DMD

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

Drug Metabolism and Disposition Fast Forward
First published on June 21, 2006; DOI: 10.1124/dmd.106.010934

0090-9556/06/3409-1615-1623$20.00
DMD 34:1615-1623, 2006

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
dmd.106.010934v1
34/9/1615    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shaffer, C. L.
Right arrow Articles by Fate, G. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shaffer, C. L.
Right arrow Articles by Fate, G. D.

Using a Tritiated Compound to Elucidate Its Preclinical Metabolic and Excretory Pathways in Vivo: Exploring Tritium Exchange Risk

Christopher L. Shaffer, Mithat Gunduz1, Bruce A. Thornburgh, and Gwendolyn D. Fate

Department of Pharmacokinetics, Pharmacodynamics and Metabolism, Pfizer Global Research and Development, Pfizer Inc., Groton, Connecticut (C.L.S., M.G.) and Kalamazoo, Michigan (B.A.T., G.D.F.)

(Received May 3, 2006; accepted June 15, 2006)


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The metabolism and excretion of N-(3R)-1-azabicyclo[2.2.2]oct-3-ylfuro[2,3-c]pyridine-5-carboxamide (1), an agonist of the {alpha}7 nicotinic acetylcholinergic receptor, were determined in both Sprague-Dawley rats and beagle dogs using [3H]1. Initially, 3-tritio-furanopyridine 1 ([3H]1a) was evaluated in pilot mass balance studies by determining total radioactivity recovery and pharmacokinetics in lyophilized excreta and nonlyophilized plasma, respectively. Lower mass balance and much greater circulatory radioactivity exposures were observed in rats than in dogs, with urinary tritiated water (HTO) only detected in rats. The 133-h half-life in rats, possibly due to very slowly eliminated metabolites, was more likely attributable to HTO formed from [3H]1a because of site-specific chemical and/or metabolic 3H instability, which was confirmed by urinary HTO. In contrast, dog data supported 3H stability within [3H]1a. Conflicting cross-species data with [3H]1a suggested species-specific metabolic fates for 1, requiring a 3H form of 1 resistant to 3H loss in rats. Therefore, tritiation of 1 at its furanopyridine C7, a site predicted to be both chemically and metabolically stable, yielded 7-tritio-N-(3R)-1-azabicyclo[2.2.2]oct-3-ylfuro[2,3-c]pyridine-5-carboxamide ditrifluoroacetate ([3H]1b), which allowed in both species the determination of all excretory pathways, total radioactivity pharmacokinetics, and major excretory and circulatory metabolites with complete radioactivity recovery without HTO generation. Definitive metabolite elucidation for 1 using [3H]1b confirmed the suspected species-dependent metabolic susceptibility for 3H loss from [3H]1a in rats, but not dogs, since the majority of rat metabolites resulted from furanopyridine biotransformation. The described studies explore the evaluation of tritium exchange risk from a mechanistic biotransformation perspective and highlight the need for careful deliberation when considering and designing 3H compounds for radiolabeled metabolism studies.

Radioisotope use in biopharmaceutical research has been invaluable in definitively elucidating test compound excretory and metabolic pathways in animals (Dalvie, 2000Go). The most common radionuclide used for these studies is 14C because of its synthetic versatility, safety relative to other radioisotopes, and favorable nuclear properties dictating optimal half-life and specific activity (McCarthy, 2000Go). However, in certain instances, 3H has been used instead of (or in addition to) 14C to attain in vitro (Guroff et al., 1967Go; Kler et al., 1992Go; Linnet, 2004Go) and in vivo (Prakash et al., 1997Go; Ehlhardt et al., 1998Go; Koller-Lucae et al., 1999Go; Rosenborg et al., 1999Go; Gray et al., 2001Go) biotransformation data. Due to the general synthetic ease, low cost, and rapid turnaround of 3H incorporation (Saljoughian and Williams, 2000Go), such high specific activity compounds are desirable when radiolabeled material is needed quickly to answer particular metabolism-related questions during drug discovery or development.

Although a compound's 14C atom may become separated from the substructure retained by the majority of metabolites because of its incorporation at a metabolically labile site (Chasseaud et al., 1974Go; Hawkins et al., 1977Go; Larsson and Lund, 1981Go), there is no concern of its passive chemical exchange leading to nonradiolabeled compound and/or metabolites. Conversely, due to the intrinsic properties of the hydrogen atom, 3H exchange within a molecule may occur readily in aqueous physiological environments of varying pH by chemical (e.g., {alpha}-H exchange during keto-enol tautomerization) or metabolic means (Lewis et al., 1988Go). In either case, 3H exchange ultimately affords tritiated water (HTO), the molecular reporter of 3H loss, resulting in both the absence of radiotracer capabilities from the test compound and/or its metabolites and ambiguous biotransformation data. Hence, the predominant risk associated with using 3H-labeled compounds in biological systems is the potential chemical and metabolic instability of the 3H atom itself at its specific site within the test molecule. For instance, when profiling in vitro or in vivo samples containing 3H metabolites requiring identification, formed HTO might be detected radiochromatographically within the solvent front, potentially resulting in its erroneous identification as a highly polar (and possibly significant) metabolite. Likewise, ambiguity might arise when calculating 3H recovery from biofluids undergoing organic extraction; low organic phase 3H levels may be ascribed to highly polar metabolites, HTO, or both. For total radioactivity PK analyses, 3H exchange causes similar data interpretation complexities as a result of tritium's three-component exponential function biological half-life, ranging from 10 to 300 days in humans (Robertson, 1973Go). If 3H exchange occurs, the total radioactivity AUC is useless because its terminal phase can be attributable to HTO and not necessarily drug-related material (Kim et al., 2004Go). Therefore, to use 3H compounds effectively for drug metabolism studies, one must not only strategically select the theoretically most chemically and metabolically stable molecular site for 3H incorporation but also seek empirical confirmation by quantifying HTO within all samples via lyophilization to determine the true inertness of the 3H atom.


Figure 1
View larger version (9K):
[in this window]
[in a new window]
  		FIG. 1. Chemical structures for [3H]1a and [3H]1b. T, 3H.

