. Pooled human 0-8 hr urine aliquots (5 ml) from the 40 mg dose group were adjusted to pH 5.0, with 0.1 M sodium acetate buffer (pH 5.0, 5.0 ml). Samples were hydrolyzed with NEE-154 glusulase, -glucuronidase, or sulfatase for 20-24 hr at 37°C. Control samples contained boiled enzyme. After hydrolysis, samples were centrifuged and analyzed directly by TSP/MS.

Biosynthesis of 7-OH-THA Glucuronide. Rat liver microsomes (2 mg/ml) were preincubated with CHAPS (6 mM) for 30 min at 4°C. Incubation of CHAPS-treated microsomes and 7-OH-THA (1 mM) was conducted in 0.1 M Tris buffer (pH 7.4) containing 10 mM MgCl₂ at 37°C for 5 min before addition of UDPGA (4 mM). After a 60-min incubation period, the sample was quenched with glacial acetic acid (5 ml) and subjected to solid-phase extraction (BondElut C18, 3 ml). The column was preconditioned with methanol (2 × 3 ml) and water (2 × 3 ml). After sample application, the column was washed with water (2 × 3 ml) and eluted with methanol, which was evaporated to dryness, and the residue was reconstituted in mobile phase for TSP/MS analysis.

Urine Extraction. A 40-ml aliquot of dog urine (from a 24-hr pool) was made alkaline by addition of sodium hydroxide (1 N, 4 ml), extracted with ethyl acetate (240 ml), washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. Extraction efficiency was ~60% and contained all of the major metabolites. The resulting residue was reconstituted in HPLC mobile phase for metabolite isolation.

Semipreparative HPLC Isolation of Metabolites. Semipreparative HPLC fractionation was accomplished with a Hypersil Phenyl 5 µm column (6.2 × 250 mm) (Keystone Scientific, Inc., Bellefonte, PA) in series with an Upchurch Uptight precolumn (Upchurch Scientific, Inc., Oak Harbor, WA) packed with C₁₈ pellicular media (30-40 µm). The mobile phase consisted of acetonitrile and 0.05 M ammonium formate (pH 3.1). Components were eluted with a linear gradient starting at 0% acetonitrile and increasing to a final acetonitrile concentration of 20% over 60 min and maintained for an additional 30 min. Analytes were eluted at 1.5 ml/min with UV monitoring at 325 nm. Aliquots of collected fractions were analyzed for radioactivity and rechromatographed on an isocratic analytical system to assess radiochemical and UV purity. Isocratic purity checks were performed with an ALLTECH Econosphere CN 5 µm column (4.6 × 250 mm) (Alltech Associates, Inc., Deerfield, IL) with the described precolumn and buffer containing 5% acetonitrile. Analytes were monitored at 250 nm and eluted at 1 ml/min. Fractions free of UV interferences with identical retention times were pooled and lyophilized. Further purification of impure fractions was accomplished by gradient semipreparative HPLC with an ALLTECH Econosphere CN 10-12 µm (10 × 250 mm) column in series with the above precolumn and mobile phase. Components were eluted with a linear gradient starting at 0% acetonitrile for 10 min and increasing to an acetonitrile concentration of 5% over 20 min, which was maintained for an additional 20 min. Analytes were monitored at 325 nm and eluted at a flow rate of 5 ml/min.

NMR Spectroscopy. ¹HNMR data were collected on a Varian XL300 spectrometer operating at 300 MHz for proton using a M600 computer operating on the 5.2 software release or a Bruker AMX-500 operating at 500 MHz for proton with UXNMRP software. Deuteromethanol was used as the solvent.

