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Departments of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Groton, Connecticut
(Received April 7, 2008; Accepted May 29, 2008)
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Abstract |
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3 h). LAS was extensively metabolized in both rats and monkeys because no unchanged drug was detected in urine and/or bile. Based on area under the curve(0–24) values, >78% of the circulating radioactivity was due to the metabolites. A total of 22 metabolites were tentatively identified by liquid chromatography-tandem mass spectrometry. Based on the structures of the metabolites, six metabolic pathways of LAS were identified: hydroxylation at the tetraline ring, hydroxylation at the aromatic ring attached to tetraline, methylation of the catechol intermediates by catechol-O-methyl transferase, oxidation at the pyrrolidine ring, and direct conjugation with glucuronic acid and sulfuric acid. LAS and its glucuronide conjugate (M7) were the major circulating drug-related moieties in both rats and monkeys. However, there were notable species-related qualitative and quantitative differences in the metabolic profiles. The catechol (M21) and its sulfate conjugate (M10) were observed only in monkeys, whereas the glucuronide conjugate of the methylated catechol (M8) and hydroxy-LAS (M9) were detected only in rats.
; Gambacciani and Ciaponi, 2000; Riggs et al., 2002; Sowers et al., 2003a,b). It is well documented that estrogen replacement therapy (ERT) is effective in preventing bone loss, and epidemiologic studies have demonstrated that ERT reduces the incidence of bone fractures (Witt and Lousberg, 1997). However, recent findings from the Women's Health Initiative study have shown that the risk of long-term ERT may overweigh benefits (Rossouw et al., 2002; Chlebowski et al., 2003; Hodis et al., 2003; Manson et al., 2003; Wassertheil-Smoller et al., 2003). Therefore, there is a growing interest in identifying an estrogen mimetic that possesses the beneficial effects and is devoid of the associated risks and negative side effects associated with ERT.
It has been reported that SERMs prevent bone loss, increase bone mineral density and decrease serum cholesterol in postmenopausal women (Cosman and Lindsay, 1999). In addition, clinical studies have demonstrated that SERMs also decrease the risk of invasive breast cancer in postmenopausal women and in women at higher risk for breast cancer (Fisher et al., 1998; Cummings et al., 1999). Lasofoxifene [LAS; 6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalen-2-ol. tartrate] is a SERM and, like other compounds in this class (tamoxifen, raloxifene, etc.), will provide benefit to postmenopausal women in three ways: protecting bone mineral density and averting osteoporotic fractures, lowering serum low-density lipoprotein-cholesterol, and reducing the risk of breast cancers. LAS is an ER agonist in bone and cholesterol regulation and an ER antagonist in the mammary gland and uterus. LAS binds with high affinity to the human ER
(Rosati et al., 1998), prevents bone loss, and maintains bone mass and strength in ovariectomized rats without stimulating the uterus (Ke et al., 1998, 2004). In both nonclinical and clinical studies, LAS has shown a favorable safety profile and demonstrated a proven efficacy in preventing bone loss and lowering cholesterol levels (Gennari et al., 2006), and it is now under submission for regulatory approval.
Preclinical pharmacokinetic studies in rats and monkeys suggested that LAS is completely absorbed and readily distributed into extravascular tissue. Based on its clearance values, it is likely that first pass hepatic metabolism is largely responsible to the attenuation of moderate bioavailability in rats (25%) and monkeys (55%). In humans, LAS is eliminated by both phase I oxidative metabolism mediated primarily by CYP3A4 and CYP2D6 isozymes and by phase II conjugation (Prakash et al., 2008). Metabolic pathways of drug candidates in animals, used for safety evaluation studies, are required to ensure that the selected animal species are exposed to all major metabolites (>10% parent AUC) formed in humans (Baillie et al., 2002; http://www.fda.gov/cder/guidance/index.htm). Additional toxicological testing on metabolites that have higher exposure in humans than preclinical species may be required (http://www.fda.gov/cder/guidance/index.htm). The objectives of the present study were to characterize the disposition of LAS in rats and monkeys and to identify and quantify its metabolites in urine, plasma, bile, and/or feces. A single dose of [14C]LAS was orally administered to intact and bile duct-cannulated Sprague-Dawley rats (1 mg/kg) and cynomolgus monkeys (5 mg/kg). The urine, bile, plasma, and feces were collected. The metabolites were separated on reverse-phase HPLC and analyzed by LC-MS and LC-MS/MS.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Radiolabeled Drug and Reference Compounds. [14C]LAS, uniformly labeled with C-14 at the phenyl ring attached to the tetraline moiety (specific activity, 40.3 mCi/mmol) (Fig. 1), was synthesized by the Radiosynthesis Group at Pfizer Global Research and Development (Groton, CT) using the procedure as described (Rosati et al., 1998). It showed a chemical purity of 97.2% and a radiopurity of 99.6% as determined by HPLC using an in-line radioactivity detector.
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Rats. A group of Sprague-Dawley rats (n = 3 per gender) was dosed by oral gavage a single 1 mg (free base)/kg dose of [14C]LAS for mass balance determination. The dose was administered orally as a suspension in 0.5% (w/v) methylcellulose to each rat with an 18-gauge x 2-inch (with 2.25-mm ball) animal feeding needle affixed to a 3-ml disposable syringe. The gavage tube was weighed before and after dosing to determine the actual dose given. For biliary excretion experiments, another group of two male and two female bile duct-cannulated rats was dosed with a single 1 mg (free base)/kg dose of [14C]LAS as described above. Each rat received an approximate dose of 40 µCi of radiolabeled material. Urine and feces were collected from intact animals for 6 days at 0 to 24, 24 to 48, 48 to 72, 72 to 96, 96 to 120, and 120 to 144 h after dose administration. The first fecal sample was collected from 0 to 48 h postdose because animals failed to produce substantial feces in the 0- to 24-h time period. Bile samples were collected from bile duct-cannulated animals at 0 to 24 and 24 to 48 h after administration. The weight of bile, urine, and feces obtained at different time points was recorded, and the samples were divided and stored at –20°C in the dark until analysis.
