1521-009X/41/4/878–887$25.00 http://dx.doi.org/10.1124/dmd.112.050591DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 41:878–887, April 2013Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics

Absorption, Elimination, and Metabolism of CS-1036, a Novela-Amylase Inhibitor in Rats and Monkeys, and the Relationship

between Gastrointestinal Distribution and Suppression of GlucoseAbsorptions

Tomohiro Honda, Yoko Kaneno-Urasaki, Takahiro Murai, Masayo Kakuta, Hatsumi Nasu,Eiko Namba, Tetsufumi Koga, Akira Okuno, and Takashi Izumi

Drug Metabolism and Pharmacokinetics Research Laboratories, R&D Division (T.H., Y.K.-U., T.I.), Corporate Strategy Department,Corporate Strategy Division (T.M.), Biological Research Laboratories, R&D Division (M.K., E.N., T.K.), Biologics Research

Laboratories, R&D Division (H.N.), and R&D Planning Department, R&D Division (A.O.), Daiichi Sankyo Co., Ltd., Tokyo, Japan

Received December 6, 2012; accepted February 1, 2013

ABSTRACT

The absorption, metabolism, and excretion of (2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-b-D-glucopyranosyl)-a-D-glucopyranoside (CS-1036), a novel and potent pancreatic andsalivary a-amylase inhibitor, were evaluated in F344/DuCrlCrlj ratsand cynomolgus monkeys. The total body clearance and volumeof distribution of CS-1036 were low (2.67–3.44 ml/min/kg and0.218–0.237 l/kg for rats and 2.25–2.84 ml/min/kg and 0.217–0.271 l/kgfor monkeys). After intravenous administration of [14C]CS-1036 torats and monkeys, radioactivity was mainly excreted into urine(77.2% for rats and 81.1% for monkeys). After oral administration,most of the radioactivity was recovered from feces (80.28% for ratsand 88.13% for monkeys) with a low oral bioavailability (1.73–2.44%for rats and 0.983–1.20% for monkeys). In rats, intestinal secretion

is suggested to be involved in the fecal excretion as a minorcomponent because fecal excretion after intravenous adminis-tration was observed (15.66%) and biliary excretion was almostnegligible. Although intestinal flora was involved in CS-1036 me-tabolism, CS-1036 was the main component in feces (70.3% forrats and 48.7% for monkeys) and in the intestinal contents (33–68%for rats up to 2 hours after the dose) after oral administration. InZucker diabetic fatty rats, CS-1036 showed a suppressive effect onplasma glucose elevation after starch loading with a 50% effectivedose at 0.015 mg/kg. In summary, CS-1036 showed optimal phar-macokinetic profiles: low oral absorption and favorable stabilityin gastrointestinal lumen, resulting in suppression of postprandialhyperglycemia by a-amylase inhibition.

Introduction

CS-1036, (2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl4-O-(6-deoxy-b-D-glucopyranosyl)-a-D-glucopyranoside (Fig. 1), isa novel and potent inhibitor of pancreatic and salivary a-amylase inrats and humans (Honda et al., 2004). CS-1036 is expected to inhibitstarch digestion in diets via a-amylase inhibition by oral administra-tion, which then leads to the suppression of postprandial glucoseabsorption. As commercially available oral antidiabetic agents withsimilar mechanisms, the a-glucosidase inhibitors (a-GIs) acarbose,voglibose, and miglitol also suppress postprandial hyperglycemia bythe inhibition of a-glucosidase expressed on intestinal brush bordermembranes (Martin and Montgomery, 1996; Hara and Hotta, 1997;

Scott and Spencer, 2000). For the pharmacokinetic (PK) properties ofa-GIs, orally administered miglitol is absorbed with a high oralbioavailability (Foral . 60%) and excreted into urine in unchangedform (Ahr et al., 1997). On the other hand, acarbose exhibits a lowabsorption and is metabolized by digestive enzymes and intestinalflora; urinary excretion of the unchanged form is below 3.4% of thedose (Ahr et al., 1989). The pharmacologic targets of CS-1036 aresalivary and pancreatic a-amylase, which are secreted into saliva andpancreatic juices, respectively. Because the administration route ofCS-1036 is oral, CS-1036 is expected to inhibit a-amylase in thegastrointestinal tract. From this perspective, low absorption andfavorable stability are considered the optimal profile for CS-1036,which differs from the PK profile of the a-GIs.In this study, the absorption, metabolism, gastrointestinal distribu-

tion, and excretion of CS-1036 were investigated in F344/DuCrlCrljdx.doi.org/10.1124/dmd.112.050591.s This article has supplemental material available at dmd.aspetjournals.org.

ABBREVIATIONS: a-GI, a-glucosidase inhibitor; AUC, area under the plasma concentration versus time curve; AUClast, area under the plasmaconcentration versus time curve up to the last quantifiable time; AUC0–inf, area under the plasma concentration versus time curve up to infinity;DAUCPG, area under the curve of change of plasma glucose level normalized by the plasma glucose level at 0 hours as a baseline; DAUCPG_min, areaunder the curve of change of plasma glucose level normalized by the plasma glucose level at 0 h as a baseline in the nonstarch control group; BDC,bile duct-cannulated; CI, confidence interval; CL, total body clearance; Cmax, maximum plasma concentration; CS-1036, (2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-b-D-glucopyranosyl)-a-D-glucopyranoside; DAB, 1,4-dideoxy-1,4-imino-D-arabinitol; Foral, oralbioavailability; F344, F344/DuCrlCrlj; g, sigmoidicity factor; GP, glycogen phosphorylase; HPLC, high-performance liquid chromatography; IC50,50% inhibitory concentration; l, terminal elimination rate constant; LC-MS, liquid chromatography–mass spectrometry; LC-MS/MS, liquidchromatography–tandem mass spectrometry; PK, pharmacokinetic; PYF, peptone yeast extract Fildes solution; RB, blood/plasma ratio; tmax, timeto reach maximum plasma concentration; Vss, volume of distribution at steady state; ZDF, Zucker diabetic fatty.

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(F344) rats and cynomolgus monkeys. To confirm the assumption thatintestinal concentrations of CS-1036 affect its inhibitory effects onstarch digestion, the suppression of plasma glucose elevation wasinvestigated in Zucker diabetic fatty (ZDF, ZDF/Crl-Leprfa) rats afterstarch loading.

