Entero-hepatic Circulation of SM in the Rat: Elucidation of the Participating Major Metabolites

Entero-hepatic Circulation of SM-10888 in the Rat: Elucidation of the Participating Major Metabolites Masashi YABUKI, Kazuhiko IBA* Iwao NAKATSUKA, Akira YOSHITAKE Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd. *Development Research Laboratories II, Sumitomo Pharmaceuticals Research Center 1-98, 3-chome, Kasugade-Naka, Konohana-Ku, Osaka 554, Japan Summary The entero-hepatic circulation of 9-amino-8-fluoro-1,2,3,4-tetrahydro-2,4-methano acridine citrate (SM-10888) in rats was investigated after oral administration and/or duo denal infusion of a pooled bile sample. 1. Pooled bile obtained from a rat receiving an oral dose of 14C-SM-10888 (5 mg/kg) was infused into the duodenum of bile-duct cannulated rats. At 72 hr after starting infusion, percentage values for infused radioactivity excreted into the urine (23%) and bile (41%) in dicated that at least 64% of the radioactivity in the bile was reabsorbed. 2. Infused bile contained the N-glucuronide of SM-10888 (SMG, 15%) and the Oglucuronide of the hydroxylated metabolite (M3G, 42%). In the urine and bile from rats receiving infusion of the pooled bile sample, the M3G was the major component. 3. After oral administration of 14C-SM-10888, M3G level in the urine from non-operat ed rats (24% of dose) was greater than from bile-duct cannulated rats (10% of dose), reflecting reabsorption of metabolites in the bile and excretion as M3G. 4. SMG and M3G, present in the infused bile sample, liberated unconjugated SM 10888 and M3 after incubation with intestinal contents. Therefore it was assumed that - the enterohepatic circulation included hydrolysis of SMG and M3G in the bile to SM-10888 and M3 by intestinal flora, reabsorption from gastrointestinal tract and subsequent further metabolism to M3G. Key words : SM-10888, Entero-hepatic circulation, Metabolism, Rats Introduction 9-amino-8-fluoro-1,2,3,4-tetrahydro-2,4-methanoacridine citrate (SM-10888) is a potent choline sterase inhibitorl,2). Previous studies using 14C labelled compound3,4) have revealed its basic disposition and metabolic characteristics in rats after oral dosing. Dosed radioactivity was found to be absorbed from the gastrointestinal tract rapidly and completely, and excreted within 168 hr after administration into the urine (52.2% of dose) and the feces (44.2% of dose), the latter being due to biliary excretion. After bile-duct cannulation the biliary excretion greatly exceeded that into the urine (bile: 65.1%, urine: 29.1%), suggesting that a portion of the radioactivity in the bile had been otherwise absorbed into the blood circulation, i.e. an entero-hepatic circulation existed. In the present report we document evidence of recirculation gained from experiments with infusion of pooled bile sample into the duodenum. Since metabolism studies revealed the main metabolites in rats to be hydroxylated SM-10888 (M3), the N glucuronide of SM-10888 (SMG) and the 0-glucuronide of M3 (M3G) (Fig. 1), and the main compo nents in the bile to be SMG and M3G4), we also analyzed these metabolites. The urine and the bile, both

Fig. 1 Chemical structures of SM-10888 and its metabolites of bile donor and donee, were examined in order to show which metabolites were associated with the en tero-hepatic circulation. Materials and Methods Materials SM-10888 and the authentic standard for metabolite M3 were synthesized by Sumitomo Pharmaceuti cals Co., Ltd. The radiolabeled compound (14C-SM-10888) (Fig. 1) was synthesized by Sumitomo Chemical Co., Ltd. The radiochemical purity determined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) was over 99 0. The chemical purity by HPLC was over 99%. The specific activity was 0.80 GBq/mmol (2.14 MBq/mg). Animals Seven-week old male Sprague-Dawley rats (Charles River Japan) weighing 254-319 g were used. After purchase as six week olds, animals were given diet (CE-2, Clea Japan) and water ad libitum, and housed in a temperature (23±2 C), and humidity (55±10%)-controlled environment with a 12 hr light-dark (light: 8:00-20:00) cycle. Animals were housed for one week, and those not demonstrating any abnormalities were submitted to experimentation. Intraduodenal Infusion of Bile Sample The bile sample used for intraduodenal infusion was obtained from the previous biliary excretion study3>, in which 14C-SM-10888 was orally given at a dose of 5 mg/kg to a bile-duct cannulated rat and the bile collected. The sample contained the pooled bile excreted over the 24 hr period after compound administration. It was stored frozen at -20 C until used in the infusion study. Rats underwent cannulation of both their bile-duct and duodenum, allowing bile drainage and introduc tion, respectively, through polyethylene tubes (PE-10, Clay Adams, USA). The animals were main tained in Bollman cages (KN-326, Natsume, Japan) and electrolytes (Solita-T3, Shimizu Pharmaceuti cals, Japan) given. Two milliliters of bile sample were infused into each duodenum using a peristaltic