For pharmacokinetic experiments, a third group of jugular vein-cannulated rats (n = 2/gender) was dosed by oral gavage a single 1 mg (free base)/kg of [14C]LAS. The dose was administered as a suspension in 0.5% methylcellulose at a concentration of 0.227 mg/ml. Blood (400 µl) was collected in heparinized tubes at 0, 0.33, 0.67, 1, 2, 4, 8, 12, and 24 h after the dose. A fourth group of animals (n = 6 per gender) was dosed for the identification of circulating metabolites. Blood was collected in heparinized tubes by decapitation of two males and two females at 1, 4, and 8 h postdose. Blood samples were centrifuged at 1000g for 10 min to obtain the plasma. Plasma was transferred to clean tubes and stored at –20°C until analysis.
For whole-body autoradioluminography (WBAL) experiments, a fifth group of Long-Evans rats (n = 5 per gender) received 13.8 ± 0.07 mg/kg containing 136 ± 0.7 µCi/kg oral dose of [14C]LAS. One rat of each gender was euthanatized by CO2 asphyxiation at 1, 3, 8, 12, and 168 h after dose administration. Immediately following euthanasia, each rat was prepared for WBAL by immersion into a freezing chamber (–75°C) containing dry ice and hexanes for 10 min.
Monkey Study. For the mass balance study, two male and two female cynomolgus monkeys (4–7 kg) were housed in separate stainless steel metabolic cages. The animals were fasted for 12 h prior to dose administration and were fed after 4 h postdose. A 24-h collection of control urine was obtained from the monkeys prior to dosing. On the day of the study, each animal received by oral gavage a single 1 ml/kg base equivalent dose of [14C]LAS followed by 10 ml of water. The dose of [14C]LAS was prepared as a suspension in 0.5% (w/v) methylcellulose at a concentration of 5 mg/ml containing 41 µCi/ml radio carbon. Urine and feces samples were quantitatively collected for 7 days from each animal at 0 to 24, 24 to 48, 48 to 72 and 72 to 96, 96 to 120, 120 to 144, and 144 to 168 h postdose and final cage washing. The weight of urine and feces obtained at different time points was recorded and stored at –20°C in the dark until analysis.
Two weeks following the first administration of [14C]LAS, the monkeys were administered a second dose of [14C]LAS by oral gavage for pharmacokinetic study. Blood (6 ml/time point) was collected in heparinized tubes from the jugular vein of each animal at 0, 0.5, 1, 2, 6, 10, 24, and 48 h postdose. The blood samples were centrifuged, and the plasma was transferred to clean tubes and stored at –20°C until analysis.
Determination of Radioactivity. The radioactivity in urine, bile, and plasma was determined by liquid scintillation counting. Aliquots of urine, bile, and plasma (10–50 µl) in triplicate, for each sampling time point, was mixed with 5 ml of Ecolite (+) scintillation cocktail (MP Biomedicals) and counted in a Packard no. 2500 TR liquid scintillation counter.
Fecal samples from rats were placed in Falcon tubes (50 ml) and homogenized in an equal amount of water to a thick slurry using a Brinkmann Polytron lab homogenizer (Brinkmann Instruments, Westbury, NY). For monkeys, the fecal samples at each time point were suspended in equivalent volumes of water and then homogenized using a Stomacher 400 Lab Blender (Cook Laboratory Products, Alexandria, VA). Aliquots (100–400 mg) of the homogenates were air-dried over night and combusted using a United Packard Oxidizer model 307. The radioactivity in combustion products was determined by trapping the liberated [14C]CO2 in Carbosorb and Permafluor E+ followed by liquid scintillation counting. Combustion efficiency was determined by combustion of [14C]standard in an identical manner.
A set of commercially sealed liquid scintillation standards and the control matrix were used to verify the performance of the quench curves. The samples collected prior to dosing were also counted to obtain a background count rate. The amount of radioactivity in the dose was expressed as 100%, and the radioactivity in urine and feces at each sampling time was expressed as the percentage of dose excreted in the respective matrices at that sampling time. The amount of radioactivity in plasma was expressed as nanogram equivalents of parent drug per milliliter and was calculated by using the specific activity of the administered dose.
Pharmacokinetic Analysis. Plasma concentrations of unchanged LAS were determined by a validated HPLC-MS/MS assay. The dynamic range for the assay was 0.25 to 60 ng/ml using a 0.25-ml plasma sample. A minivalidation of the assay was performed before analyzing the samples. Pharmacokinetic parameters were calculated by noncompartmental analysis using WinNonlin-Pro version 3.2 (Pharsight, Mountain View, CA).
