Increased Hepatobiliary Clearance of Unconjugated Thyroxine Determines DMP 904-Induced Alterations in Thyroid Hormone Homeostasis in Rats
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《毒物学科学杂志》
Departments of Metabolism and Pharmacokinetics, Discovery Toxicology and Biotransformation, Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, 5 Research Parkway, Wallingford, Connecticut, 06492-7660/Route 206 and Province Line Road, Princeton, New Jersey 08543
ABSTRACT
4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1,5-a]-pyrimidine (DMP 904) is a potent and selective antagonist of corticotropin releasing factor receptor-1 (CRF1 receptor) with an efficacious anxiolytic profile in preclinical animal models. In subchronic toxicity studies in Sprague-Dawley rats, DMP 904 produced thyroid follicular cell hypertrophy and hyperplasia, and a low incidence of follicular cell adenoma. The current investigations were designed to determine the mode of action by which DMP 904 disrupts thyroid homeostasis in male rats. Five-day treatment with DMP 904 (300 mg/kg/day) dramatically lowered serum thyroxine (T4) to levels below detectable limits (<1 μg/dl) by 72 h, with concurrent decreases in triiodothyronine (T3, about a 70% decrease) and increases in thyroid stimulating hormone (TSH; about a three-fold increase). DMP 904 increased [125I]T4 total body clearance (Cltb) (38.21 ± 10.45 ml/h) compared to control (5.61 ± 0.59 ml/h) and phenobarbital-treated rats (7.92 ± 1.62 ml/h). This increase in Cltb was associated with a significant increase in biliary clearance (Clbile) of unconjugated [125I]T4 (nearly 80-times control rates) and increased liver:blood ratios of T4, suggestive of enhanced hepatic uptake of T4. A single dose of DMP 904 (200 mg/kg) increased mRNA levels of hepatic cytochrome P450s (CYP 3A1 and CYP 2B1) and UDP-glucuronosyltransferases (UGT 1A1 and UGT 1A2). DMP 904 also induced mRNAs of the canalicular transporter, multi-drug resistance protein-2 (Mrp2) and sinusoidal transporters, organic anion transporting proteins (Oatp1 and Oatp2) within 24 h. Western blot analysis confirmed DMP 904 related increases in Oatp2 protein expression. Collectively, these data suggest that DMP 904 is an agonist of the constitutive androstane receptor (CAR) and pregnane X receptor (PXR) and that the decreased serum levels of T4 and T3 resulted from increased hepatobiliary clearance. However, DMP 904 is distinguished from other compounds associated with similar effects on thyroid hormone homeostasis because its effects were primarily related to increased biliary excretion of unconjugated T4.
Key Words: thyroxine; triiodothyronine; rat; Oatp2; biliary clearance; thyroid stimulating hormone; hepatic transporters.
INTRODUCTION
The disruption of thyroid hormone homeostasis by a variety of xenobiotics has been associated with thyroid follicular cell hypertrophy, hyperplasia, and the development of thyroid tumors in rats (Capen, 1996; Hill et al., 1989). These observed changes are attributed to reductions in blood levels of thyroid hormone causing a sustained increase in levels of thyroid stimulating hormone (TSH). When compared to non-rodent species, rats, especially males, are more sensitive to xenobiotic-induced alterations in thyroid hormone levels (Capen, 1996; Meek et al., 2003). Prolonged stimulation of the thyroid by TSH in rats can cause an increase in thyroid cell proliferation ultimately resulting in the formation of thyroid tumors (Hill et al., 1989; Hood et al., 1999; Vansell et al., 2004).
Thyroid toxicants affect circulating levels of thyroid hormone by either direct action on the thyroid gland or by increasing peripheral elimination of thyroid hormone. Thyrotrophic effects of agents such as propylthiouracil (PTU) result from a direct action on the thyroid gland via inhibition of thyroid peroxidase which impairs thyroid hormone synthesis (Capen, 1996). In contrast, xenobiotics such as phenobarbital (PB; McClain et al., 1989), pregnenolone-16-cabonitrile (PCN; Liu et al., 1995), SC-37211 (a novel imidazole; Comer et al., 1985), simvastatin (Smith et al., 1991), omeprazole, and lansoprazole (Masubuchi et al., 1997), have been shown to increase the peripheral elimination of thyroxine. Agents such as PB and PCN are activators of CAR and PXR, respectively, and the increase in thyroxine clearance has been attributed to microsomal enzyme induction, particularly induction of thyroxine (T4) glucuronidation with a subsequent enhancement of biliary excretion of the conjugated hormone (Barter and Klaassen, 1994; McClain et al., 1989; Vansell and Klaassen, 2001).
4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1,5-a]-pyrimidine (DMP 904) is a potent and selective CRF1 receptor antagonist with an anxiolytic profile in preclinical animal models (Fig. 1; Gilligan et al., 2000a; Lelas et al., 2004). Evidence from both preclinical and clinical investigations suggests that CRF1 receptor antagonists may be effective in the treatment of neurological disorders such as anxiety and depression (Gilligan et al., 2000b). In three-month toxicity studies in Sprague-Dawley rats, DMP 904 produced thyroid follicular cell hypertrophy and hyperplasia at doses 20 mg/kg/day in male and female rats, and a low incidence of follicular cell adenoma at 200 mg/kg/day in males (Gemzik et al., 2002). DMP 904 was non-genotoxic in standard assays and did not cause DNA damage in Comet assays with primary rat hepatocytes treated with DMP 904 or hepatocytes isolated from DMP 904 treated rats (Gemzik et al., 2002). Therefore, the purpose of the present work was to test the hypothesis that DMP 904 alters thyroid hormone homeostasis in rats by a mechanism involving increased hepatobiliary clearance of the hormone and to identify the major factors contributing to these changes.
MATERIALS AND METHODS
Chemicals.
DMP 904 was synthesized at Dupont Pharmaceuticals, with a purity of >99%. PTU and PB were purchased from Sigma-Aldrich Co. (St. Louis, MO). [125I]T4 was purchased from PerkinElmer Life Sciences Inc. (Boston, MA). For all studies, [125I]T4 was purified by Sephadex LH-20 (Sigma-Aldrich Co., St. Louis, MO) chromatography as described by Rutgers et al. (1989) just prior to use. All other reagents were the highest grade available.
Animals.
Male Sprague-Dawley rats (Charles River Laboratories Inc., Wilmington, MA) approximately 8–10 weeks old were used throughout. Rats were housed in environmentally controlled rooms to maintain constant temperature and humidity and were allowed free access to food (LabDiet Certified Rodent Diet 5002; PMI Nutrition International, St. Louis, MO) and water.
Five-day thyroid hormone time-course study.
This study was designed to determine the effects of DMP 904 and PTU (as a positive control) on serum levels of T3, T4, and TSH. Three groups of rats (n = 35/group) were orally administered vehicle (0.5 % aqueous methylcellulose), PTU (200 mg/kg/day), or DMP 904 (300 mg/kg/day), once a day for five consecutive days. The dosage of DMP 904 was selected as that which caused the maximum effect on thyroid hormone levels in preliminary studies. Serum (approximately 600 μl) was isolated at 12, 24, 48, 72, 96, and 120 h following the first dose from 10 to 15 rats/group/time point and frozen at –20°C. A maximum of three serum samples was collected from each rat (two collected under anesthesia from the orbital plexus and one terminal at necropsy from the abdominal vena cava). With the exception of the 12 h time point, all serum samples were collected just prior to the administration of the daily dose. Serum levels of T3, T4, and TSH were measured by radioimmunoassay (T3 and T4; Diagnostic Products Corp., Los Angeles, CA, and TSH; Amersham Pharmacia Biotech, Piscataway, NJ). After collection of the final serum sample, rats were necropsied and livers and thyroid glands were removed, weighed, fixed in formalin, and examined histologically after staining with hematoxylin and eosin.
Ex vivo determination of T4 unbound fraction.
The unbound fraction of T4 in rat serum was determined ex vivo by equilibrium dialysis (Dianorm; Munich, Germany) at 37°C using serum samples isolated at 120 h post-dose from the five-day thyroid hormone time-course study described above. Samples from each treatment group (n = 10/group) were pooled, [125I]T4 (675 μCi/ml) was added, and the serum samples were subjected to equilibration against isotonic phosphate buffer (pH 7.4) at 37°C. Following the equilibration period, concentrations of [125I]T4 in the serum and buffer samples were assessed using the same methodology described below for the bile and plasma. All samples were analyzed in duplicate. The unbound fraction of thyroxine was calculated as the quotient of the [125I]T4 concentration in the dialysis buffer divided by the concentration in the serum following dialysis.