Materials and Methods

Materials

CS-1036 and its metabolites M1, M2, and M3 (Supplemental Figs. 1, 2, and3) were synthesized at Daiichi Sankyo Co., Ltd. (Tokyo, Japan) according tothe published procedures (Honda et al., 2004). The internal standards, 2H5-CS-1036 and 2H5-M1 (Fig. 1), for the quantification of CS-1036 and M1 were alsosynthesized at Daiichi Sankyo Co., Ltd. [14C]CS-1036 (41.9, 52.2, 42.2, and63.5 mCi/mg: four different lots) was synthesized at GE Healthcare UK Ltd.(Buckinghamshire, UK) and Sekisui Medical Co., Ltd. (Tokyo, Japan). Theradiochemical purities of [14C]CS-1036 were guaranteed to be more than 98%at synthesis and more than 95% in the experiments by high-performance liquidchromatography (HPLC) with radioactive flow detection. All other reagents andsolvents used were commercially available and were of extra pure, guaranteed,HPLC or liquid chromatography-mass spectrometry (LC-MS) grade.

Animals

All animal studies were conducted with approval in accordance with theguidelines of the Institutional Animal Care and Use Committee of DaiichiSankyo. Male F344 rats at 7 weeks of age and male ZDF rats at 6 weeks of agewere purchased from Charles River Laboratories Japan, Inc. (Kanagawa,Japan). F344 rats were used after acclimatization of at least 5 days. The ZDFrats were acclimatized until they were 10 weeks of age. Male cynomolgusmonkeys purchased from Japan Laboratory Animals, Inc. (Tokyo, Japan) orGuangxi Grandforest Scientific Primate Company, Ltd. (Guangxi, China) wereused at 2–5 years of age after quarantine and acclimatization for more than 6weeks. Diets were freely accessed by rats and were supplied once daily tomonkeys. Water was provided ad libitum throughout the experiments. All theanimals were fasted overnight before administration and through to 4–8 hoursafter dosing.

Pharmacokinetics of CS-1036 in Rats and Monkeys

CS-1036 (0.3, 1.0, 3.0, and 10.0 mg/kg) was administered intravenously ororally to fasted F344 rats (146–185 g, n = 4 each) and monkeys (3.48–4.29 kg,n = 4 each). The monkey study was conducted with 2-week washout periodsbetween doses. Blood was collected at designated time points up to 48 hoursafter the dose. Plasma was obtained by centrifugation at 4°C and was stored at270°C until analysis.

Mass Balance Study in Rats

[14C]CS-1036 (1 mg/kg, 41.9 mCi/kg) was administered intravenously viathe tail vein (152–156 g, n = 4) or orally (154–159 g, n = 4) to fasted F344 rats.After administration, the rats were housed individually in metabolic cages, andurine and feces were collected at designated intervals up to 168 hours after thedose. Carbon dioxide in the expired air was trapped by the mixture of 2-aminoethanol/2-methoxyethanol = 1/1 (v/v) at designated intervals up to 48hours after the dose in rats.

Biliary Excretion of Radioactivity in Bile Duct-Cannulated (BDC) Rats

Fasted F344 rats were subjected to cannulation with a flexible polyethylenetube (SP-31; Natsume Seisakusho Co., Ltd., Tokyo, Japan) into the commonbile duct and fixed by placing a ligature around the tube to prevent dislocationunder diethyl ether anesthesia. After recovery from the anesthesia, the dosingsolution at a dose of 1 mg/kg (41.9 mCi/kg) of [14C]CS-1036 was administratedintravenously via the tail vein (161–165 g, n = 4) or by oral gavage (157–162 g,n = 4). The rats were individually accommodated in Bollman cages, and bilewas collected in tubes at 0–6, 6–24, 24–30, and 30–48 hours in a water bath setat 4°C.

Mass Balance Study in Monkeys

[14C]CS-1036 (3 mg/kg, 191 and 156.5 mCi/kg for intravenous and oraladministration, respectively) was administered intravenously via the saphenousvein (3.85–4.75 kg, n = 4) or orally by a stomach catheter (3.77–4.18 kg, n = 4)to fasted monkeys. After administration, urine, feces, and cage-washingsolution with water were collected at designated intervals up to 336 hours afterthe dose.

Sample Preparation for Qualitative In Vivo Metabolite Analysis in Urineand Feces

[14C]CS-1036 (3 mg/kg, 126 mCi/kg) was administered orally by a stomachcatheter to fasted F344 rats (148–176 g, n = 3) and monkeys (2.44–3.14 kg,n = 3). Urine and feces were collected up to 24 and 48 hours after the dose fromrats and monkeys, respectively. Urine was applied to solid phase extraction usingOasis MCX cartridges (60 mg, 3 ml; Waters Corp., Milford, MA), which hadbeen preconditioned with 3 ml of methanol and 3 ml of water. The cartridgeswere washed with 1 ml of water followed by the elution of radioactivecomponents with 3 ml of 28% ammonia solution/methanol (5/95, v/v). Feceswere homogenized with water (50 ml for rats and 4-fold volume of feces formonkeys), and extracted with an equal volume of methanol for rats and with 2-fold volume of methanol for monkeys. The fecal extracts were applied to OasisHLB cartridges (60 mg, 3 ml; Waters Corp.), which had been preconditionedwith 3 ml of methanol and 3 ml of water. The radioactive components wereeluted with 0.5 ml of water followed by 3 ml of methanol. Pretreated urinary andfecal samples were lyophilized and reconstituted with 0.1–0.4 ml of water.

Sample Preparation for In Vitro Qualitative Metabolite Analysis

Hepatic, Renal, and Intestinal S9 Fractions. [14C]CS-1036 (final con-centration: 22.7 mM, 0.42 mCi/ml) was incubated at 37°C for 1 hour in 2 mgprotein/ml of hepatic, renal, and intestinal S9 fractions from rats, monkeys, andhumans in 100 mM potassium phosphate buffer (pH 7.4) with an NADPH-generating system (2.5 mM b-NADP, 25 mM glucose-6-phosphate, 0.5 units/mlglucose-6-phosphate dehydrogenase, 10 mM magnesium chloride). After incu-bation, 0.2 ml of the reaction mixture was mixed with 0.2 ml of acetonitrilefor the termination of the reaction. The supernatant obtained by centrifugationwas subjected to metabolite analysis.