 
With these issues in mind, 1, an agonist of the {alpha}7 nicotinic acetylcholinergic receptor (Wishka et al., 2006Go), was selectively monotritiated for radiolabeled mass balance studies in Sprague-Dawley rats and beagle dogs. Initially, [3H]1a (Fig. 1) was generated because of synthetic ease, and its suitability in both species was evaluated in pilot studies by determining 3H recovery in lyophilized urine and feces and 3H AUC using nonlyophilized plasma. Only 80% mass balance was achieved in rats with urinary HTO, accounting for 4% of recovered dose, whereas 95% mass balance was attained in dogs with no detection of HTO. Furthermore, [3H]1a afforded total radioactivity half-lives of 133 and 5 h in rats and dogs, respectively (Fig. 2). The incredibly long half-life in rats was indicative of very slowly eliminated metabolite(s) and/or HTO formed from drug-derived material attributable to site-specific chemical and/or metabolic 3H instability (Fig. 3); the latter was confirmed by urinary HTO. Conversely, dog data suggested 3H stability within [3H]1a as a result of no metabolism of 1 (and the chemical stability of 3H within 1) and/or the conversion of 1 to 3H-retaining metabolites (Fig. 3). Conflicting species-dependent 3H recovery and PK data implicated different metabolic fates for 1 in rats and dogs and demonstrated the need for a 3H form of 1 impervious to 3H loss in rats. Therefore, 3H incorporation at a site predicted to be both chemically and metabolically stable yielded [3H]1b (Fig. 1). Using [3H]1b in rats and dogs, all excretory pathways, total radioactivity PK, and major excretory and circulatory metabolites were identified definitively with complete radioactivity recovery in both species without HTO generation. Studies characterizing and rationalizing the metabolites of 1 are described herein that ultimately explain species-dependent 3H loss with [3H]1a, but not [3H]1b.


Figure 2
View larger version (14K):
[in this window]
[in a new window]
  		FIG. 2. Semilogarithmic (top) and linear (bottom) plots of nonlyophilized plasma total radioactivity concentrations in rats (diamonds) and dogs (triangles) after oral administration of [3H]1a.

 

Figure 3
View larger version (10K):
[in this window]
[in a new window]
  		FIG. 3. Hypothetical chemical- and metabolism-mediated 3H exchange pathways for [3H]1a in vivo. T+, triton; MX, primary metabolite; MY, secondary metabolite.

 


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and Reagents. Compounds 1 (Wishka et al., 2006Go), 2, 3, and 4 were made by the Synthesis Group at Pfizer Global Research and Development (PGRD; Kalamazoo, MI); [3H]1a·2HCl (10 Ci/mmol, 99.9% radiochemical purity) and [3H]1b·2 trifluoroacetic acid (17 Ci/mmol, 100% radiochemical purity) were synthesized (B. D. Maxwell, D. G. Wishka, K. M. Yates, and B. N. Rogers, manuscript submitted) by the Radiochemical Synthesis Group at PGRD. The chemical purity of all synthetic compounds was >99%. Chemicals and solvents of reagent or HPLC grade were supplied by Aldrich Fine Chemical Co. (Milwaukee, WI), Cambridge Isotope Laboratories, Inc. (Cambridge, MA), Fisher Scientific (Pittsburgh, PA), and the J. T. Baker Chemical Co. (Phillipsburg, NJ). All excreta and plasma were collected gravimetrically and stored at –20°C until analysis.

Characterization of Synthetic Standards 1, 2, 3, and 4. The syntheses and full characterization of 1 (Wishka et al., 2006Go) and [3H]1a and [3H]1b (B. D. Maxwell, D. G. Wishka, K. M. Yates, and B. N. Rogers, manuscript submitted) are reported elsewhere. For the characterization of metabolite authentic standards 2, 3, and 4, an OXFORD AS400 spectrometer (OXFORD Instruments, Eynsham Witney, Oxon, UK) was used to obtain 1H NMR spectra at 400 MHz and 1H-decoupled 13C NMR spectra at 100.5 MHz. Proton chemical shifts are reported in ppm ({delta}) relative to tetramethylsilane as inferred from shifts of residual protons in used deuterated solvents (i.e., 4.80 ppm for D2O, 2.50 ppm for d6-DMSO and 3.31 ppm for CD3OD); 13C chemical shifts are in ppm relative to internal solvent carbons (i.e., 39.51 ppm for d6-DMSO and 49.15 ppm for CD3OD).

Compound 2. 1H NMR (400 MHz, D2O) {delta} 1.96 (m, 1H), 2.09 (m, 2H), 2.24 (m, 1H), 2.38 (m, 1H), 3.26 to 3.43 (m, 5H), 3.60 (s, 2H), 3.82 (t, J = 9.2 Hz, 1H), 4.44 (m, 1H), 7.78 (s, 1H), 8.12 (s, 1H); 13C NMR (100.5 MHz, D2O) {delta} 16.96, 21.35, 24.09, 39.35, 44.91, 46.19, 46.63, 52.25, 125.51, 133.98, 137.08, 140.57, 155.26, 167.52, 178.69; ESI: [M+H]+ = 306; elemental analysis calcd for C15H19N3O4 C: 58.35, H: 6.37, N: 13.53; found, C: 58.49, H: 6.38, N: 13.57.

Compound 3. 1H NMR (400 MHz, d6-DMSO) {delta} 1.70 (m, 1H), 1.90 (m, 2H), 2.04 (m, 1H), 2.16 (m, 1H), 3.14 to 3.37 (m, 5H), 3.62 (t, J = 11.4 Hz, 1H), 4.33 (m, 1H), 8.08 (s, 1H), 8.18 (s, 1H), 8.72 (s, 1H), 8.74 (s, 1H), 9.41 (s, 1H); 13C NMR (100.5 MHz, d6-DMSO) {delta} 17.19, 21.51, 24.56, 43.88, 45.22, 45.60, 50.73, 122.18, 124.62, 136.79, 139.26, 163.61, 164.77, 169.10; ESI: [M+H]+ = 292; elemental analysis calcd for C14H17N3O4 C: 57.53, H: 5.90, N: 14.38; found, C: 57.26, H: 5.88, N: 14.26.

Compound 4. 1H NMR (400 MHz, CD3OD) {delta} 2.06 (m, 1H), 2.19 (m, 2H), 2.26 (m, 1H), 2.35 (m, 1H), 3.38 to 3.51 (m, 5H), 3.81 (t, J = 11.6 Hz, 1H), 7.11 (d, J = 2.1 Hz, 1H), 8.11 (d, J = 1.7 Hz, 1H), 8.43 (s, 1H), 8.91 (s, 1H); 13C NMR (100.5 MHz, CD3OD) {delta} 22.26, 25.35, 26.66, 63.12, 63.48, 68.82, 72.32, 108.15, 117.26, 133.73, 136.75, 144.51, 151.69, 155.23, 167.30; ESI: [M+H]+ = 288; elemental analysis calcd for C15H17N3O3 C: 58.71, H: 6.31, N: 13.66; found, C: 58.76, H: 6.39, N: 13.70.