Thyroxine biliary clearance study.
This study was designed to evaluate the time course of [125I]T4 in plasma and bile following administered to rats after five days of treatment with DMP 904 or PB (as a positive control). Three groups of jugular- and bile duct-cannulated rats (n = 3/group) were administered vehicle (0.5% aqueous methylcellulose), DMP 904 (200 mg/kg/day), or PB (100 mg/kg/day by oral gavage for five days. The 200 mg/kg dosage of DMP 904 was selected in this and subsequent studies since experiments performed after the five-day thyroid hormone time-course study suggested that this dosage had similar effects on thyroid hormone levels as the 300 mg/kg dose. At 4 h after the administration of the last po dose of test articles, [125I]T4 (25 μCi) was administered iv via the lateral tail vein. Plasma was collected at predose, 0.1, 0.5, 1, 2, 4, 6, 8, 12, 24, 32, and 48 h following the administration of [125I]T4. Cumulative bile samples were collected at 0–1, 1–2, 2–4, 4–8, 8–12, and 12–24 h after the [125I]T4 dose. All samples were frozen at –20°C and assayed for [125I], [125I]T4, [125I]T4-glucuronide, [125I]T4-sulphate, [125I]T3, [125I]T3-glucuronide, and [125I]T3-sulphate.
Analysis of bile and plasma samples.
Total radioactivity in bile and plasma samples and aliquots of the administered doses were assessed using a Packard Cobra gamma counter (PerkinElmer Life Sciences Inc., Boston, MA). Concentrations of radioactivity for each sample were expressed as % dose/ml. The contribution of the individual radioactive components (i.e., free iodine, T4, T4-glucuronide, T4-sulphate, T3, T3-glucuronide, and T3-sulphate) to the total radioactivity in each sample was determined by first separating the individual components using HPLC (Rutgers et al., 1989), with measurement of each of the component peaks using an in-line radiometric detector (Packard FLO-ONE Model A-500 Radiomatic detector, PerkinElmer Life Sciences Inc., Boston, MA). The percent contribution of each component to the total amount of radioactivity in each sample was determined from the radioactivity in each component peak.
Whole body autoradiography (WBA) study.
The purpose of this study was to assess whole body distribution of [125I]T4 after five days of treatment with DMP 904. Two groups of three rats were administered either vehicle (0.5% aqueous methylcellulose) or 200 mg/kg/day of DMP 904 orally for five days. At 4 h after the administration of the last po dose of the test articles, [125I]T4 (1.8 μCi/ rat) was administered iv via lateral tail vein. Rats were sacrificed by CO2 inhalation at 0.25, 0.5, and 1 h following administration of the [125I]T4 dose. Blood (approximately 1 ml) was collected at necropsy from the vena cava and distribution of radioactivity was determined by phosphor quantitative whole body autoradiography as described by Ullberg (1977). Briefly, each rat carcass was immediately frozen by immersion in a hexane/dry ice bath (–70°C) for 10 min. Carcasses were drained, blotted dry, and placed on dry ice for 2 h. Frozen rat carcasses were embedded along with section thickness quality control standards (14C-spiked rat blood) in carboxymethylcellulose (Chay and Pohland, 1994) and frozen at approximately –70°C. Sections (about 30 microns thick) were collected at eight levels of interest in the sagittal plane (all major tissues, organs, and fluids were included in these levels) using a Leica CM3600 Cryomicrotome (Leica Microsystems, Deerfield, IL) at approximately –20°C. Sections were lyophilized, mounted on a black cardboard support, wrapped with Saran Wrap and exposed to phosphor imaging plates (IPs; BASIII, Fuji Photo Film Co., LTD., Japan) for 25 h. Exposed IPs were scanned into the WBA imaging system via a FLA 3000 BioImaging Analyzer (Fuji Biomedical Products, Fuji Photo Film Co., Ltd., Japan) and digital images of the radioactivity in each section were obtained using M5+ MCID software (Imaging Research Inc., St. Catharine's, Ontario). Approximate tissue concentrations were interpolated from a single point standard calibration curve using the MCID image analysis system, which was produced using the concentration of radioactivity in the blood of each animal as determined by liquid scintillation counting. Tissue concentrations were determined as nanocuries per gram of tissue. Tissue concentrations were obtained for liver, intestine and blood, and liver:blood and small intestine:blood ratios were determined. It should be noted that some de-iodination was evident as high concentrations of radioactivity were observed in the thyroid glands of all animals. Thus, the WBA data are to be considered as semi-quantitative.
Real-time polymerase chain reaction (PCR) analysis.
The purpose of this study was to evaluate the effects of DMP 904 on hepatic expression of cytochrome P450 enzymes, UDP-glucuronosyltransferases and various basolateral and sinusoidal transporters. Two groups of fifteen male rats were administered a single po dose of vehicle (0.5% aqueous methylcellulose) or DMP 904 (200 mg/kg). Liver samples (n = 5/time point) were collected at 3, 6, and 24 h following dosing. Liver samples were collected, immediately frozen in liquid nitrogen, and stored frozen at –80°C pending analysis. The following mRNAs were determined by PCR: CYP 2B1, CYP 3A1, UGT 1A1, UGT 1A2, UGT 1A6, Mrp2, Oatp1, 2 and 4, and Na+/taurocholate cotransporting polypeptide (Ntcp). Total RNA was extracted using Triazol (Invitrogen Corporation, Carlsbad, CA) and purified using a Qiagen's RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instruction. Complete removal of DNA was achieved by using a Qiagen's RNase-Free DNase Set. Five μg of RNA was reverse transcribed to cDNA using a High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). Real-time PCR was performed with an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) using 2X SYBR Green master mix (Eurogentec, San Diego, CA). Primers were designed for selected genes using Primer Express (v2.0, Applied Biosystems), and checked for specificity by BLAST searches. In addition, primers were only used when they gave rise to a single amplicon as revealed by melting curve analysis. Sequences of forward and reverse primers for target genes purchased from Sigma Genosys (The Woodlands, TX) and Invitrogen Corporation are listed in Table 1. Twenty ng of cDNA samples were amplified in duplicate. 18s rRNA was used as an endogenous control to normalize the mRNA target for the differences in the amount of total RNA added to each reaction. Standard curves were constructed for the target mRNA and the endogenous control (18s rRNA) by serial dilution (60, 20, 6.67, 2.22, 0.74, 0.25, and 0.082 ng cDNA) of the mixture of cDNA samples obtained from rat livers collected at 24 h following DMP 904 treatment. The amount of target gene and endogenous control in samples was determined by linear regression analysis, and the target mRNA abundance was expressed as the ng target gene/ng 18s rRNA ratio.
Western blot analysis.
Two groups of three rats were administered either vehicle (0.5% aqueous methylcellulose) or DMP 904 (200 mg/kg/day) orally for three days. Livers were collected, and membrane fractions were prepared as described by Johnson and Klaassen (2002). Membrane proteins (30 μg) were separated by SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and probed with polyclonal antibodies recognizing rat Oatp1 and Oatp2 (Alpha Diagnostic International Inc., San Antonio, TX). Proteins were visualized using a ECF Western blotting kit (Amersham Biosciences, Piscataway, NJ). The fluorescence was scanned on Phosphoimager (Molecular Dynamics, Sunnyvale, CA) and the fluorescent signal corresponding to Oatp1 and Oatp2 were selected and quantified with ImageQuant software, version 5.2a (Molecular Dynamics, Sunnyvale, CA).
Data analysis.
All pharmacokinetic parameters were calculated by standard methods as described in Gibaldi and Perrier (1982). When statistical analyses were conducted, data were compared with an ANOVA followed by Dunnett's test using JMP v 5.0.1 statistical software (SAS Institute Inc., Cary, NC). The significance level was p < 0.05.