Anaerobic Culture Broth of Rat Cecal Contents and Feces from Monkeysand Humans. After the F344 rats were sacrificed, the cecum was removed, andthe cecal contents were collected (n = 4). The feces were collected using a sterileswab from the monkeys (n = 4) and healthy male volunteers (n = 5, 29–54 yearsof age). The collected cecum and feces were maintained with oxygen-absorbingand carbon dioxide-generating agents in a hermetically sealed pouch at roomtemperature until use. The rat cecal contents and the monkey and human feceswere suspended in 5 ml of peptone yeast extract Fildes solution (PYF) brothwithin 24 hours of collection. The suspensions of rat cecal contents and the

Fig. 1. Chemical structures of [14C]CS-1036 (A), internal standard for CS-1036(B), and internal standard for M1 (C).

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monkey and human feces were cultured individually at 35°C for 3 days in anincubator attached to an anaerobic chamber. Each 0.3 ml of the culture broth wastransferred to 30 ml of PYF broth. The subcultured PYF broth was incubated at35°C for 2 days in an incubator attached to the anaerobic chamber. Then[14C]CS-1036 (final concentration: 22.7 mM, 63.5 mCi/ml) was incubated in PYFculture broths of rat cecal contents and monkey and human feces at 35°C for 24hours under anaerobic conditions. The reaction was terminated by three cycles offreeze-thaw processes, and the same volume of acetonitrile was added. Thesupernatant (0.1 ml) obtained by centrifugation of the sample was dried up usinga centrifugal evaporator, and the residues were reconstituted by 50 ml of water foranalysis.

Sample Preparation for Quantitative Analysis of Radioactivity in Urineand Feces

[14C]CS-1036 (1 mg/kg, 42.2 mCi/kg) was administered orally to fastedF344 rats (164–168 g, n = 3). Urine and feces were collected up to 24 hoursafter the dose administration. In monkeys, samples for in vivo qualitativemetabolite analysis were also used for quantitative metabolite analysis in urineand feces. Rat feces were homogenized with 9-fold volumes of water. The fecalhomogenate was mixed with the same volume of methanol for rats and 2-foldvolumes of methanol for monkeys. The supernatant after centrifugation wasused for the analysis.

Sample Preparation for Quantitative Analysis of Radioactivity inGastrointestinal Contents in Rats

[14C]CS-1036 (1 mg/kg, 42.4 mCi/kg) was administered orally to fastedF344 rats (148–168 g, n = 3). The stomach, duodenum and jejunum, ileum, andcecum were observationally isolated, and the gastrointestinal contents werecollected at 0.5, 1.0, 2.0, 4.0, 6.0, and 8.0 hours after the dose administration bywashing with 5 ml of water 3 times (total: 15 ml). Each of the gastrointestinalcontent collections was homogenized, mixed with the same volume ofmethanol, and centrifuged (4°C, 1,600g, 10 minutes). The resulting supernatant

was collected, and the residual extract was freeze-dried. The resulting residuesfrom the contents of the stomach, duodenum and jejunum, and ileum werereconstituted by 50% methanol (0.2 ml), respectively. Each resulting solutionwas centrifuged (4°C, 10,000g, 10 minutes) to obtain the supernatant as anHPLC sample.

As for the cecal contents, each resulting residue that had been obtained byfreeze-drying was mixed with 50% methanol (0.2 ml) and centrifuged (4°C,10,000g, 10 minutes); the resulting supernatant was collected. The extractionprocedures were repeated 3 times. The three batches of supernatants werecombined and evaporated under a nitrogen stream. The resulting residues fromthe cecal contents were dissolved in 50% methanol (0.2 ml), and each resultingsolution was centrifuged (4°C, 10,000g, 10 minutes) to obtain the supernatantas an HPLC sample.

Effect of CS-1036 on Plasma Glucose Level after Starch Loading in ZDFRats

CS-1036 (0, 0.001, 0.003, 0.01, 0.03, and 0.1 mg/kg) in the starchsuspensions (2 g/kg) was administered orally to fasted ZDF rats (300–366 g,n = 8 each). For the nonstarch control, water (10 ml/kg) was administered toZDF rats (314–361 g, n = 8). Blood was collected 15 minutes beforeadministration and at 0.5, 1.0, 2.0, and 4.0 hours after the dose. Plasma wasobtained by centrifugation at room temperature and was stored at 4°C untilmeasurement of the plasma glucose levels. The plasma glucose level obtainedat 15 minutes before the dose administration was regarded as the 0-hour level.

Blood/Plasma Ratio of [14C]CS-1036

Aliquots of 5 ml of [14C]CS-1036 solution (final concentrations: 113, 227,1130, 2270, and 11300 nM) were mixed with 495 ml of rat, monkey, or humanblood and were incubated for 5 minutes at 37°C. The radioactivity of the blood(0.1 ml) and plasma (0.1 ml), obtained by centrifugation at 4°C, was measured,and the blood/plasma ratio (RB) was calculated by dividing the bloodradioactivity by the plasma radioactivity.

Fig. 2. Plasma concentration profiles of CS-1036 after single intravenous and oral adminis-tration of CS-1036 to rats and monkeys ata dose of 10 mg/kg. The data are expressed asmean 6 S.D. (n = 3 or 4).

TABLE 1

Pharmacokinetic parameters of CS-1036 after a single intravenous administration of CS-1036 to rats and monkeys

Values are expressed as mean 6 S.D. (n = 4).

Species Dose AUClast AUC0-inf CL Vss t1/2

mg/kg ng·h/ml ng·h/ml ml/min/kg l/kg h

Rat 0.3 1860 6 290 1870 6 290 2.73 6 0.46 0.218 6 0.038 14.6 6 3.11 4920 6 670 4930 6 670 3.44 6 0.51 0.237 6 0.038 18.4 6 2.63 18,700 6 1300 18,800 6 1300 2.67 6 0.17 0.219 6 0.020 23.6 6 6.6

10 53,000 6 5600 53,200 6 5600 3.16 6 0.37 0.222 6 0.025 21.5 6 1.6Monkey 0.3 2000 6 350 2010 6 350 2.55 6 0.47 0.233 6 0.029 2.51 6 1.06

1 5870 6 310 5890 6 310 2.84 6 0.15 0.271 6 0.024 21.4 6 10.13 18,100 6 800 18,200 6 800 2.75 6 0.12 0.262 6 0.025 25.1 6 2.1