In Vivo Studies with [3H]1a and [3H]1b. The in-life portions of both rat and dog studies using [3H]1a were conducted at PGRD, whereas those for rats and dogs with [3H]1b were performed at Covance, Inc. (Madison, WI) and Charles River Laboratories International, Inc. (Worcester, MA), respectively. All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, and all study animals were fasted overnight before compound administration and for 4 h postdose. Individual animal doses were calculated based on respective pretreatment body weights and a dose volume appropriate for the specific species. The actual amount of dose solution administered to each animal was determined by weighing the loaded dosing syringe before and after it was dispensed.

[3H]1a in Sprague-Dawley Rats. A single dose (8 mg/kg, 400 µCi) of [3H]1a in citrate buffer (50 mM, pH 5) was administered via oral gavage to intact male rats. The study included two groups of rats: in group 1 (two males), urine, feces, and cage rinse were collected from animals predose and in 24-h intervals over 4 days postdose; and, in group 2 (one per time point), blood samples from animals euthanized predose and at 0.5, 2, and 8 h postdose were collected by exsanguination after cardiac puncture into tubes containing Na2EDTA and processed to obtain plasma.

[3H]1a in Beagle Dogs. A single dose (15 mg/kg, 640 µCi) of [3H]1a in citrate buffer (50 mM, pH 5) was administered orally to a single intact male dog. From this one dog, excreta and cage debris/rinse were collected predose and in 24-h intervals over 2 days, whereas blood samples (approximately 6 ml) were collected into heparinized tubes via venipuncture of a cephalic or jugular vein predose and at 0.5, 1, 2, 4, 8, and 24 h postdose and processed to obtain plasma.

[3H]1b in Sprague-Dawley Rats. A single dose (5 mg/kg, 25 µCi) of [3H]1b in citrate buffer (50 mM, pH 5) was administered via oral gavage to each intact rat. The study included two groups of rats: in group 1 (four per sex), from intact animals, urine was collected predose and from 0 to 12 and 12 to 24 h during day 1 and in 24-h intervals from 24 to 168 h postdose. From the same animals, feces and cage rinse were collected predose and in 24-h intervals from 0 to 168 h postdose, and in group 2 (one/sex/time point), blood samples from intact animals euthanized predose and at 0.5, 1, 2, 4, 8, and 24 h postdose were collected by cardiac puncture into tubes containing Na2EDTA and processed to obtain plasma.

[3H]1b in Beagle Dogs. A single dose (10 mg/kg, 480 µCi) of [3H]1b in citrate buffer (50 mM, pH 5) was administered orally to intact dogs (two per sex) from which excreta and cage debris/rinse were collected as described for [3H]1b in rats. Blood samples (approximately 6 ml) were collected into heparinized tubes via venipuncture of a cephalic or jugular vein predose and at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h postdose and processed to obtain plasma. Control plasma was harvested from blood collected from untreated dogs.

Determination of Radioactivity within Urine and Plasma from Animals Receiving [3H]1a. Due to the exploratory nature of the [3H]1a studies, only urine was subjected to lyophilization to determine its HTO concentration. Therefore, duplicate nonlyophilized gravimetric aliquots of urine (0.5 g) and plasma (0.1 g) from each time point were mixed with Ultima Gold (PerkinElmer Life and Analytical Sciences, Inc., Wellesley, MA) liquid scintillation cocktail (15 ml) and counted for 2 min by a Packard Tri-Carb 1900CA or 2300TR liquid scintillation counter (Packard BioScience, Co., Meriden, CT). In parallel, duplicate gravimetric aliquots (2 g) of urine from each time point were frozen, lyophilized using a FreeZone 4.5 Benchtop Freeze Dry System (Labconco Corp., Kansas City, MO), reconstituted in Milli-Q H2O (0.2 g) and liquid scintillation cocktail (15 ml), and analyzed for total radioactivity by LSC for 2 min. The difference in total radioactivity within each urine sample before and after lyophilization was attributed to HTO. Scintillation counter data were automatically corrected for counting efficiency using an external standardization technique and an instrument-stored quench curve generated from a series of sealed quench standards.

Determination of Radioactivity within Urine and Plasma from Animals Receiving [3H]1b. The following procedure was undertaken to quantify both total radioactivity and HTO within each urine and plasma sample: triplicate gravimetric aliquots (0.2–0.5 g for urine, 0.05 g for plasma) from each time point were mixed with TruCount scintillation cocktail (15 ml) and counted for 2 min by a model LS 6000 or LS 6500 liquid scintillation counter (Beckman Coulter, Inc., Fullerton, CA). In parallel, triplicate gravimetric aliquots of similar mass from each time point were frozen, lyophilized, reconstituted, and analyzed for total radioactivity as described previously. The difference in total radioactivity within each sample without and with lyophilization was ascribed to HTO. Scintillation counter data were automatically corrected for counting efficiency as described for [3H]1a.

Determination of Radioactivity within Feces and Cage Debris/Rinse from Animals Receiving [3H]1a or [3H]1b. Fecal samples were homogenized with Milli-Q H2O [20% (w/w), feces/H2O] using a probe-type or Stomacher homogenizer. Cage debris/rinse samples (collected in 50% reagent alcohol in H2O) were homogenized directly with a probe-type homogenizer. Triplicate gravimetric aliquots (0.4–0.7 g for either homogenate) were transferred into tared cones and pads, weighed, dried for a minimum of 24 h at ambient temperature, and combusted before radioanalysis. Sample combustion was performed using a Packard Instruments model A0387 sample oxidizer (Packard BioScience). Combustion efficiency using a 3H standard was determined daily before the combustion of study samples, and the measured radioactivity content in feces and cage debris/rinse was adjusted using daily combustion efficiency values. Liberated HTO was trapped in Monophase-S (Perkin Elmer Life and Analytical Sciences), mixed in Perma-Fluor-E scintillation fluid (Packard BioScience), and quantified in a model LS 6000 or LS 6500 liquid scintillation counter (Beckman Coulter, Inc.) for 10 min. All combustion-related scintillation counter data were corrected for counting efficiency as explained previously.

Pharmacokinetic Calculations. PK parameters were calculated for each gender (rats) or animal (dogs) by noncompartmental analyses using WinNonlin version 3.2 (Pharsight Corp., Mountain View, CA). Values used to determine total radioactivity PK parameters were calculated by converting the raw data generated by LSC to concentrations (ng-Eq/ml) using the specific activity of administered [3H]1a or [3H]1b. The AUC0-tlast was calculated using the linear trapezoidal method, kel was determined by linear regression of the log concentration-versus-time data during the last observable elimination phase, and half-life (t1/2) was calculated as 0.693/kel. Both Cmax and the time of its occurrence (Tmax) were taken directly from the concentration-versus-time data. Means and standard deviations were calculated when half or greater of the values exceeded the lower limit of quantification for total radioactivity (2 and 10 ng-Eq/ml for rats and dogs, respectively). A value of 0 was used when a measured value was less than the lower limit of quantification.

