Distribution of (7 )-21-[4-[(diethylamino) Methyl]-2-methoxyphenoxy]-7- Methyl-19-norpregna-1,3,5(10)-trien-3-ol-20-[c]2-hydroxy-1,2,3- Propanetricarboxylate ([c]tas-108) and Its Metabolites after Single Oral Administration to Rats Bearing 7,12-dimethylbenz( )anthracene- Induced Mammary Tumor

(7 )-21-[4-[(Diethylamino)methyl]-2-methoxyphenoxy]-7-methyl19-norpregna-1,3,5(10)-trien-3-ol 2-hydroxy-1,2,3-propanetricarboxylate (TAS-108) is a novel steroidal antiestrogen, modulating the differential recruitment of transcriptional cofactors by liganded estrogen receptors and representing a promising agent for the treatment of breast cancer. To understand better the relationships between the drug exposure and the efficacy or toxicity of TAS-108, we investigated the metabolism and distribution of TAS-108 after oral administration of [C]TAS-108 to rats bearing a 7,12dimethylbenz( )anthracene-induced mammary carcinoma. The metabolites (7 )-21-[4-[(ethylamino)methyl]-2-methoxyphenoxy]-7-methyl-19-norpregna-1,3,5(10)-trien-3-ol (deEt-TAS108), (7 )-21-[4-[(diethylamino)methyl]-2-methoxyphenoxy]-7methyl-19-norpregna-1,3,5(10)-trien-3-ol-N-oxide (TAS-108-Noxide), and 3-methoxy-4-[(7 )-7-methyl-19-norpregna-1,3,5(10)trien-3-ol-21-yl]oxybenzoic acid (TAS-108-COOH) were identified as the major metabolites in the plasma, and in addition, (7 )-21-[4-[(ethylamino)methyl]-2-methoxyphenoxy]-3methoxy-7-methyl-19-norpregna-1,3,5(10)-triene (O-Me-deEtTAS-108) was identified as a novel metabolite in this study. The time-concentration profiles of TAS-108 and its metabolites in the plasma were compared with those in the tumor and uterus of the rats. Radioactivity was found at a high level in various organs including lung, liver, spleen, ovary, and many glands at 12 h and was relatively higher in tumor tissue than in plasma. On the other hand, the levels of radioactivity in the brain and eyeball were very low or not detectable. TAS-108, deEt-TAS-108, and O-Me-deEt-TAS-108 were extensively distributed in the rat tissues and the tumor, with corresponding tissue/plasma ratios for Cmax and area under the curve in the range of 7 to 100. In contrast, TAS-108-COOH and TAS-108-N-oxide were hardly distributed to the tissues and thus may not contribute to the efficacy or toxicity of TAS-108. Thus, TAS-108, deEt-TAS-108, and O-Me-deEt-TAS-108, being distributed highly in tumor tissue, may be more important for the efficacy and toxicity of TAS-108 in vivo than TAS-108-COOH and TAS-108-N-oxide. A novel steroidal antiestrogenic compound, TAS-108 (SR16234) has been selected through the biological screening of various newly synthesized steroidal compounds in a collaborative effort between Taiho Pharmaceutical Co., Ltd. and SRI International (Menlo Park, CA). TAS-108 has been shown to be a ligand for estrogen receptors and , and its binding affinities are comparable with those of 17 -estradiol (E2; Yamamoto et al., 2003). The mode of action of TAS-108 on a molecular basis is different from that of E2, the nonsteroidal selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene, and the steroidal SERM fulvestrant. The characteristic properties of TAS-108 could present novel biological responses if the concentration of TAS-108 or its metabolites reaches Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.105.005504. ABBREVIATIONS: TAS-108, (7 )-21-[4-[(diethylamino)methyl]-2-methoxyphenoxy]-7-methyl-19-norpregna-1,3,5(10)-trien-3-ol 2-hydroxy-1,2,3-propanetricarboxylate (SR16234); E2, 17 -estradiol; SERM, selective estrogen receptor modulator; TAT-59, 4-[(E)-1-[4-(dimethylaminoethoxy)]-phenyl)-1butenyl]phenyl dihydrogen phosphate; TAS-108[M 4], 20,21-C2-21,21-D2-labeled TAS-108; [ C]TAS-108, (7 )-21-[4-[(diethylamino)methyl]-2methoxyphenoxy]-7-methyl-19-norpregna-1,3,5(10)-trien-3-ol-20-[C]2-hydroxy-1,2,3-propanetricarboxylate; deET-TAS-108, (7 )-21-[4-[(ethylamino)methyl]-2-methoxyphenoxy]-7-methyl-19-norpregna-1,3,5(10)-trien-3-ol; TAS-108-N-oxide, (7 )-21-[4-[(diethylamino)methyl]-2-methoxyphenoxy]-7methyl-19-norpregna-1,3,5(10)-trien-3-ol-N-oxide; O-Me-deET-TAS-108, (7 )-21-[4-[(ethylamino)methyl]-2-methoxyphenoxy]-3-methoxy-7-methyl-19norpregna-1,3,5(10)-triene; SAM, S-adenosyl-L-methionine; HPLC, high-performance liquid chromatography; DMBA, 7,12-dimethylbenz( )anthracene; LC/MS/MS, liquid chromatography/tandem mass spectrometry; AUC, area under the curve; m/z, mass-to-charge ratio; T/P, tissue/plasma. 