RESULTS
Liver weights were increased 1.6- or 1.3-fold (relative to body weight) in rats after five daily doses of DMP 904 or PTU, respectively. Both molecules produced thyroid follicular cell hypertrophy and hyperplasia, and increased follicular cell mitoses (results not shown). In separate experiments with DMP 904 in rats, hepatocellular hypertrophy and hyperplasia accompanied the increased liver weight (results not shown).
The time course for alterations in serum thyroid hormone levels during five days of treatment with DMP 904 is presented in Figure 2. DMP 904 decreased T4 serum concentrations within the first 12 h after dosing and hormone levels were below the limit of detection of the radioimmunoassay (<1 μg/dl) after three days. Similarly, T3 serum levels for the DMP 904-treated group decreased over the time course to approximately 30% of the control after five days of dosing. Concurrent with the changes in serum T3 and T4 levels, there was an increase in TSH (62%) at 12 h following the first dose of DMP 904, and by five days, TSH levels were nearly three-times control levels. Thyroid hormone and TSH levels for PTU-treated rats followed a trend similar to that observed for the DMP 904-treated rats. However, the magnitude of the changes in thyroid hormone concentrations observed for the PTU-treated group was less than that observed in the DMP 904-treated animals (Fig. 2).
Experiments were conducted to determine whether DMP 904 had a direct effect on the thyroid gland and on the synthesis of thyroid hormone. These results indicated that DMP 904 did not inhibit porcine thyroid peroxidase activity (tested up to 0.3 mM) or reduce iodine uptake and organification in rats (data not shown). Therefore, the effects on serum hormone levels were considered likely to represent an extra-thyroidal effect.
To determine whether the dramatic decrease in serum T4 following DMP 904 treatment was associated with changes in free circulating hormone levels, ex vivo determination of T4 unbound fraction was assessed in pooled rat serum. The average unbound fraction in the two replicates was determined to be 0.048% for the control group, 0.045% for the PTU-treated group, and 0.100% for the DMP 904-treated group. No statistical comparisons between treatment groups were made since pooled serum was used in the determinations of T4 unbound fraction.
Figure 3 shows mean semi logarithmic plots of [125I] T4 plasma concentration vs. time following iv administration of 25 μCi of [125I] T4 to rats pretreated with either control, PB, or DMP 904 for five days, and Table 2 summarizes the plasma pharmacokinetic parameters estimated from these data. The plasma half-life (t1/2) and mean residence time (MRT) were similar between the control and both treatment groups. In contrast, the major finding from this analysis was that mean total body clearance (Cltb) of [125I] T4 for the DMP 904-treated group was nearly seven-times greater than control animals. Although Cltb in the PB-treated group was slightly greater than control rats, the difference was not significant. The Vdss (steady-state volume of distribution) was larger in the DMP 904 treated group; however, given the large standard deviation, this difference was not statistically different from controls (Table 2).
The average bile flow rate was determined for the three treatment groups during the first 8-h period following dosing of [125I] T4 in order to assess any effect of the different treatments on bile flow. Bile flow rate for the control group was 1.27 ± 0.31 ml/h, whereas that for PB and DMP 904 were nearly two-times higher at 2.02 ± 1.00 ml/h and 2.05 ± 0.31 ml/h, respectively. However, these differences were not statistically significant from control rats.
Table 3 depicts the biliary mass balance data for the three treatment groups. The amount of unconjugated [125I] T4 excreted in bile by the DMP 904-treated rats was approximately 11-times higher than that observed for controls and represented nearly 40% of the administered dose. In contrast, the amount of unconjugated [125I] T4 excreted by the PB-treated group was not different from the control. However, in PB-treated rats, a significantly higher amount of [125I] T4-glucuronide (about 50% of the administered dose) was observed in bile whereas DMP 904 did not increase the percent of the total dose excreted as [125I] T4-glucuronide. Total amounts of [125I] labeled T3, T3-glucuronide, T3-sulphate, and T4-sulphate observed in bile for all treatments groups accounted for < 1% of the administered dose.
Table 4 summarizes the biliary clearance (Clbile) of [125I] T4 and [125I] T4-glucuronide. Consistent with the mass balance data above, the [125I] T4 Clbile estimate for DMP 904-treated animals was nearly 80-times higher than control and approximately 35-times higher than PB-treated rats. The [125I] T4-glucuronide Clbile estimates for PB- and DMP 904-treated rats were both greater than three-times the control estimate. However, the difference was only significant for DMP 904-treated rats.
A representative whole body radiograph (WBA) of a sagittal plane from a control and a DMP 904-treated rat 1 h following the administration of [125I] T4 is shown in Figure 4A. Estimated concentrations of [125I] appeared to be higher in the liver and small intestine of the DMP 904-treated animal. A high concentration of [125I] in the WBA was observed in the thyroid of all animals from both groups and likely resulted from deiodination and subsequent concentration of free [125I] in the thyroid. Liver:blood ratios and small intestine:blood ratios estimated from WBA analyses are presented in Figures 4B and 4C, respectively. Liver:blood ratios were approximately 1 in control rats, but DMP 904 treatment increased the liver:blood ratio over time (Fig. 4B). The small intestine:blood ratio was initially less than 1 in control rats, but increased over time for both control and DMP 904-treated animals. The magnitude of the increase, however, was greater for DMP 904 treated rats than controls.
Many agents that alter hepatic clearance of T4 in rats activate CAR or PXR and induce a variety of hepatic microsomal enzymes. Figure 5 shows the time course of changes in mRNA for a number of hepatic enzymes following a single oral dose (200 mg/kg) of DMP 904. Significant increases in mRNA levels were observed for CYP 2B1, CYP 3A1, and UGT 1A1 by 3 h after dosing, and UGT 1A2 was increased by 6 h after dosing. At 24 h, the mRNA for both CYP 3A1 and CYP 2B1 was greater than 20-times higher in livers from DMP 904-treated than control rats. The increase in mRNA levels for UGTs 1A1 and 1A2 were also significantly elevated, albeit to a lesser magnitude of change. No changes were observed in the mRNA expression of UGT 1A6.
Figure 6 shows the time course of alterations in mRNA levels of hepatic transporters. The most pronounced changes in transporter gene expression following treatment with DMP 904 were observed for Oatp2. Oatp2 mRNA levels were approximately two-times higher in livers from DMP 904-treated rats at 3 h, and by 24 h, mRNA levels of Oatp2 were about four-times higher. Oatp1 mRNA was also significantly increased at 24 h. In contrast, two other hepatic uptake transporters, Oatp4 and Ntcp, showed small but significant decreases in gene expression at 24 h. mRNA for the canalicular transporter Mrp2 increased, with 2-fold and 1.4-fold changes noted at 6 and 24 h, respectively.
Western blot analysis was used to specifically assess increases in Oatp1 and Oatp2 protein expression in liver. Consistent with the real-time PCR results, DMP 904 treatment resulted in the induction of Oatp2 protein levels in rat liver based upon the Western blot analysis shown in Figure 7. However, no difference was observed in protein levels of Oatp1.
DISCUSSION
A variety of xenobiotics are known to alter thyroid hormone homeostasis in rats through a mechanism involving increased hepatic clearance of the thyroid hormone (Curran and DeGroot, 1991). In most cases, biliary clearance of the conjugated hormone, particularly the glucuronide conjugate, is increased, and its excretion into bile increases the elimination of the hormone (Barter and Klaassen, 1994; Vansell and Klaassen, 2002a). To date, most compounds that alter hepatic clearance of thyroxine decrease serum hormone levels within a few days of dosing (Barter and Klaassen, 1994). In the present work, DMP 904 treatment also decreased serum thyroid hormone levels and increased biliary clearance of the glucuronide conjugate of the hormone. DMP 904 treatment also induced a variety of hepatic microsomal enzymes and xenobiotic transporters, suggesting that the mechanism underlying the decreased levels of serum thyroid hormones is likely to involve increased hepatic clearance of thyroxine in a manner analogous to prototypical compounds such as PB and PCN. However, DMP 904 is distinguished from compounds such as PB and PCN in two ways. First, the compound rapidly decreased serum hormone levels within 12 h of dosing, with T4 reduced to non-detectable levels after three days of dosing. Second, although biliary clearance of the glucuronide conjugate was increased, the major component excreted into bile after DMP 904 treatment was the unconjugated hormone.