Preparation of [3H]1b Samples for Metabolite Profiling and Identification. At each step during the sample preparation of all biological matrices, total radioactivity levels were determined by LSC for recovery calculations. After preparation, all samples were analyzed as described below by LC-MS/MS with radiometric detection. Predose and blank samples served as controls for determining background radioactivity and endogenous, nondrug-related ions observed within respective matrices or their extracts by LC-MS/MS.

Urine. LSC analysis of both pre- and postlyophilization urine samples for each time point from each animal found no significant difference (i.e., ±5%) in the amount of radioactivity contained within each sample suggesting that no appreciable amount of HTO was contained within this matrix. Thus, nonlyophilized urine samples from each rat (collected from 0–48 h postdose representing >93% of total urine radioactivity) and dog (0–24 or 0–48 h samples, >95% of total urine radioactivity) were pooled proportional to the amount of urine in each sampling period to afford the analytical sample.

Feces. Due to minimal amounts of fecal radioactivity recovered from dogs (<3% of dose), only rat feces were profiled. Fecal homogenates (6 to 14 g) from each rat collected from 0 to 48 h postdose, representing >94% of total fecal radioactivity were pooled proportional to the amount of feces in each sampling period. Pooled homogenates were diluted with acetonitrile (MeCN; 2 ml/g homogenate), vortex-mixed, and centrifuged (1811 rcf for 10 min), and the resulting supernatants were isolated. If necessary, the remaining fecal pellets were extracted further with 40% H2O in MeCN (4 x 6 ml) until >90% of the radioactivity from each pooled sample was recovered. The supernatants were concentrated to near dryness and reconstituted in 30% MeCN in 10 mM ammonium formate, pH 3.4 (750 µl; solvent A) for analysis. Although fecal homogenates did not undergo lyophilization for the determination of HTO content, HTO within analytical samples was evaluated by monitoring the elution of a peak within or close to the solvent front of the radiochromatogram. In all rat fecal extracts analyzed, no radioactive peak corresponding to ≥1% of the administered dose was observed within the radiochromatogram during the first 15 min after injection.

Rat Plasma. Nonlyophilized plasma from blood samples collected at 0.5, 1, 2, 4, and 8 h postdose were used for circulatory metabolite profiling and identification because approximately 90% of the lyophilized total radioactivity AUC0–24 was captured by its AUC0–8, and approximately 80% of the total nonlyophilized radioactivity AUC0–8 was captured by its lyophilized AUC0–8, suggesting little contribution to the nonlyophilized total radioactivity AUC0–8 by HTO. Plasma samples were pooled within gender according to the method of Hamilton et al. (1981Go); i.e., 17, 25, 50, 100, and 67 µl, respectively, of plasma from each time point sample were combined to afford 259 µl of pooled plasma for each gender profile. To remove dissolved proteins, the male and female pooled plasma samples were diluted with MeCN (1.04 ml), vortex-mixed for 30 min, and centrifuged (2465 rcf for 10 min), and the resulting supernatants, which contained >83% of the radioactivity from each pooled plasma sample, were isolated. Each supernatant was concentrated to near dryness at 35°C under N2, and reconstituted in solvent A (150 µl) for analysis.

Dog Plasma. Nonlyophilized plasma from blood samples collected at 0.25, 0.5, 1, 2, 3, 4, and 6 h postdose were used for circulatory metabolite profiling and identification because approximately 90% of the total radioactivity AUC0–12 was captured by its AUC0–6 using either nonlyophilized or lyophilized sample data, and approximately 100% of the total nonlyophilized radioactivity AUC0–6 was captured by its lyophilized AUC0–6, suggesting no HTO contribution to the nonlyophilized total radioactivity AUC0–6. Plasma samples were pooled as described above such that 125, 375, 750, 1000, 1000, 1500, and 1000 µl, respectively, of plasma from each time point sample per animal were combined. Thus, because two dogs per sex were dosed, a total of 11.5 ml of pooled plasma was obtained for each gender profile. The pooled plasma samples were diluted with MeCN (23 ml), vortex-mixed for 30 min, and centrifuged (2465 rcf for 10 min), and the resulting supernatants, which contained >94% of the radioactivity from each pooled plasma sample, were isolated. Each supernatant was concentrated to near dryness at 35°C under N2, and reconstituted in solvent A (300 µl) for analysis.

Metabolite Profiling and Identification Using [3H]1b Samples. Samples were analyzed by an LC-MS/MS, comprised of a PE Sciex API-3000 tandem quadrupole mass spectrometer with a Turbo Ionspray interface (Perkin Elmer Life and Analytical Sciences), two Shimadzu LC-10A HPLC pumps (Shimadzu USA, Columbia, MD), and a CTC PAL Autosampler (LEAP Technologies, Carrboro, NC), in series with a Beta-RAM radiometric detector (IN/US Systems, Inc., Tampa, FL) containing a liquid scintillant cell (500 µl). Analytes within sample aliquots (20–100 µl) were eluted on a Phenomenex Luna Phenyl-hexyl analytical column (5 µ, 4.6 x 250 mm) at 1 ml/min with 10 mM ammonium formate, pH 3.4 (solvent B), and MeCN (solvent C). The following two-step gradient was used: 0 to 10 min, 2% solvent C in solvent B; 10 to 30 min, 2 to 35% C in B; and 30 to 32 min, 35 to 90% C in B. After the elution of 1 and its metabolites, the column was washed with 90% C in B for 3 min and then returned over 3 min to 2% C in B where it remained for 7 min before the next injection. For each matrix, >94% of the radioactivity injected onto the column eluted during the first 32 min of the gradient program. HPLC effluent was split 1:9 between the mass spectrometer and the radiometric flow detector; liquid scintillation cocktail flowed at 3 ml/min to the radiometric detector. Mass spectral data were collected using positive ionization in full, precursor ion, neutral loss, product ion, and multiple-reaction monitoring scanning modes. Instrument settings and potentials were adjusted to provide optimal data in each mode. Masschrom version 1.1.1 (Perkin Elmer Life and Analytical Sciences) and Winflow version 1.4 (IN/US Systems, Inc.) software were used for the acquisition and processing of mass spectral and radiochromatographic data, respectively.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Excretion of Total Radioactivity in Rats and Dogs Administered [3H]1a or [3H]1b. Due to no readily apparent gender-related differences in overall excretory routes or mass recoveries in either species, averages (with standard deviations if applicable) of combined male and female radioactivity recovery values in rats and dogs after [3H]1b administration are listed in Table 1; [3H]1a pilot study mass balance values are also tabulated for comparison and only consist of two rats and one dog. The excretion of total radioactivity derived from either [3H]1a or [3H]1b was rapid in both species; on average, >70% of the administered radioactivity was excreted within the first 24 h predominantly renally. For [3H]1a, only 80% mass balance was achieved in rats with urinary HTO accounting for 4% of recovered dose, whereas 95% mass balance was attained in dogs with no detection of HTO. For [3H]1b, mass balance was achieved in both rats and dogs with <1% of the dose detected as HTO in cumulative excreta from either species.