10 74,300 6 7300 74,500 6 7300 2.25 6 0.22 0.217 6 0.019 30.0 6 4.6

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Sample Analysis

Quantification of CS-1036 and M1. Plasma concentrations of CS-1036 andM1 were determined by liquid chromatography–tandem mass spectrometry(LC-MS/MS). For the PK study, the plasma sample (50 ml), including studysamples or control plasma (for standard and quality controls), was mixed with50 ml of IS (100 ng/ml of 2H5-CS-1036 and 100 ng/ml of 2H5-M1 in 50%methanol), 50 ml of 50% methanol for study samples or standard or qualitycontrol samples in 50% methanol and 0.8 ml of water. The sample then wassubjected to a solid-phase extraction (100 mg, VersaPlate SCX; AgilentTechnologies, Santa Clara, CA). The extracted sample was evaporated bynitrogen stream, and the residue was dissolved in 180 ml of mobile phase A (asdescribed later), and 20 ml of the sample was injected to an LC-MS/MS systemconsisting of an API4000 mass spectrometer (Applied Biosystems, FosterCity, CA) coupled to an HPLC system (Agilent 1100 series HPLC, AgilentTechnologies; or 2795 separations module; Waters Corp.). The analytes wereseparated on a Capcell-pak UG80 SCX (5 mm, 2.0 � 75 mm; Shiseido Corp.,Tokyo, Japan), at a column oven temperature of 40°C. The flow rate was 0.4ml/min with a gradient of 10 mM ammonium acetate in methanol/water (50/50,v/v, A) and 10 mM ammonium acetate in methanol/water (95/5, v/v, B). Thegradient condition of mobile phase B was 0% from 0–4.5 minutes (constant),0% to 100% for 4.5–5.0 minutes (linear), 100% for 5.0–5.5 minutes (constant),and 0% from 5.5–12.0 minutes (constant). Ionization was conducted in thepositive ion electrospray ionization mode at a source temperature of 600°C withnitrogen nebulizing and heating gas. CS-1036 and its internal standard wereanalyzed in the multiple reaction monitoring mode using the mass transitions ofm/z 442.4 → 134.0 and 447.4 → 139.0, respectively. M1 and its internalstandard were analyzed in multiple reaction monitoring mode using the masstransitions of m/z 134.2 → 68.1 and 139.2 → 72.1, respectively.

Radioactivity Analysis. Plasma (0.1 ml), urine (0.1–1 ml), and bile (25–100ml) were mixed with tissue solubilizer (1 ml of NCS-II; GE Healthcare UKLtd.; or 1 ml of tissue solubilizer for BIOMERIT; Nacalai Tesque, Kyoto,Japan) and scintillation cocktail (10 ml of Hionic-Fluor; PerkinElmer,

Waltham, MA; or Cleasol I; Nacalai Tesque); they were then analyzed witha liquid scintillation analyzer (Tri-carb 307, 2300TR, or 2900TR counter;PerkinElmer). Blood (0.1 ml) was mixed with 30% hydrogen peroxide (0.1 ml),a tissue solubilizer (1 ml of NCS-II), and a scintillation cocktail (10 ml ofHionic-Fluor), and it then was analyzed with a liquid scintillation analyzer.Feces were homogenized with 50–160 ml or 4-fold volume of water. Next, 0.5ml of the fecal homogenate and gastrointestinal tract content homogenate wasmixed with a tissue solubilizer (1 ml of NCS-II or tissue solubilizer forBIOMERIT) and a scintillation cocktail (10 or 15 ml of Hionic-Fluor), and wasthen analyzed with a liquid scintillation counter. For fecal metabolite profilingstudies in rats, the fecal sample was mixed with water of approximately 9-foldvolume of the sample weight and then homogenized. The fecal homogenate(0.5 ml) was placed on a combust pad and weighed; then it was combusted witha sample oxidizer. The resulting 14CO2 was absorbed in Carbo-Sorb E (6 ml;PerkinElmer), mixed with a scintillation cocktail (9 ml, Permafluor E+;PerkinElmer), and then analyzed with the liquid scintillation analyzer. Thesolvent trapped expired air (1 ml) was mixed with a scintillation cocktail (10 mlof Hionic-Fluor) and then analyzed with a liquid scintillation counter.

Qualitative and Quantitative Metabolite Profiling by HPLC withRadioactivity Detector and LC-MS. Metabolites in urine, feces, gastrointes-tinal contents, and in vitro samples were analyzed using an HPLC system(Alliance 2695 separations module coupled with a 2996 photodiode arraydetector; Waters Corp.) equipped with a mass spectrometer (Q-TOF Ultimamass spectrometer; Waters Corp.), or an LC-10Avp HPLC system (ShimadzuCorp., Kyoto, Japan) or L-2000 HPLC system (Hitachi High-TechnologiesCorp., Tokyo, Japan) equipped with a Radiomatic 625TR radioactivity detector(PerkinElmer), and a mass spectrometer (Accela-LTQ Orbitrap XL; ThermoFisher Scientific, Waltham, MA). The analytical conditions for HPLC andradioactivity detector were as follows: analytical column, Atlantis HILIC Silica(5 mm, 4.6 � 250 mm; Waters Corp.); column oven temperature, 30°C; mobilephase A, 0.1% (v/v) trifluoroacetic acid in water; mobile phase B, 0.1% (v/v)trifluoroacetic acid in acetonitrile; flow rate, 1 ml/min; gradient of mobile phaseB, 95% from zero to 5 minutes (constant), 95 to 75% for 5 to 25 minutes

TABLE 2

Pharmacokinetic parameters of CS-1036 after a single oral administration of CS-1036 to rats and monkeys

Values are expressed as mean 6 S.D. (n = 3 or 4), and a dash denotes not applicable.

Species Dose AUClast AUC0-inf tmax Cmax t1/2 Foral (%)

mg/kg ng·h/ml ng·h/ml h ng/ml h

Rat 0.3 42.1 6 13.1 45.6 6 17.2 0.500 6 0.354 16.2 6 2.4 2.69 6 2.55 2.441 110 6 26 115 6 30 0.417 6 0.144 45.5 6 5.5 1.81 6 0.31 2.333 324 6 25 325 6 29 0.375 6 0.144 113 6 15 4.16 6 1.94 1.73

10 1050 6 140 1080 6 150 0.438 6 0.375 285 6 75 18.3 6 6.7 2.03Monkey 0.3 15.0 6 4.2 — 1.75 6 0.50 5.23 6 1.24 — —

1 52.0 6 11.4 57.9 6 8.2 1.75 6 0.50 15.3 6 4.2 1.29 6 0.19 0.9833 186 6 67 191 6 71 1.75 6 0.50 44.8 6 11.0 2.12 6 0.47 1.05

10 869 6 300 894 6 305 1.75 6 0.50 152 6 77 12.6 6 5.0 1.20

TABLE 3

Excretion of radioactivity after single intravenous and oral administrations of [14C]CS-1036 to rats and monkeys

Values are expressed as mean 6 S.D. (n = 4), and a dash denotes not tested.