The results of the biliary clearance study with [125I] T4 indicated that the severe reductions in T4 serum levels in DMP 904-treated rats were due to a nearly seven-fold increase in T4 Cltb. This large increase in T4 Cltb could not be explained by the modest increase in T4 unbound fraction observed following DMP 904 administration at a comparable dose, because at most, a two-fold increase in T4 unbound fraction occurred following DMP 904 treatment which would only account for two-fold increase in T4 Cltb.
Many investigators report biliary T4 and T4-glucuronide elimination as excretion rates (Masubuchi et al., 1997; McClain et al., 1989; Vansell and Klaassen, 2001), whereas, in the present work, biliary elimination of unconjugated T4 and T4-glucuronide was evaluated as biliary clearances. The additive nature of clearances allows for consideration of the contribution of each change in T4 disposition to overall changes in T4 Cltb. Accordingly, the observed increase in biliary clearance (about three-fold) of the glucuronide conjugate in PB-treated animals is consistent with induction of uridine diphosphate glucuronosyltransferases (UGTs) followed by enhanced biliary excretion of the conjugated hormone as previously described (Barter and Klaassen, 1992, 1994; McClain et al., 1989). In direct contrast, DMP 904-treated rats excreted about 16% of the dose as the glucuronide conjugate, with nearly 40% of the dose excreted as the unconjugated hormone. The large percentage of the dose recovered as unconjugated hormone is a direct result of the nearly 80-fold increase in the biliary clearance of unconjugated T4 following DMP 904-treatment. A DMP 904-related increase in T4-glucuronidation capability was not obvious from the biliary mass balance results since the percent of administered [125I] T4 recovered as the glucuronide conjugate was not increased. However, since T4-glucuronide is excreted almost entirely in the bile (Rutgers et al., 1989; Vansell and Klaassen, 2001), the increase in T4-glucuronide Clbile by DMP 904 suggests that DMP 904 treatment results in the induction of UGTs involved in T4 metabolism. This conclusion is consistent with the observed increase in UGT 1A1 mRNA levels associated with DMP 904 treatment since UGT 1A1 is thought to be involved in the T4 glucuronidation (Vansell and Klaassen, 2002a). Together, changes in total body clearance of T4 brought about by DMP 904 treatment appears to be the result of increased biliary clearance of both unconjugated T4 and T4-glucuronide along with an increase in the unbound fraction of T4. The largest contribution to the increase in T4 Cltb is due to an increase in biliary clearance of unconjugated T4. In light of the observation that DMP 904 treatment results in only modest changes in T4 unbound fraction, it is unlikely that the large contribution of unconjugated T4 Clbile results solely from changes in protein binding of T4. Rather, it is more likely that induction or upregulation of higher affinity elimination processes other than those involved in generation and excretion of the glucuronide conjugate play a role in the marked increased in T4 Clbile. The results of the whole body autoradiography (WBA) study are consistent with these observations from the biliary clearance study. The time-dependent increase in the concentration of [125I] into the liver of DMP 904 treated rats suggest that the marked reductions in serum T4 may result largely from an increased uptake of T4 into the liver. Rapid concentration of [125I] in the liver is consistent with the increase in Vdss. The larger increase in the small intestine:blood ratio with time in the DMP 904 treated rats compared to control animals is also consistent with the large increase in the biliary excretion of unconjugated T4 observed in the biliary clearance study. In combination, the data from the biliary clearance and WBA studies suggest that DMP 904 treatment results in both the induction of UGTs and induction or upregulation of a higher affinity T4 hepatic uptake process.
The results presented here for DMP 904, showing an important role for Clbile of unconjugated T4 in altering thyroid hormone homeostasis, are not unique as a similar phenomenon involving both increased uptake and increased biliary excretion of unconjugated hormone was observed reported for two novel antihistamines, SK&F 93479 (lupitidine) and SK&F 93944 (temelastine) (Poole et al., 1989, 1990). Although the magnitude was not as severe as observed for DMP 904, both SKF compounds produced thyroid pathology in rats (Atterwill et al., 1989; Brown et al., 1987), and both increased biliary excretion of T4, with the unconjugated hormone accounting for the majority of the excreted T4 (Poole et al., 1989, 1990). Furthermore, changes in T4 elimination for both compounds were accompanied by an increased hepatocellular accumulation of T4 via an energy dependent process with no apparent effect on T3 uptake (Poole et al., 1989, 1990). Currently, basolateral transporters involved in the hepatic uptake of T4 are known to include Oatp1, Oatp2, Oatp4, and Ntcp (Abe et al., 1998; Cattori et al., 2000; Friesema et al., 1999). DMP 904 induced mRNA for both Oatp1 and Oatp2, with Oatp2 mRNA increased as early as 3 h after dosing. However, only Oatp2 protein expression levels appeared to be increased by DMP 904 suggesting that Oatp2 is likely to be involved in the observed increase in T4 hepatic uptake.
In contrast to hepatic uptake of thyroid hormone, less is known regarding the transport of thyroxine out of the hepatocyte and into the bile. Ribeiro et al. (1996) observed the existence of a saturable and temperature sensitive transporter in rat hepatoma cells that appeared to be involved in the efflux of thyroid hormones and was inhibited by verapamil. Verapamil is a potent inhibitor of the ATP-binding cassette transporter superfamily. Mrp2, a member of this family, is the main canalicular transporter responsible for the secretion of organic anions in the bile (Kipp and Arias, 2002). Our experiments show that DMP 904 treatment increased hepatic levels of mRNA for Mrp2. It is plausible that Mrp2 plays a role in the observed increase in the biliary clearance of unconjugated [125I]T4, but more work to directly characterize the transporters involved in the biliary excretion of thyroid hormone is needed to adequately address this question.
From our investigation, it is not known whether DMP 904 treatment directly affects T3 elimination. However, since the majority of the circulating T3 is formed from deiodination of T4 in peripheral tissues (Oppenheimer and Surks, 1974), DMP 904 is likely to be, at the least, indirectly responsible for the observed reduction in T3 levels due to the depletion of T4 from the body. Furthermore, induction of Oatp2 may increase T3 hepatic uptake since the uptake affinity of Oatp2 is similar for both T3 and T4 (Abe et al., 1998). Vansell and Klaassen (2002b) postulated that it is the decrease of circulating T3 that is responsible for drug induced TSH increases. Again, a more direct evaluation of DMP 904-related effects on T3 would be required to address this issue.
DMP 904 caused thyroid follicular cell hypertrophy and hyperplasia and a low incidence of follicular cell adenoma in rats when dosed at 200 mg/kg/day for three months (Gemzik et al., 2002). There was no evidence from our studies to suggest that the observed effect of DMP 904 on the thyroid is related to its pharmacological activity as a CRF1 receptor antagonist. The present results suggest that DMP 904 is likely to act via an extra-thyroidal mechanism to cause prolonged stimulation of TSH, a factor known to promote thyroid tumor development in rats (Hiasa et al., 1982; Hill et al., 1989; McClain et al., 1988; Vansell et al., 2004). However, induction of thyroid tumors in rats by a mechanism involving increased hepatobiliary clearance is not likely to be predictive of a similar risk in humans, based largely on quantitative differences in the physiology and biochemistry of the thyroid pituitary axis in rats (Cohen et al., 2004; Meek et al., 2003). The lack of human relevance is also supported by the fact that many agents that reduce circulating thyroid hormone levels by extra-hepatic mechanisms in rats fail to do so in mice (Craft et al., 2002; Viollon-Abadie et al., 1999) as well as man (Ohnhaus et al., 1981).
In conclusion, the present results provide insight into the mechanisms by which DMP 904 disrupts thyroid homeostasis. The data suggest that an increase in the biliary clearance of unconjugated T4 plays a major role in the large increase in T4 total body clearance following DMP 904 treatment to rats. Collectively, the data suggest that DMP 904 disrupts thyroid hormone homeostasis primarily via alterations in hepatic transport of T4 in addition to apparent increases in T4 glucuronidation capacity. This combined mode of action of DMP 904 may explain its pronounced effect on thyroid homeostasis in rats including production of a low incidence of thyroid adenoma following three months of treatment.
NOTES
Portions of this data were presented at the 41st annual meeting of the Society of Toxicology, March 2002, Nashville, TN, and at the 42nd annual meeting of the Society of Toxicology, March 2003, Salt Lake City, UT.