	Results

Abstract Introduction Results Discussion References

Disposition in Rats, Dogs, and Humans. Excretion. The recoveries of radioactivity in urine and feces for rats, dogs, and humans are shown in table 1. Urine was the major route of excretion in all three species, with the majority of radioactivity recovered within 24 hr. Cumulative mean percentage recovery (±SD) of dose in urine and feces of rats administered a single oral 2 mg/kg (13 µCi) dose of [¹⁴C]THA was 73.0% (1.9) and 23.3% (3.4) over the 168-hr collection interval, with a cumulative mean total recovery of 96% (3.3). After administration of the single 2 mg/kg (116 µCi) oral dose to dogs, cumulative mean percentage recovery (±SD) in urine and feces was 63.6% (9.6) and 26.4% (7.3), respectively, over the 96-hr collection interval with a cumulative total recovery of 90% (11.1). Cumulative mean percentage recovery (±SD) of dose in urine and feces from human subjects administered a single 10 mg (100 µCi) oral solution dose of [¹⁴C]THA was 56.5% (8.9) and 22.9% (3.9), respectively, with a cumulative mean total recovery of 79.4% (5.5) over the 96-hr collection period. After a single 40 mg (100 µCi) solution dose, 28 days later, cumulative mean percentage recovery (±SD) in urine and feces was 54.1% (5.6) and 20.8% (4.8), respectively, with a cumulative mean total recovery of 74.9% (3.7) over 96 hr.

Metabolic Profiling. Urine collected over the initial 24-hr period from rats, dogs, and humans after dosing with [¹⁴C]THA was profiled by gradient HPLC with online radioactivity detection as well as TSP/MS. Quantitation of metabolites, expressed as percentage dose and percentage urinary radioactivity, is depicted in table 2. In all three species, plasma profiles were similar to respective urinary profiles. Representative 0- to 4-hr radiochromatograms of rat, dog, and human urine are shown in fig. 1.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Representative 0- to 4-hr radiochromatograms of rat (top), dog (middle), and human (40 mg dose) (bottom) urine after oral administration of [14C]THA.

Rats. Metabolic profiling of 0- to 24-hr urine from rats administered a single 2 mg/kg dose of [¹⁴C]THA showed primarily 1-, 2-, and 4-OH-THA accounting for ~78% of urinary radioactivity. The major component, representing 66% of urinary radioactivity, had similar HPLC retention characteristics as a 1-OH-THA reference standard. Also present was unchanged parent (6% of urinary radioactivity) and several small polar metabolites. There were no apparent gender effects observed in the metabolic profiles.

Dogs. Present in the 0- to 24-hr profile of dog urine after a single oral 2 mg/kg dose of [¹⁴C]THA were seven polar components comprising 4.2%, 8.2%, 2.8%, 4.0%, 6.2%, 20.5%, and 2.0% of urinary radioactivity, respectively. Also present was 1-OH-THA representing 33.1% of urinary radioactivity, with only trace quantities of 2- and 4-OH-THA and unchanged parent. No apparent gender differences were observed in metabolic profiles.

Humans. Metabolite profiling of 0- to 24-hr urine from subjects administered a single oral 10 mg solution dose of [¹⁴C]THA showed the presence of seven polar radiolabeled components accounting for 5.6%, 8.4%, 8.0%, 6.3%, 6.7%, 5.6%, and 8.6% of urinary radioactivity. A peak with similar retention characteristics to 1-OH-THA (6.0% of urinary radioactivity), as well as trace amounts of 2- and 4-OH-THA and unchanged parent, were also observed. HPLC radioactivity profiling of 0- to 24-hr urine after a 40 mg solution dose of [¹⁴C]THA showed a similar profile to that after the 10 mg dose with components 1-7 accounting for 5.0%, 9.8%, 7.2%, 5.2%, 7.6%, 5.6%, and 10.2% of urinary radioactivity, respectively. Also observed was 1-OH-THA (8.6% of urinary radioactivity) and trace quantities of 2- and 4-OH-THA and unchanged parent.

Reactive Metabolites: Hydroxylamine and N-acetyl-THA. The potential presence of either a hydroxylamine or N-acetylated metabolite of THA in plasma or urine was addressed by chromatographic and mass spectral comparisons to synthetic reference compounds. No evidence of N-hydroxyl-THA or N-acetyl-THA was found in the plasma or urine of rats, dogs, or humans after oral administration of [¹⁴C]THA.