Species Dose/Route Collection PeriodExcretion of Radioactivity

Urine Feces Expired Air Bile Cage Washing Total

h % of dose

Rat 1 mg/kg, i.v. 0–48 70.49 6 7.96 15.66 6 8.04 0.82 6 1.05 — — 86.96 6 5.920–168 77.21 6 7.62 17.82 6 8.83 — — — 95.85 6 4.60

1 mg/kg, oral 0–48 13.34 6 3.53 77.59 6 2.84 2.34 6 0.74 — — 93.26 6 1.530–168 15.32 6 3.09 80.28 6 3.88 — — — 97.94 6 0.26

BDC rat 1 mg/kg, i.v. 0–48 — — — 0.37 6 0.08 — 0.37 6 0.081 mg/kg, oral 0–48 — — — 0.59 6 0.04 — 0.59 6 0.04

Monkey 3 mg/kg, i.v. 0–48 80.13 6 4.76 0.46 6 0.16 — — 13.70 6 4.19 94.29 6 1.08. 0–336 81.13 6 4.92 0.66 6 0.18 — — 14.50 6 4.04 96.29 6 1.24

3 mg/kg, oral 0–48 4.54 6 2.23 78.50 6 8.09 — — 0.76 6 0.37 83.80 6 8.880–336 5.67 6 2.33 88.13 6 3.04 — — 1.35 6 0.79 95.15 6 1.48

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(linear), 75% from 25 to 30 minutes; injection volume, 10 ml; liquid scintilla-tor for radioactivity detector, Ultima Flo M or Flo-Scint II (PerkinElmer);and scintillator flow rate, 3 ml/min. The LC-MS conditions for qualitativemetabolite analysis were as follows: ionization, positive ion electrospray ion-ization mode; capillary voltage, 3 kV; cone voltage, 40 V; collision energy, 10eV; source temperature, 100°C; and desolvation gas temperature, 300°C. Thespectral data were acquired in a mass range from m/z 50–1000. The LC-MSconditions for quantitative metabolite analysis were as follows: ionization,positive ion electrospray ionization mode; spray voltage, 5 kV; capillaryvoltage, 27 V; and capillary temperature, 350°C.

Plasma Glucose. The plasma glucose levels (mg/dl) were measured with anautomatic glucose analyzer (Glucoroder-GXT; A&T Corp., Kanagawa, Japan).

Pharmacokinetic Analysis

The PK parameters of CS-1036 and its metabolite M1 in plasma werecalculated using WinNonlin Professional (version 4.0.1; Pharsight Corp.,Mountain View, CA) based on a noncompartmental method. The maximumplasma concentration (Cmax) and the time to reach Cmax (tmax) were obtainedfrom the observed data. The t1/2 was calculated by the regression analysis ofthree or more log-transformed data points in the terminal phase. The area underthe plasma concentration versus time curve (AUC) up to the last quantifiabletime (AUClast) was calculated by the trapezoidal method. The AUC up toinfinity (AUC0–inf) was determined by use of the equation:

AUC0-inf ¼ AUClast þ Ctz=l

where Ctz is the last measurable concentration and l is the terminal eliminationrate constant. For intravenous administration, the total body clearance (CL) wascalculated as the ratio of the dose and AUC0–inf. The volume of distribution ata steady state (Vss) was calculated as the dose AUMC/(AUC0–inf)

2, whereAUMC is the area under the first moment of the plasma concentration-timecurve. The Foral for each dose was calculated by dividing the AUC0–inf for theoral dose by that for the intravenous dose at the same dose level.

Evaluation of Plasma Glucose Levels

The changes of plasma glucose levels after the dose administration werecalculated by subtracting the plasma glucose level at 0 hours from that at eachtime point in the corresponding animal. The area under the curve of change inthe plasma glucose level (DAUCPG), which was normalized by the plasma

glucose level at 0 hours as a baseline, was calculated by the trapezoidal method.The ED50 and its 95% confidence intervals (CI) were estimated according to thesigmoid Emax model described here, where the DAUCPG in the starch controlgroup and the DAUCPG in the nonstarch control group were substituted for theupper limit (DAUCPG_max) and lower limit (DAUCPG_min) of the model,respectively. The sigmoidicity factor (g) and its 95% CI were also estimated bySAS System Release 8.2 (SAS Institute, Inc., Cary, NC):

DAUCPG ¼ DAUCPG min þ ðDAUCPG max 2DAUCPG minÞ � ½dosage�g½ED50�g þ ½dosage�g

Statistical Analysis

For the plasma glucose levels and the changes of plasma glucose levels, thesummary statistics (n, arithmetic mean, S.E., and 95% CI) were calculated ateach time point for each group. The DAUCPG in the groups treated with CS-1036 was compared with that of the starch control group by a Dunnett test. P,0.05 was considered statistically significant.

Results

Pharmacokinetics of CS-1036 in Rats and Monkeys. The plasmaconcentration–time profiles of CS-1036 in rats and monkeys afterintravenous and oral administration (10 mg/kg) are shown in Fig. 2.The PK parameters of CS-1036 in rats and monkeys after intravenousand oral administration are shown in Tables 1 and 2, respectively. Forintravenous administration, CS-1036 was disposed in a biphasicmanner in both species. The CL and Vss were similarly low for bothspecies: 2.67–3.44 ml/min/kg and 0.218–0.237 l/kg for rats and2.25–2.84 ml/min/kg and 0.217–0.271 l/kg for monkeys. The AUCexhibited a dose-proportional increase in rats and monkeys (0.3–10mg/kg). For oral administration, AUC and Cmax also exhibited a dose-dependent increase in rats and monkeys (0.3–10 mg/kg) although thedose-normalized AUC and Cmax in rats after oral administration wereslightly higher at 0.3 mg/kg and the dose-normalized AUC inmonkeys after oral administration tended to increase with each dose.The tmax was 0.375–0.5 hours for rats and 1.75 hours for monkeys.

Fig. 3. Representative radiochromatograms of the metabolites in rat and monkey urine and feces after oral administration of [14C]CS-1036.