1 Present address: Genentech, Inc., South San Francisco, CA.
3 Present address: Quest Pharmaceutical Services, Newark, DE.
4 Present address: Endo Pharmaceuticals, Chadds Ford, PA.
5 Present address: Incyte Corporation, Wilmington, DE.
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ABSTRACT
4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1,5-a]-pyrimidine (DMP 904) is a potent and selective antagonist of corticotropin releasing factor receptor-1 (CRF1 receptor) with an efficacious anxiolytic profile in preclinical animal models. In subchronic toxicity studies in Sprague-Dawley rats, DMP 904 produced thyroid follicular cell hypertrophy and hyperplasia, and a low incidence of follicular cell adenoma. The current investigations were designed to determine the mode of action by which DMP 904 disrupts thyroid homeostasis in male rats. Five-day treatment with DMP 904 (300 mg/kg/day) dramatically lowered serum thyroxine (T4) to levels below detectable limits (<1 μg/dl) by 72 h, with concurrent decreases in triiodothyronine (T3, about a 70% decrease) and increases in thyroid stimulating hormone (TSH; about a three-fold increase). DMP 904 increased [125I]T4 total body clearance (Cltb) (38.21 ± 10.45 ml/h) compared to control (5.61 ± 0.59 ml/h) and phenobarbital-treated rats (7.92 ± 1.62 ml/h). This increase in Cltb was associated with a significant increase in biliary clearance (Clbile) of unconjugated [125I]T4 (nearly 80-times control rates) and increased liver:blood ratios of T4, suggestive of enhanced hepatic uptake of T4. A single dose of DMP 904 (200 mg/kg) increased mRNA levels of hepatic cytochrome P450s (CYP 3A1 and CYP 2B1) and UDP-glucuronosyltransferases (UGT 1A1 and UGT 1A2). DMP 904 also induced mRNAs of the canalicular transporter, multi-drug resistance protein-2 (Mrp2) and sinusoidal transporters, organic anion transporting proteins (Oatp1 and Oatp2) within 24 h. Western blot analysis confirmed DMP 904 related increases in Oatp2 protein expression. Collectively, these data suggest that DMP 904 is an agonist of the constitutive androstane receptor (CAR) and pregnane X receptor (PXR) and that the decreased serum levels of T4 and T3 resulted from increased hepatobiliary clearance. However, DMP 904 is distinguished from other compounds associated with similar effects on thyroid hormone homeostasis because its effects were primarily related to increased biliary excretion of unconjugated T4.
Key Words: thyroxine; triiodothyronine; rat; Oatp2; biliary clearance; thyroid stimulating hormone; hepatic transporters.
INTRODUCTION
The disruption of thyroid hormone homeostasis by a variety of xenobiotics has been associated with thyroid follicular cell hypertrophy, hyperplasia, and the development of thyroid tumors in rats (Capen, 1996; Hill et al., 1989). These observed changes are attributed to reductions in blood levels of thyroid hormone causing a sustained increase in levels of thyroid stimulating hormone (TSH). When compared to non-rodent species, rats, especially males, are more sensitive to xenobiotic-induced alterations in thyroid hormone levels (Capen, 1996; Meek et al., 2003). Prolonged stimulation of the thyroid by TSH in rats can cause an increase in thyroid cell proliferation ultimately resulting in the formation of thyroid tumors (Hill et al., 1989; Hood et al., 1999; Vansell et al., 2004).
Thyroid toxicants affect circulating levels of thyroid hormone by either direct action on the thyroid gland or by increasing peripheral elimination of thyroid hormone. Thyrotrophic effects of agents such as propylthiouracil (PTU) result from a direct action on the thyroid gland via inhibition of thyroid peroxidase which impairs thyroid hormone synthesis (Capen, 1996). In contrast, xenobiotics such as phenobarbital (PB; McClain et al., 1989), pregnenolone-16-cabonitrile (PCN; Liu et al., 1995), SC-37211 (a novel imidazole; Comer et al., 1985), simvastatin (Smith et al., 1991), omeprazole, and lansoprazole (Masubuchi et al., 1997), have been shown to increase the peripheral elimination of thyroxine. Agents such as PB and PCN are activators of CAR and PXR, respectively, and the increase in thyroxine clearance has been attributed to microsomal enzyme induction, particularly induction of thyroxine (T4) glucuronidation with a subsequent enhancement of biliary excretion of the conjugated hormone (Barter and Klaassen, 1994; McClain et al., 1989; Vansell and Klaassen, 2001).
4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1,5-a]-pyrimidine (DMP 904) is a potent and selective CRF1 receptor antagonist with an anxiolytic profile in preclinical animal models (Fig. 1; Gilligan et al., 2000a; Lelas et al., 2004). Evidence from both preclinical and clinical investigations suggests that CRF1 receptor antagonists may be effective in the treatment of neurological disorders such as anxiety and depression (Gilligan et al., 2000b). In three-month toxicity studies in Sprague-Dawley rats, DMP 904 produced thyroid follicular cell hypertrophy and hyperplasia at doses 20 mg/kg/day in male and female rats, and a low incidence of follicular cell adenoma at 200 mg/kg/day in males (Gemzik et al., 2002). DMP 904 was non-genotoxic in standard assays and did not cause DNA damage in Comet assays with primary rat hepatocytes treated with DMP 904 or hepatocytes isolated from DMP 904 treated rats (Gemzik et al., 2002). Therefore, the purpose of the present work was to test the hypothesis that DMP 904 alters thyroid hormone homeostasis in rats by a mechanism involving increased hepatobiliary clearance of the hormone and to identify the major factors contributing to these changes.
MATERIALS AND METHODS
Chemicals.
DMP 904 was synthesized at Dupont Pharmaceuticals, with a purity of >99%. PTU and PB were purchased from Sigma-Aldrich Co. (St. Louis, MO). [125I]T4 was purchased from PerkinElmer Life Sciences Inc. (Boston, MA). For all studies, [125I]T4 was purified by Sephadex LH-20 (Sigma-Aldrich Co., St. Louis, MO) chromatography as described by Rutgers et al. (1989) just prior to use. All other reagents were the highest grade available.
Animals.
Male Sprague-Dawley rats (Charles River Laboratories Inc., Wilmington, MA) approximately 8–10 weeks old were used throughout. Rats were housed in environmentally controlled rooms to maintain constant temperature and humidity and were allowed free access to food (LabDiet Certified Rodent Diet 5002; PMI Nutrition International, St. Louis, MO) and water.
Five-day thyroid hormone time-course study.
This study was designed to determine the effects of DMP 904 and PTU (as a positive control) on serum levels of T3, T4, and TSH. Three groups of rats (n = 35/group) were orally administered vehicle (0.5 % aqueous methylcellulose), PTU (200 mg/kg/day), or DMP 904 (300 mg/kg/day), once a day for five consecutive days. The dosage of DMP 904 was selected as that which caused the maximum effect on thyroid hormone levels in preliminary studies. Serum (approximately 600 μl) was isolated at 12, 24, 48, 72, 96, and 120 h following the first dose from 10 to 15 rats/group/time point and frozen at –20°C. A maximum of three serum samples was collected from each rat (two collected under anesthesia from the orbital plexus and one terminal at necropsy from the abdominal vena cava). With the exception of the 12 h time point, all serum samples were collected just prior to the administration of the daily dose. Serum levels of T3, T4, and TSH were measured by radioimmunoassay (T3 and T4; Diagnostic Products Corp., Los Angeles, CA, and TSH; Amersham Pharmacia Biotech, Piscataway, NJ). After collection of the final serum sample, rats were necropsied and livers and thyroid glands were removed, weighed, fixed in formalin, and examined histologically after staining with hematoxylin and eosin.
Ex vivo determination of T4 unbound fraction.
The unbound fraction of T4 in rat serum was determined ex vivo by equilibrium dialysis (Dianorm; Munich, Germany) at 37°C using serum samples isolated at 120 h post-dose from the five-day thyroid hormone time-course study described above. Samples from each treatment group (n = 10/group) were pooled, [125I]T4 (675 μCi/ml) was added, and the serum samples were subjected to equilibration against isotonic phosphate buffer (pH 7.4) at 37°C. Following the equilibration period, concentrations of [125I]T4 in the serum and buffer samples were assessed using the same methodology described below for the bile and plasma. All samples were analyzed in duplicate. The unbound fraction of thyroxine was calculated as the quotient of the [125I]T4 concentration in the dialysis buffer divided by the concentration in the serum following dialysis.