Isolation and Identification of Dog Urinary Metabolites. Some 60% of the dose was recovered in 0- to 24-hr dog urine. Dogs were administered multiple doses of THA, followed by [¹⁴C]THA for the purposes of metabolite identification. Urine was extracted and subjected to semipreparative fractionation and purification. Purified fractions were analyzed by TSP/MS and when sufficient sample was available by ¹H-NMR analysis. TSP/MS data are shown in table 3, whereas ¹H-NMR data are depicted in table 4. Components 1(C1) and 4(C4) could not be identified, due to insufficient material.

Component 2 (C2). Photodiode array analysis of C2 showed a hypsochromic shift in the UV spectrum indicating a loss of aromaticity for this metabolite (fig. 2). ¹H-NMR analysis showed the absence of proton signals in the aromatic region consistent with the UV data (data not shown). TSP/MS data for C2 gave a protonated molecule [M + H]⁺ at m/z 233 with ions at m/z 215 [M-H₂O]⁺ and m/z 199 [M-H₂O-NH₂]⁺. The molecular weight of the protonated molecule is consistent with the addition of an oxygen atom followed by a molecule of water to THA. This data, coupled with the absence of aromatic signals for this metabolite, suggests it to be a dihydrodiol derivative of THA. Due to the limited quantity of this metabolite, studies to assess regiochemical assignments were not conducted.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Photodiode array spectra of THA metabolites. Overlay of 1-OH-THA reference standard and C6 (top). Spectrum of C2 (bottom).

Component 3 (C3). UV data obtained from C3 indicated that the aromatic chromophore was intact. TSP/MS data showed a presumed protonated molecule [M + H]⁺ at m/z 231 corresponding to addition of two oxygen atoms. Also present was an ion at m/z 213 [M-H₂O]⁺. The aforementioned data are consistent with a dihydroxy metabolite of THA.

Component 5 (C5). Photodiode array analysis of C5 showed retention of the aromatic chromophore. TSP/MS analysis displayed a presumed [M + H]⁺ at m/z 231, with ions at m/z 213 [M-H₂O]⁺ and m/z 197 [M-H₂O-NH₂]⁺ These data are suggestive of a dihydroxy metabolite of THA.

Component 6 (C6). UV data indicated that the aromatic chromophore of C6 was intact (fig. 2). TSP/MS analysis showed a presumed [M + H]⁺ at m/z 231 with ions at m/z 213 [M-H₂O]⁺ and m/z 195 [M-2H₂O]⁺. Based on mass spectral information, a dihydroxy metabolite of THA was proposed. To characterize further the regiochemistry of the two hydroxyl groups in this major metabolite, selective decoupling of individual protons in the ¹H-NMR spectrum was undertaken. Irradiation at individual resonance frequencies causes perturbation of resonance lines from protons 2-3 bonds away. Irradiation of the resonance at 5.02 ppm affected only the two most shielded methylene resonances at 2.14 and 2.45 ppm. Irradiation of these methylene resonances affected the methine peaks at 4.25 and 5.02 ppm. Irradiation of the methine at 4.25 ppm caused perturbation of all four methylene resonances at 2.14, 2.45, 3.02, and 3.20 ppm, establishing that the methine at 4.25 ppm is located between two methylene groups. This eliminates all possible dihydroxy substitution patterns except for 1,3-dihydroxy-THA and 2,4-dihydroxy-THA. Of these two possibilities, 1,3-dihydroxy-THA fits the data best based on the following chemical shift arguments (see table 4): first, the methine at 5.02 ppm is most consistent with 1-hydroxylation and not C4-hydroxylation. The methylene protons at 3.02 and 3.20 ppm coincide with the C4 methylene protons in THA and not the C1 methylene protons in THA. If the metabolite were 2,4-dihydroxy-THA, two highly shielded methylenes (four protons) would be expected at ~2.0-2.1 ppm corresponding to the C1H₂ and the C3H₂ positions. Hydroxylation at C4 causes a large shift (0.1 ppm) in the aromatic proton at C5, whereas C1 hydroxylation has minimal effect on the position of C5H. The chemical shift of C5H in the metabolite is identical to that in THA. Thus, the structure of C6 is most consistent with 1,3-dihydroxy-THA. In addition, based on published coupling constants, the stereochemistry of this metabolite can be identified as the cis regioisomer (16).