View this table:
[in this window]
[in a new window]
  		TABLE 1 Cross-species mass recoveries (% of dose) and excretory routes after oral administration of [3H]1a or [3H]1b

 

Pharmacokinetics of Total Radioactivity in Rats Administered [3H]1a or [3H]1b. For [3H]1a-derived data, total radioactivity plasma concentration-versus-time curves are plotted in Fig. 2, and PK parameters are listed in Table 2. For [3H]1b-related data, all reported PK parameters (Table 2) are an average of genders as a result of no readily apparent gender-related differences in systemic exposures to total radioactivity; average total radioactivity plasma concentration-versus-time curves for both nonlyophilized and lyophilized plasma are plotted in Fig. 4. For [3H]1b, mean total radioactivity Tmax and Cmax were identical for nonlyophilized and lyophilized plasma samples, whereas the nonlyophilized plasma t1/2 was greater (31 versus 24 h). Interestingly, [3H]1b total radioactivity AUC ratios of lyophilized versus nonlyophilized plasma decreased for AUC0–8 (0.8), AUC0–24 (0.5), and AUC0-{infty} (0.2), suggestive of HTO within nonlyophilized plasma from blood drawn at later time points postdose, which rationalizes the longer nonlyophilized plasma t1/2. If HTO was indeed present in plasma, it must have arisen from a minimal amount of drug-related material since <1% of the [3H]1b dose detected in rat urine was attributable to HTO, and overall mass recovery was high (93%).


View this table:
[in this window]
[in a new window]
  		TABLE 2 Cross-species pharmacokinetics summary for total radioactivity after oral administration of [3H]1a or [3H]1b

 

Figure 4
View larger version (14K):
[in this window]
[in a new window]
  		FIG. 4. Semilogarithmic (top) and linear (bottom) plots of nonlyophilized (filled diamonds) and lyophilized (unfilled diamonds) plasma total radioactivity concentrations in rats after oral administration of [3H]1b.

 

A comparison of [3H]1a (8 mg/kg) to [3H]1b (5 mg/kg) nonlyophilized plasma total radioactivity PK parameters clearly shows a significant difference in total radioactivity exposure (Table 2). Although Tmax was identical and Cmax was dose-proportional for both radioisomers, total radioactivity AUC ratios for [3H]1a versus [3H]1b increased nonlinearly for AUC0–8 (4.5) and AUC0-{infty} (28). Therefore, nonlyophilized plasma total radioactivity half-lives for [3H]1a and [3H]1b were 133 and 31 h, respectively, with the former being consistent with that of 85 to 98 h, reported previously for HTO in rats (Richmond et al., 1962Go; Foy, 1964Go; Wheeler et al., 1972Go). Although greater 3H exposure in rats attained by [3H]1a relative to [3H]1b could hypothetically be due to an isotope effect, altering the metabolic profile to longer-lived and/or more slowly cleared metabolite(s), a combination of PK data indicative of substantial systemic HTO, the presence of urinary HTO, and incomplete mass balance for [3H]1a versus [3H]1b implicated, instead, HTO arising from the unique loss of tritium originally within [3H]1a.

Pharmacokinetics of Total Radioactivity in Dogs Administered [3H]1a or [3H]1b. For [3H]1a, total radioactivity plasma concentration-versus-time curves are plotted in Fig. 2, and PK parameters are listed in Table 2. For [3H]1b-related data, all reported PK parameters (Table 2) are an average of genders as a result of no readily apparent gender-related differences in systemic exposures to total radioactivity; average total radioactivity plasma concentration-versus-time curves for both nonlyophilized and lyophilized plasma are plotted in Fig. 5. For [3H]1b, mean total radioactivity Tmax, Cmax, and t1/2 values were essentially identical for nonlyophilized and lyophilized plasma samples, confirming the absence of HTO in excreta. In addition, nonlyophilized plasma Cmax and AUC values for [3H]1a (15 mg/kg) were 1.5-fold greater than those for [3H]1b (10 mg/kg), consistent with linear PK for total radioactivity; identical Tmax and similar t1/2 were also observed. These PK data, supplemented by the absence of HTO in excreta and full mass recovery in both dog studies, confirmed the chemical and metabolic 3H stability within both [3H]1a and [3H]1b in dogs.


Figure 5
View larger version (13K):
[in this window]
[in a new window]
  		FIG. 5. Semilogarithmic (top) and linear (bottom) plots of mean nonlyophilized (filled triangles) and lyophilized (unfilled triangles) plasma total radioactivity concentrations in dogs after oral administration of [3H]1b.

 

Structural Rationalization of 1 and Its Metabolites. Compound 1 had a protonated molecular ion of m/z 272 and an LC tR of approximately 24.4 min. The CID product ion spectrum of m/z 272 contained fragment ions with m/z 163, 146, 118, 110, and 82 (Fig. 6). Therefore, precursor ion scanning of diagnostic fragment ions m/z 118 and 110 determined if metabolites of 1 were modified on its quinuclidine or furanopyridine moieties, respectively. A summary of all metabolite LC-MS/MS data is found in Table 3. The identification of a metabolite as a synthetic standard was determined by the compounds' indistinguishable CID spectra and LC tR, as well as an increase in metabolite MS peak height upon the addition of the authentic standard to the analytical sample.


Figure 6
View larger version (9K):
[in this window]
[in a new window]
  		FIG. 6. Collision-induced dissociation fragmentation pathway for protonated 1 (m/z 272).

 

View this table:
[in this window]
[in a new window]
  		TABLE 3 Chromatographic and mass spectral data for 1 and its metabolites

 

Quantitative Profile of [3H]1b and Its Metabolites in Rat and Dog Excreta and Plasma. In addition to 1, six metabolites were observed in rat urine and five in rat feces (Table 4). Three metabolites were tentatively identified as pyridinyl-{alpha}-hydroxy acetic acid (M1), dihydroxy-ethyl-pyridinol (M2), and hydroxyl-ethyl-pyridinol (M3), whereas the other three metabolites were 2, 3, and 4. In dog urine, 1, 2, and 4 were detected. On average in rat and dog urine, 40.0 and 9.3%, respectively, of the dose was unchanged 1.