Thyroxine biliary clearance study.
This study was designed to evaluate the time course of [125I]T4 in plasma and bile following administered to rats after five days of treatment with DMP 904 or PB (as a positive control). Three groups of jugular- and bile duct-cannulated rats (n = 3/group) were administered vehicle (0.5% aqueous methylcellulose), DMP 904 (200 mg/kg/day), or PB (100 mg/kg/day by oral gavage for five days. The 200 mg/kg dosage of DMP 904 was selected in this and subsequent studies since experiments performed after the five-day thyroid hormone time-course study suggested that this dosage had similar effects on thyroid hormone levels as the 300 mg/kg dose. At 4 h after the administration of the last po dose of test articles, [125I]T4 (25 μCi) was administered iv via the lateral tail vein. Plasma was collected at predose, 0.1, 0.5, 1, 2, 4, 6, 8, 12, 24, 32, and 48 h following the administration of [125I]T4. Cumulative bile samples were collected at 0–1, 1–2, 2–4, 4–8, 8–12, and 12–24 h after the [125I]T4 dose. All samples were frozen at –20°C and assayed for [125I], [125I]T4, [125I]T4-glucuronide, [125I]T4-sulphate, [125I]T3, [125I]T3-glucuronide, and [125I]T3-sulphate.
Analysis of bile and plasma samples.
Total radioactivity in bile and plasma samples and aliquots of the administered doses were assessed using a Packard Cobra gamma counter (PerkinElmer Life Sciences Inc., Boston, MA). Concentrations of radioactivity for each sample were expressed as % dose/ml. The contribution of the individual radioactive components (i.e., free iodine, T4, T4-glucuronide, T4-sulphate, T3, T3-glucuronide, and T3-sulphate) to the total radioactivity in each sample was determined by first separating the individual components using HPLC (Rutgers et al., 1989), with measurement of each of the component peaks using an in-line radiometric detector (Packard FLO-ONE Model A-500 Radiomatic detector, PerkinElmer Life Sciences Inc., Boston, MA). The percent contribution of each component to the total amount of radioactivity in each sample was determined from the radioactivity in each component peak.
Whole body autoradiography (WBA) study.
The purpose of this study was to assess whole body distribution of [125I]T4 after five days of treatment with DMP 904. Two groups of three rats were administered either vehicle (0.5% aqueous methylcellulose) or 200 mg/kg/day of DMP 904 orally for five days. At 4 h after the administration of the last po dose of the test articles, [125I]T4 (1.8 μCi/ rat) was administered iv via lateral tail vein. Rats were sacrificed by CO2 inhalation at 0.25, 0.5, and 1 h following administration of the [125I]T4 dose. Blood (approximately 1 ml) was collected at necropsy from the vena cava and distribution of radioactivity was determined by phosphor quantitative whole body autoradiography as described by Ullberg (1977). Briefly, each rat carcass was immediately frozen by immersion in a hexane/dry ice bath (–70°C) for 10 min. Carcasses were drained, blotted dry, and placed on dry ice for 2 h. Frozen rat carcasses were embedded along with section thickness quality control standards (14C-spiked rat blood) in carboxymethylcellulose (Chay and Pohland, 1994) and frozen at approximately –70°C. Sections (about 30 microns thick) were collected at eight levels of interest in the sagittal plane (all major tissues, organs, and fluids were included in these levels) using a Leica CM3600 Cryomicrotome (Leica Microsystems, Deerfield, IL) at approximately –20°C. Sections were lyophilized, mounted on a black cardboard support, wrapped with Saran Wrap and exposed to phosphor imaging plates (IPs; BASIII, Fuji Photo Film Co., LTD., Japan) for 25 h. Exposed IPs were scanned into the WBA imaging system via a FLA 3000 BioImaging Analyzer (Fuji Biomedical Products, Fuji Photo Film Co., Ltd., Japan) and digital images of the radioactivity in each section were obtained using M5+ MCID software (Imaging Research Inc., St. Catharine's, Ontario). Approximate tissue concentrations were interpolated from a single point standard calibration curve using the MCID image analysis system, which was produced using the concentration of radioactivity in the blood of each animal as determined by liquid scintillation counting. Tissue concentrations were determined as nanocuries per gram of tissue. Tissue concentrations were obtained for liver, intestine and blood, and liver:blood and small intestine:blood ratios were determined. It should be noted that some de-iodination was evident as high concentrations of radioactivity were observed in the thyroid glands of all animals. Thus, the WBA data are to be considered as semi-quantitative.
Real-time polymerase chain reaction (PCR) analysis.
The purpose of this study was to evaluate the effects of DMP 904 on hepatic expression of cytochrome P450 enzymes, UDP-glucuronosyltransferases and various basolateral and sinusoidal transporters. Two groups of fifteen male rats were administered a single po dose of vehicle (0.5% aqueous methylcellulose) or DMP 904 (200 mg/kg). Liver samples (n = 5/time point) were collected at 3, 6, and 24 h following dosing. Liver samples were collected, immediately frozen in liquid nitrogen, and stored frozen at –80°C pending analysis. The following mRNAs were determined by PCR: CYP 2B1, CYP 3A1, UGT 1A1, UGT 1A2, UGT 1A6, Mrp2, Oatp1, 2 and 4, and Na+/taurocholate cotransporting polypeptide (Ntcp). Total RNA was extracted using Triazol (Invitrogen Corporation, Carlsbad, CA) and purified using a Qiagen's RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instruction. Complete removal of DNA was achieved by using a Qiagen's RNase-Free DNase Set. Five μg of RNA was reverse transcribed to cDNA using a High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). Real-time PCR was performed with an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) using 2X SYBR Green master mix (Eurogentec, San Diego, CA). Primers were designed for selected genes using Primer Express (v2.0, Applied Biosystems), and checked for specificity by BLAST searches. In addition, primers were only used when they gave rise to a single amplicon as revealed by melting curve analysis. Sequences of forward and reverse primers for target genes purchased from Sigma Genosys (The Woodlands, TX) and Invitrogen Corporation are listed in Table 1. Twenty ng of cDNA samples were amplified in duplicate. 18s rRNA was used as an endogenous control to normalize the mRNA target for the differences in the amount of total RNA added to each reaction. Standard curves were constructed for the target mRNA and the endogenous control (18s rRNA) by serial dilution (60, 20, 6.67, 2.22, 0.74, 0.25, and 0.082 ng cDNA) of the mixture of cDNA samples obtained from rat livers collected at 24 h following DMP 904 treatment. The amount of target gene and endogenous control in samples was determined by linear regression analysis, and the target mRNA abundance was expressed as the ng target gene/ng 18s rRNA ratio.
Western blot analysis.
Two groups of three rats were administered either vehicle (0.5% aqueous methylcellulose) or DMP 904 (200 mg/kg/day) orally for three days. Livers were collected, and membrane fractions were prepared as described by Johnson and Klaassen (2002). Membrane proteins (30 μg) were separated by SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and probed with polyclonal antibodies recognizing rat Oatp1 and Oatp2 (Alpha Diagnostic International Inc., San Antonio, TX). Proteins were visualized using a ECF Western blotting kit (Amersham Biosciences, Piscataway, NJ). The fluorescence was scanned on Phosphoimager (Molecular Dynamics, Sunnyvale, CA) and the fluorescent signal corresponding to Oatp1 and Oatp2 were selected and quantified with ImageQuant software, version 5.2a (Molecular Dynamics, Sunnyvale, CA).
Data analysis.
All pharmacokinetic parameters were calculated by standard methods as described in Gibaldi and Perrier (1982). When statistical analyses were conducted, data were compared with an ANOVA followed by Dunnett's test using JMP v 5.0.1 statistical software (SAS Institute Inc., Cary, NC). The significance level was p < 0.05.
RESULTS
Liver weights were increased 1.6- or 1.3-fold (relative to body weight) in rats after five daily doses of DMP 904 or PTU, respectively. Both molecules produced thyroid follicular cell hypertrophy and hyperplasia, and increased follicular cell mitoses (results not shown). In separate experiments with DMP 904 in rats, hepatocellular hypertrophy and hyperplasia accompanied the increased liver weight (results not shown).