1-OH-THA Isolate. The major metabolite fraction coeluting with 1-OH-THA reference material was isolated as described and subjected to TSP/MS and ¹H-NMR comparisons to reference standard. Fragmentation and chemical shift information of the isolate were consistent with that observed for 1-OH-THA reference material.

Identification of Human Urinary Glucuronide Metabolite, Component 7 (C7). UV analysis of C7 revealed the presence of at least two metabolites containing a bathochromic shift indicative of aromatic hydroxylation. Treatment of human urine with glusulase resulted in the disappearance of C7 accompanied by the presence of two new components eluting at 72 and 74 min. Incubation of urine with purified sulfatase enzyme resulted in no change in metabolic profile, whereas incubation with glucuronidase resulted in cleavage of the conjugates. TSP/MS analysis of the aglycone eluting at 74 min showed a protonated molecular ion [M+H]⁺ at m/z 215. Confirmation of the aglycone metabolite as 7-OH-THA was made by comparison of chromatographic and spectral properties of the metabolite with synthetic reference material. Additional support was gained by incubation of 7-OH-THA reference material with detergent-treated rat liver microsomes in the presence of UDPGA. Analysis of the postreaction incubate showed a peak with identical chromatographic and photodiode array properties to that of the conjugate observed in vivo. TSP/MS analysis of the aglycone metabolite eluting at 72 min also showed a protonated molecular ion [M+H]⁺ at m/z 215.

	Discussion

Abstract Introduction Results Discussion References

Radioactivity was well and rapidly absorbed in rats, dogs, and humans after single oral doses of [¹⁴C]THA, with the majority of the dose excreted in 0- to 24-hr urine. A pathway depicting the metabolic fate of THA is shown in scheme 1.

View larger version (19K): [in this window] [in a new window]   Scheme 1.   Pathway depicting metabolic fate of THA.

HPLC gradient radioactivity and TSP/MS analysis of plasma and urine after [¹⁴C]THA administration to rats, dogs, and humans showed profiles that were qualitatively similar; however, a marked species difference in the quantitative amounts of the metabolites was apparent. In rats after a single 2 mg/kg dose of [¹⁴C]THA, 60% of the dose was identified as 1-, 2-, 4-OH-THA, and THA in urine. The major metabolite identified was 1-OH-THA representing 46% of dose, whereas unchanged parent accounted for 5% of dose. 2-OH-THA represented 7% of dose and 4-OH-THA accounted for ~2% of dose. Also present were several polar components representing 6% of the administered dose. These data are consistent with results reported by Hsu et al. (15), in which ~60% of a 20 mg/kg oral dose was eliminated as 1-, 2-, 4-OH-THA, and THA over a 48-hr collection period. However, the major metabolite observed by Hsu et al. was 2-OH-THA (29% of dose), with 1-OH-THA representing 22%. This apparent discrepancy in the amounts of 1-OH-THA excreted was investigated by us separately (17) and would seem to be a result of chromatographic interference from a new monohydroxy metabolite namely, 3-OH-THA. In dogs, urinary metabolic profiling showed an increase in excretion of polar metabolites compared with rats (29% vs. 6%) with 1-, 2-, 4-OH-THA, and THA accounting for just 21% of dose. THA was excreted in trace amounts. 1-OH-THA (20% of dose) was identified as the major metabolite, whereas 3-OH-THA was not observed in dog urine. Also present was a major metabolite, labeled C6 accounting for 12% of dose. Mass spectral data showed this metabolite to be a dihydroxylated derivative of THA, whereas selective decoupling experiments indicated the structure of C6 as cis-1,3-dihydroxy-THA. These data suggest that, if formed, 3-OH-THA undergoes more efficient sequential metabolism to a cis-1,3-dihydroxy-THA derivative in dogs, compared with rats. Also observed in dog urine were several additional mono- and dihydroxylated isomers of unknown regiochemistry, as well as a dihydrodiol derivative. The finding of a dihydrodiol metabolite in dog may have implications for reactive metabolite chemistry in this species, although in vitro studies in rat and human microsomes argue against an arene oxide pathway in THA bioactivation (6, 12). Human urinary metabolic profiling after single 10 or 40 mg doses of [¹⁴C]THA showed results more similar to that in dogs than in rats with no single major metabolite observed. Polar components accounted for 26% and 25% of dose after 10 and 40 mg doses, respectively, whereas 1-, 2-, 4-OH-THA, and THA accounted for only 4% and 6% of dose, respectively. 1-OH-THA comprised <5% of dose in both treatments, whereas THA was excreted in only trace quantities. Spectroscopic analysis of isolated 1-OH-THA from human urine in a separate study (17) did show the presence, in the 1-OH-THA isolate, of 3-OH-THA. Before the studies described herein, limited information has been published on the metabolism of THA in humans. Previous studies conducted with nonradiolabeled drug identified 1-OH-THA as a major metabolite (18).