0090-9556/06/3402-331–338$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 2 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 5504/3080290 DMD 34:331–338, 2006 Printed in U.S.A. 331 at A PE T Jornals on A uust 7, 2017 dm d.aspurnals.org D ow nladed from an adequate level in the target tissue. Recently, Yamamoto et al. (2005) reported that TAS-108 at a dosage of 1 to 3 mg/kg/day strongly inhibited tumor growth in rats bearing DMBA-induced mammary carcinoma, the endogenous E2 model. It is well known that in this model the mammary tumor growth, which depends on endogenous E2, is suppressed by recently developed antiestrogenic agents. TAS-108 also strongly inhibited tumor growth in the exogenous E2 model, in which tumor growth of MCF-7 xenografts is induced, in mice at a dosage of 1 mg/kg/day without the manifestation of toxicity. On the other hand, in ovariectomized rats administered TAS-108 orally, TAS-108 demonstrated a much weaker estrogenic effect on uterine weight compared with tamoxifen. The tissue distribution of tamoxifen has been examined in various animal studies (Fromson et al., 1973a; Major et al., 1976; Ruenitz and Bagley, 1985; Wilking et al., 1982; Robinson et al., 1991; Toko et al., 1995) and clinical studies (Fromson et al., 1973b; Lien et al., 1991; Pujol et al., 1995). Although the ratios of tissue versus plasma concentration varied in the reports, the ratios ranged from 8 to 70 for most tissues. As with tamoxifen, nonsteroidal SERMs such as tremifen and TAT-59 (Toko et al., 1995) showed higher concentrations in the tissues than in the plasma or serum. Moreover, tamoxifen is metabolized, and some of the metabolites have been detected in the tumor and tissues. In particular, N-demethyltamoxifen was detected at a higher level in tumor tissue than in plasma in rats and patients (Lien et al., 1989, 1991) and showed a binding affinity similar to that of tamoxifen. The metabolites of tamoxifen have been shown to contribute to the inhibitory effect of the drug. To understand better the relative contribution of TAS-108 and its metabolites to the efficacy or toxicity of TAS-108, we investigated the metabolism and the distribution of TAS-108 after [C]TAS-108 had been administered to rats bearing DMBA-induced mammary carcinoma. The time-concentration profile in plasma was compared with that in tumor tissue, which is the target for the activity of TAS-108, and with that in the uterus, the organ that could show adverse side effects. Additionally, we identified several metabolites of TAS-108 and investigated their pharmacokinetic properties in the rats. Materials and Methods Materials. TAS-108 (SR16234) and 20,21-C2-21,21-D2-labeled TAS-108 (TAS-108[M 4]) were synthesized at Taiho Pharmaceutical Co., Ltd. (Tokyo, Japan). C-labeled TAS-108 ([C]-TAS-108), which is (7 )-21-[4[(diethylamino)methyl]-2-methoxyphenoxy]-7-methyl-19-norpregna-1,3,5(10)trien-3-ol-20-[C]2-hydroxy-1,2,3-propanetricarboxylate (712 kBq/mg), was synthesized at the ADME/TOX Research Institute, Daiichi Pure Chemicals Co., Ltd. (Ibaraki, Japan). The purity of the labeled and unlabeled compounds was 98.6%. The standard materials [deEt-TAS-108 [(7 )-21-[4-[(ethylamino)methyl]-2methoxyphenoxy]-7-methyl-19-norpregna-1,3,5(10)-trien-3-ol], TAS-108-COOH [3-methoxy-4-[(7 )-7-methyl-19-norpregna-1,3,5(10)-trien-3-ol-21-yl]oxybenzoic acid], TAS-108-N-oxide [(7 )-21-[4-[(diethylamino)methyl]-2-methoxyphenoxy]-7-methyl-19-norpregna-1,3,5(10)-trien-3-ol-N-oxide], and O-MedeEt-TAS-108 [(7 )-21-[4-[(ethylamino)methyl]-2-methoxyphenoxy]-3-methoxy-7-methyl-19-norpregna-1,3,5(10)-triene)]], synthesized by Taiho Pharmaceutical Co., Ltd., were used as reference standards for identification of the parent compound and its metabolites. The compound 20,21-C2-21,21-D2-labeled O-Me-deEt-TAS-108 was used as the internal standard for the determination of O-Me-deEt-TAS-108 and was synthesized by Taiho Pharmaceutical Co., Ltd. Carboxymethyl cellulose sodium, disodium hydrogenphosphate, trifluoroacetic acid, and S-adenosyl-L-methionine (SAM) were purchased from Sigma-Aldrich (St. Louis, MO). Methanol and acetonitrile were from Kanto Chemical Co. (Tokyo, Japan). Reagents of HPLC grade were used for HPLC. Animals. Female CD (Sprague-Dawley) rats 7 weeks of age were purchased