The time course for alterations in serum thyroid hormone levels during five days of treatment with DMP 904 is presented in Figure 2. DMP 904 decreased T4 serum concentrations within the first 12 h after dosing and hormone levels were below the limit of detection of the radioimmunoassay (<1 μg/dl) after three days. Similarly, T3 serum levels for the DMP 904-treated group decreased over the time course to approximately 30% of the control after five days of dosing. Concurrent with the changes in serum T3 and T4 levels, there was an increase in TSH (62%) at 12 h following the first dose of DMP 904, and by five days, TSH levels were nearly three-times control levels. Thyroid hormone and TSH levels for PTU-treated rats followed a trend similar to that observed for the DMP 904-treated rats. However, the magnitude of the changes in thyroid hormone concentrations observed for the PTU-treated group was less than that observed in the DMP 904-treated animals (Fig. 2).
Experiments were conducted to determine whether DMP 904 had a direct effect on the thyroid gland and on the synthesis of thyroid hormone. These results indicated that DMP 904 did not inhibit porcine thyroid peroxidase activity (tested up to 0.3 mM) or reduce iodine uptake and organification in rats (data not shown). Therefore, the effects on serum hormone levels were considered likely to represent an extra-thyroidal effect.
To determine whether the dramatic decrease in serum T4 following DMP 904 treatment was associated with changes in free circulating hormone levels, ex vivo determination of T4 unbound fraction was assessed in pooled rat serum. The average unbound fraction in the two replicates was determined to be 0.048% for the control group, 0.045% for the PTU-treated group, and 0.100% for the DMP 904-treated group. No statistical comparisons between treatment groups were made since pooled serum was used in the determinations of T4 unbound fraction.
Figure 3 shows mean semi logarithmic plots of [125I] T4 plasma concentration vs. time following iv administration of 25 μCi of [125I] T4 to rats pretreated with either control, PB, or DMP 904 for five days, and Table 2 summarizes the plasma pharmacokinetic parameters estimated from these data. The plasma half-life (t1/2) and mean residence time (MRT) were similar between the control and both treatment groups. In contrast, the major finding from this analysis was that mean total body clearance (Cltb) of [125I] T4 for the DMP 904-treated group was nearly seven-times greater than control animals. Although Cltb in the PB-treated group was slightly greater than control rats, the difference was not significant. The Vdss (steady-state volume of distribution) was larger in the DMP 904 treated group; however, given the large standard deviation, this difference was not statistically different from controls (Table 2).
The average bile flow rate was determined for the three treatment groups during the first 8-h period following dosing of [125I] T4 in order to assess any effect of the different treatments on bile flow. Bile flow rate for the control group was 1.27 ± 0.31 ml/h, whereas that for PB and DMP 904 were nearly two-times higher at 2.02 ± 1.00 ml/h and 2.05 ± 0.31 ml/h, respectively. However, these differences were not statistically significant from control rats.
Table 3 depicts the biliary mass balance data for the three treatment groups. The amount of unconjugated [125I] T4 excreted in bile by the DMP 904-treated rats was approximately 11-times higher than that observed for controls and represented nearly 40% of the administered dose. In contrast, the amount of unconjugated [125I] T4 excreted by the PB-treated group was not different from the control. However, in PB-treated rats, a significantly higher amount of [125I] T4-glucuronide (about 50% of the administered dose) was observed in bile whereas DMP 904 did not increase the percent of the total dose excreted as [125I] T4-glucuronide. Total amounts of [125I] labeled T3, T3-glucuronide, T3-sulphate, and T4-sulphate observed in bile for all treatments groups accounted for < 1% of the administered dose.
Table 4 summarizes the biliary clearance (Clbile) of [125I] T4 and [125I] T4-glucuronide. Consistent with the mass balance data above, the [125I] T4 Clbile estimate for DMP 904-treated animals was nearly 80-times higher than control and approximately 35-times higher than PB-treated rats. The [125I] T4-glucuronide Clbile estimates for PB- and DMP 904-treated rats were both greater than three-times the control estimate. However, the difference was only significant for DMP 904-treated rats.
A representative whole body radiograph (WBA) of a sagittal plane from a control and a DMP 904-treated rat 1 h following the administration of [125I] T4 is shown in Figure 4A. Estimated concentrations of [125I] appeared to be higher in the liver and small intestine of the DMP 904-treated animal. A high concentration of [125I] in the WBA was observed in the thyroid of all animals from both groups and likely resulted from deiodination and subsequent concentration of free [125I] in the thyroid. Liver:blood ratios and small intestine:blood ratios estimated from WBA analyses are presented in Figures 4B and 4C, respectively. Liver:blood ratios were approximately 1 in control rats, but DMP 904 treatment increased the liver:blood ratio over time (Fig. 4B). The small intestine:blood ratio was initially less than 1 in control rats, but increased over time for both control and DMP 904-treated animals. The magnitude of the increase, however, was greater for DMP 904 treated rats than controls.
Many agents that alter hepatic clearance of T4 in rats activate CAR or PXR and induce a variety of hepatic microsomal enzymes. Figure 5 shows the time course of changes in mRNA for a number of hepatic enzymes following a single oral dose (200 mg/kg) of DMP 904. Significant increases in mRNA levels were observed for CYP 2B1, CYP 3A1, and UGT 1A1 by 3 h after dosing, and UGT 1A2 was increased by 6 h after dosing. At 24 h, the mRNA for both CYP 3A1 and CYP 2B1 was greater than 20-times higher in livers from DMP 904-treated than control rats. The increase in mRNA levels for UGTs 1A1 and 1A2 were also significantly elevated, albeit to a lesser magnitude of change. No changes were observed in the mRNA expression of UGT 1A6.
Figure 6 shows the time course of alterations in mRNA levels of hepatic transporters. The most pronounced changes in transporter gene expression following treatment with DMP 904 were observed for Oatp2. Oatp2 mRNA levels were approximately two-times higher in livers from DMP 904-treated rats at 3 h, and by 24 h, mRNA levels of Oatp2 were about four-times higher. Oatp1 mRNA was also significantly increased at 24 h. In contrast, two other hepatic uptake transporters, Oatp4 and Ntcp, showed small but significant decreases in gene expression at 24 h. mRNA for the canalicular transporter Mrp2 increased, with 2-fold and 1.4-fold changes noted at 6 and 24 h, respectively.
Western blot analysis was used to specifically assess increases in Oatp1 and Oatp2 protein expression in liver. Consistent with the real-time PCR results, DMP 904 treatment resulted in the induction of Oatp2 protein levels in rat liver based upon the Western blot analysis shown in Figure 7. However, no difference was observed in protein levels of Oatp1.
DISCUSSION
A variety of xenobiotics are known to alter thyroid hormone homeostasis in rats through a mechanism involving increased hepatic clearance of the thyroid hormone (Curran and DeGroot, 1991). In most cases, biliary clearance of the conjugated hormone, particularly the glucuronide conjugate, is increased, and its excretion into bile increases the elimination of the hormone (Barter and Klaassen, 1994; Vansell and Klaassen, 2002a). To date, most compounds that alter hepatic clearance of thyroxine decrease serum hormone levels within a few days of dosing (Barter and Klaassen, 1994). In the present work, DMP 904 treatment also decreased serum thyroid hormone levels and increased biliary clearance of the glucuronide conjugate of the hormone. DMP 904 treatment also induced a variety of hepatic microsomal enzymes and xenobiotic transporters, suggesting that the mechanism underlying the decreased levels of serum thyroid hormones is likely to involve increased hepatic clearance of thyroxine in a manner analogous to prototypical compounds such as PB and PCN. However, DMP 904 is distinguished from compounds such as PB and PCN in two ways. First, the compound rapidly decreased serum hormone levels within 12 h of dosing, with T4 reduced to non-detectable levels after three days of dosing. Second, although biliary clearance of the glucuronide conjugate was increased, the major component excreted into bile after DMP 904 treatment was the unconjugated hormone.
The results of the biliary clearance study with [125I] T4 indicated that the severe reductions in T4 serum levels in DMP 904-treated rats were due to a nearly seven-fold increase in T4 Cltb. This large increase in T4 Cltb could not be explained by the modest increase in T4 unbound fraction observed following DMP 904 administration at a comparable dose, because at most, a two-fold increase in T4 unbound fraction occurred following DMP 904 treatment which would only account for two-fold increase in T4 Cltb.