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The mean Foral was 1.73%–2.44% for rats and 0.983%–1.20% formonkeys.Excretion of Radioactivity. The excretion of radioactivity after

intravenous and oral administration of [14C]CS-1036 to rats and mon-keys is summarized in Table 3. After intravenous administration of[14C]CS-1036 to rats and monkeys, most of the radioactivity wasexcreted into urine: 77.21% of the dose up to 168 hours after the dose

for rats and 81.13% of the dose up to 336 hours after the dose formonkeys. Species differences of fecal excretion of radioactivity wereobserved, and the radioactivity recovered from feces was higher in rats(17.82% of the dose) than monkeys (0.66% of the dose). After oraladministration, most of the radioactivity was recovered from feces forboth species. In monkeys, the fecal excretion was 88.13% of the doseup to 336 hours after the dose mainly as a result of unabsorbedradioactivity. In rats, the fecal excretion was 80.28% of the dose up to168 hours after the dose, in which the component of intestinalsecretion might be included in addition to the unabsorbed radioactivitybecause fecal excretion was observed (15.66% of the dose) afterintravenous administration and biliary excretion in BDC rats wasalmost negligible (0.37% of the intravenous administration and 0.59%of the oral administration up to 48 hours after the dose). The urinaryexcretion of radioactivity was 15.32% and 5.67% of the dose for ratsand monkeys, respectively. In rats, the radioactivity excreted into theexpired air was higher after oral administration (2.34% of the dose)than intravenous administration (0.82% of the dose).In Vivo Metabolite Identification. Representative radiochromato-

grams of the metabolites in rat and monkey urine and feces after oraladministration of [14C]CS-1036 are shown in Fig. 3. In rat and monkeyurine, M1 was detected as a metabolite. The positive ion LC-MS spectrumof M1 showed a protonated molecule [M+H]+ at m/z 134, and wasconsistent with its authentic standard. M1 was identified as 1,4-dideoxy-1,4-imino-D-arabinitol (DAB). In rat and monkey feces, M1 and M2were detected as metabolites. The positive ion LC-MS spectrum of M2showed a protonated molecule [M+H]+ at m/z at 176, and was consistentwith its authentic standard. M2 was identified as N-acetylated M1.In Vitro Metabolism of CS-1036. To identify whether the liver,

kidney, and small intestine are responsible for CS-1036 biotransfor-mation, an in vitro metabolism study using these S9 fractions wasperformed. Incubation of [14C]CS-1036 with the hepatic, renal, orintestinal S9 fraction from rats, monkeys, or humans in the presence ofNADPH did not produce any metabolites (Supplemental Fig. 4).Therefore, we supposed that in vivo biotransformation of CS-1036—namely, hydrolysis followed by N-acetylation—might be mediated bythe intestinal flora in the intestine. To confirm this, CS-1036 wasincubated in a culture broth derived from rat cecal contents andmonkey and human feces under anaerobic conditions. After 24 hoursof incubation, M1 was produced as a common metabolite of CS-1036for rats, monkeys, and humans (Fig. 4). M3 was observed only in theculture broth of rat cecal contents and was identified as an N-acetylmetabolite of CS-1036 via LC-MS/MS analysis (Supplemental Fig. 5).Quantitative Plasma Profiles of M1 in Rats and Monkeys. After

oral administration of [14C]CS-1036 to rats and monkeys, we did notobserve metabolites in the radiochromatogram of the plasma samples

Fig. 4. Representative radiochromatograms obtained by analysis of the culturebroths of rat cecal contents, monkey feces, and human feces after incubation of[14C]CS-1036 under anaerobic conditions for 24 hours.

TABLE 4

Pharmacokinetic parameters of M1 after single intravenous and oral administrations of CS-1036 to rats and monkeys

Values are expressed as mean 6 S.D. (n = 3 or 4) for rats and monkeys.

Species Route Dose tmax Cmax AUClast Ratio of Molar-Based AUClast

mg/kg h ng/ml ng·h/ml % of CS-1036

Rat i.v. 3 0.354 6 0.438 0.774 6 0.231 0.387 6 0.169 0.00672 6 0.0025710 0.125 6 0.083 2.67 6 0.57 2.82 6 1.59 0.0172 6 0.0086

oral 0.3 1.25 6 0.50 1.71 6 0.21 2.64 6 1.32 22.1 6 12.61 1.33 6 0.58 10.6 6 5.1 26.1 6 18.6 73.4 6 34.93 0.688 6 0.375 20.9 6 3.5 49.2 6 12.2 49.9 6 9.6

10 0.938 6 0.774 39.8 6 6.5 103 6 33 32.2 6 8.4Monkey oral 1 3.50 6 1.91 1.40 6 0.52 9.66 6 4.93 60.8 6 26.6

3 1.75 6 0.50 4.86 6 1.29 40.2 6 11.0 83.5 6 54.010 1.25 6 0.50 20.7 6 7.0 146 6 22 62.5 6 28.1

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owing to the detection limit (unpublished data), but M1 was observedin urinary samples. Thereafter, we determined M1 concentrations inplasma by LC-MS/MS using the reference standard of M1 and itsinternal standard. The PK parameters of M1 in rats and monkeys areshown in Table 4. After a single intravenous administration of CS-1036, M1 was detected at low levels in rats at doses of 3 and 10 mg/kg.M1 was not detected at the lower doses (0.3 and 1 mg/kg) in ratsor at any dose levels (0.3–10 mg/kg) in monkeys. After a singleoral administration of CS-1036, we observed the the tmax of M1 at0.688–1.33 hours after the dose in rats and at 1.25–3.5 hours after the

dose in monkeys. The molar-based ratio of the AUC of M1 to that ofCS-1036 ranged from 22.1 to 83.5% after oral administration of CS-1036 to rats and monkeys.Quantitative Metabolite Profiles of Urine and Feces. The quan-

titative metabolite profiles in urine and feces up to 24 hours for ratsand up to 48 hours for monkeys after oral administration of [14C]CS-1036 are summarized in Table 5. In urine, CS-1036 (1.4% of the dosefor rats and 2.9% of the dose for monkeys) and M1 (5.3% of the dosefor rats and 2.2% of the dose for monkeys) were detected. In feces,a species difference in the metabolite formation was observed; M1 andM2 were detected not in rats but in monkeys (9.7 and 17.8% of thedose, respectively), although M1 and M2 were identified in rat fecesduring the in vivo metabolite identification.Quantitative Metabolite Profiles of Gastrointestinal Contents in