from Charles River Japan, Inc. (Hino Farm or Atsugi Farm; Tokyo, Japan). For investigating the distribution of TAS-108 in tumorous animals, the rats were treated orally with 7,12-dimethylbenz( )anthracene (DMBA) at a dose of 20 mg/ml/rat to induce mammary tumors. The length of the major axis of each mammary tumor was measured, and only those animals with a tumor of 17.4 to 29.0 mm were selected for this study. The body weights at the dosing of [C]TAS-108 ranged from 289.0 to 340.8 g. Whole-Body Autoradiography. DMBA-induced mammary tumor-bearing rats were euthanized with an overdose of ether at 2, 6, or 12 h (n 1) after a single oral administration of [C]TAS-108 at a dose of 10 mg/kg. The hair was rapidly clipped, and the nasal cavity and anus were filled with 5% carboxymethyl cellulose sodium. The carcass was frozen in a dry ice-acetone mixture. Approximately 35m-thick sections were sliced and collected onto adhesive tape and freeze dried. Each frozen section was covered with a protective membrane and placed in contact with an imaging plate. The imaging plate was then exposed at room temperature for 16 h in a lead-shielded box. After exposure, the radioactive images on the imaging plate were analyzed by using a bio-image analysis system (BAS2500; Fuji Film Co., Ltd., Tokyo, Japan). The radioactive images were expressed in color grades from 0 to 255 to prepare radioluminograms. Concentration of Radioactivity in Plasma, Tumor, and Uterus. After a single oral administration of [C]TAS-108 to DMBA-induced mammary tumor-bearing rats at a dose of 10 mg/kg, three animals at each time point, 2, 6, 12, 18, 24, 48, and 96 h, were sacrificed by exsanguination from the abdominal aorta under ether anesthesia. Blood samples were withdrawn into a vacuum tube containing heparin sodium and centrifuged (1800g, 4°C, 15 min) to separate the plasma. The whole tumors and uterus of each animal were weighed and homogenized with an approximately 4-fold volume of saline. The samples (0.1 ml of plasma, 0.5 ml of tumor homogenate, and 0.5 ml of uterus homogenate) were extracted twice with a 5-fold volume of methanol and centrifuged (1800g, 4°C, 15 min) to separate the extracted radioactivity from the nonextracted material. Radioactivity in the extraction residue and in the supernatant of the each sample was measured to determine the extraction ratio. The remaining methanol extract was evaporated to dryness with a nitrogen gas stream, and the residue was reconstituted in the solvent containing TAS-108 and the synthetic metabolite standards. The solution was centrifuged (10,000g, 4°C, 15 min), after which the supernatant was subjected to HPLC analysis. HPLC Analysis of TAS-108 and Its Metabolites. Chromatography was performed on an Inertsil ODS-3 column (5 m, 4.6 250 mm; GL Sciences Inc., Tokyo, Japan) by using a Shimadzu LC-10A HPLC system (Shimadzu, Kyoto, Japan). Mobile phases A and B were 20 mM disodium hydrogen phosphate (pH 6.0) and acetonitrile, respectively. The column was run initially at 45% B, and then a linear gradient from 45% B to 60% B was used between 0 to 30 min, followed by a second linear gradient from 60% B to 90% B applied up to 50 min. Finally, an isocratic condition at 90% B was maintained for 10 min. The flow rate was maintained at 1.0 ml/min and the column temperature at 40°C. UV detection was performed at 205 nm. Fractions of the HPLC eluate were collected at intervals of 18 s by using a RETRIEVER IV fraction collector (ISCO, Uppsala, Sweden), and radioactivity in each fraction was determined by liquid scintillation counting after the addition of 4 ml of scintillator Hionic-Fluor (PerkinElmer Life and Analytical Sciences, Boston, MA). Concentrations of radioactivity in the plasma, tumor, and uterus were expressed as equivalents of TAS-108 (nanograms of equivalent per milliliters or grams). Identification of O-Me-deEt-TAS-108 by LC/MS/MS. We investigated the concentrations of the metabolite O-Me-deEt-TAS-108 in the liver from two normal rats after administration of a mixture of TAS-108 and TAS-108[M 4] (1:1) at the dosage of 30 mg/kg/day for 3 days. The liver was weighed and homogenized in an approximately 4-fold volume of saline. The homogenate was prepared in the same manner as described for the tumor and uterus. Each sample was subjected to preparative HPLC to obtain the fraction containing the