View this table:
[in this window]
[in a new window]
  		TABLE 4 Cross-species comparison of excretory and circulatory metabolite profiles after oral administration of [3H]1b

 

In addition to 1, metabolites 2, M3, 3, and 4 were identified in rat plasma, whereas only 2 and 4 were observed in dog plasma (Table 4). On average in plasma, 1 comprised 29.7 and 10.1% of total circulatory radioactivity in rats and dogs, respectively.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Due to the cited caveats for using 3H compounds in radiolabeled biotransformation studies, 3H stability within [3H]1a was first evaluated in rat and dog pilot studies by monitoring its 3H recoveries in excreta (Table 1) and nonlyophilized plasma radioactivity PK curves (Fig. 2). These studies generated contrasting results, with rat and dog total 3H half-lives of 133 and 5 h, respectively. Although the extensive half-life in rats could hypothetically be attributed to long-lived circulatory metabolites, it seemed more reasonably explained by HTO, which has a reported half-life of 85 to 98 h in rats (Richmond et al., 1962Go; Foy, 1964Go; Wheeler et al., 1972Go), formed by 3H loss from [3H]1a; this was confirmed by urinary HTO and low overall mass recovery. The short total radioactivity half-life and high mass recovery in dogs, both indicative of no HTO generation as confirmed by lyophilized urine, further suggested that 3H loss in rats was due to a species-specific difference in the metabolism of [3H]1a. These data and the known metabolic vulnerability of furans (Dalvie et al., 2002Go) prompted the radiosynthesis of [3H]1b with strategic monotritiation at a site envisaged to be both chemically and metabolically inert. Subsequent mass balance studies were conducted with [3H]1b in both species with expected lower, yet very relevant, 3H exchange risk that was evaluated by HTO quantification within urine and plasma via lyophilization.

The metabolic and excretory pathways of 1 in rats and dogs were determined definitively using [3H]1b, with ≥86% of administered radioactivity recovered on average in both species (Table 1). Lyophilization demonstrated in both species that HTO contributed to <1% of the dose recovered in urine. It also detected HTO in rat plasma after 8 h postdose but not in dog plasma over 24 h (Table 2). For rats, the insignificant amount of HTO in urine, the predominant route of radioactivity excretion, suggested essentially complete 3H retention within [3H]1b in vivo. Nonetheless, enough HTO (beyond experimental error) was present in rat plasma to show meaningful differences in radioactivity PK curves for nonlyophilized versus lyophilized samples (Fig. 4); HTO formation from [3H]1b is proposed by H/3H exchange via resonance of oxygen lone pair electrons with C7 in pyridinol metabolites. The longer turnover half-time of HTO relative to 1 and its metabolites resulted in an increasing HTO-related contribution to total circulatory radioactivity over time. This phenomenon manifested in an extended terminal phase of the nonlyophilized total radioactivity plasma PK curve, which is conspicuously absent in that from lyophilized plasma because of the physical removal of HTO during the freeze-drying process. This observation stresses the need to lyophilize all plasma samples when conducting 3H-labeled drug metabolism studies since lyophilized samples ensure the true AUC and kel of non-HTO-related radioactivity.

Proposed metabolic pathways, incorporating customary furan biotransformation mechanisms (Schmid et al., 1980Go; Kobayashi et al., 1987Go; Sahali-Sahly et al., 1996Go), of 1 in rats and dogs are presented in Fig. 7. Within both species, the two metabolically susceptible sites in 1 were its quinuclidine to afford 4 and its furanopyridine to generate all other metabolites. Definitive metabolite elucidation for 1 using [3H]1b explained the 3H instability within [3H]1a observed in rats but not dogs. For [3H]1a, the rationale for 3H loss before identifying its metabolites with [3H]1b was chemical exchange within 1 and/or its metabolites (Fig. 3, paths a and c) and/or metabolism-mediated exchange during the biotransformation of 1 and/or its metabolites (Fig. 3, paths b and d). These possibilities for [3H]1a are explored below using definitive [3H]1b results while considering the different sites of monotritiation within each radioisomer.


Figure 7
View larger version (19K):
[in this window]
[in a new window]
  		FIG. 7. An overview of the proposed metabolic and mechanistic pathways for 1 in rats and dogs. Bold numerical designations are for structures that were detected analytically; all other structures were not detected and are solely putative metabolite intermediates. [O], enzyme-mediated oxidation; H2, enzyme-mediated reduction; Ta, site of 3H within [3H]1a; Tb, site of 3H within [3H]1b.

 
For the evaluation of chemically mediated 3H exchange within [3H]1a, hypothetical "electron-pushing" suggests its C3 3H could exchange with solvent protons to afford nonradiolabeled 1 and HTO (Fig. 3, path a). Retrospectively, this is an important consideration, because it was determined with [3H]1b that 1 accounted for 41 and 9% of the dose excreted in rats and dogs, respectively. However, although such chemical exchange could have contributed significantly to 3H loss for [3H]1a in vivo, it proved minimal (if any) based on the dog pilot study with [3H]1a, which generated a total radioactivity half-life of 5 h and no HTO within excreta. Since the furanopyridine of [3H]1a is unchanged in 4, which comprised 11 and 63% of the excreted dose in rats and dogs, respectively, it too maintained its C3 3H. For 2, if it retained 3H after its formation as predicted, then 3H loss should not occur since carboxylic acid {alpha}-Hs typically are not acidic enough for solvent exchange. Likewise, passive 3H exchange would not be expected within M1. For putative metabolites M2 and M3, the extent of C3 3H exchange is unknown, although it can be considered mechanistically (Fig. 7). Based on proposed extensive intramolecular hydrogen bonding within M2 that might prevent chemically mediated 3H loss, 3H exchange is believed to most likely occur for M3. Thus, per [3H]1b data (Table 4), 14% of administered [3H]1a would be at risk for passive chemical exchange.

From a metabolism-mediated perspective, 3H loss within [3H]1a would unquestionably occur upon its conversion to 3 and conceivably occur during its transformation to M1 via 2 and to M2 via M3 (Fig. 7). For 3, 3H loss is apparent as a result of C3 within [3H]1a being quaternary in 3; in studies with [3H]1b, 3 accounted for 3.9% of the excreted dose, confirming this as a 3H exchange pathway for [3H]1a in rats. If 2 completely retained the [3H]1a radiolabel as predicted, 3H would also be expected to remain predominantly within M1 if it arose directly from 2 based on the mechanism of cytochrome P450-mediated aliphatic hydroxylations (Groves and McClusky, 1976Go). Due to kinetic deuterium isotope effects for such biotransformations (Atkinson et al., 1994Go) and the inherently stronger bond strength of C–3H versus C–H (Hu et al., 2003Go), hydroxylation of 2 to M1 is expected to occur mainly (probably exclusively) via removal of the carboxylic acid {alpha}-H resulting in mostly (or complete) 3H retention. Because this hydroxylation may not occur entirely selectively, 3H loss might occur during formation of M1 from 2, but such 3H exchange concerns diminish if M1 arises from the aldehyde tautomer of a dihydrodiol intermediate (Fig. 7). For M2 and M3, the projected extent of 3H loss from [3H]1a during their formation is more difficult to assess because their structures are unconfirmed. However, if these molecules and proposed formation pathways are indeed correct, 3H loss would only predominantly occur as M3 is converted to M2 via an epoxide pathway than by direct aliphatic hydroxylation as discussed above; M2 and M3 comprised 17% of [3H]1b-derived material excreted in rats. Thus, if 3H loss occurred during the formation of all metabolites for which it is mechanistically conceivable, then per [3H]1b rat data (Table 4), 11% of dosed [3H]1a would be at risk for metabolism-mediated 3H exchange.