Rats. Quantitative metabolite profiles of gastrointestinal contents afteroral administration of [14C]CS-1036 (1 mg/kg) are shown in Fig. 5. Asshown in Fig. 5A, radioactivity was mainly distributed in the ileumcontents (67.7% of the dose) at 1 hour after the dose, in the ileum andcecal contents (33.9 and 49.9% of the dose, respectively) at 2 hoursafter the dose, and in the cecal contents (49.4–72.7% of the dose) after4 hours after the dose. The radioactivity-time profiles of CS-1036 ineach gastrointestinal section almost overlapped with those of the totalradioactivity (Fig. 5B). M1 was below 1% of the dose in the stomachand intestinal contents at each time point, but it increased in a time-dependent manner in cecal contents to 7.1% of the dose at 8 hoursafter the dose (Fig. 5C). Although M2 was not observed in stomach orintestinal contents at any time points, small amounts of M2 (1.7–2.1%

TABLE 5

Composition of radioactive components in urine and feces after a single oraladministration of [14C]CS-1036 to rats and monkeys

The ratio of CS-1036 and its metabolites was determined by the ratio of excreted radioactivityand the peak area ratio of each component on the radiochromatogram. Values are expressed asmean 6 S.D. (n = 3).

Species Dose Collection Time ComponentExcretion of Radioactivity

Urine Feces

mg/kg h % of dose

Rat 1 24 CS-1036 1.4 6 0.4 70.3 6 10.8M1 5.3 6 1.3 ND

Monkey 3 48 CS-1036 2.9 6 2.1 48.7 6 6.7M1 2.2 6 0.6 9.7 6 1.4M2 ND 17.8 6 11.1

ND: not determined.

Fig. 5. Total radioactivity (A), CS-1036 (B) and M1 (C) time-profiles in stomach, duodenum and jejunum, ileum, and cecal contents after single oral administration of[14C]CS-1036 to rats. The data are expressed as mean 6 S.D. (n = 3).

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of the dose) were observed in cecal contents at 6 and 8 hours after thedose.Suppressive Effect on Plasma Glucose Elevation after Starch

Loading in ZDF Rats. As shown in Fig. 6, CS-1036 suppressedplasma glucose elevation after starch loading in ZDF rats in a dose-dependent manner. The plasma glucose elevations in the groups thatwere orally treated with CS-1036 at doses of 0.01, 0.03, and 0.1 mg/kgwere significantly suppressed compared with the control group (P ,0.05). The ED50 was estimated to be 0.015 mg/kg according to thesigmoid Emax model.RB of [14C]CS-1036. The RB of [14C]CS-1036 was 0.575–0.581

in rats, 0.595–0.603 in monkeys, and 0.521–0.538 in humans. Noconcentration dependency was observed in the range of 113–11300 nM.

Discussion

After intravenous administration of [14C]CS-1036 to rats andmonkeys, the radioactivity was mainly excreted via the urinary route(77.21% for rats and 81.13% for monkeys). These results suggest thatthe renal clearance is a major fraction of the CL of CS-1036. In bothanimals, the CL of CS-1036 was almost constant at the dose range of0.3–1.0 mg/kg and the blood clearances of CS-1036 (CL/RB: 4.6–6.0ml/min/kg for rats and 3.7–4.8 ml/min/kg for monkeys) were 6- to 8-fold lower than the renal blood flows (36.8 ml/min/kg for rats and27.6 ml/min/kg for monkeys) (Davies and Morris, 1993). The Vss of

CS-1036 in rats and monkeys was also small and constant at thestudied doses (0.218–0.237 l/kg for rats and 0.217–0.271 l/kg formonkeys), which was comparable with the extracellular fluid volume:0.3 l/kg for rats and 0.2 l/kg for monkeys (Davies and Morris, 1993).Because the lipophilicity is reported to correlate positively with the Vss

(Poulin and Theil, 2002), the highly hydrophilic physicochemicalproperty (estimated ClogP by Pallas: –4.57) of CS-1036 might lead toa low Vss.After oral administration, the main route of excretion was feces

(80.28% of the dose for rats, and 88.13% of the dose for monkeys),and the main component in feces was CS-1036 (70.3% of the dose upto 24 hours after the dose for rats, and 48.7% of the dose up to 48hours after the dose for monkeys). On the other hand, the urinaryexcretion of CS-1036 was low in both animals (1.4% of the dose up to24 hours after the dose for rats, and 2.9% of the dose up to 48 hoursafter the dose for monkeys), and was comparable to Foral of bothanimals (1.73–2.44% for rats, and 0.983–1.20% for monkeys). Theseresults suggest that most of the unabsorbed radioactivity is recoveredfrom feces and that the absorbed radioactivity is mainly excreted intourine. The low Foral of CS-1036 has a possibly strong relation to thehighly hydrophilic trisaccharide structure of CS-1036. Kestose, whichis also a trisaccharide compound as CS-1036, was reported to have notransmural potential in rats (Tsuji et al., 1986).The biliary excretion was negligible in BDC rats for both

intravenous and oral administration (below 1% of the dose), althoughthe fecal excretion (15.66% of the dose) after intravenous adminis-tration was much higher than the biliary excretion. This discrepancymay suggest the contribution of intestinal secretion to the fecalexcretion of CS-1036. In some drugs, intestinal efflux transporterssuch as multidrug resistance 1 (MDR1), breast cancer resistanceprotein (BCRP), and multidrug resistance protein 2 (MRP2) areinvolved in the intestinal secretion (Lowes and Simmons, 2002;Haslam et al., 2011). However, the contribution of intestinal effluxtransporters to the intestinal secretion of CS-1036 might be negligiblebecause CS-1036 is more hydrophilic than the substrates of intestinalefflux transporters. Although the mechanism of intestinal secretion isunknown, we consider intestinal secretion of CS-1036 to be a specificphenomenon in rats because fecal excretion was below 1% of the doseafter intravenous administration of [14C]CS-1036 to monkeys.After oral administration of [14C]CS-1036, M1 and M2, the N-

acetyl form of M1, were identified as metabolites in rat cecal contentsand monkey feces, and only M1 was identified as a metabolite in ratand monkey urine. The in vitro metabolism study using hepatic, renal,and intestinal S9 fractions exhibited no metabolite observation afterincubation of [14C]CS-1036 in these S9 fractions, suggesting thathepatic, renal, and intestinal enzymes were not involved in themetabolite formation of CS-1036. M2 was observed in addition to M1in feces, and M1 in plasma was higher after oral administration thanintravenous administration to rats and monkeys. Furthermore, M1 wasproduced in a culture broth of rat cecal contents, monkey feces, andhuman feces under anaerobic conditions. M2 was not observed, butM3, the N-acetyl form of CS-1036, was identified in the culture brothof rat cecal contents. Intestinal flora is reported to have the potentialfor N-acetylation in humans, rats, mice, and dogs (King et al., 1990;Okumura et al., 1995). These facts suggest that CS-1036 wasmetabolized by enzymes of intestinal flora. In addition, M1 and M2were higher in monkey feces than in rat feces, indicating that themetabolite formation of CS-1036 by intestinal flora was higher inmonkeys than in rats.Based on the in vivo and in vitro metabolite information, the

metabolic pathways of CS-1036 by intestinal flora are proposed inFig. 7. As observed in CS-1036, acarbose, which is an a-GI and