Whole-Body Autoradioluminography. The whole-body cryosectioning technique developed by Ullberg (1977) was utilized to prepare whole-body cryosections for autoradioluminography. Quantification of whole-body cryo-images described by Potchoiba et al. (1995, 1998) was used to determine the concentration of [14C]LAS radioequivalents in tissues. Briefly, frozen whole-body specimens were embedded onto microtome stages using 3% (w/v) carboxymethylcellulose (Sigma-Aldrich, St. Louis, MO). Sagittal 25-µm cryosections were obtained onto type 810 Scotch tape (3M, St. Paul, MN) at –18°C. Cryosections were then freeze-dried at –18°C for a minimum of 72 h. Each freeze-dried cryosection was identified using radioactive ink and covered with mylar (Fralock, Valencia, CA) prior to being apposed to storage phosphor screens for 4 days. Storage phosphor screens were scanned with a Molecular Dynamics STORM860 PhosphorImager (GE Healthcare, Chalfont St. Giles, UK) to acquire computerized images. The concentration of [14C]LAS radioequivalents in tissues was determined by reference to standard curve calibrators prepared with [14C]glucose (PerkinElmer Life and Analytical Sciences) in rat whole blood (Rockland Immunochemicals, Gilbertsville, PA) evaluated with the MicroComputer Imaging Device (InterFocus Imaging; Linton, Cambridge, UK). [14C]LAS radioequivalents were expressed as microgram equivalents per gram. The lower quantification limit (LLOQ) for this WBAL assay was 0.6 µg Eq/g. [14C]LAS radioequivalents were calculated by averaging tissue concentrations measured at different sectioning levels and/or from replicate cryosections obtained from the same sectioning level. Cryosection quality control samples were prepared by fortifying rat blood with known amounts of [14C]glucose at four concentrations of 3.7, 7.1, 13, and 26 µg Eq/g. Triplicate aliquots of these four cryosection quality control samples were embedded along with each study rat in a frozen block of 3% carboxymethylcellulose to assess the accuracy and precision of the WBAL assay.
Extraction of Metabolites from Biological Samples. The majority of radioactivity recovered in rat urine was excreted during the first 72-h postdose. Therefore, urine samples collected at 0 to 24, 24 to 48, and 48 to 72 h postdose were pooled on the basis of weight, and the pooled samples were used for profiling and identification of metabolites. Pooled urine (3 ml) from each animal was centrifuged, and the supernatant was transferred to a clean tube and concentrated under nitrogen in a Turbo Vap LV evaporator (Caliper Life Sciences, Hopkinton, MA). The residue was dissolved in 1 ml of HPLC mobile phase, and an aliquot (80–100 µl) was injected onto the HPLC column without further purification. Urine samples from monkeys were centrifuged and were applied to a preconditioned Water C-18 Sep-Pak column. The column was washed with water, and the metabolites were eluted with methanol. The recovery of radioactivity was >85% after extraction. The methanolic eluant was concentrated under nitrogen using a Turbo Vap evaporator, reconstituted in methanol/water (50:50, v/v).
The fecal homogenates (1–3 g) from 0 to 72 h postdose for both rats and monkeys were pooled on the basis of weights collected at each time interval, and the pooled samples were diluted with acetonitrile (6 ml). The suspension was stirred overnight on a magnetic stirrer and centrifuged at 1500g for 10 min. The supernatants were removed, and the extraction was repeated with methanol and methanol/water (50:50). All supernatants were combined and small aliquots were counted. The overall recovery of radioactivity in feces was about 78–85% after extraction for both species. The organic solvent was evaporated using the Turbo Vap LV evaporator. The residue was dissolved in 1 ml of methanol/ammonium acetate (1:1). An aliquot (20–50 µl) was injected on HPLC. Bile (50 µl) was injected on HPLC without further purification.
For rats, plasma (5 ml pooled, 1, 4, and 8 h) was diluted with 30 ml of acetonitrile, and the precipitated protein was removed by centrifugation. The pellet was washed with an additional 2 ml of acetonitrile, and both the supernatants were combined. The extraction recovery of the radioactivity in plasma was about 80 to 88%. The supernatant was concentrated under nitrogen in a Turbo Vap LV evaporator, and the residue was reconstituted in 500 µl of methanol/ammonium acetate (1:1). An aliquot (100 µl) was injected onto the HPLC column without further sample purification.
For monkeys, plasma (9 ml, 0–24 h pool, 1 ml from each time point) was diluted with 4 volumes of acetonitrile, and the precipitated protein was removed by centrifugation. The pellet was washed with an additional 5 ml of acetonitrile, and the supernatants from the two washes were combined. The extraction recovery of the radioactivity in plasma was about 78 to 85%. The supernatant was concentrated in a Turbo Vap LV evaporator, and the residue was reconstituted in 400 µl of methanol/20 mM ammonium acetate (1:1). An aliquot (80 µl) was injected on the LC-MS.
HPLC. The HPLC system consisted of an HP-1050 solvent delivery system, an HP-1050 membrane-degasser, an HP-1050 autoinjector (Hewlett Packard, Palo Alto, CA), an LDC analytical UV detector, and a radioactivity monitor (β-RAM; IN/US, Tampa, FL). Chromatography was carried out on a Beckman Ultrasphere C-18 column (4.6 x 250 mm, 5 µm) with a binary mixture of 10 mM ammonium acetate (solvent A) and methanol (solvent B). The mobile phase initially consisted of solvent A/solvent B (80:20), and it was then linearly programmed to solvent A/solvent B (20:80) over 30 min. Chromatography was carried out under isocratic conditions for 5 min and then programmed to solvent A/solvent B (5:95) and held for 5 min. The mobile phase composition returned to the starting solvent mixture over 5 min. The system was allowed to equilibrate for approximately 15 min before making the next injection. A flow rate of 0.7 ml/min was used for all analyses, except for the profiling of feces extracts, where 1.0 ml/min was used.
For profiling urine, feces, and plasma from monkeys, the mobile phase initially consisted of solvent A/solvent B (50:50), and it was linearly programmed to solvent A/solvent B (20:80) over 20 min followed by a linear increase to solvent A/solvent B (5:95) over 5 min. Chromatography was carried out under isocratic conditions for 10 min, then linearly programmed back to starting solvent mixture over 5 min. The system was allowed to equilibrate for 10 min before making the next injection.