The aforementioned data are consistent with studies by Turcan et al. (19) on the disposition of [¹⁴C]1-OH-THA, a primary metabolite of THA as shown herein, in rats, dogs, and humans. During the course of these investigations, metabolic profiling of urine after administration of [¹⁴C]1-OH-THA resulted in the identification of several hydroxylated metabolites, including isomers at the 3- and 7-positions, as well as dihydrodiol derivatives; however, no apparent species differences in metabolism were noted.

The primary enzyme involved in human THA metabolism has been suggested to be CYP1A2 (6, 11-13). This enzyme is inducible by cigarette smoking (20, 21) and indeed smoking has been shown to enhance THA clearance (22). Significant intersubject variability in the expression of CYP1A2 has been reported by several investigators (23, 24), as well as ethnic and gender differences (25). Clinical studies with THA have reported wide interindividual variation in pharmacokinetic parameters (7-9). In the clinical results presented herein, we also see variability in the metabolic profiles (table 2), the significance of which is not clear at the present time.

Species differences in CYP1A catalytic activity have been noted with rats showing relatively low activity, dogs showing moderate activity, and humans possessing higher activity (26). In vitro studies investigating species variation in the metabolism and bioactivation of THA have shown THA to be biotransformed in rats, dogs, and humans to protein reactive and cytotoxic metabolites, with rats producing the lowest levels, dogs intermediate, and humans the highest (11). During the course of these investigations, the authors showed similar microsomal THA metabolic profiles between the species investigated, with differences observed attributed to quantitative differences in the rates of metabolism. These in vitro data correlate well with the aforementioned in vivo data; nevertheless, the relevance of CYP1A differences to the in vivo differences described herein remains to be established.

Hepatotoxicity, expressed as ALT elevations, is a relatively common (20-50%) asymptomatic adverse event in Alzheimer's disease patients after several weeks of treatment with THA (1, 27-30) that is not seen in rats or dogs (4). The N-hydroxylamine derivative of THA, a potentially hepatotoxic metabolite, which might be expected based on THA's aromatic amine structure (31, 32), was tentatively identified as a metabolite in rat microsomes by in situ electrochemical detection (33). However, this metabolite was not observed in plasma or urinary metabolic profiles of rats, dogs, or humans in our investigations wherein the reference standard was available. Another possible metabolic pathway for THA that could lead to hepatotoxicity is N-acetylation (34, 35); however, no evidence of N-acetyl-THA was found in our studies. It is intriguing to speculate that THA metabolism directly plays a role in the susceptibility of patients to THA-induced hepatotoxicity. However, a direct relationship to CYP1A2 activity does not seem to be the case based on results reported by Fontana et al. (36). The authors investigated the relationship between ALT elevations and CYP1A2 activity in Alzheimer's patients using the caffeine breath test as the CYP1A2 probe and found no direct correlation between CYP1A2 levels and ALT elevations. These data would suggest that susceptibility to THA-induced ALT elevations involves more than just CYP1A2 enzyme activity and are consistent with a multifactorial mechanism for THA hepatotoxicity (5). Additional studies are needed to assess the role of other enzyme systems; in particular, detoxication enzymes, in the process of THA-induced hepatotoxicity.