pump (SJ-1220, Atto Corporation, Japan) at a flow rate of 1 ml/hr for 2 hr, and successively pooled drug free bile obtained from rats, which received no drugs, was infused at 1 ml/hr for 22 hr. The infu sion flow rate (1 ml/hr) was employed considering the bile flow rate of ca. 1 ml/hr observed in the previ ous study3), which was also consistent with the present study findings (data not shown). Bile, urine and feces samples were collected separately at 1, 2, 4, 6, 24, 48 and 72 hr after the beginning of infusion. The dose infused was 54µg SM-10888 eq./head/2 hr. Determination of Radioactivity in the Excreta Feces samples were mixed with 5%carboxymethyl cellulose (Kanto Chemical, Japan) solution and ca. 0.2 aliquots were combusted with a sample oxidizer (306, Packard, USA). Aliquots of urine and bile (ca. 0.2 ml) were mixed with EMULSIFIER SCINTILLATOR (Packard, USA). The radioactivities were determined using a liquid scintillation spectrometer (TRI-CARB 4640, Packard, USA). Quantification of Metabolites in the Urine and Bile Urine and bile excreted within 24 hr after oral administration (obtained from the previous study3)) or after starting infusion were pooled. These samples and the bile sample used for infusion were applied directly, with the authentic standards, to TLC plates (Kieselgel 60 F254 0.25 mm thickness, Merck, Ger many), and developed with the solvent system: chloroform/methanol/triethylamine, 40: 5 : 1 (v/v). Au toradiograms were obtained with a bioimage analyzer (BAS 2000, Fuji Film, Japan). Non-radioactive spots were detected under UV light (254 nm). Spots corresponding to the metabolites were scraped from the plate, mixed with EMULSIFIER SCINTILLATOR (Packard), and radioactivities measured. Incubation of Bile Sample with Intestinal Contents The same bile sample used for infusion was incubated with rat intestinal contents in vitro according to the method of Booth et a15). Intestinal contents of rats (mixture from duodenum to caecum), which had received no drugs, were collected, mixed with 0.2 M phosphate buffer (ph 7.0), and filtrated through gauze to give a 25% (w/v) suspension. To glass vials containing 0.1 ml of bile sample, 1 ml of this filtrate or phosphate buffer was added, and the vials were sealed with air-tight silicon caps. After thoroughly displacing the inner air with a nitrogen stream using disposable needles, the vials were incu bated at 38 C with gentle shaking for 18 hr. After centrifugation were analyzed by TLC in the same manner described above. Results (3,000 rpm, 10 min, 4 C), supernatants Data for cumulative excretion of radioactivity into the urine, bile and feces of bile-duct cannulated rats receiving infusion of bile sample are summarized in Table 1. For comparison, cumulative excretion into the excreta from non-operated or bile-duct cannulated rats, receiving an oral dose of 14C-SM-10888 (5 mg/kg), are listed in the same table (data cited from a previous study')). In the oral administration study, cumulative excretion within 72 hr was 51.3% into the urine and 43.5% into the feces in the non-operated rats, whereas it was 29.1% into the urine and 65.1% into the bile in the bile-duct cannulated animals. In the bile infusion study, 23.1% of the dosed radioactivity was

Table 1 Cumulative excretion of radioactivity into excreta (% of total administered) after a single oral adminis tration (14C-SM-10888, 5 mg/kg) or after the beginning of 2 hr infusion of bile a) Values are means±s.e. of data for five animals. Data are cited from a previous study3). b) Infusion bile was collected over 24 hr from a bile-duct cannulated rat receiving an oral dose of 14C-SM-10888 (5 mg/kg). c) Values are means ±S.E. of data for four animals. excreted into the urine, 40.7% into the bile and 22.3% into the feces within 72 hr after the beginning of infusion. In all cases, excretion was almost complete at the 24 hr time point. The metabolites contained in the urine and the bile excreted after administration in each experiment and in the bile sample used for infusion, were analyzed by TLC and the results summarized in Table 2. In the urine samples obtained after oral administration, from both non-operated and bile-duct cannulated rats, M3G was the major component. The excreted amount of this metabolite was greater in non-operat ed rats (24.2% of dose) than in bile-duct cannulated rats (10.3% of dose). The respective values for other metabolites, including the parent compound, M3, SMG and other unknown metabolites were almost the same under both conditions. In the bile samples after oral administration and the infusion sam ple, M3G and SMG were the major components. On the other hand, in the urine and bile samples ob tained from bile infused rats, M3G predominated (urine: 14.3% of dose, bile: 30.3% of dose) and almost all of the other metabolites were below the detection limit. When the bile sample used for infusion was incubated with or without rat intestinal contents and the contained metabolites analyzed by TLC, a pronounced difference was found as shown in Fig. 2. After in cubation with the intestinal contents SM-10888 and M3 (the aglycones of SMG and M3G, respectively), were found, while without intestinal contents they could hardly be detected. Discussion Since inter-individual differences in metabolite composition in the bile after oral administration of SM-10888 were found to be small (data not shown), it was considered appropriate to evaluate entero