Citing the rationale outlined above for those metabolites that may lose their radiolabel by chemical (M3) or metabolic (M1, M2, and 3) means, a maximum of 25 and 0% of administered [3H]1a would be predicted to undergo 3H loss in rats and dogs, respectively. In rats, although either [3H]1a or [3H]1b afforded approximately 70% dose recovery in urine, fecal dose recovery for [3H]1b was 17 percentage points greater than that for [3H]1a (Table 1), which equals the fecal [3H]1b-related recovery comprised by M1, M2, M3, and 3 (Table 4). Assuming that M1 remains radiolabeled, M2, M3, and 3 still comprise 13% of [3H]1b-related fecal dose. Based on the mechanistic rationale discussed above, 3H loss from [3H]1a upon formation of M2, M3, or 3, and the excretion of these nonradiolabeled metabolites into feces while HTO remained systemically beyond the excreta collection interval, may explain the difference in fecal (and total) dose recovery for [3H]1a versus [3H]1b in rats.

Strategic 3H placement within 1 to afford [3H]1b at a site predicted to be both chemically and metabolically stable demonstrated that 3H loss in [3H]1a was attributable to primarily species-specific metabolism-dependent instability since the majority of rat metabolites resulted from furanopyridine biotransformation. Interestingly, consistent with pilot [3H]1a data in dogs, the identified canine metabolites using [3H]1b confirmed no metabolism-mediated 3H exchange would be predicted for [3H]1a since nearly complete metabolism of 1 occurred at its quinuclidine. Furthermore, the detection of only 1, 2, and 4 in dogs administered [3H]1b also verified that the 3H label within [3H]1a was chemically stable in these compounds, further implicating the species-specific metabolites M2, M3, and 3 (and possibly M1) for 3H loss from [3H]1a in rats.

The presented studies underscore the importance of careful deliberation when considering 3H compounds for radiolabeled metabolism studies. Ironically, due to the intrinsic properties of the hydrogen atom, facile 3H exchange, which may readily afford a 3H compound synthetically, is often the culprit for compound unacceptability for biotransformation studies. To ascertain this "exchange risk" quantitatively, all biological samples must undergo lyophilization to determine their HTO content, the amount of which determines unequivocally the suitability of the tritiation site from a biotransformation standpoint. Although 3H-labeled compounds may instinctively be more cost effective and time efficient to synthesize than their 14C brethren, such compounds from a metabolism perspective are only as good as the chemical and metabolic stability of their tritium atom.


   Acknowledgments
 
We acknowledge Drs. Marc B. Skaddan and Brad D. Maxwell for the synthesis and purification of [3H]1a and [3H]1b, respectively, and Dr. Donn G. Wishka and Karen M. Yates for the synthesis and purification of 1, 2, 3, and 4.


   Footnotes
 
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.010934.

ABBREVIATIONS: HTO, tritiated water; PK, pharmacokinetic(s); AUC, area under the plasma concentration-time curve; 1, N-(3R)-1-azabicyclo[2.2.2]oct-3-ylfuro[2,3-c]pyridine-5-carboxamide (2E)-2-butanedioate; [3H]1a, 3-tritio-N-(3R)-1-azabicyclo[2.2.2]oct-3-ylfuro[2,3-c]pyridine-5-carboxamide hydrochloride; [3H]1b, 7-tritio-N-(3R)-1-azabicyclo[2.2.2]oct-3-ylfuro[2,3-c]pyridine-5-carboxamide ditrifluoroacetate; 2, N-(3R)-1-azabicyclo[2.2.2]oct-3-ylcarbamoyl-5-hydroxypyridin-4-yl-acetic acid; 3, N-(3R)-1-azabicyclo[2.2.2]oct-3-ylcarbamoyl-5-hydroxypyridine-4-carboxylic acid; 4, N-(3R)-1-azabicyclo[2.2.2]oct-3-ylfuro[2,3-c]pyridine-5-carboxamide-1-N-oxide; PGRD, Pfizer Global Research and Development; HPLC, high-performance liquid chromatography; DMSO, dimethyl sulfoxide; ESI, electrospray ionization; LSC, liquid scintillation counting; LC-MS/MS, liquid chromatography-tandem mass spectrometry; rcf, relative centrifugal force; LC tR, liquid chromatography retention time; CID, collision-induced dissociation.

1 Current affiliation: Novartis Institutes for BioMedical Research, Department of Metabolism and Pharmacokinetics, Cambridge, MA. Back

Address correspondence to: Dr. Christopher L. Shaffer, Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton/New London Laboratories, Pfizer Inc., Eastern Point Road, MS 8220-4186, Groton, CT 06340. E-mail: christopher.l.shaffer{at}pfizer.com


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 


Atkinson JK, Hollenberg PF, Ingold KU, Johnson CC, Le Tadic M-H, Newcomb M, and Putt DA (1994) Cytochrome P450-catalyzed hydroxylation of hydrocarbons: kinetic deuterium isotope effects for the hydroxylation of an ultrafast radical clock. Biochemistry 33: 10630–10637.[CrossRef][Medline]

Chasseaud LF, Fry BJ, and Gros PM (1974) Metabolic and pharmacokinetic study of bucloxic acid. II. Pharmacokinetic study in the baboon. Arzneim-Forsch/Drug Res 24: 1390–1397.

Dalvie D (2000) Recent advances in the applications of radioisotopes in drug metabolism, toxicology and pharmacokinetics. Curr Pharmaceut Des 6: 1009–1028.