Quantitative Assessment of Metabolites. Quantification of the metabolites was carried out by measuring radioactivity in the individual HPLC-separated peaks using a β-RAM. The β-RAM provided an integrated printout in counts per minute and percentage of the radiolabeled material, as well as peak representation. The β-RAM was operated in the homogeneous liquid scintillation counting mode, with addition of 4 ml/min Ecolite (+) scintillation cocktail to the effluent after UV detection. The radiochromatograms of metabolites in rat plasma were generated by collecting fractions at 20-s intervals and counting the fractions in a Packard no. 2200CA liquid scintillation counter.
LC-MS/MS. LC-MS/MS was conducted on either a Sciex API III HPLC-MS/MS system using ion spray or a Finnigan TSQ 7000 LC-MS/MS. The effluent from the HPLC column was split, and about 50 µl/min was introduced into the atmospheric ionization source via an ion spray interface. The remaining effluent was directed into the flow cell of the β-RAM. The β-RAM response was recorded in real time by the mass spectrometer that provided simultaneous detection of radioactivity and mass spectrometry data. The delay in response between the two detectors was about 0.2 min with the mass spectrometric response recorded earlier. The ion spray interface was operated at 4000 V, sheath pressure at 80 PSI, and capillary temperature at 200°C, and the mass spectrometer was operated in the positive mode. Collision-induced dissociation (CID) and MS/MS studies were performed using argon gas at a collision energy of 27 to 30 eV and a collision gas thickness of 2.4 x 1014 molecules/cm2 on the Sciex API III or at a pressure of 2.4 mTorr on the Finnigan TSQ 7000.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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85%, was excreted in the first 96 h after dose administration.
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Pharmacokinetics. Rats. Mean plasma concentration versus time curves of LAS and total radioactivity after a single 1 mg/kg oral dose of [14C]LAS to rats are shown in Fig. 2. Mean peak plasma concentrations of LAS were 22.2 and 64.0 ng/ml for male and female rats, respectively. For the total radioactivity, they were 90.3 and 90.9 ng Eq/ml for male and female rats, respectively (Table 4). Mean AUC(0-
) values for unchanged LAS were 172 and 334 ng/h/ml, respectively, in male and female rats. Mean AUC(0-) values for total radioactivity were 1035 and 1525 ng Eq/h/ml in male and female rats, respectively. The mean t1/2 was 8.3 and 13.2 h for parent drug and radioactivity, respectively (Table 4). Based on AUC(0-), 16.6 and 21.9% of the circulating radioactivity was attributable to unchanged drug in male and female rats, respectively. The balance, approximately 78 to 83% of the radioactivity in the circulation, was due to metabolites.
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Monkeys. Mean plasma concentration versus time curves of LAS and total radioactivity after a single 1 mg/kg oral dose of [14C]LAS to monkeys are shown in Fig. 3. The mean peak plasma concentrations of LAS were 79 and 46 ng/ml, respectively, for male and female monkeys (Table 4). The mean peak plasma concentration for total radioactivity in male monkeys was 318 ng/ml at 6 h postdose and was 458 ng/ml at 2 h postdose in female monkeys. Mean AUC(0-) values for unchanged LAS were 2035 and 2159 ng·h/ml in male and female monkeys, respectively. Mean AUC(0-) values for total radioactivity were 13832 and 13887 ng Eq/h/ml in male and female monkeys, respectively (Table 4). Based on mean AUC(0-) values, only 17.6% of the circulating radioactivity was attributable to the parent drug.
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). [14C]LAS radioequivalents were not observed in urine. Fecal excretion appeared to be the primary route of elimination.
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Metabolic Profiles. Rat urine. Urine was the minor route of elimination for LAS because urine accounted for only 1.72% of the administered dose. A major portion of the radioactivity recovered in the urine was excreted in the first 72 h postdose. Therefore, urine from the first 72 h postdose was pooled, and the pooled samples were used for profiling and identification. A representative metabolic profile in urine from rats following oral administration of [14C]LAS is shown in Fig. 4. The metabolites were quantified with on-line integration of the radiochromatographic peaks. The percentages of urinary metabolites excreted in relation to the administered dose are presented in Table 6. Eight metabolites in urine of female rats and seven metabolites in urine of male rats were identified. The major urinary metabolites were the glucuronide conjugates of LAS (M6) and the methylated catechol (M7).
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Rat feces. Most of the radioactivity was eliminated in the feces, accounting for 95% of the dose in both sexes. Feces from the first 72 h postdose were pooled, extracted, and used for profiling and identification of metabolites. A representative HPLC-radiochromatogram of fecal metabolites from rats is shown in Fig. 4. The percentage of fecal metabolites in relation to dose for male and female rats is presented in Table 6. Unchanged drug in the feces accounted for 50% of the administered dose. The major fecal metabolites were M11 (10.3%), M13 (9.3%), and M15 (8.6%).
Rat bile. In the bile duct-cannulated rats, most of the administered radioactivity (83%) was eliminated in the bile within the first 48 h postdose. The bile samples from 0 to 8 and 8 to 24 h postdose were pooled, and the pooled samples were used for the identification of metabolites. A representative HPLC radiochromatogram for biliary metabolites is shown in Fig. 4. The percentage of bile metabolites in relation to total radioactivity excreted in bile is presented in Table 7. The major metabolite (55% of the radioactivity in bile) was attributed to the glucuronide conjugate LAS. The other major biliary metabolites included M3 (6.7%), M6 (18.3%), and M11 (6.3%).
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Rat circulating metabolites. The representative reconstructed HPLC-radiochromatograms of plasma metabolites (1-, 4-, and 8-h time points pooled) for both male and female rats are given in Fig. 5. The percentage of metabolites in relation to the total radioactivity extracted from the plasma of both male and female rats is presented in Table 8. LAS and a total of 10 metabolites were identified in plasma. Metabolites were identified by ion spray LC-MS using multiple reaction monitoring. LAS (22.5%), its glucuronide conjugate (M7, 29.0%), and the glucuronide conjugate of the methyl catechol (M8, 10.5%) were the major circulating drug-related moieties in rats. The identified metabolites and unchanged drug accounted for approximately 89.7 and 86.5% of the radioactivity in plasma from female and male rats, respectively.