Many investigators report biliary T4 and T4-glucuronide elimination as excretion rates (Masubuchi et al., 1997; McClain et al., 1989; Vansell and Klaassen, 2001), whereas, in the present work, biliary elimination of unconjugated T4 and T4-glucuronide was evaluated as biliary clearances. The additive nature of clearances allows for consideration of the contribution of each change in T4 disposition to overall changes in T4 Cltb. Accordingly, the observed increase in biliary clearance (about three-fold) of the glucuronide conjugate in PB-treated animals is consistent with induction of uridine diphosphate glucuronosyltransferases (UGTs) followed by enhanced biliary excretion of the conjugated hormone as previously described (Barter and Klaassen, 1992, 1994; McClain et al., 1989). In direct contrast, DMP 904-treated rats excreted about 16% of the dose as the glucuronide conjugate, with nearly 40% of the dose excreted as the unconjugated hormone. The large percentage of the dose recovered as unconjugated hormone is a direct result of the nearly 80-fold increase in the biliary clearance of unconjugated T4 following DMP 904-treatment. A DMP 904-related increase in T4-glucuronidation capability was not obvious from the biliary mass balance results since the percent of administered [125I] T4 recovered as the glucuronide conjugate was not increased. However, since T4-glucuronide is excreted almost entirely in the bile (Rutgers et al., 1989; Vansell and Klaassen, 2001), the increase in T4-glucuronide Clbile by DMP 904 suggests that DMP 904 treatment results in the induction of UGTs involved in T4 metabolism. This conclusion is consistent with the observed increase in UGT 1A1 mRNA levels associated with DMP 904 treatment since UGT 1A1 is thought to be involved in the T4 glucuronidation (Vansell and Klaassen, 2002a). Together, changes in total body clearance of T4 brought about by DMP 904 treatment appears to be the result of increased biliary clearance of both unconjugated T4 and T4-glucuronide along with an increase in the unbound fraction of T4. The largest contribution to the increase in T4 Cltb is due to an increase in biliary clearance of unconjugated T4. In light of the observation that DMP 904 treatment results in only modest changes in T4 unbound fraction, it is unlikely that the large contribution of unconjugated T4 Clbile results solely from changes in protein binding of T4. Rather, it is more likely that induction or upregulation of higher affinity elimination processes other than those involved in generation and excretion of the glucuronide conjugate play a role in the marked increased in T4 Clbile. The results of the whole body autoradiography (WBA) study are consistent with these observations from the biliary clearance study. The time-dependent increase in the concentration of [125I] into the liver of DMP 904 treated rats suggest that the marked reductions in serum T4 may result largely from an increased uptake of T4 into the liver. Rapid concentration of [125I] in the liver is consistent with the increase in Vdss. The larger increase in the small intestine:blood ratio with time in the DMP 904 treated rats compared to control animals is also consistent with the large increase in the biliary excretion of unconjugated T4 observed in the biliary clearance study. In combination, the data from the biliary clearance and WBA studies suggest that DMP 904 treatment results in both the induction of UGTs and induction or upregulation of a higher affinity T4 hepatic uptake process.
The results presented here for DMP 904, showing an important role for Clbile of unconjugated T4 in altering thyroid hormone homeostasis, are not unique as a similar phenomenon involving both increased uptake and increased biliary excretion of unconjugated hormone was observed reported for two novel antihistamines, SK&F 93479 (lupitidine) and SK&F 93944 (temelastine) (Poole et al., 1989, 1990). Although the magnitude was not as severe as observed for DMP 904, both SKF compounds produced thyroid pathology in rats (Atterwill et al., 1989; Brown et al., 1987), and both increased biliary excretion of T4, with the unconjugated hormone accounting for the majority of the excreted T4 (Poole et al., 1989, 1990). Furthermore, changes in T4 elimination for both compounds were accompanied by an increased hepatocellular accumulation of T4 via an energy dependent process with no apparent effect on T3 uptake (Poole et al., 1989, 1990). Currently, basolateral transporters involved in the hepatic uptake of T4 are known to include Oatp1, Oatp2, Oatp4, and Ntcp (Abe et al., 1998; Cattori et al., 2000; Friesema et al., 1999). DMP 904 induced mRNA for both Oatp1 and Oatp2, with Oatp2 mRNA increased as early as 3 h after dosing. However, only Oatp2 protein expression levels appeared to be increased by DMP 904 suggesting that Oatp2 is likely to be involved in the observed increase in T4 hepatic uptake.
In contrast to hepatic uptake of thyroid hormone, less is known regarding the transport of thyroxine out of the hepatocyte and into the bile. Ribeiro et al. (1996) observed the existence of a saturable and temperature sensitive transporter in rat hepatoma cells that appeared to be involved in the efflux of thyroid hormones and was inhibited by verapamil. Verapamil is a potent inhibitor of the ATP-binding cassette transporter superfamily. Mrp2, a member of this family, is the main canalicular transporter responsible for the secretion of organic anions in the bile (Kipp and Arias, 2002). Our experiments show that DMP 904 treatment increased hepatic levels of mRNA for Mrp2. It is plausible that Mrp2 plays a role in the observed increase in the biliary clearance of unconjugated [125I]T4, but more work to directly characterize the transporters involved in the biliary excretion of thyroid hormone is needed to adequately address this question.
From our investigation, it is not known whether DMP 904 treatment directly affects T3 elimination. However, since the majority of the circulating T3 is formed from deiodination of T4 in peripheral tissues (Oppenheimer and Surks, 1974), DMP 904 is likely to be, at the least, indirectly responsible for the observed reduction in T3 levels due to the depletion of T4 from the body. Furthermore, induction of Oatp2 may increase T3 hepatic uptake since the uptake affinity of Oatp2 is similar for both T3 and T4 (Abe et al., 1998). Vansell and Klaassen (2002b) postulated that it is the decrease of circulating T3 that is responsible for drug induced TSH increases. Again, a more direct evaluation of DMP 904-related effects on T3 would be required to address this issue.
DMP 904 caused thyroid follicular cell hypertrophy and hyperplasia and a low incidence of follicular cell adenoma in rats when dosed at 200 mg/kg/day for three months (Gemzik et al., 2002). There was no evidence from our studies to suggest that the observed effect of DMP 904 on the thyroid is related to its pharmacological activity as a CRF1 receptor antagonist. The present results suggest that DMP 904 is likely to act via an extra-thyroidal mechanism to cause prolonged stimulation of TSH, a factor known to promote thyroid tumor development in rats (Hiasa et al., 1982; Hill et al., 1989; McClain et al., 1988; Vansell et al., 2004). However, induction of thyroid tumors in rats by a mechanism involving increased hepatobiliary clearance is not likely to be predictive of a similar risk in humans, based largely on quantitative differences in the physiology and biochemistry of the thyroid pituitary axis in rats (Cohen et al., 2004; Meek et al., 2003). The lack of human relevance is also supported by the fact that many agents that reduce circulating thyroid hormone levels by extra-hepatic mechanisms in rats fail to do so in mice (Craft et al., 2002; Viollon-Abadie et al., 1999) as well as man (Ohnhaus et al., 1981).
In conclusion, the present results provide insight into the mechanisms by which DMP 904 disrupts thyroid homeostasis. The data suggest that an increase in the biliary clearance of unconjugated T4 plays a major role in the large increase in T4 total body clearance following DMP 904 treatment to rats. Collectively, the data suggest that DMP 904 disrupts thyroid hormone homeostasis primarily via alterations in hepatic transport of T4 in addition to apparent increases in T4 glucuronidation capacity. This combined mode of action of DMP 904 may explain its pronounced effect on thyroid homeostasis in rats including production of a low incidence of thyroid adenoma following three months of treatment.
NOTES
Portions of this data were presented at the 41st annual meeting of the Society of Toxicology, March 2002, Nashville, TN, and at the 42nd annual meeting of the Society of Toxicology, March 2003, Salt Lake City, UT.
1 Present address: Genentech, Inc., South San Francisco, CA.
3 Present address: Quest Pharmaceutical Services, Newark, DE.
4 Present address: Endo Pharmaceuticals, Chadds Ford, PA.
5 Present address: Incyte Corporation, Wilmington, DE.
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