Dalvie DK, Kalgutkar AS, Khojasteh-Bakht SC, Obach RS, and O'Donnell JP (2002) Biotransformation reactions of five-membered aromatic heterocyclic rings. Chem Res Toxicol 15: 269–299.[CrossRef][Medline]

Ehlhardt WJ, Woodland JM, Baughman TM, Vandenbranden M, Wrighton SA, Kroin JS, Norman BH, and Maple SR (1998) Liquid chromatography/nuclear magnetic resonance spectroscopy and liquid chromatography/mass spectrometry identification of novel metabolites of multidrug resistance modulator LY335979 in rat bile and human liver microsomal incubations. Drug Metab Dispos 26: 42–51.[Abstract/Free Full Text]

Foy JM (1964) The biological half-life of tritiated water in the mouse, rat, guinea-pig and rabbit under tropical conditions and the effect of climate and saline drinking on the biological half-life of tritiated water in the rat. J Cell Comp Physiol 64: 279–282.[CrossRef]

Gray A, Wilkinson DJ, and Seddon H (2001) The use of tritium radiolabelled compounds in the investigation of the DMPK properties of new pharmaceuticals, in Synthesis and Applications of Isotopically Labelled Compounds, Volume 7 (Pleiss U and Voges R eds) pp 496–499, John Wiley & Sons, Ltd., West Sussex, UK.

Groves JT and McClusky GA (1976) Aliphatic hydroxylation via oxygen rebound. Oxygen transfer catalyzed by iron. J Am Chem Soc 98: 859–861.[CrossRef]

Guroff G, Daly JW, Jerina DM, Renson J, Witkop B, and Udenfriend S (1967) Hydroxylation-induced migration: the NIH shift. Science (Wash DC) 15: 1524–1530.

Hamilton RA, Garnett WR, and Kline BJ (1981) Determination of mean valproic acid serum levels by assay of a single pooled sample. Clin Pharmacol Ther 29: 408–413.[Medline]

Hawkins DR, Weston KT, Chasseaud LF, and Franklin ER (1977) Fate of methoprene (isopropyl (2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate) in rats. J Agr Food Chem 25: 398–403.[CrossRef]

Hu Y, Dehal SS, Hynd G, Jones GB, and Kupfer D (2003) CYP2D6-mediated catalysis of tamoxifen aromatic hydroxylation with an NIH shift: similar hydroxylation mechanism in chicken, rat and human liver microsomes. Xenobiotica 33: 141–151.[CrossRef][Medline]

Kim H, Prelusky D, Wang L, Hesk D, Palamanda J, and Nomeir AA (2004) The importance of radiochemical analysis of biological fluids before and after lyophilization from animals dosed with [3H]-labeled compounds in drug discovery. Am Pharmaceut Rev 7: 44–48.

Kler RS, Sherratt HS, and Turnbull DM (1992) The measurement of mitochondrial ß-oxidation by release of 3H2O from [9,10-3H]hexadecanoate: application to skeletal muscle and the use of inhibitors as models of metabolic disease. Biochem Med Metab Biol 47: 145–156.[CrossRef][Medline]

Kobayashi T, Sugihara J, and Harigaya S (1987) Mechanism of metabolic cleavage of a furan ring. Drug Metab Dispos 15: 877–881.[Abstract]

Koller-Lucae SKM, Suter MJ-F, Rentsch KM, Schott H, and Schwendener RA (1999) Metabolism of the new liposomal anticancer drug N4-octadecyl-1-ß-D-arabinofuranosylcytosine in mice. Drug Metab Dispos 27: 342–350.[Abstract/Free Full Text]

Larsson H and Lund J (1981) Metabolism of femoxetine. Acta Pharmacol Toxicol 48: 424–432.

Lewis CJ, Havler ME, Humphrey MJ, Lloyd-Jones JG, McCleavy MA, Muir NC, and Waltham K (1988) The pharmacokinetics and metabolism of idazoxan in the rat. Xenobiotica 18: 519–532.[Medline]

Linnet K (2004) In vitro microsomal metabolism of imipramine under conditions mimicking the in vivo steady-state situation. Hum Psychopharmacol 19: 31–36.[CrossRef][Medline]

McCarthy KE (2000) Recent advances in the design and synthesis of carbon-14 labelled pharmaceuticals from small molecule precursors. Curr Pharmaceut Des 6: 1057–1083.

Prakash C, Kamel A, Gummerus J, and Wilner K (1997) Metabolism and excretion of a new antipsychotic drug, ziprasidone, in humans. Drug Metab Dispos 25: 863–872.[Abstract/Free Full Text]

Richmond CR, Langham WH, and Trujillo TT (1962) Comparative metabolism of tritiated water by mammals. J Cell Comp Physiol 59: 45–53.[CrossRef][Medline]

Robertson JS (1973) Tritium turnover rates in mammals, in Tritium Symposium (Moghissi AA ed) pp 322–327, Messenger Graphics, Phoenix, AZ.

Rosenborg J, Larsson P, Tegner K, and Hallstrom G (1999) Mass balance and metabolism of [3H]formoterol in healthy men after combined i.v. and oral administration-mimicking inhalation. Drug Metab Dispos 27: 1104–1116.[Abstract/Free Full Text]

Sahali-Sahly Y, Balani SK, Lin JH, and Baillie TA (1996) In vitro studies on the metabolic activation of the furanopyridine L-754,394, a highly potent and selective mechanism-based inhibitor of cytochrome P450 3A4. Chem Res Toxicol 9: 1007–1012.[CrossRef][Medline]

Saljoughian M and Williams PG (2000) Recent developments in tritium incorporation for radiotracer studies. Curr Pharmaceut Des 6: 1029–1056.

Schmid J, Prox A, Reuter A, Zipp H, and Koss FW (1980) The metabolism of 8-methoxypsoralen in man. Eur J Drug Metab Pharmacokinet 5: 81–92.[Medline]

Wheeler JK, Moghissi AA, Rehnberg BF, and Colvin MC (1972) Comparison between the biological half-life of a tritiated luminous compound with that of tritiated water in rats and cats. Health Phys 22: 35–38.

Wishka DG, Walker DP, Yates KM, Reitz SC, Jia S, Myers JK, Olson KL, Jacobsen EJ, Wolfe ML, Groppi VE, et al. (2006) Discovery of PHA-543,613, an agonist of the {alpha}7 nicotinic acetylcholine receptor, for the treatment of cognitive deficits in schizophrenia: synthesis and SAR. J Med Chem 49: 4425–4436.[CrossRef][Medline]


This article has been cited by other articles:


Home page
 Drug Metab. Dispos.Home page
C. L. Shaffer, M. Gunduz, R. J. Scialis, and A. F. Fang
Metabolism and Disposition of a Selective {alpha}7 Nicotinic Acetylcholine Receptor Agonist in Humans
Drug Metab. Dispos., July 1, 2007; 35(7): 1188 - 1195.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
dmd.106.010934v1
34/9/1615    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shaffer, C. L.
Right arrow Articles by Fate, G. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shaffer, C. L.
Right arrow Articles by Fate, G. D.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
All ASPET Journals Molecular Pharmacology Pharmacological Reviews
Molecular Interventions Drug Metabolism and Disposition