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Monkey urine. A small portion of the radioactive dose was excreted in the urine (1.91%) during 0 to 168 h postdose. Therefore, urine samples containing greater than 0.1% of the dose were pooled, and the pooled samples were used for profiling and identification of metabolites. A representative metabolic profile of urinary metabolites in monkeys is shown in Fig. 6. A total of 10 metabolites were identified in monkey urine. The percentages of metabolites excreted in urine of male and female monkeys are presented in Table 9. There were no qualitative differences in the urinary metabolic profiles between male and female monkeys. The major urinary metabolites were M7 and M23. Overall, the identified metabolites accounted for 1.13% of dose in urine.
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Monkey feces. The major portion of the radioactivity (about 94%) was excreted in feces during the first 72 h after oral administration of LAS. Therefore, fecal homogenates from 0 to 72 h were pooled on the basis of sample weight for profiling and identification of metabolites. The pooled fecal homogenates were extracted and purified as described under Materials and Methods. The overall recovery of radioactivity in feces was about 85% after extraction. A representative HPLC-radiochromatogram of fecal metabolites in monkeys is given in Fig. 6. The percentages of fecal metabolites in male and female monkeys are presented in Table 9. LAS and a total of nine metabolites were detected in feces. There were no major notable gender-related differences in the fecal metabolites of male and female monkeys. Three major metabolites were (M13, 8.6%; M17, 7.8%; M21, 14.4%) and the unchanged drug (27%).
Circulating metabolites. The HPLC-radiochromatogram of the plasma metabolites (0–24 h) from one monkey is given in Fig. 7. The percentage of the metabolites in relation to the total radioactivity extracted from the plasma of both male and female monkeys is presented in Table 8. Unchanged drug and six metabolites were identified in plasma of monkeys using selected reaction monitoring and by comparison of HPLC-retention times with metabolites obtained from urine and/or feces. The ions at m/z 606/430, 590/414, 510/430, 494/414, 428/112, 414/98, and 444/98, corresponding to M5, M7, M10, M11, M15, LAS, and M17, were monitored. There appear to be no qualitative differences between the male and female monkey plasma metabolites. The major components in the plasma were LAS (19.5%), M5 (9.8%), M10 (12.0%), M11 (6.5%), and M17 (7.6%). The identified metabolites accounted for approximately 82.8 and 89.7% of the circulating radioactivity in male and female monkeys, respectively.
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; Prakash et al., 2007). Mass spectral fragmentation of LAS. LAS had a retention time of 30.4 min on the HPLC system. Full scan MS of LAS produced a protonated molecule (MH+) of m/z 414. Its CID product ion spectrum of m/z 414 gave prominent and diagnostic fragment ions at m/z 223, 192,145, 107, 98, 71, and 56 (Fig. 8; Table 10). The ion at m/z 107 was due to the hydroxylated tropylium moiety. The ion at m/z 98 resulted from the dissociation of the C-O bond between ethylpyrrolidene and phenyl tetraline moiety with charge retention at the ethylpyrrolidene. The other diagnostic fragment ions at m/z 223 and 192 were formed by cleavage of the C-C bond between phenoxyethylpyrrolidene and phenyl tetraline moiety. The product ion of m/z 223 was then dissociated to the (EE+) at m/z 145 due to loss of the benzene ring. The other ions at m/z 71 and 56 were from the pyrrolidine moiety.
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Metabolites M2 and M4. M2 and M4 were detected only in rats. Both M2 and M4 showed a protonated molecular ion at m/z 636, 222 (32 + 14 + 176) Da higher than the parent drug, indicating the addition of two oxygen atoms, a methyl group, and a glucuronic acid to the parent molecule. The CID product ion spectrum of M2 (m/z 636) showed fragment ions at m/z 460, 347, and 98 (Table 10). The fragment ion at m/z 460 (loss of 176) was indicative of the presence of a glucuronide conjugate. The significant and distinct fragment at m/z 347 indicated that the addition of the methyl group and an oxygen atom had occurred at the phenyl tetraline moiety. The ion at m/z 347, loss of 113 from the aglycone further suggested that the addition of other oxygen atom had occurred on the pyrrolidine ring. The characteristic fragment ion at m/z 98 suggested the presence of an N-oxide. Treatment of urine sample with TiCl3 resulted in the disappearance of the peak corresponding to M2. These data further suggested that M2 was an N-oxide because TiCl3 is known to reduce N-oxides. Based on these data, M2 was tentatively identified as the glucuronide conjugate of the methoxy LAS-N-oxide.
On the other hand, the CID product ion spectrum of M4 (m/z 636) gave the fragment ions at m/z 460, 363, 175, and 98 (Table 10). The fragment ions at m/z 363 and 98 suggested the addition of an oxygen atom and a methoxy group on the phenyl tetraline moiety. The ion at m/z 175 indicated that the addition of a methoxy group had occurred on the teralin moiety. Based on these data, M4 was tentatively identified as the glucuronide conjugate of hydroxy-methoxy-LAS.
Metabolites M3, M5, and M9. M3, M5, and M9 all showed a protonated molecular ion at m/z 606, 192 Da higher than the parent drug, indicating that they were glucuronide conjugates of a hydroxyl metabolite. The CID product ion spectra of both M3 and M5 (m/z 606) gave fragment ions at m/z 430, 239, 192, and 98 (Table 10). The ion at m/z 239 suggested the presence of an additional oxygen atom on the phenyl tetraline moiety. The prominent fragment ions at m/z 98 and 192 indicated that the pyrrolidine and phenoxy ethyl moieties were unchanged. Based on these fragmentations, M3 and M5 were tentatively identified as the glucuronide conjugates of hydroxyl-LAS.