Fig. 6. Change of plasma glucose level from predose (A) and AUC of change ofplasma glucose level from baseline (B) after single oral administration of CS-1036 withstarch (2 g/kg) to ZDF rats. The data are expressed as mean 6 S.E. (n = 8). TheDAUCPG in the groups treated with starch and CS-1036 was compared with that of thestarch control group by a Dunnett test. *P , 0.05 versus the starch control group.

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oligosaccharide derivative, is also reported to be metabolized by in-testinal flora, and the primary metabolites were formed by hydrolysisof O-glycosidic bonds in the molecule (Ahr et al., 1989). In addition tointestinal flora, a-glucosidase might be involved in the metabolism ofacarbose because acarbose exhibits a-glycoside bonds in its molecule.For other a-GIs, the hydrolysis of O-glycosidic bond by intestinalflora is not observed because the chemical structures of vogliboseand miglitol are monosaccharide and do not exhibit O-glycosidicbonds in their molecules (Ahr et al., 1997; Hara and Hotta, 1997). ForCS-1036, b-glucosidase from intestinal flora might be involved in themetabolism because CS-1036 exhibits a b-glycoside bond in itsmolecule and was stable in intestinal S9 fractions which containa-glucosidase (Hauri et al., 1979; Goldin, 1990).After oral administration of [14C]CS-1036, radioactivity and CS-

1036 were rapidly eliminated from stomach, duodenum, and jejunumcontents; the peak of CS-1036 in ileum contents was observed at 1hour after the dose (66% of the dose), and most of the CS-1036 in theileum contents was diminished at 4 hours after the dose. In ZDF rats,CS-1036 showed suppressive effects on the glucose elevation from 0.5to 2.0 hours after oral administration of CS-1036 at $0.01 mg/kg(ED50 5 0.015 mg/kg). Alpha-amylase, which digests starch intooligosaccharides, is mainly distributed in the intestine in rats(McGeachin and Ford, 1959). Assuming the linear PK from the resultof oral dosing in rats (0.3–10 mg/kg), the estimated intestinalconcentration calculated from the intestinal amount of CS-1036 afteran oral dose (0.01 mg/kg) and the intestinal fluid volume (McConnellet al., 2008) was estimated to be 1.5–3.1 mM from 0.5 to 2.0 hoursafter the dose, which will exceed the 50% inhibitory concentration(IC50 5 0.45 mM) to pancreatic amylase (Honda et al., 2004).This suggests that CS-1036 exerts the suppressive effects on the

glucose absorption when the concentration of CS-1036 in its site ofaction, intestinal lumen, is achieved to IC50 and over. Thus, thelow absorption and a favorable stability in gastrointestinal contentsof CS-1036 are considered to be its optimal properties as ana-amylase inhibitor. In addition to our estimation of intestinal CS-1036

concentration, some simulation tools might help us to predict theintestinal concentration in humans by taking into account the speciesdifferences in the length, transit time, and pH of each intestinalsection (Parrott and Lave, 2002; Jamei et al., 2009). Not only thetransition and concentration of CS-1036 but also the transitions ofstarch (which is the substrate of a-amylase) and a-amylase itselfare important factors for the pharmacologic activity. Precise hu-man pharmacokinetic/pharmacodynamic prediction is expected tobe possible if the species differences of intestinal transition andactivity of a-amylases secreted from saliva and pancreatic fluid arerevealed.M1 was reported as DAB, which is a potent inhibitor of hepatic

glycogen phosphorylase (GP), with IC50 of 1.0 and 1.1 mM forinhibition to basal and glucagon-stimulated glycogenolysis in rathepatocytes, respectively (Andersen et al., 1999). In ob/ob mice, theinhibition constant (Ki) of DAB on GP was reported to be 392 nM(Fosgerau et al., 2000). Although the M1 (DAB) is reported to behighly absorbed (Foral 89%) (Mackay et al., 2003), the Cmax of M1(,300 nM) after oral administration of CS-1036 at 10 mg/kg to ratswas approximately 3/4 of the in vivo Ki on GP in mice. On the otherhand, the suppressive effect of CS-1036 on glucose elevation wasobserved at 0.01 mg/kg. These facts suggest that the pharmacologiceffect of M1 via GP inhibition is almost negligible after CS-1036treatment.In conclusion, CS-1036 exhibited a low absorption, which leads

to high distribution in its site of action, the intestinal lumen. Further-more, most of CS-1036 was stable in the intestinal lumen, althougha small amount of metabolites were produced by intestinal flora. Thesuppression of glucose absorption is suggested to be closely related tothe transition of CS-1036 in the gastrointestinal system; therefore, CS-1036 showed a suppressive effect on postprandial hyperglycemia inrats.

Acknowledgments

The authors thank Takeshi Honda for the supply of compounds and KeiichiFusegawa for assistance with the animal experiments.

Authorship ContributionsParticipated in research design: Honda, Murai, Okuno, Nasu, Koga, Izumi.Conducted experiments: Honda, Kaneno-Urasaki, Murai, Okuno, Kakuta,

Nasu, Namba.Contributed new reagents or analytic tools: Honda, Kaneno-Urasaki, Murai,

Nasu.Performed data analysis: Honda, Kaneno-Urasaki, Murai.Wrote or contributed to the writing of the manuscript: Honda, Murai, Nasu,

Koga, Okuno, Izumi.

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Address correspondence to: Tomohiro Honda, Drug Metabolism and Pharma-cokinetics Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58,Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan. E-mail: honda.tomohiro.us@daiichisankyo.co.jp

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