Table 2 Metabolite composition in excreta from rats receiving an oral administration of 14C-SM-10888 or infu sion of bile sample Urine and bile were collected for 24 hr after dosing or beginning of infusion. a) Rf values of metabolites in TLC analysis described in the Materials and Methods. b) Bile for infusion was collected for 24 hr from a bile-duct cannulated rat receiving an oral dose of 14C-SM-10888 (5 mg/kg). c) Values are means±s.e. of data for five animals. Urine and bile samples were those obtained in the previous study3) d) Values are means ±S.E. of data for four animals. e) Not detected (<0.2 for urine and <0.1 for bile). hepatic circulation using a bile sample collected from one rat receiving an oral dose of 14C-SM-10888. Biliary excretion was almost complete after 24 hr and therefore collection of bile sample for infusion was over this time period. After infusion of this bile sample, percentages of total administered radioactivity recovered in the urine and the bile by 72 hr were 23 and 41%, respectively. This observation indicates that at least 64 / of the radioactivity in the bile was reabsorbed, providing direct evidence for entero-hepatic circulation. The gap in urinary excretion rates between non-operated and bile-duct cannulated rats after oral adminis tration (51.3-29.1=22.20/ at 72 hr), could be produced by this reabsorption and presumably further recirculation of radioactivity and partitioning to give greater urinary excretion. In order to elucidate which metabolites participated in entero-hepatic circulation, we analyzed the con tained metabolites in the urine and bile, and incubated the infusion bile sample with intestinal contents. As shown in Table 2, whereas in the dosed bile sample the major components were SMG (15.3%) and M3G (41.9%), in the excreta of rats which received infusion of the bile sample only M3G was a major component and SMG was little detected. This fact indicates that the metabolites in the bile suffered fur ther metabolism to finally give M3G during enterohepatic circulation. Therefore it can be assumed that,

Fig. 2 TLC autoradiogram showing metabolites in bile after incuba tion without (A) or with (B) intestinal contents in the oral administration study, urinary M3G excretion from non-operated rats became greater than from bile-duct cannulated rats, because in the former case reabsorption of bile was preserved. The in vitro incubation study further suggested that SMG and M3G in bile pouring into the duodenum are hydro lyzed by the intestinal flora to give unconjugated SM-10888 and M3, respectively. The liberated SM 10888 and M3, more lipophilic forms than their glucuronides (log (octanol/buffer) at ph 7.4; SM-10888 (2.23), M3 (1.59), SMG (-1.37), M3G (-1.72) 6)), could be absorbed from the gastrointestinal tract, and subsequently metabolized to M3G and excreted. Since the SMG and M3G in the bile used for infusion accounted for 57% of total 14C, and 64% of the infused dose was absorbed, the majority of the entero hepatic circulation might involve these two metabolites and their unconjugated counterparts. Acknowledgements The authors would like to express their appreciation to Ms. Junko Kitada for her assistance. References 1) Kazuichi Natori, Yuko Okazaki, Tsunemasa Irie and Junki Katsube: Pharmacological and Biochemical Assessment of SM-10888, a Novel Cholinesterase Inhibitor. Japan. J. Pharmacol., 53: 145-155 (1990). 2) Yuko Okazaki, Kazuichi Natori, Tsunemasa Irie and Junki Katsube: Effect of a Novel

CNS-selective Cholinesterase Inhibitor, SM-10888, on Habituation and Passive Avoidance Responses in Mice. Japan. J. Pharmacol., 53: 211-220 (1990). 3) Masashi Yabuki, Kazuhiko Iba, Iwao Nakatsuka and Akira Yoshitake: Absorption, Dis tribution and Excretion of SM-10888 in Rats. Xenobio. Metab. Disp., 8: 1169-1179 (1994). 4) M. Yabuki, T. Mine, K. Iba, I. Nakatsuka and A. Yoshitake: Metabolism of tetra hydroaminoacridine derivative (SM-10888) in rat: Structural analysis of N glucuronide of SM-10888 and O-glucuronide of hydroxylated SM-10888 by FAB-MS/ MS.Xenobiotica, 23: 1367-1375 (1993). 5) A. N. Booth, D. J. Robbins, F. T. Jones, O. H. Emerson and M. S. Masri: Xanthuretic Acid Dehydroxylation by Fecal Microflora. Proc. Soc. Exp. Biol. Med., 120: 546-548 (1965). 6) Masashi Yabuki, Takeshi Mine, Kazuhiko Iba, Iwao Nakatsuka and Akira Yoshitake: Pharmacokinetics of SM-10888 and its metabolites depending on their physicochemical properties. Drug Metab. Dispos., 22: 294-297 (1994).

<I> Entero-hepatic Circulation of SM in the Rat: Elucidation of the Participating Major Metabolites</I>