On the other hand, CID spectrum of M9 (m/z 606) gave the fragment ions at m/z 430 (aglycone), 223, 208, 98, 85, and 71 (Table 10). The characteristic fragment ion at m/z 98 suggested that the pyrrolidine ring was unsubstituted. The fragment ion at m/z 223, similar to that of the parent drug, indicated that the phenyl tetraline moiety was also unchanged. The other fragment ion at m/z 208 suggested that the oxidation had occurred at the phenoxy moiety. Based on these data, M9 was tentatively identified as a glucuronide conjugate of hydroxyl-LAS, an isomer of M3 and M5.
Metabolites M6 and M8. M6 and M8 both showed a protonated molecular ion at m/z 620, 206 (16 + 14 + 176) Da higher than the parent drug, indicating the addition of an oxygen atom, a methyl group, and a glucuronic acid. The CID product ion spectrum of both M6 and M8 (m/z 620) gave diagnostic fragment ions at m/z 444 (aglycone), 253, and 98 (Table 10). The ion at m/z 253, 30 Da higher than that of the parent compound, suggested that the tetraline moiety was substituted with an oxygen atom and a methyl group. The characteristic fragment ion at m/z 98 indicated that the pyrrolidine ring was unsubstituted. Based on these fragmentations, M6 and M8 were tentatively identified as the glucuronide conjugates of methoxy-LAS.
Metabolite M7. M7 showed a protonated molecular ion at m/z 590, 176 Da higher than the parent drug, indicating the addition of a glucuronic acid. The CID product ion spectrum of M7 (m/z 590) gave fragment ions at m/z 414, 223, 192, 145, and 98, similar to those of the parent compound (Table 10). Based on these fragmentations, M7 was identified as the LAS-glucuronide.
Metabolite M10. M10 showed a protonated molecular ion at m/z 510, 96 (16 + 80) Da higher than the parent drug, indicating that M10 was a sulfate conjugate of a hydroxyl metabolite. The CID product ion spectrum of m/z 510 gave the fragment ions at m/z 430, 239, 145, and 98 (Table 10). The prominent ion at m/z 98 suggested that the pyrrolidine ring was unsubstituted. The fragment ion at m/z 239 indicated that the addition of an oxygen atom had occurred on the phenyl tetraline moiety. The other diagnostic fragment ion at m/z 145 suggested that the tetraline ring was unsubstituted. Based on these data, M10 was tentatively identified as the sulfate conjugate of the hydroxyl-LAS.
Metabolite M11. M11 showed a protonated molecular ion at m/z 494, 80 Da higher than the parent drug, indicating that M11 was a sulfate conjugate of the parent compound. The CID product ion spectrum of m/z 494 gave the fragment ions at m/z 414, 223, 192, and 98, similar to those of the parent drug. Based on these fragmentations, M11 was identified as the sulfate conjugate of LAS.
Metabolites M12, M14, and M21. M12, M14, and M21 showed a protonated molecular ion at m/z 430, 16 Da higher than the parent drug, suggesting the addition of an oxygen atom. The CID product ion spectra of M12 and M14 (m/z 430) gave fragment ions at m/z 239, 192, 145, 107, 98, 71, and 56 (Table 10). The ion at m/z 239 indicated the hydroxylation of the phenyl tetraline moiety. The ions at m/z 145 and 107 suggested that the hydroxylation was on the phenyl ring. The fragment ions at m/z 192, 98, and 71 suggested that the pyrrolidine ethoxy phenyl portion of the molecule was unsubstituted. Based on these data, M12 and M14 were tentatively identified as hydroxy-LAS. The exact position of the hydroxy groups, however, could not be established by mass spectral data.
On the other hand, CID spectrum of M21 (m/z 430) gave fragment ions at m/z 239, 192, 161, 98, 91, 71, and 56 (Table 10). The ion at m/z 239, 16 Da higher than that of the parent drug, indicated that the addition of an oxygen atom had occurred on the phenyl tetraline moiety. Based on these data, M21 was tentatively identified as hydroxy-LAS. The exact position of the hydroxylation, however, could not be established by mass spectral data.
Metabolite M13. M13 showed a protonated molecular ion at m/z 446, 32 Da higher than the parent drug, indicating the addition of two oxygen atoms to the parent drug. The CID product ion spectrum of M13 (m/z 446) gave prominent fragment ions at m/z 223, 145, 130, and 112 (Table 10). The fragment ions at m/z 223 and 145, similar to those of the parent compound, suggested that the phenyl tetraline portion of the molecule was unsubstituted. The fragment ion at m/z 130, 32 Da higher than the fragment ion (m/z 98) of the parent drug, indicated the addition of two oxygen atoms to the pyrrolidine ring. The fragment ion at m/z 86 (loss of 44) from the ion at m/z 130 suggested the presence of a carboxyl group. Based on these data, M13 was tentatively identified as a pyrrolidine ring opened metabolite.
Metabolite M15. M15 showed a protonated molecular ion at m/z 428, 14 Da higher than the parent drug, indicating the addition of an oxygen atom with subsequent loss of two hydrogen atoms. The CID product ion spectrum of M15 (m/z 428) gave prominent fragment ions at m/z 223, 145, and 112 (Table 10). The ion at m/z 223 indicated that the substitution was remote from the phenyl-tetraline moiety, and the ion at m/z 112 suggested that the oxidation had occurred on the pyrrolidine ring. Based on these fragmentations, M15 was tentatively identified as oxo-LAS.
Metabolite M17. M17 showed a protonated molecular ion at m/z 444, 30 Da higher than the parent drug, suggesting the addition of a methoxy group to the molecule. The CID product ion spectrum of M17 (m/z 444) gave prominent fragment ions at m/z 253, 192, 175, 137, 98, 91, 71, and 56 (Table 10). The ion at m/z 253 suggested the addition of a methoxy group to the phenyl-tetraline moiety. The other fragment ions at m/z 192, 98, 71, and 56, similar to those of the parent compound, suggested that the pyrrolidine ethoxy-phenyl moiety was unsubstituted. The fragment ion at m/z 137, 30 Da higher than that of the parent drug, indicated the presence of a methoxy group on the tetraline ring. Based on these fragmentations, M17 was tentatively identified as methoxy-LAS.
Metabolites M18, M19, and M20. M18, M19, and M20 all showed a protonated molecular ion at m/z 444, 30 Da higher than the parent drug, suggesting the addition of two oxygen atoms with subsequent loss of two hydrogen atoms. The CID product ion spectrum of M18 and M19 (m/z 444) gave prominent fragment ions at m/z 239, 206, 145, 112, 107, and 84 (Table 10). The ion at m/z 239, 16 Da higher than that of the parent drug, indicated the addition of an oxygen atom to the phenyl tetraline moiety. The ion at m/z 107 suggested that the hydroxylation had occurred on the phenyl ring of the phenyl tetraline moiety. The ion at m/z 112 suggested that the oxidation had occurred on the pyrrolidine ring. Based on these fragmentations, M18 and M19 were isomers and tentatively identified as the hydroxy-LAS-lactam.
On the other hand, the CID product ion spectrum of M20 (m/z 444) gave prominent fragment ions at m/z 239, 161, 112, 91, and 69 (Table 10). The prominent fragment ions at m/z 239, 161, and 91 (tropylium) indicated that the hydroxylation had occurred on the phenyl-tetraline moiety. The ion at m/z 112 suggested that the oxidation had also occurred on the pyrrolidine ring. Based on these fragmentations, M20 was tentatively identified as hydroxy-LAS-lactam.
Metabolite M22. M22 showed a protonated molecular ion at m/z 524, 110 (30 + 80) Da higher than the parent drug, indicating the addition of an oxygen atom, a methyl group, and a sulfonate group to the parent compound. The CID product ion spectrum of M22 (m/z 524) gave diagnostic fragment ions at m/z 444, 253, 98, 91, 71, and 56 (Table 10). The fragment ion at m/z 253 suggested the addition of a methoxy group to the phenyl tetraline moiety. Based on these fragmentations, M22 was identified as the sulfate conjugate of methoxy-LAS.
Metabolites M23 and M24. M23 and M24 both showed a protonated molecular ion at m/z 622, 208 Da higher than the parent drug, suggesting the addition of two oxygen atoms and a glucuronide. The CID product ion spectra of both metabolites M23 and M24 (m/z 622) gave fragment ions at m/z 446 (aglycone), 255, 191, 107, and 98 (Table 10). The fragment ion at m/z 446 (loss of 176) indicated the presence of a glucuronide conjugate. The fragment ions at m/z 255 and 191 suggested the addition of two oxygen atoms on the phenyl tetraline moiety. The ion at m/z 107 indicated that the addition of one oxygen atom had occurred on the phenyl ring of the phenyl tetraline moiety. Based on these data, M23 and M24 were tentatively identified as glucuronide conjugates of dihydroxy-LAS.
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83% of the radioactivity administered is recovered in the bile in the first 48 h postdose, indicating that the bile was the primary route of excretion of LAS and metabolites in rats.
Absorption of LAS was rapid in both rats and monkeys, as indicated by the early appearance of radioactivity in plasma after oral administration of [14C]LAS. The plasma concentrations of total radioactivity were always much greater than for unchanged drug, suggesting the early formation of circulating metabolites. The magnitude of the difference between concentrations of unchanged drug and radioactivity indicated that at all times, the majority of the drug-related material in plasma was attributable to metabolites in both rats and monkeys. Tissue distribution study in rats suggests that [14C]LAS-related radioactivity distributed rapidly in the rat, with most tissues achieving maximal concentrations at 1 h. Tissue exposure was similar in male and female rats, with the greatest exposure in the uvea. Half-life of radioactivity was longest in the uvea (124 h) and shortest in the spleen (3 h). Radioactivity became undetectable in some tissues by 8 h postdose. Bone marrow radioactivity was 4- and 5-fold greater than myocardial blood concentrations at 1 h postdose and increased to 30-fold greater by 8 h postdose. At 168 h postdose, radioactivity could only be measured in the intestinal contents and uvea. Radioactivity was not observed in the uvea of albino rats, indicating that the uvea binding observed in this study is associated with binding to melanin present in the eye of pigmented rats. Reversible binding to melanin is commonly seen with lipophilic compounds and has been shown to generally have no toxicological significance (Leblanc et al., 1998).
The urine and bile/feces radiochromatograms from rats and monkeys indicated that LAS was readily metabolized before excretion. The major portion of administered radioactivity was excreted in urine and bile as conjugates of parent drug and its oxidative metabolites. There were no gender-related qualitative differences in the profile of metabolites. However, there were notable species-related qualitative and quantitative differences in the metabolic profiles. In addition to the parent drug, a total of 22 metabolites were tentatively identified by electrospray LC-MS/MS. The use of electrospray, a very soft ionization technique, has allowed the identification of polar phase II metabolites (Kamel and Prakash, 2006; Prakash et al., 2007). Due to low dose and the presence of a large number of metabolites, enough quantities could not be obtained for NMR. A proposed scheme for the biotransformation pathways of LAS in rats and monkeys is shown in Fig. 1.
The three major components in excreta of rats were LAS, its sulfate conjugate (M11), and oxidation at the pyrrolidine ring (M15), accounting for 50.1, 10.3, and 8.6% of the dose. LAS in feces was likely the consequence of gut microflora hydrolysis of the glucuronide and is supported by observations in bile duct-cannulated rats. Approximately 61.3% (M7 and M11) of the radioactivity in bile was excreted as sulfate and glucuronide conjugates of LAS, and 34.6% (M2, M3, M4, M5, M6, and M10) was due to metabolites that had a pathway through the oxidation at the teraline ring.
The hydroxylation at the tetraline ring formed a catechol intermediate (M21), which then underwent either conjugation with glucuronic acid and sulfuric acid to form metabolites (M3, M5, and M10) or methylation by catechol-O-methyl transferase to form metabolite (M17). Metabolite M17 was also followed by conjugation with glucuronic acid to form two metabolites, M6 and M8. These data suggested that M17 was a mixture of two regioisomeric metabolites. The hydroxylation at the tetraline ring could form two regioisomers of catechol intermediate (5-hydroxy- and 7-hydroxy-lasofoxifene). Catechol-O-methyltransferase could then generate four isomeric monomethyl ethers from these catechol intermediates, i.e., 5-methoxy-, 5-hydroxy-6-O-methyl, 7-methoxy, and 7-hydroxy-6-O-methyl-lasofoxifene. Due to low abundance of these metabolites, the exact position of methylation could not be determined. The AUC for LAS was 70% greater in the female rats than in the male rats, but as a fraction of total radioactivity, there was little difference, with lasofoxifene representing 20% of total radioactivity in male rats and 27% in female rats. LAS (22.5%), its glucuronide conjugate (M7, 29.0%), and the glucuronide conjugate of the methyl catechol (M8, 10.5%) were the major circulating drug-related moieties in rats.
The three major components in excreta of monkey were LAS (26.9%), LAS sulfate (3.3%), and the catechol (M21, 14.4%). On the assumption that LAS represents the hydrolysis product of biliary excreted LAS glucuronide, direct conjugation accounts for 30% of the metabolism in monkey. As in the rat, oxidation on the aromatic portion of the tetralin to form a catechol followed by subsequent conjugation and further oxidation (M21, M10, M3/M5, M6/M8, M23/M24, M17, and M22) was also an important pathway, accounting for 26.3% of the dose. A third important pathway in monkey was oxidation at the pyrrolidine ring, leading to several metabolites (M13, M15, M18/M19, M20) that account for 25% of the dose. LAS (18.3% of the radioactivity) and the direct glucuronide of lasofoxifene (M7, 28.1%) were the major circulating compounds. Glucuronide or sulfate conjugates (M7, M5, M11, and M10) accounted for 56% of the radioactivity. Three of the six metabolites (M5, M10, M17) were products of the catechol (M21), accounting for 24% of the radioactivity and illustrating the importance of this pathway.
There were notable species-related qualitative and quantitative differences in the metabolic profiles. The catechol (M21) and its sulfate conjugate (M10) were observed only in monkeys, whereas the glucuronide conjugate of the methylated catechol (M8, 10.5%) and hydroxy-LAS (M9, 1.4%) were detected only in rats. There are a number of compounds that demonstrate similar species specificity for the formation of the O-sulfate conjugate in nonrodents compared with rodents (Mattiuz et al., 2003). Recently, Daniels et al. (2007) have illuminated a novel metabolism-mediated ring contraction of a naphthyridine-N-oxide compound in cynomolgus monkeys, the mechanism of which appears to require both direct sulfate conjugation and oxidation in the overall reaction.
It is well known that the catechols are metabolized to highly reactive O-quinone by monooxygenase or peroxidase enzymes, metal ions, or molecular oxygen (Monks et al., 1992; Bolton et al., 2000). However, unlike other SERMs such as raloxifene and tamoxifen, no glutathione conjugate of LAS was detected in preclinical species or humans (Chen et al., 2002), suggesting that the formation of catechol is either rate limited or it undergoes rapid conjugative metabolism (O-methylation and subsequent glucuronidation or sulfation) followed by biliary excretion.
In summary, the results of this study provide the first analysis of formation, excretion, and characterization of metabolites of LAS in rats and monkeys, two species used in toxicology studies. LAS is extensively metabolized in both rats and monkeys after oral administration, and the radioactive dose is excreted mainly in feces via bile. LAS is eliminated by both phase I and phase II metabolism similar to humans. Based on the structures of the metabolites, six metabolic pathways of LAS were identified: hydroxylation at the tetraline ring, hydroxylation at the phenyl rings attached to tetraline, methylation of the catechol intermediates by catechol-O-methyl transferase, conjugation with glucuronic acid and sulfuric acid, and oxidation at the pyrrolidine ring. All the human metabolites were observed in at least one of the species (Prakash et al., 2008). In addition, the exposure of major circulating metabolites in these test species was higher relative to humans at proposed therapeutic doses, suggesting that the preclinical test species were suitable models for assessing safety (http://www.fda.gov/cder/guidance/index.htm).
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ABBREVIATIONS: ERT, estrogen replacement therapy; SERM, selective estrogen receptor modulator; LAS, lasofoxifene, 6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalen-2-ol. tartrate; ER, estrogen receptor; AUC, area(s) under the curve; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; WBAL, whole-body autoradioluminography; LLOQ, lower quantification limit; β-RAM, radioactive monitor; CID, collision-induced dissociation; CNS, central nervous system.
Address correspondence to: Dr. Chandra Prakash, Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Groton, CT 06340. E-mail: chandra.prakash{at}pfizer.com
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